Novel tumor-specific antigens for ovarian cancer and their uses - Patents.com

Abstract

Ovarian cancer, particularly high-grade serous ovarian cancer (HGSC), the leading cause of death from gynecological malignancies worldwide, has not benefited significantly from recent advances in cancer immunotherapy. While lymphocyte infiltration of HGSC correlates with increased survival, the nature of antigens capable of eliciting anti-HGSC immune responses remains unknown. Novel tumor-specific antigens (TSAs) shared by the majority of ovarian tumors are described herein. Most (>80%) of the TSAs described herein are derived from aberrantly expressed, non-mutated genomic sequences, such as intronic and intergenic sequences, that are not expressed in normal tissues. Nucleic acids, compositions, cells, and vaccines derived from these TSAs are described. Use of the TSAs, nucleic acids, compositions, cells, and vaccines for the treatment of ovarian cancer is also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 866,089, filed June 25, 2019, which is incorporated herein by reference.

[0002] The present disclosure relates generally to cancer, and more specifically to ovarian cancer-specific tumor antigens useful for T cell-based cancer immunotherapy. [Background technology]

[0003] Ovarian cancer is the leading cause of death from gynecologic malignancies worldwide, causing more than 14,000 deaths annually in the United States (1). High-grade serous ovarian cancer (HGSC) accounts for 70–80% of these deaths, and overall survival has not changed notably over several decades (2). A positive correlation between tumor-infiltrating lymphocyte (TIL) abundance and increased overall survival suggests that T cells are able to recognize biologically relevant tumor antigens in HGSC (3, 4). Moreover, compelling evidence suggests that HGSC TILs adjacent to tumor epithelial cells are actively involved in local immunoediting. Indeed, in a multimodality study of 212 HGSC samples from 38 patients, CD8 + TILs have been negatively associated with malignant cell diversity (5). In line with this, and given the therapeutic efficacy of immune checkpoint inhibitors in some tumor types, clinical trials with one or more checkpoint inhibitors are currently underway in HGSC. However, early trials with anti-PD1 have shown limited activity in HGSC (6, 7).

[0004] Considering this, there is an urgent need to identify antigens that can again induce therapeutic immune responses in ovarian tumors such as HGSC (3, 8). Such antigens could be used as vaccines (± immune checkpoint inhibitors) or as targets for T cell receptor-based approaches (cell therapy, bispecific biologics) (9).

[0005] This description refers to several documents, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention

[0006] This disclosure provides the following items 1 to 61. 1. A tumor antigen peptide comprising one of the amino acid sequences set forth in SEQ ID NOs: 1 to 103. 2. The tumor antigen peptide according to item 1, comprising one of the amino acid sequences set forth in SEQ ID NOs: 19 to 103. 3. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-A*01:01 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 21, 28, 40, 41, 66 or 88. 4. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-A*02:01 molecule and comprises an amino acid sequence according to SEQ ID NO: 1, 19, 20, 22, 30, 31, 36, 50, 52, 60, 62, 73, 84, 85, 86 or 91. 5. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-A*11:01 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 32, 54, 55, 67, 69, 81, 87, 90 or 102. 6. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-A*24:02 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 33 or 43. 7. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-A*25:01 molecule and comprises one of the amino acid sequences set forth in SEQ ID NO: 24. 8. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-A*29:02 molecule and comprises one of the amino acid sequences set forth in SEQ ID NO: 34 or 58. 9. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-A*32:01 molecule and comprises the amino acid sequence set forth in SEQ ID NO: 16. 10. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-B*07:02 molecule and comprises an amino acid sequence according to SEQ ID NO: 4, 6, 8, 9, 26, 49, 78, 92, 97, or 101. 11. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-B*08:01 molecule and comprises one of the amino acid sequences set forth in SEQ ID NOs: 23, 35, 42, 44, 46, 59, 63, 70, 74, 76, 83 or 103. 12. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-B*14:01 molecule and comprises the amino acid sequence set forth in SEQ ID NO: 53. 13. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-B*15:01 molecule and comprises an amino acid sequence according to SEQ ID NO: 2, 3 or 5. 14. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-B*18:01 molecule and comprises the amino acid sequence set forth in SEQ ID NO: 89. 15. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-B*39:01 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 47, 64, 96 or 99. 16. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-B*40:01 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 11, 12 or 13. 17. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-B*44:02 molecule and comprises the amino acid sequence set forth in SEQ ID NO: 65. 18. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-B*44:03 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 37 or 94. 19. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-C*03:03 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 10, 29, 71 or 95. 20. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-C*04:01 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 6 or 15. 21. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-C*05:01 molecule and comprises the amino acid sequence set forth in SEQ ID NO: 27. 22. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-C*06:02 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 18 or 72. 23. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-C*07:01 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 38, 61 or 93. 24. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-C*07:02 molecule and comprises the amino acid sequence set forth in SEQ ID NO:7. 25. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-C*12:03 molecule and comprises the amino acid sequence set forth in SEQ ID NO: 80. 26. The tumor antigen peptide according to item 1 or 2, wherein the tumor antigen peptide binds to an HLA-C*14:02 molecule and comprises an amino acid sequence as set forth in SEQ ID NO: 25, 57 or 79. 27. The tumor antigen peptide according to any one of items 1 to 26, which is encoded by a sequence located in a non-protein coding region of the genome and comprises an amino acid sequence according to any one of SEQ ID NOs: 1, 4, 6 to 8, 10, 13, 15 to 27 and 36 to 99, preferably SEQ ID NOs: 19 to 27 and 36 to 99. 28. The tumor antigen peptide according to item 27, wherein the non-protein coding region of the genome is an untranslated transcribed region (UTR), and the tumor antigen peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1, 8, 10, 13, 15, and 19 to 27, preferably SEQ ID NOs: 19 to 27. 