Colorectal tumor antigens and uses thereof

Abstract

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide. Novel tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) expressed by colorectal tumor cells are described herein. Synthetic long peptides, nucleic acids, compositions, cells, TCRs, antibodies and vaccines derived from these TSAs and TAAs are described. The use of the TSAs / TAAs, nucleic acids, compositions, antibodies, cells and vaccines for the prevention or treatment of cancer expressing the TSAs and / or TAAs, including CRC, is also described.

Description

[0001] TITLE

[0002] COLORECTAL TUMOR ANTIGENS AND USES THEREOF

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims the benefit of U.S. provisional patent application serial No. 63 / 596,384, filed on November 6, 2023, which is incorporated herein by reference in its entirety.

[0005] SEQUENCE LISTING

[0006] A sequence listing is submitted herewith as an XML file named G17971-00098_Seq Listing.xml, created on November 5, 2024, and having a size of ~ 474 323 bytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.

[0007] TECHNICAL FIELD

[0008] The present invention generally relates to the field of cancer, and more particularly to the treatment of cancer such as colorectal cancer (CRC).

[0009] BACKGROUND ART

[0010] Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide, with over 1.8 million cases and 881 ,000 deaths estimated in 2018 alone (1). The incidence of CRC is expected to increase as global socioeconomic changes occur, with a predicted 2.2 million cases and 1.1 million deaths occurring annually by 2030 (1 ,2). This significant disease burden highlights the necessity of developing new and effective treatments against this disease.

[0011] The positive correlation between the abundance of tumor-infiltrating lymphocytes (TILs) and increased overall survival in both colon and rectal cancer suggests that T cells can recognize biologically relevant tumor antigens in these tumors (3,4). The potential immunogenicity of these antigens made immune checkpoint inhibition (ICI) a promising treatment for cancer patients; however, early clinical trials evaluating their efficacy in CRC have yielded mixed results. Colorectal tumors characterized by deficiencies in mismatch repair proteins resulting in the accumulation of repetitive DNA sequences (microsatellites), known as microsatellite instability (MSI), have shown relative success in phase II clinical trials with anti-PD1 treatment (5). In contrast, such treatments have had very little efficacy in clinical trials against microsatellite stable (MSS) tumors that do not possess a high mutational burden, which make up approximately 80% of CRC cases (5,6).

[0012] Given the significance of the immune response in CRC and the limited success of ICI alone, a promising avenue for research in recent years has been a neoantigen-based vaccine or T cell receptor-based therapy which could be used with or without ICI and would ideally bridge the gap in treatment efficacy across MSI and MSS tumors. For example, tumor-associated antigens (TAAs), antigens overexpressed in cancer cells compared to normal cells, have been previously identified in CRC (7,8). While several TAAs have been tested in vaccine and phase I trials against CRC, most were met with ‘limited success’, likely due to the negative selection of such antigens by the thymus (9). In one study, treatment of metastatic CRC with genetically engineered anti- CEA T cells resulted in tumor regression in one patient but ‘serious inflammatory colitis’ in all patients, demonstrating that an adverse autoimmune response is another possible negative consequence of using TAAs (10).

[0013] Due to the mixed responses to TAAs, an effective neoantigen-based therapy would more likely utilize tumor-specific antigens (TSAs), which may be generated through genetic, epigenetic, and post-translational variations, including but not limited to single-nucleotide variants, aberrantly expressed transcripts, or novel splicing events, and are expressed exclusively by the tumor (11). The high prevalence of single nucleotide variants, splice variants, and INDEL mutations in CRC suggests that there is a high probability of unique antigens being presented by the major histocompatibility complex (MHC) of tumors, such that it would be possible to invoke a tumorspecific immune response (12). TSAs have recently been identified in CRC and have demonstrated some success in phase I and II vaccine trials. A 2015 vaccine trial using frameshift antigens originating from MSI-high tumors demonstrated significant and specific immune responses among all patients (13). However, as this study used antigens derived from microsatellite instability frameshifts, these findings are not applicable to the majority of CRC patients. Other studies identifying TSAs in CRC to date have focused exclusively on mutant TSAs (mTSAs) derived from coding regions of the genome (13,14). An investigation of MSS CRC organoids revealed that only 0.5% of non-silent mutations were identified as mTSAs; this was a significantly lower proportion than what was predicted by HLA-binding prediction software (15). In addition, previous studies did not employ mass spectrometry (MS) techniques to quantify the expression of those TSAs on tumor cells, which is information that could influence the therapeutic potential of targeting a given TSA (13,14).

[0014] In view of this, there is a pressing need to identify the tumor-specific antigens that can elicit therapeutic immune responses again CRC. Such antigens could be used as vaccines (± immune checkpoint inhibitors) or as targets for T-cell receptor-based approaches (cell therapy, bispecific biologies).

[0015] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0016] SUMMARY

[0017] In various aspects and embodiments, the present disclosure provides the following items 1 to 96: 1. A tumor antigen peptide (TAP) comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-269.

[0018] 2. The TAP of item 1 , wherein the TAP binds to an HLA-A*01 :01 molecule and comprises or consists of the sequence of SEQ ID NO: 20, 169, 173, 254, 265 or 266.

[0019] 3. The TAP of item 1 , wherein the TAP binds to an HLA-A*02:01 molecule and comprises or consists of the sequence of SEQ ID NO: 6, 27, 30, 44, 58, 84, 124, 132, 154-156, 160, 164, 203, 221 , 224, 243, 264 or 267.

[0020] 4. The TAP of item 1 , wherein the TAP binds to an HLA-A*02:03 molecule and comprises or consists of the sequence of SEQ ID NO: 23, 84, 89, 157, 159, 177, 190, 223 or 225.

[0021] 5. The TAP of item 1 , wherein the TAP binds to an HLA-A*03:01 molecule and comprises or consists of the sequence of SEQ ID NO: 1 , 35, 158 or 202.

[0022] 6. The TAP of item 1 , wherein the TAP binds to an HLA-A*11 :01 molecule and comprises or consists of the sequence of SEQ ID NO: 11 , 25, 32, 34, 45, 86, 87, 128, 131 , 191 , 229, or 233- 238.

[0023] 7. The TAP of item 1 , wherein the TAP binds to an HLA-A*23:01 molecule and comprises or consists of the sequence of SEQ ID NO: 74, 121 , 122, 188, 209 or 210.

[0024] 8. The TAP of item 1 , wherein the TAP binds to an HLA-A*24:02 molecule and comprises or consists of the sequence of SEQ ID NO: 33, 122, 148, 184, 200, 210, 259, or 260.

[0025] 9. The TAP of item 1 , wherein the TAP binds to an HLA-A*25:01 molecule and comprises or consists of the sequence of SEQ ID NO: 16, 17, 90, 92, 104, 112, 115, 120 or 139.

[0026] 10. The TAP of item 1 , wherein the TAP binds to an HLA-A*26:01 molecule and comprises or consists of the sequence of SEQ ID NO: 4, 61 , 103, 118, or 242.

[0027] 11 . The TAP of item 1 , wherein the TAP binds to an HLA-A*29:01 molecule and comprises or consists of the sequence of SEQ ID NO: 21 , 204, 242, 251 , or 255.

[0028] 12. The TAP of item 1 , wherein the TAP binds to an HLA-A*32:01 molecule and comprises or consists of the sequence of SEQ ID NO: 153 or 162.

[0029] 13. The TAP of item 1 , wherein the TAP binds to an HLA-A*68:01 molecule and comprises or consists of the sequence of SEQ ID NO: 15, 49, 69, 102, 116, 117, 119, 183 or 192.

[0030] 14. The TAP of item 1 , wherein the TAP binds to an HLA-B*07:02 molecule and comprises or consists of the sequence of SEQ ID NO: 38, 47, 54, 66, 88, 205, 227, 228, 245, 246, or 256.

[0031] 15. The TAP of item 1 , wherein the TAP binds to an HLA-B*8:01 molecule and comprises or consists of the sequence of SEQ ID NO: 46, 99, 143, or 182.

[0032] 16. The TAP of item 1 , wherein the TAP binds to an HLA-B*13:02 molecule and comprises or consists of the sequence of SEQ ID NO: 51 or 160.

[0033] 17. The TAP of item 1 , wherein the TAP binds to an HLA-B*14:02 molecule and comprises or consists of the sequence of SEQ ID NO: 101 , 126 or 232. 18. The TAP of item 1 , wherein the TAP binds to an HLA-B*15:02 molecule and comprises or consists of the sequence of SEQ ID NO: 63, 98, 100, 114, 133, 244, or 248.

[0034] 19. The TAP of item 1 , wherein the TAP binds to an HLA-B*15:03 molecule and comprises or consists of the sequence of SEQ ID NO: 5, 22, 53, 130, 135, 145, 158, 181 , 201 , 204, 222, 226, or 257.

[0035] 20. The TAP of item 1 , wherein the TAP binds to an HLA-B*15:25 molecule and comprises or consists of the sequence of SEQ ID NO: 9, 10, 55, 83, 158, 204, 207, 226, 257, or 258.

[0036] 21 . The TAP of item 1 , wherein the TAP binds to an HLA-B*18:01 molecule and comprises or consists of the sequence of SEQ ID NO: 12, 93-96, 105, 108, 215, 219, 239, 261 , or 263.

[0037] 22. The TAP of item 1 , wherein the TAP binds to an HLA-B*27:05 molecule and comprises or consists of the sequence of SEQ ID NO: 68 or 230.

[0038] 23. The TAP of item 1 , wherein the TAP binds to an HLA-B*35:01 molecule and comprises or consists of the sequence of SEQ ID NO: 179 or 206.

[0039] 24. The TAP of item 1 , wherein the TAP binds to an HLA-B*35:02 molecule and comprises or consists of the sequence of SEQ ID NO: 38, 113, or 171.

[0040] 25. The TAP of item 1 , wherein the TAP binds to an HLA-B*35:03 molecule and comprises or consists of the sequence of SEQ ID NO: 59, 76, 113, 125, 140, 170, 247, or 268.

[0041] 26. The TAP of item 1 , wherein the TAP binds to an HLA-B*38:01 molecule and comprises or consists of the sequence of SEQ ID NO: 65, 75, 134, 180, 189, 200, 220, 241 , or 252.

[0042] 27. The TAP of item 1 , wherein the TAP binds to an HLA-B*38:02 molecule and comprises or consists of the sequence of SEQ ID NO: 19, 189, or 252.

[0043] 28. The TAP of item 1 , wherein the TAP binds to an HLA-B*40:01 molecule and comprises or consists of the sequence of SEQ ID NO: 64, 80, 81 , 129, 142, 166, 167, 194, 195, 198, 213, 216, 218, or 250.

[0044] 29. The TAP of item 1 , wherein the TAP binds to an HLA-B*40:02 molecule and comprises or consists of the sequence of SEQ ID NO: 18, 29, 52, 56, 151 , 185, 198, 216 or 218.

[0045] 30. The TAP of item 1 , wherein the TAP binds to an HLA-B*44:02 molecule and comprises or consists of the sequence of SEQ ID NO: 97, 176, 199, or 215.

[0046] 31 . The TAP of item 1 , wherein the TAP binds to an HLA-B*44:03 molecule and comprises or consists of the sequence of SEQ ID NO: 3, 13, 43, 82, 97, 105-108, 110, 111 , 175, 186, 196, 197, 199, 212, 214, or 215.

[0047] 32. The TAP of item 1 , wherein the TAP binds to an HLA-B*45:01 molecule and comprises or consists of the sequence of SEQ ID NO: 14, 24, or 109.

[0048] 33. The TAP of item 1 , wherein the TAP binds to an HLA-B*49:01 molecule and comprises or consists of the sequence of SEQ ID NO: 152, 165, 187, 217, 240, or 262.

[0049] 34. The TAP of item 1 , wherein the TAP binds to an HLA-B*51 :01 molecule and comprises or consists of the sequence of SEQ ID NO: 91. 35. The TAP of item 1 , wherein the TAP binds to an HLA-B*52:01 molecule and comprises or consists of the sequence of SEQ ID NO: 172 or 190.

[0050] 36. The TAP of item 1 , wherein the TAP binds to an HLA-B*54:01 molecule and comprises or consists of the sequence of SEQ ID NO: 245 or 269.

[0051] 37. The TAP of item 1 , wherein the TAP binds to an HLA-B*56:01 molecule and comprises or consists of the sequence of SEQ ID NO: 7, 8, 67, 71 , 85, 137, 161 , or 245.

[0052] 38. The TAP of item 1 , wherein the TAP binds to an HLA-B*57:01 molecule and comprises or consists of the sequence of SEQ ID NO: 28, 147, 163, or 208.

[0053] 39. The TAP of item 1 , wherein the TAP binds to an HLA-C*01 :02 molecule and comprises or consists of the sequence of SEQ ID NO: 37, 40, or 193.

[0054] 40. The TAP of item 1 , wherein the TAP binds to an HLA-C*03:03 molecule and comprises or consists of the sequence of SEQ ID NO: 72.

[0055] 41 . The TAP of item 1 , wherein the TAP binds to an HLA-C*03:04 molecule and comprises or consists of the sequence of SEQ ID NO: 36, 50, 60, 70, 77, 79, 136, 141 , 149, or 174.

[0056] 42. The TAP of item 1 , wherein the TAP binds to an HLA-C*04:01 molecule and comprises or consists of the sequence of SEQ ID NO: 123, 127, 168, 178, or 253.

[0057] 43. The TAP of item 1 , wherein the TAP binds to an HLA-C*05:01 molecule and comprises or consists of the sequence of SEQ ID NO: 253.

[0058] 44. The TAP of item 1 , wherein the TAP binds to an HLA-C*06:02 molecule and comprises or consists of the sequence of SEQ ID NO: 231 or 232.

[0059] 45. The TAP of item 1 , wherein the TAP binds to an HLA-C*07:01 molecule and comprises or consists of the sequence of SEQ ID NO: 68.

[0060] 46. The TAP of item 1 , wherein the TAP binds to an HLA-C*07:04 molecule and comprises or consists of the sequence of SEQ ID NO: 39.

[0061] 47. The TAP of item 1 , wherein the TAP binds to an HLA-C*08:01 molecule and comprises or consists of the sequence of SEQ ID NO: 2, 138, 150, or 249.

[0062] 48. The TAP of item 1 , wherein the TAP binds to an HLA-C*08:02 molecule and comprises or consists of the sequence of SEQ ID NO: 144 or 253.

[0063] 49. The TAP of item 1 , wherein the TAP binds to an HLA-C*12:03 molecule and comprises or consists of the sequence of SEQ ID NO: 26, 42 or 57.

[0064] 50. The TAP of item 1 , wherein the TAP binds to an HLA-C*15:02 molecule and comprises or consists of the sequence of SEQ ID NO: 136.

[0065] 51. The TAP of item 1 , wherein the TAP binds to an HLA-C*15:05 molecule and comprises or consists of the sequence of SEQ ID NO: 41 , 62, or 136.

[0066] 52. The TAP of item 1 , wherein the TAP binds to an HLA-C*16:01 molecule and comprises or consists of the sequence of SEQ ID NO: 31 , 48, 78, 146, 211 , or 257. 53. The TAP of any one of items 1-52, which is encoded by a sequence located a non-protein coding region of the genome.

[0067] 54. The TAP of item 53, wherein said non-protein coding region of the genome is an intergenic region.

[0068] 55. The TAP of item 53, wherein said non-protein coding region of the genome is a long noncoding RNAs.

[0069] 56. A combination comprising at least two of the TAPs defined in any one of items 1-55.

[0070] 57. A synthetic long peptide (SLP) comprising at least one, for example at least 5, 10, 15 or 20, of the amino acid sequences defined in item 1 , or a nucleic acid encoding the SLP.

[0071] 58. A nucleic acid encoding the TAP defined in any one of items 1-55, the combination of item 56 or the SLP of item 57.

[0072] 59. The nucleic acid of item 58, wherein the nucleic acid is an mRNA.

[0073] 60. The nucleic acid of item 59, wherein the mRNA comprises one or more 5’-end modifications, 3’-end modifications, and / or modified nucleosides to increase stability, improve translation and / or reduce immunogenicity of the mRNA.