29. The tumor antigen peptide according to item 27, wherein the non-protein coding region of the genome is an intron, and the tumor antigen peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 16, 17, and 36 to 64, preferably SEQ ID NOs: 36 to 64. 30. The tumor antigen peptide according to item 27, wherein the non-protein coding region of the genome is an intergenic region, and the tumor antigen peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 65 to 84. 31. The tumor antigen peptide according to item 27, wherein the non-protein coding region of the genome is an exon of a non-coding RNA transcript (ncRNA), and the tumor antigen peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 4 and 85 to 92, preferably SEQ ID NOs: 85 to 92. 32. The tumor antigen peptide according to item 27, wherein the non-protein coding region of the genome is an antisense strand of a gene, and the tumor antigen peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 93 to 99. 33. A nucleic acid encoding the tumor antigen peptide according to any one of items 1 to 32. 34. The nucleic acid according to item 33, which is an mRNA or a viral vector. 35. A liposome comprising the tumor antigen peptide according to any one of items 1 to 32, or the nucleic acid according to item 33 or 34. 36. A composition comprising the tumor antigen peptide according to any one of items 1 to 32, the nucleic acid according to item 31 or 32, or the liposome according to item 33, and a pharma- ceutically acceptable carrier. 37. A vaccine comprising the tumor antigen peptide according to any one of items 1 to 32, the nucleic acid according to item 33 or 34, the liposome according to item 35, or the composition according to item 36, and an adjuvant. 38. An isolated major histocompatibility complex (MHC) class I molecule comprising a tumor antigen peptide according to any one of items 1 to 32 within its peptide-binding groove. 39. The isolated MHC class I molecule according to item 38, in the form of a multimer. 40. The isolated MHC class I molecule according to item 39, wherein the multimer is a tetramer. 41. An isolated cell comprising (i) a tumor antigen peptide according to any one of items 1 to 32, or (ii) a vector comprising a nucleotide sequence encoding the tumor antigen peptide according to any one of items 1 to 32. 42. An isolated cell expressing on its surface major histocompatibility complex (MHC) class I molecules that contain a tumor antigen peptide according to any one of items 1 to 32 in their peptide-binding groove. 43. The cell according to item 42, which is an antigen-presenting cell (APC). 44. The cell according to item 43, wherein the APC is a dendritic cell. 45. A T cell receptor (TCR) that specifically recognizes an isolated MHC class I molecule according to any one of items 38 to 40 and / or an MHC class I molecule expressed on the surface of a cell according to any one of items 42 to 44. 46. ​​An isolated CD8 expressing the TCR according to item 45 on its cell surface. + T lymphocytes. 47. At least 0.5% of CD8 as defined in item 46 + A population of cells that includes T lymphocytes. 48. A method for treating ovarian cancer in a subject, comprising administering to a subject an effective amount of (i) a tumor antigen peptide according to any one of items 1 to 32, (ii) a nucleic acid according to item 33 or 34, (iii) a liposome according to item 35, (iv) a composition according to item 36, (v) a vaccine according to item 37, (vi) a cell according to any one of items 41 to 45, or (vii) a CD8 according to item 46. + (viii) administering to a subject a T lymphocyte or a cell population described in item 47. 49. The method according to item 48, wherein the ovarian cancer is serous carcinoma. 50. The method of claim 49, wherein the serous cancer is high-grade serous carcinoma (HGSC). 51. The method of any one of items 48 to 50, further comprising administering to the subject at least one additional anti-tumor agent or therapy. 52. The method according to item 51, wherein the at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy or surgery. 53. (i) a tumor antigen peptide according to any one of items 1 to 32, (ii) a nucleic acid according to item 33 or 34, (iii) a liposome according to item 35, (iv) a composition according to item 36, (v) a vaccine according to item 37, (vi) a cell according to any one of items 41 to 45, or (vii) a CD8 according to item 46. + (viii) Use of a T lymphocyte or a cell population according to item 47 for treating ovarian cancer in a subject. 54. (i) a tumor antigen peptide according to any one of items 1 to 32, (ii) a nucleic acid according to item 33 or 34, (iii) a liposome according to item 35, (iv) a composition according to item 36, (v) a vaccine according to item 37, (vi) a cell according to any one of items 41 to 45, or (vii) a CD8 according to item 46. + (viii) Use of a T lymphocyte, or a cell population according to item 47, for the manufacture of a medicament for treating ovarian cancer in a subject. 55. The use according to item 53 or 54, wherein the ovarian cancer is serous carcinoma. 56. The use according to item 55, wherein the serous cancer is high-grade serous carcinoma (HGSC). 57. The use according to any one of items 53 to 56, further comprising the use of at least one additional anti-tumor agent or therapy. 58. The use according to item 57, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy or surgery. 59. A method for treating ovarian cancer in a subject, comprising: (i) a tumor antigen peptide according to any one of items 1 to 32; (ii) a nucleic acid according to item 33 or 34; (iii) a liposome according to item 35; (iv) a composition according to item 36; (v) a vaccine according to item 37; (vi) a cell according to any one of items 41 to 45; or (vii) a CD8 according to item 46. + or (viii) a cell population according to item 47. 60. The tumor antigen peptide, nucleic acid, liposome, composition, vaccine, cell, CD8 for use according to item 59, wherein the ovarian cancer is serous cancer. + T lymphocytes, or cell populations. 61. The tumor antigen peptide, nucleic acid, liposome, composition, vaccine, cell, CD8 for use according to item 60, wherein the serous cancer is high-grade serous carcinoma (HGSC). + T lymphocytes, or cell populations. 62. Tumor antigen peptide, nucleic acid, liposome, composition, vaccine, cell, CD8 + 62. The tumor antigen peptide, nucleic acid, liposome, composition, vaccine, cell, CD8 T lymphocyte, or cell population for use according to any one of items 59 to 61, in which the T lymphocyte or cell population is combined with at least one additional antitumor agent or therapy. + T lymphocytes, or cell populations. 63. The tumor antigen peptide, nucleic acid, liposome, composition, vaccine, cell, CD8 for use according to item 62, wherein the at least one additional antitumor agent or therapy is a chemotherapeutic agent, an immunotherapy, an immune checkpoint inhibitor, radiation therapy or surgery. + T lymphocytes, or cell populations.