[0074] 61 . The nucleic acid of item 58, wherein the nucleic acid is a DNA.

[0075] 62. The nucleic acid of item 61 , wherein the nucleic acid is a component of a viral vector.

[0076] 63. A vesicle or particle comprising the TAP, combination, SLP, or nucleic acid of any one of items 1 to 62.

[0077] 64. The vesicle or particle of item 63, which is a lipid nanoparticle (LNP).

[0078] 65. The vesicle or particle of item 63 or 64, which comprises a cationic lipid.

[0079] 66. A composition comprising the TAP, combination, SLP or nucleic acid of any one of items 1 to 62, or the vesicle or particle of any one of items 63-65, and a pharmaceutically acceptable carrier.

[0080] 67. A vaccine comprising the TAP, combination, SLP or nucleic acid of any one of items 1 to 62, the vesicle or particle of any one of items 63-65, or the composition of item 66, and an adjuvant.

[0081] 68. An isolated major histocompatibility complex (MHC) class I molecule comprising the TAP of any one of items 1-55 in its peptide binding groove.

[0082] 69. The isolated MHC class I molecule of item 68, which is in the form of a multimer.

[0083] 70. The isolated MHC class I molecule of item 69, wherein said multimer is a tetramer.

[0084] 71 . An isolated cell comprising (i) the TAP of any one of items 1 -55, (ii) the combination of item 56; (iii) the SLP of item 57; or (iv) a vector comprising a nucleotide sequence encoding the TAP of any one of items 1-55, the combination of item 56 or the SLP of item 57.

[0085] 72. An isolated cell expressing at its surface major histocompatibility complex (MHC) class I molecules comprising the TAP or combination of any one of items 1-56 in their peptide binding groove. 73. The cell of item 71 or 72, which is an antigen-presenting cell (APC).

[0086] 74. The cell of item 73, wherein said APC is a dendritic cell.

[0087] 75. A T-cell receptor (TCR) that specifically recognizes the isolated MHC class I molecule of any one of items 68-70 and / or MHC class I molecules expressed at the surface of the cell of any one of items 72-74.

[0088] 76. The TCR of item 75, which is a soluble TCR.

[0089] 77. An antibody or an antigen-binding fragment thereof that specifically binds to the isolated MHC class I molecule of any one of items 68-70 and / or MHC class I molecules expressed at the surface of the cell of any one of items 72-74.

[0090] 78. The TCR of item 75 or 76, or the antibody or antigen-binding fragment thereof according to item 77, which is a bispecific TCR or a bispecific antibody or antigen-binding fragment thereof.

[0091] 79. The TCR, antibody or antigen-binding fragment thereof according to item 78, wherein the bispecific antibody or antigen-binding fragment thereof is a single-chain diabody (scDb).

[0092] 80. The TCR, antibody or antigen-binding fragment thereof according to item 78 or 79, wherein the bispecific TCR, antibody or antigen-binding fragment thereof also specifically binds to a T cell signaling molecule.

[0093] 81 . The TCR, antibody or antigen-binding fragment thereof according to item 80, wherein the T cell signaling molecule is a CD3 chain.

[0094] 82. A chimeric antigen receptor (CAR) comprising the antibody or an antigen-binding fragment thereof of any one of items 77-81 , or a nucleic acid encoding said CAR.

[0095] 83. An isolated cell expressing at its cell surface the TCR of item 75 or the CAR of item 82.

[0096] 84. The isolated cell of item 82, which is a CD8+T lymphocyte.

[0097] 85. A cell population comprising at least 0.5% or 1% of the isolated cell as defined in item 83 or 84.

[0098] 86. A method of treating cancer in a subject comprising administering to the subject an effective amount of:

[0099] (a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1-269 or any combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1-269;

[0100] (b) at least one nucleic acid encoding the TAP, combination thereof or SLP defined in (a);

[0101] (c) a vesicle or particle comprising the TAP, combination thereof or SLP defined in (a) or the at least one nucleic acid defined in (b);

[0102] (d) a composition comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), or the vesicle or particle defined in (c), and a pharmaceutically acceptable carrier; (e) a vaccine comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), the vesicle or particle defined in (c), or the composition defined in (d), and an adjuvant;

[0103] (f) a cell expressing at its surface major histocompatibility complex (MHC) class I molecules comprising the TAP or combination thereof defined in (a) in their peptide binding groove;

[0104] (g) a cell expressing at its cell surface a T-cell receptor (TCR) that specifically recognizes MHC class I molecules expressed at the surface of the cell defined in (f); or

[0105] (h) a soluble TCR, an antibody or an antigen-binding fragment thereof that specifically binds to the MHC class I molecules expressed at the surface of the cell defined in (f).

[0106] 87. The method of item 86, wherein the cancer is colorectal cancer.

[0107] 88. The method of item 86 or 87, further comprising administering at least one additional antitumor agent or therapy to the subject.

[0108] 89. The method of item 88, wherein said at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, an immune checkpoint inhibitor, radiotherapy or surgery.

[0109] 90. Use of:

[0110] (a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1-269 or any combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1-269;

[0111] (b) at least one nucleic acid encoding the TAP, combination thereof or SLP defined in (a);

[0112] (c) a vesicle or particle comprising the TAP, combination thereof or SLP defined in (a) or the at least one nucleic acid defined in (b);

[0113] (d) a composition comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), or the vesicle or particle defined in (c), and a pharmaceutically acceptable carrier;

[0114] (e) a vaccine comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), the vesicle or particle defined in (c), or the composition defined in (d), and an adjuvant;

[0115] (f) a cell expressing at its surface major histocompatibility complex (MHC) class I molecules comprising the TAP or combination thereof defined in (a) in their peptide binding groove;

[0116] (g) a cell expressing at its cell surface a T-cell receptor (TCR) that specifically recognizes MHC class I molecules expressed at the surface of the cell defined in (f); or

[0117] (h) a soluble TCR, an antibody or an antigen-binding fragment thereof that specifically binds to the MHC class I molecules expressed at the surface of the cell defined in (f); for treating cancer in a subject, or for the manufacture of a medicament for treating cancer in a subject.

[0118] 91 . The use of item 90, wherein the cancer is colorectal cancer. 92. The use of item 90 or 91 , further comprising the use at least one additional antitumor agent or therapy to the subject.

[0119] 93. The use of item 92, wherein said at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, an immune checkpoint inhibitor, radiotherapy or surgery.

[0120] 94. An agent for use in treating cancer in a subject, wherein the agent is:

[0121] (a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1-269 or any combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1-269;

[0122] (b) at least one nucleic acid encoding the TAP, combination thereof or SLP defined in (a);

[0123] (c) a vesicle or particle comprising the TAP, combination thereof or SLP defined in (a) or the at least one nucleic acid defined in (b);

[0124] (d) a composition comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), or the vesicle or particle defined in (c), and a pharmaceutically acceptable carrier;

[0125] (e) a vaccine comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), the vesicle or particle defined in (c), or the composition defined in (d), and an adjuvant;

[0126] (f) a cell expressing at its surface major histocompatibility complex (MHC) class I molecules comprising the TAP or combination thereof defined in (a) in their peptide binding groove;

[0127] (g) a cell expressing at its cell surface a T-cell receptor (TCR) that specifically recognizes MHC class I molecules expressed at the surface of the cell defined in (f); or

[0128] (h) a soluble TCR, an antibody or an antigen-binding fragment thereof that specifically binds to the MHC class I molecules expressed at the surface of the cell defined in (f).

[0129] 95. The agent for use of item 94, wherein the cancer is colorectal cancer.

[0130] 96. The agent for use of item 94 or 95, further comprising the use at least one additional antitumor agent or therapy to the subject.

[0131] 97. The agent for use of item 96, wherein said at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, an immune checkpoint inhibitor, radiotherapy or surgery.

[0132] Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0133] BRIEF DESCRIPTION OF DRAWINGS

[0134] In the appended drawings: FIGs. 1A-B show the proteogenomic workflow for the discovery of tumor antigens (TAs) in colorectal primary tumor samples. FIG. 1A: Primary tumor biopsies obtained from 26 individuals were processed for both RNA sequencing and major histocompatibility complex class I (MHC-I) immunoprecipitation (IP). RNA sequencing data were used for both the transcriptomic characterization of the samples and the generation of customized global cancer proteome databases. MHC-I associated peptides (MAPs) isolated via IP were analyzed by LC-MS / MS and spectra were searched against the respective personalized cancer database using PEAKS X. After validating the sequences of peptide candidates their therapeutic potentials were evaluated by determining their inter-tumoral distribution. FIG. 1B: Paired-end RNA sequencing (RNA-seq) of each primary CRC specimen was used to create a personalized cancer database, consisting of a canonical cancer proteome and a cancer-specific proteome. This was achieved by generating cancer-specific kmers which, once combined into contigs, are translated into three reading frames to encompass non-canonical sequences from any genomic origin. Cancer-specific kmers were obtained following the subtraction of mTEC-derived kmers, which approximated the expression of these sequences in normal tissues.

[0135] FIGs. 2A-C show the classification of samples across different transcriptomic signatures. FIG. 2A: Expression heat map of 20 genes of the immunologic constant rejection (ICR) signature. Expression values were normalized, log2-transformed, z scored by row (7-9). Columns represent samples (n = 26) annotated with ICR, Consensus Molecular Subtypes (CMS) (10), and Microsatellite instability (MSS / MSI status) (4). FIG. 2B: RNA transcript biotype classes proportion. Transcripts were quantified using Kallisto (v0.43.0) using an index composed of the following biotypes: coding, non-coding, LINE, LTR, and SINE. Protein coding and non-coding biotypes refer to transcripts annotated in the Ensembl database (v88). Endogenous retroelements (EREs) families (LINE, LTR, SINE) were annotated with RepeatMasker (www.repeatmasker.org). FIG. 2C: Widespread CRC non-synonymous mutations found in the cohort. Freebayes (v1.0.2) was used to detect SNP from the RNA sequencing data. The list of widespread SNP was extracted from the Catalogue of Somatic Mutations in Cancer (COSMIC). Mutations present in samples are highlighted in black.

[0136] FIGs. 3A-C show the distribution of MAPs and tumor antigens across samples. FIG. 3A: Number of MAPs for each primary specimen. An average of 9292 MAPs were identified per sample for a total of 115293 unique MAPs. Samples are grouped based on their MSS and MSI- H status. FIG. 3B: Distribution of MAPs across alleles. The relative proportion of MAPs dotted line) identified in a given allele is shown on the right axis (Proportion). FIG. 3C: Number of tumor- associated antigens (TAAs) and tumor-specific antigens (TSAs) identified in each sample.

[0137] FIG. 4 shows the estimated frequency of TSAs presented by individual CRCs in four different populations. One million simulated patients were generated for each population. A TSA was considered present in a simulated patient when its mRNA was expressed, and the relevant HLA allotype was present. The frequency of TSA’s mRNA expression was calculated using TCGA-COAD RNA-seq data. HLA allotype frequencies were obtained from USA National Marrow Donor Program. Dashed lines indicate the median number of aeTSAs per tumor in each population (median = 6 for European Caucasian, Hispanic, Asian-Pacific Islanders and African American individuals).

[0138] DETAILED DISCLOSURE

[0139] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0140] The terms "comprising", "having", "including", and "containing" are to be construed as open- ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0141] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0142] The use of any and all examples, or exemplary language (“e.g.”, "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.

[0143] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.

[0144] Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).

[0145] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0146] Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.

[0147] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry). Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, 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), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1- 4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0148] In the studies described herein, the present inventors have identified TSA and TAA candidates from CRC specimens using a proteogenomic-based approach. The novel TSA and TAA candidates identified herein may be useful, e.g., for immunotherapies and vaccines against cancers expressing the TSA and TAA candidates, such as CRC.

[0149] In various aspects and embodiments, the present disclosure relates to the tumor antigen peptides described herein (e.g., in Tables 2A-2D), and / or to the use of one or more of these tumor antigen peptides for the treatment of cancer, such as CRC.

[0150] The present disclosure relates to a tumor antigen peptide (TAP), such as a CRC TAP, comprising, or consisting of, one of the following amino acid sequences:

[0151] In another aspect, the present disclosure further relates to the use of a TAP comprising, or consisting of, one of the amino acid sequences set forth in any one of SEQ ID Nos: 1-269, for the treatment of cancer, such as CRC:

[0152] In general, peptides such as tumor antigen peptides (TAPs) presented in the context of HLA class I vary in length from about 7 or 8 to about 15, or preferably 8 to 14 amino acid residues. In some embodiments of the methods of the disclosure, longer peptides comprising the TAP sequences defined herein are artificially loaded into cells such as antigen presenting cells (APCs), processed by the cells and the TAP is presented by MHC class I molecules at the surface of the APC. In this method, peptides / polypeptides longer than 15 amino acid residues can be loaded into APCs, are processed by proteases in the APC cytosol providing the corresponding TAP as defined herein for presentation. In some embodiments, the precursor peptide / polypeptide that is used to generate the TAP defined herein is for example 1000, 500, 400, 300, 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20 or 15 amino acids or less. Thus, all the methods and processes using the TAPs described herein include the use of longer peptides or polypeptides (including the native protein), i.e., tumor antigen precursor peptides / polypeptides, to induce the presentation of the “final” 8-14 TAP following processing by the cell (APCs). In some embodiments, the herein- mentioned TAP is about 8 to 14, 8 to 13, or 8 to 12 amino acids long (e.g., 8, 9, 10, 11 , 12 or 13 amino acids long), small enough for a direct fit in an HLA class I molecule. In an embodiment, the TAP comprises 20 amino acids or less, preferably 15 amino acids or less, more preferably 14 amino acids or less. In an embodiment, the TAP comprises at least 7 amino acids, preferably at least 8 amino acids or less, more preferably at least 9 amino acids.

[0153] The term "amino acid" as used herein includes both L- and D-isomers of the naturally occurring amino acids as well as other amino acids (e.g., naturally-occurring amino acids, non- naturally-occurring amino acids, amino acids which are not encoded by nucleic acid sequences, etc.) used in peptide chemistry to prepare synthetic analogs of TAPs. Examples of naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc. Other amino acids include for example non-genetically encoded forms of amino acids, amino acid analogs as well as a conservative substitution of an L-amino acid. Naturally-occurring non- genetically encoded amino acids and amino acid analogs include, for example, beta-alanine, 3- amino-propionic acid, 2,3-diaminopropionic acid, alpha-aminoisobutyric acid (Aib), 4-amino- butyric acid, / V-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t- butylalanine, t- butylglycine, / V-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1 , 2,3,4- tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, L- homoarginine (Hoarg), N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,- diaminobutyric acid (D- or L-), p-aminophenylalanine, / V-methylvaline, homocysteine, homoserine (HoSer), cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, benzyloxy-tyrosine, P-phenylalanine or 2,3-diaminobutyric acid (D- or L-), etc. These amino acids are well known in the art of biochemistry / peptide chemistry. Thus, one or more of the amino acids in the TAPs described herein (SEQ ID NOs:1-269) may be replaced by a non-genetically encoded amino acid and / or an amino acid analog. The TAPs may also be modified to improve the proteolytic stability of the peptides, for example by the incorporation of methyl-amino acids, p-amino acids or peptoids. In an embodiment, the TAP comprises only naturally-occurring amino acids.

[0154] In embodiments, the TAPs described herein include peptides with altered sequences containing substitutions of functionally equivalent amino acid residues, relative to the herein- mentioned sequences. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity (having similar physico-chemical properties) which acts as a functional equivalent, resulting in a silent alteration. Substitution for an amino acid within the 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 be included in either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions. The herein-mentioned TAP may comprise all L- amino acids, all D-amino acids or a mixture of L- and D-amino acids. In an embodiment, the herein-mentioned TAP comprises all L-amino acids. In an embodiment, in the sequences of the TAPs comprising or consisting of one of sequences of SEQ ID NOs: 1-269, the amino acid residues that do not substantially contribute to interactions with the T-cell receptor may be modified by replacement with other amino acid whose incorporation does not substantially affect T-cell reactivity and does not eliminate binding to the relevant MHC.