[0007] Other objects, advantages and features of the present invention will become apparent from a reading of the following non-restrictive description of specific embodiments, given by way of example only with reference to the accompanying drawings. [Brief description of the drawings]

[0008] [Figure 1]A schematic workflow of the TSA identification pipeline used in this study is shown. HGSC samples were processed for immunoprecipitation and RNA sequencing. Peptide sequences were identified using MS analysis, in which MAPs were identified by searching for matches in customized individual global cancer databases built from RNA-Seq data. CDS: coding sequence. [Diagram 2] Figures 2A-B show expression in normal tissues of RNAs encoding aeTSA candidates. Heatmap showing average RNA expression of aeTSA coding sequences in 27 peripheral tissues with color intensity corresponding to each tissue's expression level in mean log-transformed reads per 100 million reads (rphm). Bold boxes indicate tissues / organs above threshold RNA expression (average rphm>10). Numbers next to each peptide sequence indicate the number of tissues above threshold expression of the corresponding RNA. [Diagram 3] Figures 3A-D show that most TSAs originate from non-mutated non-exonic sequences. Figure 3A: Number of MAPs (left) and TSAs (right) identified in each sample. Figure 3B: Scatter plot shows Pearson correlation between the number of MAPs and the number of TSAs identified per sample. Figure 3C: Bar graph showing the origin of TSAs identified in the cohort studied here and in the Schuster et al. dataset. Blue shades indicate the number of TSAs resulting from in-frame exon translation (coding-in), out-of-frame exon translation (coding-out), or non-exon translation (non-coding). Figure 3D: Pie chart showing the translation frame (inner circle), detailed genomic origin (middle circle) of aeTSAs, and their reporting status (outer circle). [Figure 4] Figure 1. Expression of aeTSA coding regions across ovarian cancer samples. Heatmaps show RNA expression of each aeTSA coding region in the nine samples reported in this study (left) and in 378 samples from the TCGA-OV cohort, with color intensity indicating RNA levels in reads mapped to the region per million reads. [Diagram 5]Figures 5A-B show that copy number changes correlate with expression of several aeTSAs. Figure 5A: Heatmap shows Spearman correlation between aeTSA RNA expression levels and DNA copy number, promoter methylation or gene expression for aeTSAs located intragenic (left) or extragenic (right). Unavailable data are displayed as light grey. Figure 5B: Number of aeTSAs identified from each chromosome arm (top) with arm-level amplification score (bottom). Asterisks indicate amplifications considered significant (Q-value <0.25). [Figure 6] Figure 6A-6E show that presentation of three aeTSAs can induce spontaneous antitumor immune responses. Figure 6A-6C: Kaplan-Meier curves show survival of four groups of patients in the TCGA-OV cohort. For three individual aeTSAs, one group was able to present the aeTSA (EP) and three groups were unable to present (ED, ND and NP). ED: expression of aeTSA-encoded RNA in the absence of relevant HLA allotypes; ND: no expression of aeTSA-encoded RNA in the absence of relevant HLA allotypes; EP: expression of aeTSA-encoded RNA in the presence of relevant HLA allotypes; NP: no expression of aeTSA-encoded RNA in the presence of relevant HLA allotypes. Color shades represent 95% confidence intervals. Log-rank P values ​​are indicated. Figure 6D, Figure 6E: Abundance of T and cytotoxic cells in tumors from the four groups shown in Figure 6C. [Figure 7] Figure 3 shows the estimated frequency of aeTSAs presented by individual HGSCs in three different populations. One million simulated patients were generated for each population. An aeTSA was considered present in a simulated patient when its RNA was expressed and the associated HLA allotype was present. The frequency of aeTSA RNA expression was based on TCGA-OV RNA-Seq data (shown in Figure 4). HLA allotype frequencies were obtained from the USA National Marrow Donor Program. The red dashed lines indicate the median number of aeTSAs per tumor in each population. [Figure 8]Figures 8A-B show that dbSNP-based mTSA validation is also supported by sequencing data of paired normal samples. Figure 8A: Example of an excluded TSA candidate matching a common polymorphism reported in dbSNP. The variant nucleotide was also found in the paired normal sample. Figure 8B: Example of an mTSA where the variant is not present in dbSNP. The variant nucleotide is only detected in the paired normal with one read, which is likely a sequencing error. [Figure 9] Figure 1 shows the peripheral expression of coding sequences of TSA candidates containing germline polymorphisms. TSA candidates with single nucleotide mutations recorded in dbSNP were considered as aeTSAs if both their coding sequences and the corresponding reference sequences restricted RNA expression in peripheral tissues. Heatmap showing the average RNA expression of aeTSA coding sequences in 27 peripheral tissues, where color intensity corresponds to the expression level of each tissue in mean log-transformed reads per 100 million reads (rphm). Bold indicates tissues / organs with average rphm values ​​higher than 10. The number next to each peptide sequence indicates the number of tissues with significant RNA expression for a given peptide. Red asterisks indicate peptides that are retained as aeTSAs. [Figure 10] Graph showing correlation between number of MAPs and tumor size. Scatter plot shows Pearson correlation between tumor size (x-axis) and MAP number identified for each HLA allele (y-axis) for each sample. In this plot, only samples from Schuster et al. (largest subgroup) were used to avoid batch effects. [Figure 11] 11A-11B are graphs showing the relationship between aeTSA expression and DNA copy number variation (CNV). FIG. 11A: Enrichment analysis of significant correlations between intragenic aeTSA RNA expression and CNV, calculated with Fisher's exact test. aeTSAs are grouped based on the proportion of tumors expressing TSA (top and bottom halves). FIG. 11B: Correlation between aeTSA counts and chromosome arm amplifications. Scatter plots show the Pearson correlation between chromosome arm amplifications (x-axis) and aeTSA counts identified from each sample arm (y-axis). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The terms and symbols of genetics, molecular biology, biochemistry, and nucleic acid used herein follow standard treatises and texts in the field, such as Kornberg and Baker, DNA Replication, Second Edition (WH Freeman, New York, 1992), Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975), Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999), Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991), Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984), etc. All terms should be understood using their typical meanings established in the relevant technical field.

[0010] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Throughout this specification, unless the context requires otherwise, it will be understood that the terms "comprise", "comprises" and "comprising" imply the inclusion of a specified step or element, or group of steps or elements, but not the exclusion of any other step or element, or group of steps or elements.

[0011] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within that range, and each separate value is incorporated herein as if it were individually recited herein. Also, every subset of values ​​within a range is incorporated herein as if it were individually recited herein.

[0012] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0013] Any and all examples provided herein, or the use of exemplary language (e.g., "etc."), are intended only to better illustrate the invention and do not pose a limitation on the scope of the invention unless otherwise stated.

[0014] No language in the specification should be construed as indicating any element not claimed as essential to the practice of the invention.

[0015] As used herein, the term "about" has its ordinary meaning. The term "about" is used to indicate that a value includes the inherent variation of error for the device or method being used to determine the value, or encompasses values ​​that are close to the recited value, e.g., within 10% or 5% of the recited value (or range of values).

[0016] In the study described herein, we used a proteogenomic-based approach to identify 111 TSA candidates (103 novel and 8 previously reported candidates) from 23 HGSC tumors. The majority of these TSAs (93) originate from aberrantly expressed, non-mutated genomic sequences that are not expressed in normal tissues. These aberrantly expressed TSAs (herein referred to as aeTSAs) were shown to be primarily derived from non-exonic sequences, particularly intronic (31%) and intergenic (22%) sequences, and their expression was regulated at the transcriptional level by variations in gene copy number and DNA methylation. These aeTSAs were shared by the majority of HGSCs, and considering the frequency of aeTSA expression and HLA allele frequency, the median number of aeTSAs per tumor was estimated to be 5 in Caucasians. The novel TSA candidates identified herein may be useful for T cell-based immunotherapy of ovarian cancer.

[0017] Thus, in an embodiment, the present disclosure relates to a tumor antigen peptide (or tumor-specific peptide) comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 1 to 103, preferably SEQ ID NOs: 19 to 103 (Table 3A, Table 3B).

[0018] In an embodiment, the present disclosure relates to a tumor antigen peptide encoded by a sequence located in the untranslated transcribed region (UTR), i.e., the 3'-UTR or 5'-UTR region, comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 1, 8, 10, 13, 15, and 19-27, preferably SEQ ID NOs: 19-27. In an embodiment, the tumor antigen peptide is encoded by a sequence located in the 5'-UTR region comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 1, 10, 13, 15, and 19-23, preferably SEQ ID NOs: 19-23. In an embodiment, the tumor antigen peptide is encoded by a sequence located in the 3'-UTR region comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 8, and 24-27, preferably SEQ ID NOs: 24-27.

[0019] In an embodiment, the present disclosure relates to a tumor antigen peptide encoded by a sequence located in an intron, the sequence comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 16, 17, and 36 to 64, preferably SEQ ID NOs: 36 to 64.

[0020] In an embodiment, the present disclosure relates to a tumor antigen peptide encoded by a sequence located in an intergenic region, the sequence comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 65-84.

[0021] In another embodiment, the present disclosure relates to a tumor antigen peptide encoded by a sequence located in an exon and beginning with a frameshift, the sequence comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 6, 7, 18 and 28-35, preferably SEQ ID NOs: 28-35.

[0022] In another embodiment, the present disclosure relates to a tumor antigen peptide encoded by a non-coding RNA sequence (ncRNA) (located in an exon of a non-coding transcript) comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 4, and 85-92, preferably SEQ ID NOs: 85-92.

[0023] In another embodiment, the present disclosure relates to a tumor antigen peptide encoded by a sequence that is an antisense to a gene comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 93-99.

[0024] In another embodiment, the present disclosure relates to a tumor antigen peptide encoded by a sequence from a mucin gene, the sequence comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 100-103.