[0155] The TAP may also be modified by replacing one or more of the amide bonds of the peptide that may improve chemical stability and / or enhanced biological / pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.). Typical peptide bond replacements include esters, polyamines and derivatives thereof as well as substituted alkanes and alkenes, such as aminomethyl and ketomethylene. For example, the above-mentioned TAP 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-.

[0156] The TAP may also be N- and / or C-terminally capped or modified to prevent degradation, increase stability, affinity and / or uptake. Thus, in another aspect, the present disclosure provides a modified TAP of the formula Z1-X-Z2, wherein X is a TAP comprising, or consisting of, one of the amino acid sequences of SEQ ID NOs:1-269.

[0157] In an embodiment, the amino terminal residue (i.e., the free amino group at the N-terminal end) of the TAP is modified (e.g., for protection against degradation), for example by covalent attachment of a moiety / chemical group (Z1). Z1may be a straight chained or branched alkyl group of one to eight carbons, or an acyl group (R-CO-), wherein R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, iso-propionyl, or iso-butanyl), or an aroyl group (Ar-CO-), wherein Ar is an aryl group. In an embodiment, the acyl group is a C1-C16 or C3-C16 acyl group (linear or branched, saturated or unsaturated), in a further embodiment, a saturated Ci-Ce acyl group (linear or branched) or an unsaturated C3-C6acyl group (linear or branched), for example an acetyl group (CH3-CO-, AC). In an embodiment, Z1is absent. The carboxy terminal residue (i.e., the free carboxy group at the C-terminal end of the TAP) of the TAP may be modified (e.g., for protection against degradation), for example by amidation (replacement of the OH group by a NH2group), thus in such a case Z2is a NH2group. In an embodiment, Z2may be an hydroxamate group, a nitrile group, an amide (primary, secondary or tertiary) group, an aliphatic amine of one to ten carbons such as methyl amine, iso-butylamine, iso-valerylamine or cyclohexylamine, an aromatic or arylalkyl amine such as aniline, napthylamine, benzylamine, cinnamylamine, or phenylethylamine, an alcohol or CH2OH. In an embodiment, Z2is absent. In an embodiment, the TAP comprises one of the amino acid sequences of SEQ ID NOs: 1-269. In an embodiment, the TAP consists of one of the amino acid sequences of SEQ ID NOs: 1-269, i.e., wherein Z1and Z2are absent.

[0158] In another aspect, the present disclosure provides a TAP binding to an HLA-A*01 :01 molecule, comprising or consisting of the sequence of SEQ ID NO: 20, 169, 173, 254, 265 or 266. In another aspect, the present disclosure provides a TAP binding to an HLA-A*02:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 6, 27, 30, 44, 58, 84, 124, 132, 154-156, 160, 164, 203, 221 , 224, 243, 264 or 267.

[0159] In another aspect, the present disclosure provides a TAP binding to an HLA-A*02:03 molecule, comprising or consisting of the sequence of SEQ ID NO: 23, 84, 89, 157, 159, 177, 190, 223 or 225.

[0160] In another aspect, the present disclosure provides a TAP binding to an HLA-A*03:01 molecule, comprising or consisting of the sequence of SEQ ID NO:1 , 35, 158 or 202.

[0161] In another aspect, the present disclosure provides a TAP binding to an HLA-A*11 :01 molecule, comprising or consisting of the sequence of SEQ ID NO: 11 , 25, 32, 34, 45, 86, 87, 128, 131 , 191 , 229, or 233-238.

[0162] In another aspect, the present disclosure provides a TAP binding to an HLA-A*23:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 74, 121 , 122, 188, 209 or 210.

[0163] In another aspect, the present disclosure provides a TAP binding to an HLA-A*24:02 molecule, comprising or consisting of the sequence of SEQ ID NO:33, 122, 148, 184, 200, 210, 259, or 260.

[0164] In another aspect, the present disclosure provides a TAP binding to an HLA-A*25:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 16, 17, 90, 92, 104, 112, 115, 120 or 139.

[0165] In another aspect, the present disclosure provides a TAP binding to an HLA-A*26:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 4, 61 , 103, 118, or 242.

[0166] In another aspect, the present disclosure provides a TAP binding to an HLA-A*29:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 21 , 204, 242, 251 , or 255.

[0167] In another aspect, the present disclosure provides a TAP binding to an HLA-A*32:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 153 or 162.

[0168] In another aspect, the present disclosure provides a TAP binding to an HLA-A*68:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 15, 49, 69, 102, 116, 117, 119, 183 or 192.

[0169] In another aspect, the present disclosure provides a TAP binding to an HLA-B*07:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 38, 47, 54, 66, 88, 205, 227, 228, 245, 246, or 256.

[0170] In another aspect, the present disclosure provides a TAP binding to an HLA-B*08:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 46, 99, 143, or 182.

[0171] In another aspect, the present disclosure provides a TAP binding to an HLA-B*13:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 51 or 160.

[0172] In another aspect, the present disclosure provides a TAP binding to an HLA-B*14:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 101 , 126 or 232. In another aspect, the present disclosure provides a TAP binding to an HLA-B*15:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 63, 98, 100, 114, 133, 244, or 248.

[0173] In another aspect, the present disclosure provides a TAP binding to an HLA-B*15:03 molecule, comprising or consisting of the sequence of SEQ ID NO: 5, 22, 53, 130, 135, 145, 158, 181 , 201 , 204, 222, 226, or 257.

[0174] In another aspect, the present disclosure provides a TAP binding to an HLA-B*15:25 molecule, comprising or consisting of the sequence of SEQ ID NO: 9, 10, 55, 83, 158, 204, 207, 226, 257, or 258.

[0175] In another aspect, the present disclosure provides a TAP binding to an HLA-B*18:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 12, 93-96, 105, 108, 215, 219, 239, 261 , or 263.

[0176] In another aspect, the present disclosure provides a TAP binding to an HLA-B*27:05 molecule, comprising or consisting of the sequence of SEQ ID NO:68 or 230.

[0177] In another aspect, the present disclosure provides a TAP binding to an HLA-B*35:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 179 or 206.

[0178] In another aspect, the present disclosure provides a TAP binding to an HLA-B*35:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 38, 113, or 171.

[0179] In another aspect, the present disclosure provides a TAP binding to an HLA-B*35:03 molecule, comprising or consisting of the sequence of SEQ ID NO: 59, 76, 113, 125, 140, 170, 247, or 268.

[0180] In another aspect, the present disclosure provides a TAP binding to an HLA-B*38:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 65, 75, 134, 180, 189, 200, 220, 241 , or 252.

[0181] In another aspect, the present disclosure provides a TAP binding to an HLA-B*38:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 19, 189, or 252.

[0182] In another aspect, the present disclosure provides a TAP binding to an HLA-B*40:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 64, 80, 81 , 129, 142, 166, 167, 194, 195, 198, 213, 216, 218, or 250.

[0183] In another aspect, the present disclosure provides a TAP binding to an HLA-B*40:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 18, 29, 52, 56, 151 , 185, 198, 216 or 218.

[0184] In another aspect, the present disclosure provides a TAP binding to an HLA-B*44:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 97, 176, 199, or 215.

[0185] In another aspect, the present disclosure provides a TAP binding to an HLA-B*44:03 molecule, comprising or consisting of the sequence of SEQ ID NO: 3, 13, 43, 82, 97, 105-108, 110, 111 , 175, 186, 196, 197, 199, 212, 214, or 215. In another aspect, the present disclosure provides a TAP binding to an HLA-B*45:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 14, 24, or 109.

[0186] In another aspect, the present disclosure provides a TAP binding to an HLA-B*49:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 152, 165, 187, 217, 240, or 262.

[0187] In another aspect, the present disclosure provides a TAP binding to an HLA-B*51 :01 molecule, comprising or consisting of the sequence of SEQ ID NO: 91.

[0188] In another aspect, the present disclosure provides a TAP binding to an HLA-B*52:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 172 or 190.

[0189] In another aspect, the present disclosure provides a TAP binding to an HLA-B*54:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 245 or 269.

[0190] In another aspect, the present disclosure provides a TAP binding to an HLA-B*56:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 7, 8, 67, 71 , 85, 137, 161 , or 245.

[0191] In another aspect, the present disclosure provides a TAP binding to an HLA-B*57:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 28, 147, 163, or 208.

[0192] In another aspect, the present disclosure provides a TAP binding to an HLA-C*01 :02 molecule, comprising or consisting of the sequence of SEQ ID NO: 37, 40, or 193.

[0193] In another aspect, the present disclosure provides a TAP binding to an HLA-C*03:03 molecule, comprising or consisting of the sequence of SEQ ID NO: 72.

[0194] In another aspect, the present disclosure provides a TAP binding to an HLA-C*03:04 molecule, comprising or consisting of the sequence of SEQ ID NO: 36, 50, 60, 70, 77, 79, 136, 141 , 149, or 174.

[0195] In another aspect, the present disclosure provides a TAP binding to an HLA-C*04:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 123, 127, 168, 178, or 253.

[0196] In another aspect, the present disclosure provides a TAP binding to an HLA-C*05:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 253.

[0197] In another aspect, the present disclosure provides a TAP binding to an HLA-C*06:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 231 or 232.

[0198] In another aspect, the present disclosure provides a TAP binding to an HLA-C*07:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 68.

[0199] In another aspect, the present disclosure provides a TAP binding to an HLA-C*07:04 molecule, comprising or consisting of the sequence of SEQ ID NO: 39.

[0200] In another aspect, the present disclosure provides a TAP binding to an HLA-C*08:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 2, 138, 150, or 249.

[0201] In another aspect, the present disclosure provides a TAP binding to an HLA-C*08:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 144 or 253. In another aspect, the present disclosure provides a TAP binding to an HLA-C*12:03 molecule, comprising or consisting of the sequence of SEQ ID NO: 26, 42 or 57.

[0202] In another aspect, the present disclosure provides a TAP binding to an HLA-C*15:02 molecule, comprising or consisting of the sequence of SEQ ID NO: 136.

[0203] In another aspect, the present disclosure provides a TAP binding to an HLA-C*15:05 molecule, comprising or consisting of the sequence of SEQ ID NO: 41 , 62, or 136.

[0204] In another aspect, the present disclosure provides a TAP binding to an HLA-C*16:01 molecule, comprising or consisting of the sequence of SEQ ID NO: 31 , 48, 78, 146, 211 , or 257.

[0205] The TAPs of the disclosure may be produced by expression in a host cell comprising a nucleic acid encoding the TAPs (recombinant expression) or by chemical synthesis (e.g., solidphase peptide synthesis). Peptides can be readily synthesized by manual and / or automated solid phase procedures well known in the art. Suitable syntheses can be performed for example by utilizing "T-boc" or "Fmoc" procedures. Techniques and procedures for solid phase synthesis are described in for example Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the TAPs may be prepared by way of segment condensation, as described, for example, in Liu et al., Tetrahedron Lett. 37: 933-936, 1996; Baca et al., J. Am. Chem. Soc. 117: 1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res. 45: 209-216, 1995; Schnolzer and Kent, Science 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 Res. 31 : 322-334, 1988). Other methods useful for synthesizing the TAPs are described in Nakagawa et al., J. Am. Chem. Soc. 107: 7087-7092, 1985. In an embodiment, the TAP is chemically synthesized (synthetic peptide). Another embodiment of the present disclosure relates to a non-naturally occurring peptide wherein said peptide consists or consists essentially of an amino acid sequences defined herein and has been synthetically produced (e.g., synthesized) as a pharmaceutically acceptable salt. The salts of the TAPs according to the present disclosure differ substantially from the peptides in their state(s) in vivo, as the peptides as generated in vivo are no salts. The non-natural salt form of the peptide may modulate the solubility of the peptide, in particular in the context of pharmaceutical compositions comprising the peptides, e.g., the peptide vaccines as disclosed herein. Preferably, the salts are pharmaceutically acceptable salts of the peptides.

[0206] In an embodiment, the herein-mentioned TAP is substantially pure. A compound is "substantially pure" when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally 75%, 80% or 85%, preferably over 90% and more preferably over 95%, by weight, of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesized or produced by recombinant technology will generally be substantially free from its naturally associated components, e.g., components of its source macromolecule. A nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the nucleic acid is derived. A substantially pure compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a peptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc. In an embodiment, the TAP is in solution. In another embodiment, the TAP is in solid form, e.g., lyophilized.

[0207] In an embodiment, the TAP is encoded by a sequence located a non-protein coding region of the genome. In an embodiment, the TAP is encoded by a sequence located in an intergenic region. In another embodiment, the TAP is encoded by a non-coding RNA (ncRNA).

[0208] In another aspect, the disclosure further provides a synthetic long peptide (SLP) comprising at least one of the TAP described herein. In an embodiment, the SLP comprises at least two TAPs, wherein at least one of the TAP is a TAP as described herein. In an embodiment, the SLP comprises at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 of the TAPs described herein. In an 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 or domains that stabilize the SLP and / or that improve processing and presentation by MHC molecules, for example a sequence comprising a motif cleavable by cellular proteases such as cathepsins. In another embodiment, the SLP comprises at least one of the TAPs described herein, and a TAP that binds to MHC class II molecules. The TAPs may be directly attached to each other, or may be indirectly attached via a linker such as a short amino acid linker. In embodiments, the linker comprises about 4 to about 20 amino acids, or about 4 to about 15 amino acids, e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids. In an embodiment, the linker comprises glycine residues, serine residues, proline residues, threonine residues, or a mixture thereof. The linker may include sequences promoting the processing of the SLP to release the TAPs, such as a cathepsin-sensitive linker (e.g., a linker of 4-6 amino acids comprising the sequence LVGS (SEQ ID NO:539), ASLG (SEQ ID NO:540), PIVG (SEQ ID NO:541), LLSV (SEQ ID NO:542), VLSVG (SEQ ID NO:543) or LLSVGG (SEQ ID NO:544), see Rabu et al., Oncoimmunology. 2019; 8(4): e1560919). In an embodiment, the SLP has a length of 500, 400, 300, 200, 150, 100, 90, 80, 70, 60 or 50 amino acids or less. In a further embodiment, the SLP has a length of 20 to 50, 45 or 40 amino acids, for example from 20 or 25 amino acids to 30, 35 or 40 amino acids.

[0209] In another aspect, the disclosure further provides a nucleic acid (isolated) encoding the herein-mentioned TAPs or a tumor antigen precursor-peptide or SLP. In an embodiment, the nucleic acid comprises from about 24 nucleotides to about 1200 nucleotides, from about 24 to about 1000, 900, 800, 700, 600, 500, 400, 300 or 200 nucleotides, for example from about 24 to about 150 or 100 nucleotides, for example 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 , 54, 57, 60, 53, 66, 69, or 72 nucleotides. "Isolated", as used herein, refers to a peptide or nucleic acid molecule separated from other components that are present in the natural environment of the molecule or a naturally occurring source macromolecule (e.g., including other nucleic acids, proteins, lipids, sugars, etc.). "Synthetic", as used herein, refers to a peptide or nucleic molecule that is not isolated from its natural sources, e.g., which is produced through recombinant technology or using chemical synthesis. In an embodiment, the nucleic acid (DNA, RNA) encoding the TAP or SLP of the disclosure comprises any one of the sequences set forth in the table below or a corresponding RNA sequence. In an embodiment, the nucleic acid encoding the TAP or SLP is an mRNA molecule. In other embodiments, the nucleic acid encoding the TAP or SLP is a self-amplifying mRNA (saRNA), a trans-amplifying mRNA (taRNA) or a circular mRNA (circRNA) (see, e.g., Liu et al., Nature Reviews Cancer, Volume 23, August 2023, pages 526-543).

[0210]

[0211]

[0212] Of course, because of the degeneracy of the genetic code, the TAPs described herein may be encoded by variants of the above-noted sequences.