[0025] Generally, peptides such as tumor antigen peptides presented in the context of HLA class I are about 7 or 8 to about 15, or preferably 8 to 14 amino acid residues in length. In some embodiments of the methods of the present disclosure, longer peptides comprising the tumor antigen peptide sequences defined herein are artificially loaded into cells such as antigen presenting cells (APCs) and processed by the cells, and the tumor antigen peptides are presented by MHC class I molecules on the surface of the APCs. In this method, peptides / polypeptides longer than 15 amino acid residues (i.e., tumor antigen precursor peptides) can be loaded into the APCs and processed by proteases in the APC cytoplasm that provide the corresponding tumor antigen peptides defined herein for presentation. In some embodiments, the precursor peptides / polypeptides used to generate the tumor antigen peptides defined herein are, 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 methods and processes using tumor antigen peptides described herein include the use of longer peptides or polypeptides (including natural proteins), i.e., tumor antigen precursor peptides / polypeptides, to induce the presentation of the "final" 8-14 tumor antigen peptides after processing by cells (APCs). In some embodiments, the tumor antigen peptides described herein are about 8-14, 8-13, or 8-12 amino acids long (e.g., 8, 9, 10, 11, 12, or 13 amino acids long) and small enough to fit directly into an HLA class I molecule. In embodiments, the tumor antigen peptide comprises 20 or fewer amino acids, preferably 15 or fewer amino acids, more preferably 14 or fewer amino acids. In embodiments, the tumor antigen peptide comprises at least 7 amino acids, preferably at least 8 amino acids, more preferably at least 9 amino acids.

[0026] The term "amino acid" as used herein includes both L- and D-isomers of naturally occurring amino acids, as well as other amino acids (e.g., naturally occurring amino acids, non-naturally occurring amino acids, amino acids that are not encoded by nucleic acid sequences, etc.) that are used in peptide chemistry to prepare synthetic analogs of tumor antigen peptides. 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, as well as conservative substitutions for L-amino acids. Naturally occurring non-genetically encoded amino acids include, for example, β-alanine, 3-aminopropionic acid, 2,3-diaminopropionic acid, α-aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-naphthylalanine, pyridylalanine, 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 4-aminophenylalanine, 5-aminophenylalanine, 6-aminophenylalanine, 7-aminophenylalanine, 8-aminophenylalanine, 9-aminophenylalanine, 10-aminophenylalanine, 11-aminophenylalanine, 12-aminophenylalanine, 13-aminophenylalanine, 14-aminophenylalanine, 15-aminophenylalanine, 16-aminophenylalanine, 17-aminophenylalanine, 18-aminophenylalanine, 19-aminophenylalanine, 20-aminophenylalanine, 21-aminophenylalanine, 22-aminophenylalanine, 23-aminophenylalanine, 24-aminophenylalanine, 25-aminophenylalanine, 26-aminophenylalanine, 27-aminophenylalanine, 28-aminophenylalanine, 29-aminophenylalanine, 29-aminophenylalanine, 30-aminophenylalanine, 31-aminophenylalanine, 32-aminophenylalanine, 33-aminophenylalanine, 34-aminophenylalanine, Examples of amino acids that may be used include fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, β-2-thienylalanine, methionine sulfoxide, L-homoarginine (Hoarg), N-acetyl-lysine, 2-aminobutyric acid, 2-aminobutyric acid, 2,4-diaminobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine, homoserine (HoSer), cysteic acid, ε-aminohexanoic acid, δ-aminovaleric acid, or 2,3-diaminobutyric acid (D- or L-). These amino acids are well known in the field of biochemistry / peptide chemistry. In an embodiment, the tumor antigen peptide comprises only naturally occurring amino acids.

[0027] In embodiments, the tumor antigen peptides described herein include peptides with modified sequences that contain functionally equivalent amino acid residue substitutions compared to the sequences described herein. For example, one or more amino acid residues in the sequence may be replaced by another amino acid of similar polarity (having similar physicochemical properties), which acts as a functional equivalent and results in a silent alteration. Substitutes for amino acids in 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 can be included in either the non-polar 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 tumor antigen peptides described herein may comprise all L-amino acids, all D-amino acids, or a mixture of L-amino acids and D-amino acids. In embodiments, the tumor antigen peptides described herein comprise all L-amino acids.

[0028] In embodiments, in the sequence of a tumor antigen peptide comprising or consisting of one of the sequences disclosed in Tables 3A to 3B, amino acid residues that do not substantially contribute to the interaction with the T cell receptor may be modified by replacement with other amino acids whose incorporation does not substantially affect T cell reactivity and does not eliminate binding to the relevant MHC.

[0029] The tumor antigen peptide may also be capped or modified at the N-terminus and / or C-terminus to prevent degradation and increase stability, affinity and / or uptake. Thus, in another aspect, the present disclosure provides a tumor antigen peptide of formula Z 1 -XZ 2 In the present invention, there is provided a modified tumor antigen peptide of the formula: wherein X is a tumor antigen peptide comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 1 to 103, preferably SEQ ID NOs: 19 to 103 (Table 3A, Table 3B).

[0030] In embodiments, the amino terminal residue of the tumor antigen peptide (i.e., the free amino group at the N-terminus) is, for example, a moiety / chemical group (Z 1 ) is modified (e.g., for protection from degradation). 1 may be a straight or branched chain alkyl group of 1 to 8 carbons, or an acyl group (R-CO-), where R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, isopropionyl, or iso-butanyl), or an aroyl group (Ar-CO-), where Ar is an aryl group. In embodiments, the acyl group is a C1 to C6 16 Or C3~C 16 In a further embodiment, Z is a saturated C1-C6 acyl group (linear or branched, saturated or unsaturated), and in a further embodiment, a saturated C3-C6 acyl group (linear or branched) or an unsaturated C3-C6 acyl group (linear or branched), such as an acetyl group (CH3-CO-Ac). 1 The carboxy-terminal residue of the tumor antigen peptide (i.e., the free carboxy group at the C-terminus of the tumor antigen peptide) may be modified (e.g., for protection from degradation) by, for example, amidation (replacement of an OH group with an NH2 group), and in such cases, Z 2 is an NH group. In embodiments, Z 2may be a hydroxamate group, a nitrile group, an amide (primary, secondary or tertiary) group, an aliphatic amine of 1 to 10 carbons such as methylamine, iso-butylamine, iso-valerylamine or cyclohexylamine, an aromatic or arylalkyl amine such as aniline, naphthylamine, benzylamine, cinnamylamine or phenylethylamine, an alcohol, or CHOH. 2 In an embodiment, the tumor antigen peptide comprises one of the amino acid sequences of SEQ ID NOs: 1 to 103, preferably SEQ ID NOs: 19 to 103 (Table 3A, Table 3B). In an embodiment, the tumor antigen peptide consists of one of the amino acid sequences of SEQ ID NOs: 1 to 103, preferably SEQ ID NOs: 19 to 103 (Table 3A, Table 3B), i.e., Z 1 and Z 2 does not exist.

[0031] In another aspect, the present disclosure provides an HLA-A antibody comprising or consisting of the sequence of SEQ ID NO: 21, 28, 40, 41, 66 or 88. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 01:01 molecule.

[0032] In another embodiment, the present disclosure relates to an HLA-A antibody comprising or consisting of the sequence of SEQ ID NO: 14, 17, 45, 48, 51, 56, 75, 77, 82, 98 or 100, preferably 45, 48, 51, 56, 75, 77, 82, 98 or 100. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 02:01 molecule.

[0033] In another aspect, the present disclosure provides an HLA-A antibody comprising or consisting of the sequence of SEQ ID NO: 1, 19, 20, 22, 30, 31, 36, 50, 52, 60, 62, 73, 84, 85, 86 or 91, preferably 19, 20, 22, 30, 31, 36, 50, 52, 60, 62, 73, 84, 85, 86 or 91. *A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 03:01 molecule.

[0034] In another aspect, the present disclosure provides an HLA-A antibody comprising or consisting of the sequence of SEQ ID NO: 32, 54, 55, 67, 69, 81, 87, 90 or 102. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 11:01 molecule is provided.

[0035] In another aspect, the present disclosure relates to an HLA-A antibody comprising or consisting of the sequence of SEQ ID NO: 33 or 43. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 24:02 molecule is provided.

[0036] In another aspect, the present disclosure relates to an HLA-A antibody comprising or consisting of the sequence of SEQ ID NO:24. * Tumor antigen peptides (or tumor-specific peptides), preferably ovarian tumor antigen peptides, that bind to the 25:01 molecule are provided.

[0037] In another aspect, the present disclosure relates to an HLA-A antibody comprising or consisting of the sequence of SEQ ID NO: 34 or 58. * Provided is a tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 29:02 molecule.

[0038] In another aspect, the present disclosure relates to an HLA-A antibody comprising or consisting of the sequence of SEQ ID NO: 16. * Provided is a tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 32:01 molecule.

[0039] In another embodiment, the present disclosure relates to an HLA-B antibody comprising or consisting of the sequence of SEQ ID NO: 4, 6, 8, 9, 26, 49, 78, 92, 97 or 101, preferably 26, 49, 78, 92, 97 or 101. *A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 07:02 molecule.

[0040] In another aspect, the present disclosure provides an HLA-B antibody comprising or consisting of the sequence of SEQ ID NO: 23, 35, 42, 44, 46, 59, 63, 70, 74, 76, 83 or 103. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 08:01 molecule.

[0041] In another aspect, the present disclosure relates to an HLA-B antibody comprising or consisting of the sequence of SEQ ID NO:53. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 14:01 molecule is provided.

[0042] In another aspect, the present disclosure relates to an HLA-B antibody comprising or consisting of the sequence of SEQ ID NO: 2, 3 or 5. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 15:01 molecule is provided.

[0043] In another aspect, the present disclosure relates to an HLA-B antibody comprising or consisting of the sequence of SEQ ID NO:89. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 18:01 molecule is provided.

[0044] In another aspect, the present disclosure provides an HLA-B antibody comprising or consisting of the sequence of SEQ ID NO: 47, 64, 96 or 99. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 39:01 molecule is provided.

[0045] In another aspect, the present disclosure relates to an HLA-B antibody comprising or consisting of the sequence of SEQ ID NO: 11, 12 or 13. *A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 40:01 molecule is provided.

[0046] In another aspect, the present disclosure relates to an HLA-B antibody comprising or consisting of the sequence of SEQ ID NO:65. * Provided is a tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 44:02 molecule.

[0047] In another aspect, the present disclosure relates to an HLA-B antibody comprising or consisting of the sequence of SEQ ID NO: 37 or 94. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 44:03 molecule is provided.

[0048] In another embodiment, the present disclosure relates to an HLA-C antibody comprising or consisting of the sequence of SEQ ID NO: 10, 29, 71 or 95, preferably SEQ ID NO: 29, 71 or 95. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 03:03 molecule.

[0049] In another aspect, the present disclosure provides an HLA-C antibody comprising or consisting of the sequence of SEQ ID NO: 6 or 15. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 04:01 molecule.

[0050] In another aspect, the present disclosure provides an HLA-C antibody comprising or consisting of the sequence of SEQ ID NO:27. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 05:01 molecule.

[0051] In another embodiment, the present disclosure relates to an HLA-C antibody comprising or consisting of the sequence of SEQ ID NO: 18 or 72, preferably SEQ ID NO: or 72. *A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 06:02 molecule.

[0052] In another aspect, the present disclosure provides an HLA-C antibody comprising or consisting of the sequence of SEQ ID NO: 38, 61 or 93. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 07:01 molecule.

[0053] In another aspect, the present disclosure provides an HLA-C antibody comprising or consisting of the sequence of SEQ ID NO:7. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, is provided that binds to the 07:02 molecule.

[0054] In another aspect, the present disclosure provides an HLA-C antibody comprising or consisting of the sequence of SEQ ID NO:80. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 12:03 molecule is provided.

[0055] In another aspect, the present disclosure provides an HLA-C antibody comprising or consisting of the sequence of SEQ ID NO: 25, 57 or 79. * A tumor antigen peptide (or tumor specific peptide), preferably an ovarian tumor antigen peptide, that binds to the 14:02 molecule is provided.

[0056] In an embodiment, the tumor antigen peptide is encoded by a sequence located in the untranslated transcribed region (UTR), i.e., the 3'-UTR or 5'-UTR region. In another embodiment, the tumor antigen peptide is encoded by a sequence located in an intron. In another embodiment, the tumor antigen peptide is encoded by a sequence located in an intergenic region. In another embodiment, the tumor antigen peptide is encoded by a sequence located in an exon and derived from a frameshift.

[0057] The tumor antigen peptide of the present disclosure can be produced by expression in a host cell containing a nucleic acid encoding the tumor antigen peptide (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis). Peptides can be easily synthesized by manual and / or automated solid-phase procedures well known in the art. Suitable synthesis can be performed, for example, by utilizing the "T-boc" or "Fmoc" procedures. Techniques and procedures for solid-phase synthesis are described, for example, in Solid Phase Peptide Synthesis: A Practical Approach (E. Atherton and RC Sheppard, published by IRL, Oxford University Press, 1989). Alternatively, tumor antigen peptides can be used, for example, Liu et al., Tetrahedron Lett. 37:933-936, 1996, Baca et al., J. Am. Chem. Soc. 117: 1881-1887, 1995, Tam et al., Int. 256:221-225,1992, Liu and Tam, J.Am.Chem.Soc.116:4149-4153,1994, Liu and Tam,Proc.Natl.Acad.Sci.USA 91:6584-6588,1994, and Yamashiro and Li,Int.J.Peptide Protein Res.31:322-334,1988). Other methods useful for the synthesis of tumor antigen peptides are described in Nakagawa et al., J.Am.Chem.Soc.107:7087-7092,1985. In embodiments, tumor antigen peptides are chemically synthesized (synthetic peptides). Another embodiment of the present disclosure relates to non-naturally occurring peptides, which consist of or consist essentially of the amino acid sequences defined herein and are synthetically produced (e.g., synthesized) as pharmaceutically acceptable salts.The salt of the tumor antigen peptide according to the present disclosure is significantly different from the peptide in the in vivo state, because the peptide produced in vivo is not a salt. The non-natural salt form of the peptide can adjust the solubility of the peptide, especially in the context of pharmaceutical compositions comprising the peptide, such as the peptide vaccines disclosed herein. Preferably, the salt is a pharma-ceutically acceptable salt of the peptide.