[0213] A nucleic acid of the disclosure may be used for recombinant expression of the TAP or SLP of the disclosure, and may be included in a vector or plasmid, such as a cloning vector or an expression vector, which may be transfected into a host cell. In an embodiment, the disclosure provides a cloning, expression or viral vector or plasmid comprising a nucleic acid sequence encoding the TAP of the disclosure. Alternatively, a nucleic acid encoding a TAP of the disclosure may be incorporated into the genome of the host cell. In either case, the host cell expresses the TAP or protein encoded by the nucleic acid. The term “host cell” as used herein refers not only to the particular subject cell, but to the progeny or potential progeny of such a cell. A host cell can be any prokaryotic (e.g., E. coll) or eukaryotic cell (e.g., insect cells, yeast cells, plant cells, or mammalian cells) capable of expressing the TAPs described herein. The vector or plasmid contains the necessary elements for the transcription and translation of the inserted coding sequence, and may contain other components such as resistance genes, cloning sites, etc. Methods that are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding peptides or polypeptides and appropriate transcriptional and translational control / regulatory elements operably linked thereto. 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, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. "Operably linked" refers to a juxtaposition of components, particularly nucleotide sequences, such that the normal function of the components can be performed. Thus, a coding sequence that is operably linked to regulatory sequences refers to a configuration of nucleotide sequences wherein the coding sequences can be expressed under the regulatory control, that is, transcriptional and / or translational control, of the regulatory sequences. "Regulatory / control region" or "regulatory / control sequence", as used herein, refers to the non-coding nucleotide sequences that are involved in the regulation of the expression of a coding nucleic acid. Thus, the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like. The vector (e.g., expression vector) may have the necessary 5' upstream and 3' downstream regulatory elements such as promoter sequences such as CMV, PGK and EF-1a promoters, ribosome recognition and binding TATA box, and 3' UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell. Other suitable promoters include the constitutive promoter of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and Rous sarcoma vims promoter. Human gene promoters may also be used, including, but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In certain embodiments inducible promoters are also contemplated as part of the vectors expressing the TAP. This provides a molecular switch capable of turning on expression of the polynucleotide sequence of interest or turning off expression. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter. Examples of vectors are plasmid, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M 13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno- associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). Examples of expression vectors are Lenti-X™ Bicistronic Expression System (Neo) vectors (Contech), pCIneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pl_enti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequences of the TAPs disclosed herein can be ligated into such expression vectors for the expression of the TAP in mammalian cells.

[0214] In certain embodiments, the nucleic acids encoding the TAP of the present disclosure are provided in a viral vector. A viral vector can be those derived from adenovirus, vaccinia virus, retrovirus, lentivirus, or foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the coding sequence for the various TAPs or SLPs described herein in place of nonessential viral genes. In another embodiment, the nucleic acids encoding the TAP or SLP of the present disclosure are provided in a self-amplifying or self-replicating RNA (saRNA or srRNA) vectors. srRNAs are derived from positive-strand RNA viruses where the structural proteins have been removed and replaced with heterologous genes of interest. srRNAs have been successfully derived from flaviviruses, nodamura viruses, nidoviruses, and alphaviruses with therapeutic versions of the technology providing the structural proteins in trans to create single cycle viral replicon particles (VRPs) (see, e.g., 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). The vector and / or particle can be utilized for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0215] In embodiment, the nucleic acid (DNA, RNA) encoding the TAP or SLP of the disclosure is comprised within a vesicle or nanoparticle such as a lipid vesicle (e.g., liposome) or lipid nanoparticle (LNP), or any other suitable vehicle (e.g., mRNA packaging systems). Thus, in another aspect, the present disclosure provides a vesicle or nanoparticle, such as a lipid vesicle or nanoparticle, comprising a nucleic acid, such as an mRNA, encoding one or more of the TAP or SLP described herein.

[0216] The term liposome as used herein in accordance with its usual meaning, referring to microscopic lipid vesicles composed of a bilayer of phospholipids or any similar amphipathic lipids (e.g., sphingolipids) encapsulating an internal aqueous medium. The term “lipid nanoparticle” refers to liposome-like structure that may include one or more lipid bilayer rings surrounding an internal aqueous medium similar to liposomes, or micellar-like structures that encapsulates molecules (e.g., nucleic acids) in a non-aqueous core. Lipid nanoparticles typically contain cationic lipids, such as ionizable cationic lipids. Examples of cationic lipids that may be used for LNPs include DOTMA, DOSPA, DOTAP, DOPE, ePC, DLin- MC3-DMA, C12-200, ALC-0315, CKK-E12, Lipid H (SM-102), OF-Deg-Lin, A2-lso5-2DC18, 3060iio, BAME-O16B, TT3, 9A1 P9, FTT5, COATSOME® SS-E, COATSOME® SS-EC, COATSOME® SS-OC and COATSOME® SS-OP (see, e.g., Hou etal., Nature Reviews Materials, volume s, pages 1078-1094 (2021); Tenchov et al., ACS Nano, 15, 16982-17015 (2021).

[0217] Liposomes and lipid nanoparticles typically include other lipid components such as lipids, lipid-like materials, and polymers that can improve liposome or nanoparticle properties, such as stability, delivery efficacy, tolerability and biodistribution. These include phospholipids (e.g., phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidylglycerol) such as 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and DOPE, sterols (such as cholesterol and cholesterol derivatives), PEGylated lipids (PEG-lipids) such as 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG) and 1 ,2- distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DSG).

[0218] In an embodiment, the lipid nanoparticle according to the present disclosure comprises one or more cationic lipids, such as ionizable cationic lipids. Examples of ionizable cationic lipids include those listed in PCT publications Nos. WO 2017 / 061150 and WO 2019 / 188867, which encompassed ionizable cationic lipids commercialized under the tradenames COATSOME® SS- E, COATSOME® SS-EC, COATSOME® SS-OC and COATSOME® SS-OP.

[0219] The nucleic acid (e.g., mRNA) encoding one or more of the TAPs, may be modified, for example to increase stability and / or reduce immunogenicity. For example, the 5’ end may be capped to stabilize the molecule and decrease immunogenicity (for example, as described in US10519189 and US10494399). One or more nucleosides of the mRNA may be modified or substituted with 1 -methyl pseudo-uridine, pseudouridine (qj), A / 6-methyladenosine, 5- methylcytidine and / or 5-methyluridine to increase stability of the molecule, improve translation efficacy, and / or reduce recognition of the molecule by the innate immune system. A form of modified nucleosides are described in US9371511. Other types of modifications that may be made to the mRNA include incorporation of anti-reverse cap analog (ARCA), 5'-methyl-cytidine triphosphate (m5CTP), N6-methyl-adenosine-5'-triphosphate (m6ATP), 2-thio-uridine triphosphate (s2UTP), pseudouridine triphosphate, N1Methylpseudouridine triphosphate or 5- Methoxyuridine triphosphate (5moUTP). The mRNA may also include additional modifications to the 5'- and / or 3'-untranslated regions (UTRs) and polyadenylation (polyA) tail (see, for example, Kim et al., Molecular & cellular toxicology vol. 18,1 (2022): 1-8). The poly(A) tail preferably comprises 100-200 nucleotides, and more preferably 120-150 nucleotides, and may include modified adenosines. All these modifications and other modifications to the nucleic acid (e.g., mRNA) encoding the TAP are encompassed by the present disclosure.

[0220] In another aspect, the present disclosure provides an MHC class I molecule comprising (i.e., presenting or bound to) one or more of the CRC TAPs comprising or consisting of the sequence of SEQ ID NOs: 1-269 defined herein.

[0221] In an embodiment, the MHC class I molecule is an HLA-A*01 :01 molecule. In an embodiment, the MHC class I molecule is an HLA-A*02:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A*02:03 molecule. In an embodiment, the MHC class I molecule is an HLA-A*03:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A*11 :01 molecule. In an embodiment, the MHC class I molecule is an HLA-A*23:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A*24:02 molecule. In an embodiment, the MHC class I molecule is an HLA-A*25:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A*26:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A*29:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A*32:01 molecule. In an embodiment, the MHC class I molecule is an HLA-A*68:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*07:02 molecule. In an embodiment, the MHC class I molecule is an HLA-B*08:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*13:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*13:02 molecule. In an embodiment, the MHC class I molecule is an HLA-B*14:02 molecule. In an embodiment, the MHC class I molecule is an HLA-B*15:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*15:02 molecule. In an embodiment, the MHC class I molecule is an HLA-B*15:03 molecule. In an embodiment, the MHC class I molecule is an HLA-B*15:25 molecule. In an embodiment, the MHC class I molecule is an HLA-B*18:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*27:05 molecule. In an embodiment, the MHC class I molecule is an HLA-B*35:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*35:02 molecule. In an embodiment, the MHC class I molecule is an HLA-B*35:03 molecule. In an embodiment, the MHC class I molecule is an HLA-B*38:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*38:02 molecule. In an embodiment, the MHC class I molecule is an HLA-B*40:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*40:02 molecule. In an embodiment, the MHC class I molecule is an HLA-B*44:03 molecule. In an embodiment, the MHC class I molecule is an HLA-B*45:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*46:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*49:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*51 :01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*52:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*54:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*56:01 molecule. In an embodiment, the MHC class I molecule is an HLA-B*57:01 molecule. In an embodiment, the MHC class I molecule is an HLA-C*01 :02 molecule. In an embodiment, the MHC class I molecule is an HLA-C*03:03 molecule. In an embodiment, the MHC class I molecule is an HLA-C*03:04 molecule. In an embodiment, the MHC class I molecule is an HLA-C*04:01 molecule. In an embodiment, the MHC class I molecule is an HLA-C*05:01 molecule. In an embodiment, the MHC class I molecule is an HLA-C*06:02 molecule. In an embodiment, the MHC class I molecule is an HLA-C*07:01 molecule. In an embodiment, the MHC class I molecule is an HLA-C*07:04 molecule. In an embodiment, the MHC class I molecule is an HLA-C*08:01 molecule. In an embodiment, the MHC class I molecule is an HLA-C*08:02 molecule. In an embodiment, the MHC class I molecule is an HLA-C*12:03 molecule. In an embodiment, the MHC class I molecule is an HLA-C*15:02 molecule. In an embodiment, the MHC class I molecule is an HLA-C*15:05 molecule. In an embodiment, the MHC class I molecule is an HLA-C*16:01 molecule.

[0222] In an embodiment, the TAP (e.g., comprising or consisting of the sequence of SEQ ID NOs: 1-269 defined herein) is non-covalently bound to the MHC class I molecule (i.e., the TAP is loaded into, or non-covalently bound to the peptide binding groove / pocket of the MHC class I molecule). In another embodiment, the TAP is covalently attached / bound to the MHC class I molecule (alpha chain). In such a construct, the TAP and the MHC class I molecule (alpha chain) are produced as a synthetic fusion protein, typically with a short (e.g., 5 to 20 residues, preferably about 8-12, e.g., 10) flexible linker or spacer (e.g., a polyglycine linker). In another aspect, the disclosure provides a nucleic acid encoding a fusion protein comprising a TAP defined herein fused to an MHC class I molecule (alpha chain). In an embodiment, the MHC class I molecule (alpha chain) - peptide complex is multimerized. Accordingly, in another aspect, the present disclosure provides a multimer of MHC class I molecule loaded (covalently or not) with the herein- mentioned TAP. Such multimers may be attached to a tag, for example a fluorescent tag, which allows the detection of the multimers. A great number of strategies have been developed for the production of MHC multimers, including MHC dimers, tetramers, pentamers, octamers, etc. (reviewed 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. Thus, in another aspect, the present disclosure provides a method for detecting or purifying (isolating, enriching) CD8+T lymphocytes specific for a TAP defined herein, the method comprising contacting a cell population with a multimer of MHC class I molecule loaded (covalently or not) with the TAP; and detecting or isolating the CD8+T lymphocytes bound by the MHC class I multimers. CD8+T lymphocytes bound by the MHC class I multimers may be isolated using known methods, for example fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

[0223] In yet another aspect, the present disclosure provides a cell (e.g., a host cell), in an embodiment an isolated cell, comprising the herein-mentioned nucleic acid, vector or plasmid of the disclosure, i.e., a nucleic acid or vector encoding one or more TAPs or SLPs. In another aspect, the present disclosure provides a cell expressing at its surface an MHC class I molecule (e.g., an MHC class I molecule of one of the alleles disclosed above) bound to or presenting a TAP according to the disclosure. In one embodiment, the host cell is a eukaryotic cell, such as a mammalian cell, preferably a human cell, a cell line or an immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC), such as a dendritic cell. In one embodiment, the host cell is a primary cell, a cell line or an immortalized cell. 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 foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE- dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. supra), and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known, and may be used to deliver the vector or plasmid of the disclosure to a subject for gene therapy.

[0224] Cells such as APCs can be loaded with one or more TAPs using a variety of methods known in the art. As used herein “loading a cell” with a TAP means that RNA or DNA encoding the TAP, or the TAP, is transfected into the cells or alternatively that the APC is transformed with a nucleic acid encoding the TAP. The cell can also be loaded by contacting the cell with exogenous TAPs that can bind directly to MHC class I molecule present at the cell surface (e.g., peptide-pulsed cells). The TAPs may also be fused to a domain or motif that facilitates its presentation by MHC class I molecules, for example to an endoplasmic reticulum (ER) retrieval signal, a C-terminal Lys-Asp-Glu-Leu sequence (see Wang et al., Eur J Immunol. 2004 Dec;34(12):3582-94).

[0225] In another aspect, the present disclosure provides a composition or peptide combination / pool comprising any one of, or any combination of, the TAPs defined herein (or a nucleic acid encoding said peptide(s)). In an embodiment, the composition comprises any combination of the TAPs defined herein (any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TAPs), or a combination of nucleic acids encoding said TAPs). Compositions comprising any combination / sub-combination of the TAPs defined herein are encompassed by the present disclosure. In another embodiment, the combination or pool may comprise one or more known tumor antigens.

[0226] Thus, in another aspect, the present disclosure provides a composition comprising any one of, or any combination of, the TAPs defined herein (e.g., comprising or consisting of the sequence of SEQ ID NOs:1-269 defined herein) and a cell expressing a MHC class I molecule (e.g., a MHC class I molecule of one of the alleles disclosed above). APC for use in the present disclosure are not limited to a particular type of cell and include 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 CD8+T lymphocytes. For example, an APC can be obtained by inducing DCs from peripheral blood monocytes and then contacting (stimulating) the TAPs, either in vitro, ex vivo or in vivo. APC can also be activated to present a TAP in vivo where one or more of the TAPs of the disclosure are administered to a subject and APCs that present a TAP are induced in the body of the subject. The phrase "inducing an APC" or “stimulating an APC” includes contacting or loading a cell with one or more TAPs, or nucleic acids encoding the TAPs such that the TAPs are presented at its surface by MHC class I molecules. As noted herein, according to the present disclosure, the TAPs may be loaded indirectly for example using longer peptides / polypeptides comprising the sequence of the TAPs (including the native protein), which is then processed (e.g., by proteases) inside the APCs to generate the TAP / MHC class I complexes at the surface of the cells. After loading APCs with TAPs and allowing the APCs to present the TAPs, the APCs can be administered to a subject as a vaccine. For example, the ex vivo administration can include the steps of: (a) collecting APCs from a first subject, (b) contacting / loading the APCs of step (a) with a TAP to form MHC class l / TAP complexes at the surface of the APCs; and (c) administering the peptide-loaded APCs to a second subject in need for treatment.

[0227] The first subject and the second subject may be the same subject (e.g., autologous vaccine), or may be different subjects (e.g., allogeneic vaccine). Alternatively, according to the present disclosure, use of a TAP described herein (or a combination thereof) for manufacturing a composition (e.g., a pharmaceutical composition) for inducing antigen-presenting cells is provided. In addition, the present disclosure provides a method or process for manufacturing a pharmaceutical composition for inducing antigen-presenting cells, wherein the method or the process includes the step of admixing or formulating the TAP, or a combination thereof, with a pharmaceutically acceptable carrier. Cells such as APCs expressing a MHC class I molecule (e.g., any of the above-noted HLA molecules) loaded with any one of, or any combination of, the TAPs defined herein, may be used for stimulating / amplifying CD8+T lymphocytes, for example autologous CD8+T lymphocytes. Accordingly, in another aspect, the present disclosure provides a composition comprising any one of, or any combination of, the TAPs defined herein (or a nucleic acid or vector encoding same); a cell expressing an MHC class I molecule and a T lymphocyte, more specifically a CD8+T lymphocyte (e.g., a population of cells comprising CD8+T lymphocytes).