[0058] In an embodiment, the tumor antigen peptides described herein are substantially pure. A compound is "substantially pure" when it is separated from components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more typically 75%, 80% or 85%, preferably more than 90%, more preferably more than 95% by weight of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesized or produced by recombinant techniques will generally be substantially free of its naturally associated components, e.g., components of the macromolecule from which it is sourced. A nucleic acid molecule is substantially pure when it is not immediately contiguous (i.e., not covalently linked) with coding sequences that are 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 that encodes the peptide compound, or by chemical synthesis. Purity can be measured using any suitable method, such as column chromatography, gel electrophoresis, HPLC, etc. In an embodiment, the tumor antigen peptide is in solution. In another embodiment, the tumor antigen peptide is in a solid form, e.g., lyophilized.

[0059] In another aspect, the present disclosure further provides a nucleic acid (isolated) encoding a tumor antigen peptide or tumor antigen precursor peptide described herein. In an embodiment, the nucleic acid comprises about 21 nucleotides to about 45 nucleotides, about 24 to about 45 nucleotides, e.g., 24, 27, 30, 33, 36, 39, 42 or 45 nucleotides. "Isolated" as used herein refers to a peptide or nucleic acid molecule that is separated from other components present in the molecule's natural environment or macromolecules of the naturally occurring source (e.g., including other nucleic acids, proteins, lipids, sugars, etc.). "Synthetic" as used herein refers to a peptide or nucleic acid molecule that is not isolated from its natural source, e.g., produced through recombinant technology or using chemical synthesis. The nucleic acids of the present disclosure can be used for recombinant expression of the tumor antigen peptides of the present disclosure and can be included in a vector or plasmid, such as a cloning vector or expression vector that can be transfected into a host cell. In an embodiment, the present disclosure provides a cloning, expression, or viral vector or plasmid that includes a nucleic acid sequence encoding the tumor antigen peptide of the present disclosure. Alternatively, the nucleic acid encoding the tumor antigen peptide of the present disclosure may be integrated into the genome of the host cell. In either case, the host cell expresses the tumor antigen peptide or the protein encoded by the nucleic acid. The term "host cell" as used herein refers not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. The host cell can be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., insect cell, yeast cell, or mammalian cell) capable of expressing the tumor antigen peptide described herein. The vector or plasmid contains elements necessary for transcription and translation of the inserted coding sequence and may contain other components such as resistance genes, cloning sites, etc. Methods well known to those skilled in the art may be used to construct expression vectors containing a peptide or polypeptide coding sequence 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, NY, and Ausubel, FM et al.(1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY. "Operably linked" refers to the juxtaposition of components, particularly components that allow the normal function of the nucleotide sequence to be performed. Thus, a coding sequence operably linked to a regulatory sequence refers to a configuration of nucleotide sequences that allows the coding sequence to be expressed under the regulatory control of the regulatory sequence, i.e., transcriptional and / or translational control. "Regulatory / control region" or "regulatory / control sequence" as used herein refers to a non-coding nucleotide sequence that is involved in regulating the expression of the coding nucleic acid. Thus, the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like. In an embodiment, the nucleic acid (DNA, RNA) encoding the tumor antigen peptide of the present disclosure is contained within or attached to a liposome or any other suitable vehicle.

[0060] In another aspect, the present disclosure provides an MHC class I molecule that comprises (i.e., presents or binds) a tumor antigen peptide. In an embodiment, the MHC class I molecule is an HLA-A1 molecule, and in a further embodiment, an HLA-A*01:01 molecule. In another embodiment, the MHC class I molecule is an HLA-A2 molecule, and in a further embodiment, an HLA-A*02:01 molecule. In another embodiment, the MHC class I molecule is an HLA-A3 molecule, and in a further embodiment, an HLA-A*03:01 molecule. In another embodiment, the MHC class I molecule is an HLA-A11 molecule, and in a further embodiment, an HLA-A*11:01 molecule. In another embodiment, the MHC class I molecule is an HLA-A24 molecule, and in a further embodiment, an HLA-A*24:02 molecule. In another embodiment, the MHC class I molecule is an HLA-A25 molecule, and in a further embodiment, an HLA-A*25:01 molecule. In another embodiment, the MHC class I molecule is an HLA-A29 molecule, and in a further embodiment, an HLA-A*29:02 molecule. In another embodiment, the MHC class I molecule is an HLA-A32 molecule, and in a further embodiment, an HLA-A*32:02 molecule. In another embodiment, the MHC class I molecule is an HLA-B07 molecule, and in a further embodiment, an HLA-B*07:02 molecule. In another embodiment, the MHC class I molecule is an HLA-B08 molecule, and in a further embodiment, an HLA-B*08:01 molecule. In another embodiment, the MHC class I molecule is an HLA-B14 molecule, and in a further embodiment, an HLA-B*14:01 molecule. In another embodiment, the MHC class I molecule is an HLA-B15 molecule, and in a further embodiment, an HLA-B*15:01 molecule. In another embodiment, the MHC class I molecule is an HLA-B18 molecule, and in a further embodiment, an HLA-B*18:01 molecule. In another embodiment, the MHC class I molecule is an HLA-B39 molecule, and in a further embodiment, an HLA-B*39:01 molecule. In another embodiment, the MHC class I molecule is an HLA-B40 molecule, and in a further embodiment, an HLA-B*40:01 molecule.In another embodiment, the MHC class I molecule is an HLA-B44 molecule, and in a further embodiment, an HLA-B*44:02 molecule or an HLA-B*44:03 molecule. In another embodiment, the MHC class I molecule is an HLA-C03 molecule, and in a further embodiment, an HLA-C*03:03 molecule. In another embodiment, the MHC class I molecule is an HLA-C04 molecule, and in a further embodiment, an HLA-C*04:01 molecule. In another embodiment, the MHC class I molecule is an HLA-C05 molecule, and in a further embodiment, an HLA-C*05:01 molecule. In another embodiment, the MHC class I molecule is an HLA-C06 molecule, and in a further embodiment, an HLA-C*06:02 molecule. In another embodiment, the MHC class I molecule is an HLA-C07 molecule, and in a further embodiment, an HLA-C*07:01 or an HLA-C*07:02 molecule. In another embodiment, the MHC class I molecule is an HLA-C12 molecule, and in a further embodiment, an HLA-C*12:03 molecule. In another embodiment, the MHC class I molecule is an HLA-C14 molecule, and in a further embodiment, an HLA-C*14:02 molecule.

[0061] In an embodiment, the tumor antigen peptide is non-covalently bound to the MHC class I molecule (i.e., the tumor antigen peptide is loaded or non-covalently bound into the peptide-binding groove / pocket of the MHC class I molecule). In another embodiment, the tumor antigen peptide is covalently attached / bound to the MHC class I molecule (α chain). In such a construct, the tumor antigen peptide and the MHC class I molecule (α chain) are typically produced as a synthetic fusion protein with a short (e.g., 5-20 residues, preferably about 8-12, e.g., 10) flexible linker or spacer (e.g., polyglycine linker). In another aspect, the present disclosure provides a nucleic acid encoding a fusion protein comprising a tumor antigen peptide as defined herein fused to an MHC class I molecule (α chain). In an embodiment, the MHC class I molecule (α chain)-peptide complex is multimerized. Thus, in another aspect, the present disclosure provides multimers of MHC class I molecules loaded (covalently or non-covalently) with tumor antigen peptides as described herein. Such multimers may be bound to a tag, e.g., a fluorescent tag, that allows detection of the multimer. Numerous 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 multimers of CD8 specific for tumor antigen peptides as defined herein. + A method for detecting or purifying (isolating, enriching) T lymphocytes, comprising contacting a cell population with multimers of MHC class I molecules loaded (covalently or non-covalently) with tumor antigen peptides, and detecting CD8 + and detecting or isolating a CD8 T lymphocyte bound by an MHC class I multimer. + T lymphocytes may be isolated using known methods, for example, fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