[0228] In an embodiment, the composition further comprises a buffer, an excipient, a carrier, a diluent and / or a medium (e.g., a culture medium). In a further embodiment, the buffer, excipient, carrier, diluent and / or medium is / are pharmaceutically acceptable buffer(s), excipient(s), carrier(s), diluent(s) and / or medium (media). As used herein “pharmaceutically acceptable buffer, excipient, carrier, diluent and / or medium” includes any and all solvents, buffers, binders, lubricants, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and the like that are physiologically compatible, do not interfere with effectiveness of the biological activity of the active ingredient(s) and that are not toxic to the subject. The use of such media and agents for pharmaceutically active substances is well known in the art (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4thedition, Pharmaceutical Press, London UK). Except insofar as any conventional media or agent is incompatible with the active compound (peptides, cells), use thereof in the compositions of the disclosure is contemplated. In an embodiment, the buffer, excipient, carrier and / or medium is a non-naturally occurring buffer, excipient, carrier and / or medium. In an embodiment, one or more of the TAPs defined herein, or the nucleic acids (e.g., mRNAs) encoding said one or more TAPs, are comprised within or complexed to a lipid vesicle or liposome, e.g., a cationic liposome (see, e.g., Vitor MT et al., Recent Pat Drug Deliv Formul. 2013 Aug;7(2):99-110) or suitable other carriers.

[0229] In another aspect, the present disclosure provides a composition comprising one of more of the any one of, or any combination of, the TAPs defined herein (e.g., comprising or consisting of the sequence of SEQ ID NOs: 1-269 defined herein) (or a nucleic acid such as a mRNA encoding said peptide(s)), and a buffer, an excipient, a carrier, a diluent and / or a medium. For compositions comprising cells (e.g., APCs, T lymphocytes), the composition comprises a suitable medium that allows the maintenance of viable cells. Representative examples of such media include saline solution, Earl’s Balanced Salt Solution (Life Technologies®) or PlasmaLyte® (Baxter International®). In an embodiment, the composition (e.g., pharmaceutical composition) is an “immunogenic composition”, “vaccine composition” or “vaccine”. The term “Immunogenic composition”, “vaccine composition” or “vaccine” as used herein refers to a composition or formulation comprising one or more TAPs, nucleic acids or vaccine vector and which is capable of inducing an immune response against the one or more TAPs present therein when administered to a subject. Vaccination methods for inducing an immune response in a mammal (e.g., human) comprise use of a vaccine or vaccine vectorto be administered by any conventional route known in the vaccine field, e.g., via a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface, via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route, or topical administration (e.g., via a transdermal delivery system such as a patch). In an embodiment, the TAP (or a combination thereof) is conjugated to a carrier protein (conjugate vaccine) to increase the immunogenicity of the TAP(s). The present disclosure thus provides a composition (conjugate) comprising a TAP (or a combination thereof), or a nucleic acid encoding the TAP or combination thereof, and a carrier protein. For example, the TAP(s) or nucleic acid(s) may be conjugated or complexed to a Toll-like receptor (TLR) ligand (see, e.g., Zorn et al., Adv Immunol. 2012, 114: 177-201) or polymers / dendrimers (see, e.g., Liu et al., Biomacromolecules. 2013 Aug 12;14(8):2798-806) such as polymer-linked TLR agonists (see, e.g., Lynn et al., Nature Biotechnology 33: 1201-1210 (2015); Lynn et al., Nature Biotechnology 38: 320-332 (2020)). In an embodiment, the immunogenic composition or vaccine further comprises an adjuvant. "Adjuvant" refers to a substance which, when added to an immunogenic agent such as an antigen (TAPs, nucleic acids and / or cells according to the present disclosure), nonspecifically enhances or potentiates an immune response to the agent in the host upon exposure to the mixture. Examples of adjuvants currently used in the field of vaccines include (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gels), squalene, (2) oil-based adjuvants such as oil emulsions and surfactant based formulations, e.g., MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + QS-21), (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), AS04 ([SBAS4] aluminum salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide co-glycolide (PLG), (4) microbial derivatives (natural and synthetic), e.g., monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulators able to self-organize into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified Cholera toxin (CT) and Escherichia coli enterotoxin (LT) (genetically modified bacterial toxins to provide nontoxic adjuvant effects), (5) endogenous human immunomodulators, e.g., hGM-CSF or hlL-12 (cytokines that can be administered either as protein or plasmid encoded), Immudaptin (C3d tandem array) and / or (6) inert vehicles, such as gold particles, and the like. In an embodiment, the vaccine is an RNA vaccine.

[0230] In an embodiment, the TAP(s) (e.g., comprising or consisting of the sequence of SEQ ID NOs: 1-269), or SLPs (or a nucleic acid such as a mRNA encoding said peptide(s)) or composition comprising same is / are in lyophilized form. In another embodiment, the TAP(s), SLP(s), nucleic acid(s) or composition comprising same is / are in a liquid composition. In a further embodiment, the TAP(s) or nucleic acid(s) is / are at a concentration of about 0.01 pg / mL to about 100 pg / mL in the composition. In further embodiments, the TAP(s) or nucleic acid(s) is / are at a concentration of about 0.2 pg / mL to about 50 pg / mL, about 0.5 pg / mL to about 10, 20, 30, 40 or 50 pg / mL, about 1 pg / mL to about 10 pg / mL, or about 2 pg / mL, in the composition.

[0231] As noted herein, cells such as APCs that express an MHO class I molecule loaded with or bound to any one of, or any combination of, the TAPs defined herein, may be used for stimulating / amplifying CD8+T lymphocytes in vivo or ex vivo. Accordingly, in another aspect, the present disclosure provides T cell receptor (TOR) molecules capable of interacting with or binding the herein-mentioned MHO class I molecule / TAP complex, and nucleic acid molecules encoding such TOR molecules, and vectors comprising such nucleic acid molecules. A TOR according to the present disclosure is capable of specifically interacting with or binding a TAP loaded on, or presented by, an MHO class I molecule, preferably at the surface of a living cell in vitro or in vivo. The term TCR as used herein refers to an immunoglobulin superfamily member having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al, Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell and generally is comprised of a heterodimer having a and p chains (also known as TCRa and TCR|3, respectively). Like immunoglobulins, the extracellular portion of TCR chains (e.g., a-chain, p-chain) contain two immunoglobulin regions, a variable region (e.g., TCR variable a region or a and TCR variable p region or P; typically amino acids 1 to 116 based on Rabat numbering at the N-terminus), and one constant region (e.g., TCR constant domain a or Ca and typically amino acids 117 to 259 based on Rabat, TCR constant domain p or cp, typically amino acids 117 to 295 based on Rabat) adjacent to the cell membrane. Also, like immunoglobulins, the variable domains contain complementary determining regions (CDRs, 3 in each chain) separated by framework regions (FRs). In certain embodiments, a TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex.

[0232] A TCR and in particular nucleic acids encoding a TCR of the disclosure may for instance be applied to genetically transform / modify T lymphocytes (e.g., CD8+T lymphocytes) or other types of lymphocytes generating new T lymphocyte clones that specifically recognize an MHC class l / TAP complex. In a particular embodiment, T lymphocytes (e.g., CD8+T lymphocytes) obtained from a patient are transformed to express one or more TCRs that recognize a TAP and the transformed cells are administered to the patient (autologous cell transfusion). In a particular embodiment, T lymphocytes (e.g., CD8+T lymphocytes) obtained from a donor are transformed to express one or more TCRs that recognize a TAP and the transformed cells are administered to a recipient (allogenic cell transfusion). In another embodiment, the disclosure provides a T lymphocyte e.g., a CD8+T lymphocyte transformed / transfected by a vector or plasmid encoding a TAP-specific TCR. In a further embodiment the disclosure provides a method of treating a patient with autologous or allogenic cells transformed with a TAP-specific TCR. In certain embodiments, TCRs are expressed in primary T cells (e.g., cytotoxic T cells) by replacing an endogenous locus, e.g., an endogenous TRAC and / or TRBC locus, using, e.g., CRISPR, TALEN, zinc finger nuclease, or other targeted disruption systems.

[0233] In another embodiment, the present disclosure provides a nucleic acid encoding the abovenoted TCR. In a further embodiment, the nucleic acid is present in a vector, such as the vectors described above.

[0234] In yet a further embodiment the use of a tumor antigen-specific TCR in the manufacture of autologous or allogenic cells for the treatment of cancer, such as CRC, is provided.

[0235] In some embodiments, patients treated with the compositions (e.g., pharmaceutical compositions) of the disclosure are treated prior to or following treatment with an anti-tumor agent and / or immunotherapy (e.g., CAR therapy, immune checkpoint inhibitor therapy). Compositions of the disclosure include: allogenic T lymphocytes (e.g., CD8+T lymphocyte) activated ex vivo against a TAP; allogenic or autologous APC vaccines loaded with a TAP; vaccines including TAPs of nucleic acids (e.g. mRNA) encoding TAPs and allogenic or autologous T lymphocytes (e.g., CD8+T lymphocyte) or lymphocytes transformed with a tumor antigen-specific TCR. The method to provide T lymphocyte clones capable of recognizing a TAP according to the disclosure may be generated for and can be specifically targeted to tumor cells expressing the TAP in a subject (e.g., graft recipient), for example an allogenic T lymphocyte and / or donor lymphocyte infusion (DLI) recipient. Hence the disclosure provides a CD8+T lymphocyte encoding and expressing a T cell receptor capable of specifically recognizing or binding a TAP / MHC class I molecule complex. Said T lymphocyte (e.g., CD8+T lymphocyte) may be a recombinant (engineered) or a naturally selected T lymphocyte. This specification thus provides at least two methods for producing CD8+T lymphocytes of the disclosure, comprising the step of bringing undifferentiated lymphocytes into contact with a TAP / MHC class I molecule complex (typically expressed at the surface of cells, such as APCs) under conditions conducive of triggering T cell activation and expansion, which may be done in vitro or in vivo (i.e. in a patient administered with a APC vaccine wherein the APC is loaded with a TAP or in a patient treated with a TAP vaccine). Using a combination or pool of TAPs bound to MHC class I molecules, it is possible to generate a population CD8+T lymphocytes capable of recognizing a plurality of TAPs. Alternatively, tumor antigen-specific or targeted T lymphocytes may be produced / generated in vitro or ex vivo by cloning one or more nucleic acids (genes) encoding a TCR (more specifically the alpha and beta chains) that specifically binds to a MHC class I molecule / TAP complex (i.e. engineered or recombinant CD8+T lymphocytes). Nucleic acids encoding a TAP-specific TCR of the disclosure, may be obtained using methods known in the art from a T lymphocyte activated against a TAP ex vivo (e.g., with an APC loaded with a TAP); or from an individual exhibiting an immune response against peptide / MHC molecule complex. TAP-specific TCRs of the disclosure may be recombinantly expressed in a host cell and / or a host lymphocyte obtained from a graft recipient or graft donor, and optionally differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). The nucleic acid(s) (transgene(s)) encoding the TCR alpha and beta chains may be introduced into a T cells (e.g., from a subject to be treated or another individual) using any suitable methods such as transfection (e.g., electroporation) or transduction (e.g., using viral vector). The engineered CD8+T lymphocytes expressing a TCR specific for a TAP may be expanded in vitro using well known culturing methods.

[0236] The present disclosure provides methods for making the immune effector cells which express the TCRs as described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells, e.g., immune effector cells isolated from a subject, such as a subject having a colorectal cancer, such that the immune effector cells express one or more TCR as described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a TCR. In this regard, the immune effector cells may be cultured before or after being genetically modified (i.e., transduced or transfected to express a TCR as described herein).

[0237] Prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells may be obtained from a subject. In particular, the immune effector cells for use with the TCRs as described herein comprise T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cell can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL™ separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. In one embodiment of the invention, the cells are washed with PBS. In an alternative embodiment, the washed solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As would be appreciated by those of ordinary skill in the art, a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated flow-through centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. In certain embodiments, the undesirable components of the apheresis sample may be removed in the cell directly resuspended culture media. In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation ofT cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD8+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11 b, CD16, HLA-DR, and CD4. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present disclosure. PBMC may be used directly for genetic modification with the TCRs using methods as described herein. In certain embodiments, after isolation of PBMC, T lymphocytes are further isolated and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion.

[0238] The present disclosure provides isolated immune cells such as T lymphocytes (e.g., CD8+T lymphocytes) that are specifically induced, activated and / or amplified (expanded) by a TAP (i.e., a TAP bound to MHC class I molecules expressed at the surface of cell), or a combination of TAPs. The present disclosure also provides a composition comprising CD8+T lymphocytes capable of recognizing a TAP, or a combination thereof, according to the disclosure (i.e., one or more TAPs bound to MHC class I molecules) and said TAP(s).

[0239] In another aspect, the present disclosure provides a cell population or cell culture (e.g., a CD8+T lymphocyte population) enriched in T lymphocytes (e.g., CD8+T lymphocytes) that specifically recognize one or more MHC class I molecule / TAP complex(es) as described herein. Such enriched population may be obtained by performing an ex vivo expansion of specific T lymphocytes (e.g., TILs) using cells such as APCs that express MHC class I molecules loaded with (e.g., presenting) one or more of the TAPs disclosed herein. “Enriched” as used herein means that the proportion of tumor antigen-specific T lymphocytes (e.g., CD8+T lymphocytes) in the population is significantly higher relative to a native population of cells, i.e., which has not been subjected to a step of ex v / vo-expansion of specific T lymphocytes. In an embodiment, the cell population is a TIL population or is derived from TILs, e.g., TILs isolated from a patient and expanded ex vivo. In a further embodiment, the proportion of TAP-specific T lymphocytes (e.g., CD8+T lymphocytes) in the cell population is at least about 0.5%, for example at least about 1%, 1.5%, 2% or 3%. In some embodiments, the proportion of TAP-specific T lymphocytes (e.g., CD8+T lymphocytes) in the cell population is about 0.5 to about 10%, about 0.5 to about 8%, about 0.5 to about 5%, about 0.5 to about 4%, about 0.5 to about 3%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5% or about 3% to about 4%. Such cell population or culture (e.g., a CD8+T lymphocyte population) enriched in T lymphocytes (e.g., CD8+T lymphocytes) that specifically recognizes one or more MHC class I molecule / peptide (TAP) complex(es) of interest may be used in tumor antigen-based cancer immunotherapy, as detailed below. In some embodiments, the population of TAP-specific T lymphocytes (e.g., CD8+T lymphocytes) is further enriched, for example using affinity- based systems such as multimers of MHC class I molecule loaded (covalently or not) with the TAP(s) defined herein. Thus, the present disclosure provides a purified or isolated population of TAP-specific T lymphocytes (e.g., CD8+T lymphocytes), e.g., in which the proportion of TAP-specific T lymphocytes (e.g., CD8+T lymphocytes) is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof (e.g., a TCR mimic or TCR-like antibody), or a soluble TCR, that specifically binds to a complex comprising a TAP as described herein bound to an HLA molecule, such as the HLA molecules defined herein. The term “antibody or antigen-binding fragment thereof’ as used herein refers to any type of antibody / antibody fragment including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies and antibody fragments so long as they exhibit the desired antigenic specificity / binding activity. Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof. 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., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments, single-chain diabodies (scDbs), bispecific T cell engagers (BiTEs), dual affinity retargeting molecules (DARTs), bivalent scFv-Fcs, and trivalent scFv-Fcs. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to,Hregions (H, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. In an embodiment, the antibody or antigen-binding fragment thereof is a single-chain antibody, preferably a single-chain Fv (scFv). In an embodiment, the antibody or antigen-binding fragment thereof comprises at least one constant domain, e.g., a constant domain of a light and / or heavy chain, or a fragment thereof. In a further embodiment, the antibody or antigen-binding fragment thereof comprises a Fragment crystallizable (Fc) fragment of the constant heavy chain of an antibody. In an embodiment, the antibody or antigen-binding fragment is a scFv comprising a Fc fragment (scFV- Fc). In an embodiment, the scFv component is connected to the Fc fragment by a linker, for example a hinge. The presence of an Fc region is useful to induce a complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), or antibody-dependent cellular cytotoxicity (ADCC) response against a tumor cell.