[0062] In yet another aspect, the present disclosure provides a cell (e.g., a host cell), in embodiments an isolated cell, comprising a nucleic acid, vector or plasmid of the present disclosure described herein, i.e., a nucleic acid or vector encoding one or more tumor antigen peptides. In another aspect, the present disclosure provides a cell expressing an MHC class I molecule (e.g., an MHC class I molecule of one of the alleles disclosed above) that binds to or presents a tumor antigen peptide according to the present disclosure. In one embodiment, the host cell is a eukaryotic cell, e.g., a mammalian cell, preferably a human cell, cell line or immortalized cell. In another embodiment, the cell is an antigen presenting cell (APC). In one embodiment, the host cell is a primary cell, cell line or immortalized cell. In another embodiment, the cell is an antigen presenting cell (APC). The nucleic acid and vector can be introduced into the cell via conventional transformation or transfection techniques. The terms "transformation" and "transfection" refer to techniques for introducing foreign nucleic acid into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation, microinjection and virus-mediated transfection. Suitable methods for transforming or transfecting host cells can be found, for example, in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing nucleic acid into mammalian cells in vivo are also known and can be used to deliver the vector or plasmid of the present disclosure to a subject for gene therapy.

[0063] Various methods known in the art can be used to load cells, such as APCs, with one or more tumor antigen peptides. As used herein, "loading cells" with tumor antigen peptides means that tumor antigen peptides or RNA or DNA encoding tumor antigen peptides are transfected into cells, or alternatively, APCs are transformed with nucleic acids encoding tumor antigen peptides. Cells can also be loaded by contacting cells with exogenous tumor antigen peptides that can directly bind to MHC class I molecules present on the cell surface (e.g., peptide-pulsed cells). Tumor antigen peptides can also be fused to domains or motifs that facilitate their presentation by MHC class I molecules, such as endoplasmic reticulum (ER) retrieval signals, C-terminal Lys-Asp-Glu-Leu sequences (see Wang et al. Eur J Immunol. 2004 Dec:34(12):3582-94).

[0064] In another aspect, the present disclosure provides a composition or peptide combination / pool comprising any one or any combination of the tumor antigen peptides (or nucleic acids encoding said peptides) defined herein. In an embodiment, the composition comprises any combination of the tumor antigen peptides defined herein (any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tumor antigen peptides) or a combination of nucleic acids encoding said tumor antigen peptides). The composition comprising any combination / subcombination of the tumor antigen peptides defined herein is encompassed by the present disclosure. In another embodiment, the combination or pool may comprise one or more known tumor antigens.

[0065] Thus, in another aspect, the present disclosure provides a composition comprising any one of the tumor antigen peptides defined herein, or any combination thereof, and a cell expressing an MHC class I molecule (e.g., an MHC class I molecule of one of the alleles disclosed above). APCs for use in the present disclosure are not limited to a particular type of cell, and may be any type of cell, including CD8 +It includes professional APCs such as dendritic cells (DCs), Langerhans cells, macrophages, and B cells, which are known to present proteinaceous antigens on their cell surface so that they can be recognized by T lymphocytes. For example, APCs can be obtained by inducing DCs from peripheral blood mononuclear cells, either in vitro, ex vivo, or in vivo, and then contacting (stimulating) tumor antigen peptides. APCs can also be activated to present tumor antigen peptides in vivo, where one or more of the tumor antigen peptides of the present disclosure are administered to a subject, and APCs presenting tumor antigen peptides are induced in the subject's body. The phrases "inducing APCs" or "stimulating APCs" include contacting or loading cells with one or more tumor antigen peptides, or nucleic acids encoding tumor antigen peptides, so that the tumor antigen peptides are presented on their surface by MHC class I molecules. As described herein, according to the present disclosure, tumor antigen peptides can be indirectly loaded, for example, using a longer peptide / polypeptide that includes the sequence of tumor antigen peptide (including natural protein), and then processed (e.g., by protease) inside APC to generate tumor antigen peptide / MHC class I complex on the cell surface. After loading APC with tumor antigen peptide and allowing APC to present tumor antigen peptide, APC can be administered to a subject as a vaccine. For example, ex vivo administration can include the following steps: (a) harvesting APC from a first subject; (b) contacting / loading APC of step (a) with tumor antigen peptide to form MHC class I / tumor antigen peptide complex on the surface of APC; and (c) administering peptide-loaded APC to a second subject in need of treatment.

[0066] The first subject and the second subject may be the same subject (e.g., autologous vaccine) or different subjects (e.g., allogeneic vaccine).Alternatively, the present disclosure provides the use of the tumor antigen peptides (or combinations thereof) described herein for producing a composition (e.g., pharmaceutical composition) for inducing antigen-presenting cells.In addition, the present disclosure provides a method or process for producing a pharmaceutical composition for inducing antigen-presenting cells, the method or process includes mixing or formulating the tumor antigen peptides, or combinations thereof, with a pharma-ceutically acceptable carrier. Cells such as APCs expressing MHC class I molecules (e.g., HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A25, HLA-A29, HLA-A32, HLA-B07, HLA-B08, HLA-B14, HLA-B15, HLA-B18, HLA-B39, HLA-B40, HLA-B44, HLA-C03, HLA-C04, HLA-C05, HLA-C06, HLA-C07, HLA-C12, or HLA-C14 molecules) loaded with any one, or any combination, of the tumor antigen peptides defined herein, are expressed by CD8 + T lymphocytes, e.g., autologous CD8 + It can be used to stimulate / expand T lymphocytes. Thus, in another aspect, the present disclosure relates to a method for the treatment of tumors comprising administering to a patient a tumor antigen peptide (or a nucleic acid or vector encoding the same) as defined herein, any one of the peptides, or any combination thereof, an MHC class I molecule, and a T lymphocyte, more specifically, a CD8 + Cells expressing T lymphocytes (e.g., CD8 + The present invention provides a composition comprising a population of cells comprising T lymphocytes.

[0067] In an embodiment, the composition further comprises a buffer, excipient, carrier, diluent, and / or medium (e.g., culture medium). In a further embodiment, the buffer, excipient, carrier, diluent, and / or medium is a pharma- ceutically acceptable buffer, excipient, carrier, diluent, and / or medium (culture medium). As used herein, "pharma-ceutically acceptable buffer, excipient, carrier, diluent, and / or medium" includes any and all solvents, buffers, binders, lubricants, fillers, thickeners, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizers, release retardants, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and the like, that are physiologically compatible, do not interfere with the effectiveness of the biological activity of the active ingredient, and are non-toxic to the subject. The use of such media and agents for pharma-ceutically active substances is well known in the art (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4). th edition, Pharmaceutical Press, London UK). Any conventional medium or agent is contemplated for use in the compositions of the present disclosure, except where the active compound (peptide, cells) is incompatible with them. In embodiments, the buffer, excipient, carrier, and / or medium is a non-naturally occurring buffer, excipient, carrier, and / or medium. In embodiments, one or more of the tumor antigen peptides defined herein, or the nucleic acid (e.g., mRNA) encoding said one or more tumor antigen peptides, are contained within or complexed to liposomes, e.g., cationic liposomes (see, e.g., Vitor MT et al., Recent Pat Drug Deliv Formul. 2013 Aug; 7(2): 99-110).