[0240] In an embodiment, the antibody or antigen-binding fragment thereof is a multispecific antibody or an antigen-binding fragment thereof, such as a bispecific antibody or an antigenbinding fragment thereof, wherein at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognize(s) a complex comprising a TAP as described herein bound to an HLA molecule. In an embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognize(s) an immune cell effector molecule. The term “immune cell effector molecule" refers to a molecule (e.g., protein) expressed by an immune cell and whose engagement by the multispecific antibody or antibody fragment leads to activation of the immune cells. Examples of immune cell effector molecules include the CD3 signaling complex in T cells such as CD8 T cells and the various activating receptors on NK cells (NKG2D, KIR2DS, NKp44, etc.). In a further embodiment, at least one of the antigen-binding domains of the multispecific antibody or antibody fragment recognize(s) and engage(s) the CD3 signaling complex in T cells (e.g., anti-CD3). In a further embodiment, the multispecific antibody or antibody fragment is a single-chain diabody (scDb). In a further embodiment, the scDb comprises a first antibody fragment (e.g., scFv) that binds to a complex comprising a TAP as described herein bound to an HLA molecule and a second antibody fragment (e.g., scFv) that binds to and engages an immune cell effector molecule, such as the CD3 signaling complex in T cells (e.g., anti-CD3 scFv). Such constructs may be used for example to induce the cytotoxic T cell-mediated killing of tumor cells expressing the tumor antigen / MHC complex recognized by the multispecific antibody or antibody fragment. Antibodies or antigen-binding fragments thereof may also be used as a chimeric antigen receptor (CAR) to produce CAR T cells, CAR NK cells, etc. CAR combines a ligand-binding domain (e.g., antibody or antibody fragment) that provides specificity for a desired antigen (e.g., MHC / TAP complex) with an activating intracellular domain (or signal transducing domain) portion, such as a T cell or NK cell activating domain, providing a primary activation signal. Antigen-binding fragments of antibodies, and more particularly scFv, capable of binding to molecules expressed by tumor cells are commonly used as ligand-binding domains in CAR.

[0241] In an embodiment, the soluble TCR is a soluble therapeutic bispecific TCR (see, e.g., Robinson et al., FEBS J. 2021 Nov;288(21):6159-6173; Dilchert et al., Antibodies (Basel). 2022 May 10;11 (2):34).

[0242] In an embodiment, the soluble TCR, antibody or antibody fragment (e.g., TCR-mimic) is attached to an antitumor agent to form an antibody-drug conjugate (ADC). Such ADC permits to target the antitumor agent to tumor cells expressing one or more of the TAPs described herein (see, e.g., Shen et al., Asian J Pharm Sci. 2020 Nov;15(6):777-785).

[0243] The present disclosure also provides a nucleic acid such as an mRNA encoding the soluble TCR, antibody, antibody fragment or CAR described herein. Such nucleic acids may be formulated into suitable vehicles such as lipid nanoparticles are described above, and may be used in the treatment of cancers such as CRC, as described below.

[0244] Thus, in another aspect, the present disclosure provides a host cell, preferably an immune cell such as a T cell or NK cell, expressing the antibody or antibody fragment (e.g., scFv) described herein.

[0245] The present disclosure further relates to a pharmaceutical composition or vaccine comprising the above-noted immune cell (CD8+T lymphocytes, CAR T cell) or population of TAP- specific CD8+T lymphocytes. Such pharmaceutical composition or vaccine may comprise one or more pharmaceutically acceptable excipients and / or adjuvants, as described above.

[0246] In another aspect, the present disclosure further relates to the use of any of the TAP comprising or consisting of any of the sequences of SEQ ID NOs:1-269, nucleic acid, expression vector, T cell receptor, antibody / antibody fragment, cell (e.g., T lymphocyte, APC, CAR T cell), and / or composition according to the present disclosure, or any combination thereof, as a medicament or in the manufacture of a medicament for the treatment of a cancer expressing the TAP, for example a CRC. The present disclosure 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) according to the present disclosure, or any combination thereof, for use in the treatment of a cancer expressing the TAP, for example a CRC, e.g., as a CRC cancer vaccine. The TAP sequences identified herein may be used for the production of synthetic peptides to be used i) for in vitro priming and expansion of tumor antigen-specific T cells to be injected into tumor patients and / or ii) as vaccines to induce or boost the anti-tumor T cell response in cancer patients, e.g., CRC patients.

[0247] In another aspect, the present disclosure provides the use of a TAP or SLP described herein (e.g., comprising or consisting of any of the sequences of SEQ ID NOs:1-269), or a combination thereof (e.g., a peptide pool), or of one or more nucleic acid(s) encoding the TAP(s) or SLP(s), as a vaccine for treating a cancer expressing the TAP, for example a CRC, in a subject. The present disclosure also provides the TAP or SLP described herein, or a combination thereof (e.g., a peptide pool), or of one or more nucleic acid(s) encoding the TAP(s) or SLP(s), for use as a vaccine for treating a cancer expressing the TAP, for example a CRC, in a subject. In an embodiment, the subject is a recipient of TAP-specific T lymphocytes (e.g., CD8+T lymphocytes). Accordingly, in another aspect, the present disclosure provides a method of treating a cancer expressing the TAP, for example a CRC (e.g., of reducing the number of tumor cells, killing tumor cells), said method comprising administering (infusing) to a subject in need thereof an effective amount of T lymphocytes (e.g., CD8+T lymphocytes) recognizing (i.e., expressing a TCR that binds) one or more MHC class I molecule / TAP complexes (expressed at the surface of a cell such as an APC). In an embodiment, the method further comprises administering an effective amount of the TAP or SLP, or a combination thereof, or of one or more nucleic acid(s) encoding the TAP(s), and / or a cell (e.g., an APC such as a dendritic cell) expressing MHC class I molecule(s) loaded with the TAP(s) or SLP(s), to said subject after administration / infusion of said CD8+T lymphocytes. In yet a further embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of a dendritic cell loaded with one or more TAPs. In yet a further embodiment the method comprises administering to a patient in need thereof a therapeutically effective amount of an allogenic or autologous cell that expresses a recombinant TCR that binds to a TAP presented by an MHC class I molecule.

[0248] In another aspect, the present disclosure provides the use of T lymphocytes (e.g., CD8+T lymphocytes) that recognize one or more MHC class I molecules loaded with (presenting) a TAP, or a combination thereof, for treating a cancer expressing the TAP, for example a CRC (e.g., of reducing the number of tumor cells, killing tumor cells) in a subject. In another aspect, the present disclosure provides the use of T lymphocytes (e.g., CD8+T lymphocytes) that recognize one or more MHC class I molecules loaded with (presenting) a TAP, or a combination thereof, for the preparation / manufacture of a medicament for treating a cancer expressing the TAP, for example a CRC (e.g., for reducing the number of tumor cells, killing tumor cells) in a subject. In another aspect, the present disclosure provides T lymphocytes (e.g., CD8+T lymphocytes) that recognize one or more MHC class I molecule(s) loaded with (presenting) a TAP, or a combination thereof, for use in the treatment of a cancer expressing the TAP, for example a CRC (e.g., for reducing the number of tumor cells, killing tumor cells), in a subject. In a further embodiment, the use further comprises the use of an effective amount of a TAP (or a combination thereof), or of one or more nucleic acid(s) encoding the TAP(s), and / or of a cell (e.g., an APC) that expresses one or more MHC class I molecule(s) loaded with (presenting) a TAP, after the use of said TAP-specific T lymphocytes.

[0249] The present disclosure also provides a method of generating an immune response against tumor cells expressing human class I MHC molecules loaded with any of the TAP disclosed herein (e.g., comprising or consisting of any of the sequences of SEQ ID NOs:1-269) or combination thereof in a subject, the method comprising administering cytotoxic T lymphocytes that specifically recognizes the class I MHC molecules loaded with the TAP or combination of TAPs. The present disclosure also provides the use of cytotoxic T lymphocytes that specifically recognizes class I MHC molecules loaded with any of the TAP or combination of TAPs disclosed herein for generating an immune response against tumor cells expressing the human class I MHC molecules loaded with the TAP or combination thereof.

[0250] In an embodiment, the cancer is colorectal cancer. In a further embodiment, the cancer is colon cancer. In another embodiment, the cancer is rectal cancer. In an embodiment, the colorectal cancer is characterized by microsatellite instability (MSI). In an embodiment, the colorectal cancer is characterized by microsatellite stability (MSS). In an embodiment, the colorectal cancer is characterized by RAS (e.g., KRAS) and / or RAF mutations. In another embodiment, the colorectal cancer is resistant / refractory to chemotherapy. In another embodiment, the colorectal cancer is resistant / refractory to EGFR inhibitors.

[0251] In an embodiment, the methods or uses described herein further comprise determining the HLA class I alleles expressed by the patient prior to the treatment / use, and administering or using TAPs that bind to one or more of the HLA class I alleles expressed by the patient. For example, if it is determined that the patient expresses HLA-A*02:01 and HLA-B07*02, any combinations of (i) the TAPs of SEQ ID NO: 19, 34, 47, 105, 112, 132-134, 138, 142, 184, 204, 206, 224, and / or 240 (that bind to HLA-A*0201) and (ii) the TAPs of SEQ ID NO: 37, 43, 72, 187, 209, 210, 225, 226, and / or 235 (that bind to HLA-B07*02) may be administered or used in the patient.

[0252] In an embodiment, the TAP, nucleic acid, expression vector, T cell receptor, antibody / antibody fragment, cell (e.g., T lymphocyte, CAR T or NK cell, APC), and / or composition according to the present disclosure, or any combination thereof, may be used in combination with one or more additional active agents or therapies to treat cancer (e.g., CRC), such as chemotherapy (e.g., vinca alkaloids, agents that disrupt microtubule formation (such as colchicines and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agent (such as tyrosine kinase inhibitors), transitional metal complexes, proteasome inhibitors, antimetabolites (such as nucleoside analogs), alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (such as all-trans retinoic acids or a derivatives thereof), geldanamycin or a derivative thereof (such as 17-AAG), surgery, immune checkpoint inhibitors or immunotherapeutic agents (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 an embodiment, the TAP, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocyte, APC), and / or composition according to the present disclosure is administered / used in combination with an immune checkpoint inhibitor. In an embodiment, the TAP, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocyte, APC), and / or composition according to the present disclosure is administered / used in combination one or more therapies used for the treatment of CRC (e.g., surgery, chemotherapy (e.g., using 5-fluorouracil, capecitabine, oxaliplatin, irinotecan, raltitrexed, trifluridine, tipiracil), radiation therapy, bevacizumab, cetuximab, panitumumab, regorafenib.

[0253] The additional therapy may be administered prior to, concurrent with, or after the administration of the TAP, SLP, nucleic acid, expression vector, T cell receptor, antibody / antibody fragment, cell (e.g., T lymphocyte, CAR T or NK cell, APC), and / or composition according to the present disclosure.

[0254] EXAMPLES

[0255] The present disclosure is illustrated in further details by the following non-limiting examples.

[0256] Example 1 : Materials and Methods

[0257] Primary tissues.

[0258] Primary human colon adenocarcinoma tumor samples were purchased from BiolVT (n=15) and Reprocell (n=11). Tissue samples were taken from patients receiving surgery as the first line of treatment and were flash-frozen in liquid nitrogen. More information about primary tissue samples is depicted in Tables 1A and 1B.

[0259] Table 1A

[0260] | Sample ID | Sex | Age | Ethnicity | Clinical Diagnosis (Specimen) |

[0261] Table 1B

[0262] The proteogenomic workflow for the discovery of tumor antigens in colorectal primary tumor samples is depicted in FIGs. 1A-B. RNA extraction and sequencing.

[0263] Total RNA was isolated from primary tissues (29-51 mg) using the All Prep DNA / RNA / miRNA Universal kit (Qiagen) as recommended by the manufacturer. RNA sequencing. The RNA quantification was performed using a QuBIT (Life Technologies), and the RNA quality was assessed using a Bioanalyzer Nano (Agilent). cDNA library preparation was done using the KAPA Hyperprep RNAseq stranded kit (KAPA) with polyA capture. Libraries were quantified by QuBit, and the average library length was evaluated with the BioAnalyzer DNA1000. All libraries were diluted to 10 nM and normalized by qPCR using the KAPA library quantification kit (KAPA). Libraries were pooled to equimolar concentration. Sequencing was performed with the Illumina Novaseq S4 using PE100 sequencing. A mean of 200 million paired-end reads were generated. Library preparation was performed at the Genomic Platform of the Institute for Research in Immunology and Cancer (I RIC) and sequencing was done at McGill Genome Centre. Transcriptomics.

[0264] Bioinformatic analyses. Sequences were trimmed using Trimmomatic version 0.35 (7) and aligned to the reference human genome version GRCh38 (gene annotation from Gencode version 26, based on Ensembl 88) using STAR version 2.75.1a 1 b (2) running with default parameters except for --alignSJoverhangMin, --alignMatesGapMax, --alignlntronMax, and alignSJstitchMismatchNmax parameters for which default values were replaced by 10, 200,000, 200,000 and “5 -1 5 5”, respectively, to generate bam files.

[0265] HLA genotyping. HLA genotyping of tissues was performed using OptiType v1 .3.5, an HLA genotyping tool that uses next generation sequencing data to predict a sample’s HLA alleles (https: / / github.com / FRED-2 / OptiType) (3). Information is provided in Tables 1A and 1B.

[0266] Microsatellite instability detection. MSI status of the primary tumor samples was evaluated using the MSIsensor-pro-1.2.0 program using the GRCh38 TCGA baseline, (github.com / xjtu- omics / msisensor-pro / )(4).

[0267] Transcriptomic signatures. Transcripts were quantified using Kallisto v0.43.0 (5) with an index built from Ensembl v88 annotations. All sample transcript expression values were merged and converted to gene expression using tximport v1.28.0 (6). The gene expression matrix was then used to feed R scripts (7-9) and generate the immunologic constant of rejection (ICR) (8) and consensus molecular subtypes (CMS) (70).

[0268] Transcript biotype analysis. The proportion of various biotypes in the transcriptome of tissue samples was determined as previously described (77). Briefly, transcripts and repetitive elements were annotated using a Kallisto index containing Ensembl annotated transcripts supplemented with genetic repeat identifications from the UCSC Table Browser GRCh38 repeat masker database (72). Transcript expression was quantified in TPM.

[0269] Mutation profiles. Single nucleotide variants were called using freebayes v1 .0.2 with the BAM file generated by STAR. List of CRC frequently found mutations was obtained from COSMIC (73).

[0270] Database generation.

[0271] Global cancer databases were constructed as previously described (14). In brief, each database is composed of two parts: the personalized canonical proteome and the cancer-specific proteome. Canonical proteome. For each sample, single-base mutations with a minimum alternate count setting of 5 were identified using freeBayes v1.0.2-16-gd466dde75. Transcript expression was quantified in transcripts per million (tpm) with kallisto v0.43.0 (5) in stranded mode with “-b 100” and other default parameters. pyGeno v2.0.0 was then used to insert high-quality samplespecific single-base mutations (freeBayes quality > 20) in the reference exome and export sample-specific sequences of known proteins generated by expressed transcripts (tpm > 0) to generate fasta files of personalized canonical proteomes.

[0272] Cancer-specific proteome. This was conducted as previously described (74), with the following exceptions: 8 mTEC samples (GEO accessions GSE127825, GSE127826) were used instead of 6 mTECs, the k-mer occurrence allowed in mTECs was 1 instead of 0, and polypeptides were linked with “JJ” linkers before concatenation to the personalized canonical proteome. Briefly, the R1 and R2 fastq files of each sample were trimmed as reported above, and the reverse mapping reads were reverse complemented using the fastx_reverse_complement function of the FASTX-T oolkit v0.0.14. K-mer databases (24 or 33-long) were generated using Jellyfish v2.2.377. A single k-mer database was generated for each tumor sample, while the eight mTEC samples were combined in a unique database by concatenating their fastq files. Because the duration of k-mer assembly increases exponentially above 30 million k-mers, each cancer 33-nucleotide-long k-mer database was filtered based on a sample-specific threshold on occurrence (the number of times that a given k-mer is present in the database) to reach a maximum of 30 million k-mers for the assembly step. After this filtering, k-mers present more than once in the mTECs k-mer database were removed from each sample database. The remaining k-mers were assembled into contigs with NEKTAR, an in-house developed software. Briefly, one of the submitted 33- nucleotide-long k-mer is randomly selected as a seed that is extended from both ends with consecutive k-mers overlapping by 32 nucleotides on the same strand (-r option disabled, as stranded sets of k-mers were used). The assembly process stops when either no k-mers can be assembled or when more than one k-mer fits (-a 1 option for linear assembly). Then a new seed is selected, and the assembly process resumes until all k-mers from the submitted list have been used once. Finally, the contigs were 3-frame translated using an in-house python script and split amino acid sequences at internal stop codons. The resulting polypeptides were linked with “JJ” linkers and concatenated with the respective personalized canonical proteome for each sample, is generated using an alignment-free RNA-seq workflow called k-mer profiling because current mappers and variant callers poorly identify structural variants. This second dataset enabled the detection of peptides encoded by any reading frame of any genomic origin (including structural variants), as long as they were cancer specific (that is, absent from normal cells). MHO llhimedullary thymic epithelial cells (mTEChi) cells were used as a “normal control”. To identify RNA sequences that were cancer specific, cancer RNA-seq reads were chopped into 33-nucleotide long sequences, called k-mers with Jellyfish 2.2.3 (75), from which k-mers present in syngeneic mTEChicells were removed. Redundancy inherent to the k-mer space was removed by assembling overlapping cancer-specific k-mers into longer sequences, called contigs, which were 3-frame translated in silica. The canonical and cancer-specific proteomes were then concatenated to create a global cancer database, one for each analyzed sample.

[0273] Isolation of MAPs

[0274] The W6 / 32 antibodies (BioXcell) were coupled to CNBR-activated Sepharose 4B beads (Cytivia) as described in Sirois et al, (76) and the beads were stored at 4°C in PBS pH 7.2 and 0.02% NaN3until use. CRC adenocarcinoma primary samples (573-1346 mg) were cut into small pieces (cubes, ~3 mm in size) and 6 ml of ice-cold PBS containing protein inhibitor cocktail (Sigma) was added. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (I KA-Labortechnik) set at a speed of 20 000 rpm. Then, 700 pl of ice-cold 10X lysis buffer (5% w / v CHAPS) was added to each sample. After 60-minute incubation with tumbling at 4°C, tissue samples were spun at 16,600g for 20 minutes at 4°C. Supernatants were transferred into new tubes containing 1 mg of W6 / 32 antibody covalently-cross-linked to Sepharose beads and incubated with tumbling for 3 hours at 4°C. The samples were transferred into poly prep chromatography columns (Biorad) and the liquid mixture was eluted by gravity. Sepharose beads were first washed with 11 mL PBS, then with 11 mL of 0.1X PBS and finally with 11 mL of water. MHC I complexes were eluted from Sepharose™ beads by acidic treatment using 1% trifluoroacetic acid (TFA). Filtrates containing peptides were separated from MHC I subunits (HLA molecules and p-2 macroglobulin) using home-made stage tips packed with two 1 mm diameter octadecyl (C-18) solid-phase extraction disks (EMPORE). Stage tips were prewashed first with methanol then with 80% acetonitrile (ACN) in 0.1% TFA, followed by 0.1 % TFA and finally with 1 % TFA. Samples were loaded onto the stage tips and the peptides were retained on the stage tips while the HLA molecules and p-2 macroglobulin were found in the flow through. Stage tips were washed with 1 % TFA and then with 0.1% TFA and peptides were eluted with 30% ACN in 0.1%TFA. The peptides were dried using vacuum centrifugation and then stored at -20°C until MS analysis.

[0275] Mass spectrometry analyses

[0276] Dried peptide extracts were resuspended in 4% FA and loaded on an lonOpticks Aurora 25-cm Cis column. Peptides were eluted using a 106 min linear gradient from 0% to 38% ACN (0.2% FA) and a 300 nL / min flow rate on a Neo Vanquish nano LC system. Samples were analyzed with an Orbitrap T ribrid Ascend mass spectrometer (Thermo Fisher Scientific) in positive ion mode with Nanoflex source at 3kV. Each full MS spectrum, acquired with a 120000 resolution was followed by 40 MS / MS spectra, where the most abundant multiply charged ions were selected for MS / MS sequencing with a resolution of 30 000, a normalized automatic gain control target of 100%, an injection time of 59 ms, and collisional energy of 27%. LC-MS instrument was controlled using Xcalibur version 4.6 (Thermo Fisher Scientific). MAP Identification

[0277] Database searches were conducted using the PeaksXPro software, version 10.6 (Bioinformatics Solutions Inc.) (17). Error tolerances for precursor mass and fragment ions were set to 10.0 ppm and 0.01 Da, respectively. Variable modifications included Oxidation (M), and Deamidation (NQ). Peaks searches were then loaded into MAPDP (78), which was used to apply the following filters: selecting peptides of 8-11 amino acids in length, with rank eluted ligand threshold < 2% based on NetMHCpan-4.1 b predictions, using a 5% false discovery rate (FDR). FDR was calculated using the decoy hits imported from Peaks, which employ the decoy-fusion strategy ( 9).

[0278] Selection of tumor antigens (TAs)

[0279] To identify TA candidates, each MAP and its coding sequence(s) were queried in mTEC and the relevant cancer canonical proteomes, as well as in mTEC and and the relevant cancer 24-nucleotide-long k-mer databases, respectively, as previously described (14). MAPs were retained as TA candidates if they were not found in the mTEC canonical proteome or if all possible MAP-coding sequences (MCS) for a given MAP i) were expressed below 2 KPHM (minimum occurrence of the MCS’s 24-nucleotide-long k-mer set per hundred million reads) in mTECs, and ii) had a KPHM fold change superior or equal to 10 in cancer compared to mTECs. Since leucine and isoleucine variants are not distinguishable by standard MS approaches, TA candidates for which an existing variant was flagged as a non-TA candidate were discarded unless they had a higher RNA expression than the variant in the respective tumor samples.

[0280] Then, BamQuery (20) was used in “peptide” mode to evaluate the genomic location and biotypes of TA candidates and the expression of their coding sequences in benign and cancer tissues. Genomic locations and biotypes were manually validated using the UCSC genome browser. Unmutated TAs listed herein are the peptides meeting the following criteria:

[0281] The 95th percentile RNA expression value in TCGA-COAD samples (n=483) is at least two times higher than the 95th percentile RNA expression value in GTEx (except testis and colon) and mTEC samples. In addition, the following filters were used for the selection of TAs: a) For aeTSAs: The aeTSA’s source RNA is expressed below 8.55 reads per hundred million (RPHM) in more than 90% normal samples from mTECs, blood and bone marrow cells, and each GTEx tissue except the testis. Also, the mean expression is at least two times higher in TCGA-COAD compared to normal GTEx colon. b) For TAAs: The TAA’s source RNA can be expressed above 8.55 reads per hundred million (RPHM) in >10% of samples from any normal tissues (GTEx, mTECs and / or blood and bone marrow cells), but the mean expression is at least two times higher in TCGA-COAD compared to mTECs and each GTEx tissue (except testis).

[0282] Assessment of intertumoral sharing To examine the intertumoral distribution of TSA and TAA sequences in other CRC tumors, the log(RPHM+1) expression of the peptide coding sequences in 483 colon adenocarcinoma (COAD) samples from TCGA was determined (see ‘Quantification of MAP coding sequences in RNA-Seq data’).

[0283] MS validation of TAs

[0284] 1) LC-MS / MS data were searched against the relevant database using Comet 2022.01 rev 0 (27), using the same parameters used in PEAKS. A 5% FDR as determined by Percolator v3.4 (22) (features: mass, mass error, charge, sequence length, ions matched and total, Comet e-value I Xcorr I deltaCN I sp score) was applied to all peptide-spectrum matches.

[0285] 2) Prosit tool with the non-tryptic HCD 2020 model (23, 24) was used to evaluate the correlation between predicted vs. experimental spectra of each TA. TAs with Prosit spectral angle value above 0.6 were considered as good candidates.

[0286] 3) MS spectra of all TA candidates that were found in the Comet database search and / or that passed Prosit filtering were submitted to an in-house manual validation to remove any false identification.

[0287] 4) TSAs that were found in the Comet database search and / or that passed Prosit filtering but that were not manually validated were further validated by MS, using synthetic analogs.

[0288] Thus, all TAs listed herein were re-identified by a second MS search algorithm (Comet) and / or by Prosit and were further validated manually (TAAs and TSAs) or using synthetic analogs (TSAs only).

[0289] Presentation of aeTSAs across populations

[0290] The frequency of TSAs presented by individual CRC in four different populations was estimated as previously described (25) with slight modifications. First, a list of the most frequent HLA alleles was generated. In total, 187 HLA alleles were added to this simulation, which collectively cover > 99% of the alleles of individuals within the TCGA-COAD cohort. From this, binding of individual TSAs against their primary allele (i.e. alleles on which TSAs were initially identified, in the sample of origin) or against any of the 187 alleles (promiscuous alleles) was assessed using NetMHCpan-4.1 b. HLA alleles were considered as TSA binders if an eluted ligand likelihood prediction rank < 2% on primary alleles (strong and weak binders) or < 0.5 % on promiscuous alleles (strong binders only) was calculated for at least one TSA.

[0291] Then, bioinformatic simulations were based on two parameters.

[0292] 1) The likelihood of TSA expression was based on the proportion of tumors expressing the corresponding RNA in the TCGA-COAD cohort. An RNA was considered as expressed if the rphm value of the MCS in TCGA was above 2x the 95th percentile value in all GTEx tissues, except testis.

[0293] 2) The HLA allele frequencies were retrieved from USA National Marrow Donor Program for the European-American, African American, Asian-Pacific Islanders and Hispanic populations.

[0294] Finally, patient’s HLA genotype with six HLA class I alleles was simulated based on the reported frequencies in the given population. Because it was assumed that the six HLA alleles were independent events, some HLA loci were homozygous in simulated patients. A TSA was considered to be presented in a simulated patient when the MCS was expressed, and the relevant HLA allele was present. Expression of each TSA was considered to be an independent event. One million simulated patients and their TSA presentation status were generated for each of the four populations and used to plot the distribution.

[0295] Example 2: Identification of TSAs and TAAs in CRC samples The characteristics of the TSAs and TAAs identified herein are disclosed in Tables 2A-2D and FIGs. 2A-2C, 3A-3C and 4.

[0296] Table 2A: Characteristics of the TSAs identified herein

[0297] Primary HLA-allele: HLA corresponding to bound peptide and correlated with primary specimen

[0298] Sample: Primary sample in which tumor antigen found

[0299] MCS: MHC l-associated peptide coding sequence Table 2B: Characteristics of the TSAs identified herein (continued)

[0300] MCS_alignment: MCS genomic location

[0301] Biotype: Transcript classification

[0302] Overlap ERE: Indicate if MCS overlaps with endogenous retroelement (ERE)

[0303] %Samples_TCGA_COAD_2GTEx95th: Proportion of colon adenocarcinoma from TCGA (n= 151) where MCS transcript show 2-fold higher expression than the 95th percentile of all GTEX samples (except for testis)

[0304] Table 2C: Characteristics of the TAAs identified herein

[0305] Table 2D: Characteristics of the TAAs identified herein (continued)

[0306]

[0307] Example 3: Confirmation of the immunogenicity of representative TSAs

[0308] It is recognized that two specific features are important for predicting the immunogenicity of MHC-associated peptides (MAPs):

[0309] (1) high MHC-binding affinity; and

[0310] (2) lack of expression by medullary thymic epithelial cells (mTECs)

[0311] The tumor antigen peptides identified in Example 2 were eluted by immunoprecipitation from MHC class I molecules present at the surface of ovarian tumor cells isolated from CRC patients, and thus it is implicit that they strongly bind to MHC class I molecules because otherwise they could not have been identified by the method used by the present inventors. Furthermore, the binding of the identified peptides to MHC class I molecules was assessed using the NetMHCpan-4.1 b tool, and only peptides predicted to have an MHC allele affinity rank eluted ligand < 2% were selected (see Example 1 , “MAP identification” section).

[0312] Regarding the expression by mTECs, this factor is important because mTECs express MAPs present in all normal cells of the organism, and it has been reported that MAP expression negatively correlates with expression in TECs. One criterion for selecting the tumor antigens tumor antigen peptides identified in Example is the lack of expression in TECs (see, Example 1 , “Selection of tumor antigens” (TAs) section).

[0313] The immunogenicity of representative tumor antigen peptides identified in Example 2, AAAKQSLPK (SEQ ID NO:1), DEFRQLKTL (SEQ ID NO:9), GSSNPLTLK (SEQ ID NO:17), KSVYYFIMK (SEQ ID NO:23), KVIPGWSK (SEQ ID NO:24), KVLGGNLTK (SEQ ID NO:25), and RPAVLPLPEL (SEQ ID NO:43) was tested using an in vitro stimulation assay. Monocyte- derived dendritic cells (mDCs) were generated from frozen PBMCs, based on a previously described protocol (26). Briefly, monocytes were obtained from a CD14+ enriched PBMC fraction using the EasySep™ Human CD14 Positive Selection Kit II from StemCell Technologies. Monocytes were cultured in CellGenix medium supplemented with: human serum (BiolVT), IL-4 (Peprotech) and GM-CSF (Peprotech). DCs were then matured with CellGenix medium supplemented with human serum (BiolVT), IL-4 (Peprotech), LPS (Sigma Aldrich), IFNy (Peprotech), GM-CSF (Peprotech) and peptide (10 pM / ml, SB peptide). The matured DCs were then cocultured with enriched naive CD8 T cells for 16 days. On day 16, CD8 T cells were analyzed for responses to aeTSAs via an IFNy ELISpot kit (MabTech) following manufacturer’s guidelines. As a positive control in the expansions, a known immunogenic peptide (MelanA binding HLA-A*02:01) was included for HLA-A2+donors.

[0314] The results depicted in Table 3 demonstrate that all tested TSAs induce the expansion of antigen-specific CD8 T cells following ex vivo stimulation, thus confirming their immunogenicity.

[0315] Table 3: Expansion of antigen-specific CD8 T cells

[0316] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" 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 corresponding plural references unless the context clearly dictates otherwise.

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Claims

WHAT IS CLAIMED IS:

1. A tumor antigen peptide (TAP) comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-269.

2. The TAP of claim 1 , wherein the TAP binds to an HLA-A*01 :01 molecule and comprises or consists of the sequence of SEQ ID NO: 20, 169, 173, 254, 265 or 266.

3. The TAP of claim 1 , wherein the TAP binds to an HLA-A*02:01 molecule and comprises or consists of the sequence of SEQ ID NO: 6, 27, 30, 44, 58, 84, 124, 132, 154-156, 160, 164, 203, 221 , 224, 243, 264 or 267.

4. The TAP of claim 1 , wherein the TAP binds to an HLA-A*02:03 molecule and comprises or consists of the sequence of SEQ ID NO: 23, 84, 89, 157, 159, 177, 190, 223 or 225.

5. The TAP of claim 1 , wherein the TAP binds to an HLA-A*03:01 molecule and comprises or consists of the sequence of SEQ ID NO: 1 , 35, 158 or 202.

6. The TAP of claim 1 , wherein the TAP binds to an HLA-A*11 :01 molecule and comprises or consists of the sequence of SEQ ID NO: 11 , 25, 32, 34, 45, 86, 87, 128, 131 , 191 , 229, or 233- 238.

7. The TAP of claim 1 , wherein the TAP binds to an HLA-A*23:01 molecule and comprises or consists of the sequence of SEQ ID NO: 74, 121 , 122, 188, 209 or 210.

8. The TAP of claim 1 , wherein the TAP binds to an HLA-A*24:02 molecule and comprises or consists of the sequence of SEQ ID NO: 33, 122, 148, 184, 200, 210, 259, or 260.

9. The TAP of claim 1 , wherein the TAP binds to an HLA-A*25:01 molecule and comprises or consists of the sequence of SEQ ID NO: 16, 17, 90, 92, 104, 112, 115, 120 or 139.

10. The TAP of claim 1 , wherein the TAP binds to an HLA-A*26:01 molecule and comprises or consists of the sequence of SEQ ID NO: 4, 61 , 103, 118, or 242.11 . The TAP of claim 1 , wherein the TAP binds to an HLA-A*29:01 molecule and comprises or consists of the sequence of SEQ ID NO: 21 , 204, 242, 251 , or 255.

12. The TAP of claim 1 , wherein the TAP binds to an HLA-A*32:01 molecule and comprises or consists of the sequence of SEQ ID NO: 153 or 162.

13. The TAP of claim 1 , wherein the TAP binds to an HLA-A*68:01 molecule and comprises or consists of the sequence of SEQ ID NO: 15, 49, 69, 102, 116, 117, 119, 183 or 192.

14. The TAP of claim 1 , wherein the TAP binds to an HLA-B*07:02 molecule and comprises or consists of the sequence of SEQ ID NO: 38, 47, 54, 66, 88, 205, 227, 228, 245, 246, or 256.

15. The TAP of claim 1 , wherein the TAP binds to an HLA-B*8:01 molecule and comprises or consists of the sequence of SEQ ID NO: 46, 99, 143, or 182.

16. The TAP of claim 1 , wherein the TAP binds to an HLA-B*13:02 molecule and comprises or consists of the sequence of SEQ ID NO: 51 or 160.

17. The TAP of claim 1 , wherein the TAP binds to an HLA-B*14:02 molecule and comprises or consists of the sequence of SEQ ID NO: 101 , 126 or 232.

18. The TAP of claim 1 , wherein the TAP binds to an HLA-B*15:02 molecule and comprises or consists of the sequence of SEQ ID NO: 63, 98, 100, 114, 133, 244, or 248.

19. The TAP of claim 1 , wherein the TAP binds to an HLA-B*15:03 molecule and comprises or consists of the sequence of SEQ ID NO: 5, 22, 53, 130, 135, 145, 158, 181 , 201 , 204, 222, 226, or 257.

20. The TAP of claim 1 , wherein the TAP binds to an HLA-B*15:25 molecule and comprises or consists of the sequence of SEQ ID NO: 9, 10, 55, 83, 158, 204, 207, 226, 257, or 258.21 . The TAP of claim 1 , wherein the TAP binds to an HLA-B*18:01 molecule and comprises or consists of the sequence of SEQ ID NO: 12, 93-96, 105, 108, 215, 219, 239, 261 , or 263.

22. The TAP of claim 1 , wherein the TAP binds to an HLA-B*27:05 molecule and comprises or consists of the sequence of SEQ ID NO: 68 or 230.

23. The TAP of claim 1 , wherein the TAP binds to an HLA-B*35:01 molecule and comprises or consists of the sequence of SEQ ID NO: 179 or 206.

24. The TAP of claim 1 , wherein the TAP binds to an HLA-B*35:02 molecule and comprises or consists of the sequence of SEQ ID NO: 38, 113, or 171.

25. The TAP of claim 1 , wherein the TAP binds to an HLA-B*35:03 molecule and comprises or consists of the sequence of SEQ ID NO: 59, 76, 113, 125, 140, 170, 247, or 268.

26. The TAP of claim 1 , wherein the TAP binds to an HLA-B*38:01 molecule and comprises or consists of the sequence of SEQ ID NO: 65, 75, 134, 180, 189, 200, 220, 241 , or 252.

27. The TAP of claim 1 , wherein the TAP binds to an HLA-B*38:02 molecule and comprises or consists of the sequence of SEQ ID NO: 19, 189, or 252.

28. The TAP of claim 1 , wherein the TAP binds to an HLA-B*40:01 molecule and comprises or consists of the sequence of SEQ ID NO: 64, 80, 81 , 129, 142, 166, 167, 194, 195, 198, 213, 216, 218, or 250.

29. The TAP of claim 1 , wherein the TAP binds to an HLA-B*40:02 molecule and comprises or consists of the sequence of SEQ ID NO: 18, 29, 52, 56, 151 , 185, 198, 216 or 218.

30. The TAP of claim 1 , wherein the TAP binds to an HLA-B*44:02 molecule and comprises or consists of the sequence of SEQ ID NO: 97, 176, 199, or 215.31 . The TAP of claim 1 , wherein the TAP binds to an HLA-B*44:03 molecule and comprises or consists of the sequence of SEQ ID NO: 3, 13, 43, 82, 97, 105-108, 110, 111 , 175, 186, 196, 197, 199, 212, 214, or 215.

32. The TAP of claim 1 , wherein the TAP binds to an HLA-B*45:01 molecule and comprises or consists of the sequence of SEQ ID NO: 14, 24, or 109.

33. The TAP of claim 1 , wherein the TAP binds to an HLA-B*49:01 molecule and comprises or consists of the sequence of SEQ ID NO: 152, 165, 187, 217, 240, or 262.

34. The TAP of claim 1 , wherein the TAP binds to an HLA-B*51 :01 molecule and comprises or consists of the sequence of SEQ ID NO: 91.

35. The TAP of claim 1 , wherein the TAP binds to an HLA-B*52:01 molecule and comprises or consists of the sequence of SEQ ID NO: 172 or 190.

36. The TAP of claim 1 , wherein the TAP binds to an HLA-B*54:01 molecule and comprises or consists of the sequence of SEQ ID NO: 245 or 269.

37. The TAP of claim 1 , wherein the TAP binds to an HLA-B*56:01 molecule and comprises or consists of the sequence of SEQ ID NO: 7, 8, 67, 71 , 85, 137, 161 , or 245.

38. The TAP of claim 1 , wherein the TAP binds to an HLA-B*57:01 molecule and comprises or consists of the sequence of SEQ ID NO: 28, 147, 163, or 208.

39. The TAP of claim 1 , wherein the TAP binds to an HLA-C*01 :02 molecule and comprises or consists of the sequence of SEQ ID NO: 37, 40, or 193.

40. The TAP of claim 1 , wherein the TAP binds to an HLA-C*03:03 molecule and comprises or consists of the sequence of SEQ ID NO: 72.41 . The TAP of claim 1 , wherein the TAP binds to an HLA-C*03:04 molecule and comprises or consists of the sequence of SEQ ID NO: 36, 50, 60, 70, 77, 79, 136, 141 , 149, or 174.

42. The TAP of claim 1 , wherein the TAP binds to an HLA-C*04:01 molecule and comprises or consists of the sequence of SEQ ID NO: 123, 127, 168, 178, or 253.

43. The TAP of claim 1 , wherein the TAP binds to an HLA-C*05:01 molecule and comprises or consists of the sequence of SEQ ID NO: 253.

44. The TAP of claim 1 , wherein the TAP binds to an HLA-C*06:02 molecule and comprises or consists of the sequence of SEQ ID NO: 231 or 232.

45. The TAP of claim 1 , wherein the TAP binds to an HLA-C*07:01 molecule and comprises or consists of the sequence of SEQ ID NO: 68.

46. The TAP of claim 1 , wherein the TAP binds to an HLA-C*07:04 molecule and comprises or consists of the sequence of SEQ ID NO: 39.

47. The TAP of claim 1 , wherein the TAP binds to an HLA-C*08:01 molecule and comprises or consists of the sequence of SEQ ID NO: 2, 138, 150, or 249.

48. The TAP of claim 1 , wherein the TAP binds to an HLA-C*08:02 molecule and comprises or consists of the sequence of SEQ ID NO: 144 or 253.

49. The TAP of claim 1 , wherein the TAP binds to an HLA-C*12:03 molecule and comprises or consists of the sequence of SEQ ID NO: 26, 42 or 57.

50. The TAP of claim 1 , wherein the TAP binds to an HLA-C*15:02 molecule and comprises or consists of the sequence of SEQ ID NO: 136.51 . The TAP of claim 1 , wherein the TAP binds to an HLA-C*15:05 molecule and comprises or consists of the sequence of SEQ ID NO: 41 , 62, or 136.

52. The TAP of claim 1 , wherein the TAP binds to an HLA-C*16:01 molecule and comprises or consists of the sequence of SEQ ID NO: 31 , 48, 78, 146, 211 , or 257.

53. The TAP of any one of claims 1-52, which is encoded by a sequence located a non-protein coding region of the genome.

54. The TAP of claim 53, wherein said non-protein coding region of the genome is an intergenic region.

55. The TAP of claim 53, wherein said non-protein coding region of the genome is a long noncoding RNAs.

56. A combination comprising at least two of the TAPs defined in any one of claims 1-55.

57. A synthetic long peptide (SLP) comprising at least one, for example at least 5, 10, 15 or 20, of the amino acid sequences defined in claim 1 , or a nucleic acid encoding the SLP.

58. A nucleic acid encoding the TAP defined in any one of claims 1 -55, the combination of claim 56 or the SLP of claim 57.

59. The nucleic acid of claim 58, wherein the nucleic acid is an mRNA.

60. The nucleic acid of claim 59, wherein the mRNA comprises one or more 5’-end modifications, 3’-end modifications, and / or modified nucleosides to increase stability, improve translation and / or reduce immunogenicity of the mRNA.61 . The nucleic acid of claim 58, wherein the nucleic acid is a DNA.

62. The nucleic acid of claim 61 , wherein the nucleic acid is a component of a viral vector.

63. A vesicle or particle comprising the TAP, combination, SLP, or nucleic acid of any one of claims 1 to 62.

64. The vesicle or particle of claim 63, which is a lipid nanoparticle (LNP).

65. The vesicle or particle of claim 63 or 64, which comprises a cationic lipid.

66. A composition comprising the TAP, combination, SLP or nucleic acid of any one of claims 1 to 62, or the vesicle or particle of any one of claims 63-65, and a pharmaceutically acceptable carrier.

67. A vaccine comprising the TAP, combination, SLP or nucleic acid of any one of claims 1 to 62, the vesicle or particle of any one of claims 63-65, or the composition of claim 66, and an adjuvant.

68. An isolated major histocompatibility complex (MHC) class I molecule comprising the TAP of any one of claims 1-55 in its peptide binding groove.

69. The isolated MHC class I molecule of claim 68, which is in the form of a multimer.

70. The isolated MHC class I molecule of claim 69, wherein said multimer is a tetramer.

71. An isolated cell comprising (i) the TAP of any one of claims 1-55, (ii) the combination of claim 56; (iii) the SLP of claim 57; or (iv) a vector comprising a nucleotide sequence encoding the TAP of any one of claims 1-55, the combination of claim 56 or the SLP of claim 57.

72. An isolated cell expressing at its surface major histocompatibility complex (MHC) class I molecules comprising the TAP or combination of any one of claims 1-56 in their peptide binding groove.

73. The cell of claim 71 or 72, which is an antigen-presenting cell (APC).

74. The cell of claim 73, wherein said APC is a dendritic cell.

75. A T-cell receptor (TCR) that specifically recognizes the isolated MHC class I molecule of any one of claims 68-70 and / or MHC class I molecules expressed at the surface of the cell of any one of claims 72-74.

76. The TCR of claim 75, which is a soluble TCR.

77. An antibody or an antigen-binding fragment thereof that specifically binds to the isolated MHC class I molecule of any one of claims 68-70 and / or MHC class I molecules expressed at the surface of the cell of any one of claims 72-74.

78. The TCR of claim 75 or 76, or the antibody or antigen-binding fragment thereof according to claim 77, which is a bispecific TCR or a bispecific antibody or antigen-binding fragment thereof.

79. The TCR, antibody or antigen-binding fragment thereof according to claim 78, wherein the bispecific antibody or antigen-binding fragment thereof is a single-chain diabody (scDb).

80. The TCR, antibody or antigen-binding fragment thereof according to claim 78 or 79, wherein the bispecific TCR, antibody or antigen-binding fragment thereof also specifically binds to a T cell signaling molecule.81 . The TCR, antibody or antigen-binding fragment thereof according to claim 80, wherein the T cell signaling molecule is a CD3 chain.

82. A chimeric antigen receptor (CAR) comprising the antibody or an antigen-binding fragment thereof of any one of claims 77-81 , or a nucleic acid encoding said CAR.

83. An isolated cell expressing at its cell surface the TCR of claim 75 or the CAR of claim 82.

84. The isolated cell of claim 82, which is a CD8+T lymphocyte.

85. A cell population comprising at least 0.5% or 1% of the isolated cell as defined in claim 83 or 84.

86. A method of treating cancer in a subject comprising administering to the subject an effective amount of:(a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1-269 or any combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1-269;(b) at least one nucleic acid encoding the TAP, combination thereof or SLP defined in (a);(c) a vesicle or particle comprising the TAP, combination thereof or SLP defined in (a) or the at least one nucleic acid defined in (b);(d) a composition comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), or the vesicle or particle defined in (c), and a pharmaceutically acceptable carrier;(e) a vaccine comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), the vesicle or particle defined in (c), or the composition defined in (d), and an adjuvant;(f) a cell expressing at its surface major histocompatibility complex (MHC) class I molecules comprising the TAP or combination thereof defined in (a) in their peptide binding groove;(g) a cell expressing at its cell surface a T-cell receptor (TCR) that specifically recognizes MHC class I molecules expressed at the surface of the cell defined in (f); or(h) a soluble TCR, an antibody or an antigen-binding fragment thereof that specifically binds to the MHC class I molecules expressed at the surface of the cell defined in (f).

87. The method of claim 86, wherein the cancer is colorectal cancer.

88. The method of claim 86 or 87, further comprising administering at least one additional antitumor agent or therapy to the subject.

89. The method of claim 88, wherein said at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, an immune checkpoint inhibitor, radiotherapy or surgery.

90. Use of:(a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1-269 or any combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1-269;(b) at least one nucleic acid encoding the TAP, combination thereof or SLP defined in (a);(c) a vesicle or particle comprising the TAP, combination thereof or SLP defined in (a) or the at least one nucleic acid defined in (b);(d) a composition comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), or the vesicle or particle defined in (c), and a pharmaceutically acceptable carrier;(e) a vaccine comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), the vesicle or particle defined in (c), or the composition defined in (d), and an adjuvant;(f) a cell expressing at its surface major histocompatibility complex (MHC) class I molecules comprising the TAP or combination thereof defined in (a) in their peptide binding groove;(g) a cell expressing at its cell surface a T-cell receptor (TCR) that specifically recognizes MHC class I molecules expressed at the surface of the cell defined in (f); or(h) a soluble TCR, an antibody or an antigen-binding fragment thereof that specifically binds to the MHC class I molecules expressed at the surface of the cell defined in (f); for treating cancer in a subject, or for the manufacture of a medicament for treating cancer in a subject.91 . The use of claim 90, wherein the cancer is colorectal cancer.

92. The use of claim 90 or 91 , further comprising the use at least one additional antitumor agent or therapy to the subject.

93. The use of claim 92, wherein said at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, an immune checkpoint inhibitor, radiotherapy or surgery.

94. An agent for use in treating cancer in a subject, wherein the agent is:(a) a TAP comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 1-269 or any combination thereof, or a synthetic long peptide (SLP) comprising at least one of the sequences set forth in SEQ ID NOs: 1-269;(b) at least one nucleic acid encoding the TAP, combination thereof or SLP defined in (a);(c) a vesicle or particle comprising the TAP, combination thereof or SLP defined in (a) or the at least one nucleic acid defined in (b);(d) a composition comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), or the vesicle or particle defined in (c), and a pharmaceutically acceptable carrier;(e) a vaccine comprising the TAP, combination thereof or SLP defined in (a), the at least one nucleic acid defined in (b), the vesicle or particle defined in (c), or the composition defined in (d), and an adjuvant;(f) a cell expressing at its surface major histocompatibility complex (MHC) class I molecules comprising the TAP or combination thereof defined in (a) in their peptide binding groove;(g) a cell expressing at its cell surface a T-cell receptor (TCR) that specifically recognizes MHC class I molecules expressed at the surface of the cell defined in (f); or(h) a soluble TCR, an antibody or an antigen-binding fragment thereof that specifically binds to the MHC class I molecules expressed at the surface of the cell defined in (f).

95. The agent for use of claim 94, wherein the cancer is colorectal cancer.

96. The agent for use of claim 94 or 95, further comprising the use at least one additional antitumor agent or therapy to the subject.

97. The agent for use of claim 96, wherein said at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, an immune checkpoint inhibitor, radiotherapy or surgery.

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