[0068] In another aspect, the present disclosure provides a composition comprising any one or any combination of tumor antigen peptides (or nucleic acids encoding said peptides) defined herein, and one or more of buffers, excipients, carriers, diluents and / or media. For compositions comprising cells (e.g., APCs, T lymphocytes), the composition comprises a suitable media that allows for the maintenance of viable cells. Representative examples of such media include saline, 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 that comprises one or more tumor antigen peptides or vaccine vectors and can induce an immune response against one or more tumor antigen peptides present therein when administered to a subject. Vaccination methods for inducing immune responses in mammals include the use of vaccines or vaccine vectors administered by any conventional route known in the vaccine field, for example, via mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary) surfaces, via parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) routes, or by topical administration (e.g., via transdermal delivery systems such as patches). In embodiments, tumor antigen peptides (or combinations thereof) are conjugated to carrier proteins (conjugate vaccines) to increase the immunogenicity of tumor antigen peptides. Thus, the present disclosure provides compositions (conjugates) comprising tumor antigen peptides (or combinations thereof) or nucleic acids encoding tumor antigen peptides or combinations thereof, and carrier proteins.For example, the tumor antigen peptide or nucleic acid may be conjugated or complexed to a Toll-like receptor (TLR) ligand (see, e.g., Zom et al., Adv Immunol. 2012, 114:177-201) or a polymer / dendrimer (see, e.g., Liu et al., Biomacromolecules. 2013 Aug 12;14(8):2798-806). In an embodiment, the immunogenic composition or vaccine further comprises an adjuvant. "Adjuvant" refers to a substance that, when added to an immunogenic agent such as an antigen (a tumor antigen peptide, a nucleic acid and / or a cell according to the present disclosure), non-specifically enhances or enhances the immune response to the agent in the host upon exposure to the mixture.Examples of adjuvants currently used in the vaccine field include: (1) mineral salts (aluminum salts such as aluminum phosphate and aluminum hydroxide, calcium phosphate gels), squalene; (2) oil-based adjuvants (oil emulsions and surfactant-based formulations, etc.), e.g., MF59 (microfluidized detergent-stabilized oil-in-water emulsion), QS21 (purified saponin), AS02[SBAS2] (oil-in-water emulsion + MPL + QS-21); (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza hemagglutinin), AS04 (aluminum salt of [SBAS4] with MPL), ISCOMS (structural complexes of saponin and lipids), polylactide-co-glycolides (PLGs); (4) microbial derivatives (natural and and synthetic), such as monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharides), DC_Chol (lipidic immunostimulants that can self-assemble into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (bacterial toxin immunoglobulins genetically modified to provide a non-toxic adjuvant effect), (5) endogenous human immunomodulators, such as hGM-CSF or hIL-12 (cytokines that can be administered either as proteins or encoded plasmids), Immuduaptin (C3d tandem arrays), and / or (6) inert vehicles such as gold particles.

[0069] In an embodiment, the tumor antigen peptide or the composition comprising the same is in a lyophilized form. In another embodiment, the tumor antigen peptide or the composition comprising the same is a liquid composition. In a further embodiment, the tumor antigen peptide is at a concentration of about 0.01 μg / mL to about 100 μg / mL in the composition. In a further embodiment, the tumor antigen peptide is at a concentration of about 0.2 μg / mL to about 50 μg / mL, about 0.5 μg / mL to about 10, 20, 30, 40, or 50 μg / mL, about 1 μg / mL to about 10 μg / mL, or about 2 μg / mL in the composition.

[0070] As described herein, cells such as APCs expressing MHC class I molecules loaded with or binding to any one or any combination of the tumor antigen peptides defined herein can be used to stimulate / expand CD8+ T lymphocytes in vivo or ex vivo. Thus, in another aspect, the present disclosure provides T cell receptors (TCRs) capable of interacting with or binding to the MHC class I molecule / tumor antigen peptide complexes described herein, and nucleic acid molecules encoding such TCR molecules, as well as vectors comprising such nucleic acid molecules. The TCRs according to the present disclosure can specifically interact with or bind to tumor antigen peptides loaded on or presented by MHC class I molecules, preferably on the surface of living cells in vitro or in vivo. The TCRs, particularly the nucleic acids encoding the TCRs of the present disclosure, can be used to stimulate / expand T lymphocytes (e.g., CD8 T cells) that generate new T lymphocyte clones that specifically recognize the MHC class I / tumor antigen peptide complexes. + T lymphocytes) or other types of lymphocytes. In certain embodiments, T lymphocytes (e.g., CD8 + T lymphocytes) are transformed to express one or more TCRs that recognize tumor antigen peptides, and the transformed cells are administered to the patient (autologous cell transfusion). In certain embodiments, T lymphocytes (e.g., CD8 + T lymphocytes (e.g., CD8 T cells) are transformed to express one or more TCRs that recognize tumor antigen peptides, and the transformed cells are administered to the recipient (allogeneic cell transfusion). In another embodiment, the present disclosure relates to T lymphocytes, e.g., CD8 T cells, transformed / transfected with a vector or plasmid encoding a tumor antigen peptide-specific TCR. + Provided is a T lymphocyte. In a further embodiment, the present disclosure provides a method for treating a patient with autologous or allogeneic cells transformed with tumor antigen peptide-specific TCR. In yet another embodiment, the present disclosure provides the use of tumor antigen-specific TCR in the manufacture of autologous or allogeneic cells for the treatment of cancer.

[0071] In some embodiments, patients treated with the compositions (e.g., pharmaceutical compositions) of the present disclosure are treated prior to or following treatment with allogeneic stem cell transplantation (ASCL), allogeneic lymphocyte infusion, or autologous lymphocyte infusion. The compositions of the present disclosure are administered to patients receiving allogeneic T lymphocytes (e.g., CD8 + T lymphocytes), allogeneic or autologous APC vaccines loaded with tumor antigen peptides, tumor antigen peptide vaccines, and allogeneic or autologous T lymphocytes transformed with tumor antigen-specific TCRs (e.g., CD8 + The present disclosure provides a method for providing a T-lymphocyte clone capable of recognizing a tumor antigen peptide according to the present disclosure, which may be generated for and specifically target tumor cells expressing the tumor antigen peptide in a subject (e.g., a transplant recipient), e.g., an ASCT and / or donor lymphocyte infusion (DLI) recipient. Thus, the present disclosure provides a method for providing a CD8 T-lymphocyte clone capable of recognizing a tumor antigen peptide / MHC class I molecule complex that encodes and expresses a T cell receptor that can specifically recognize or bind the tumor antigen peptide / MHC class I molecule complex. + A T lymphocyte is provided. The T lymphocyte (e.g., CD8 + The CD8 T lymphocytes may be recombinant (engineered) or naturally selected T lymphocytes. Thus, the present disclosure relates to a method for the treatment of CD8 T lymphocytes comprising contacting undifferentiated lymphocytes with a tumor antigen peptide / MHC class I molecule complex (typically expressed on the surface of a cell, such as an APC) under conditions favorable for causing T cell activation and proliferation, which may be performed in vitro or in vivo (i.e., in a patient administered an APC vaccine in which the APC is loaded with a tumor antigen peptide, or in a patient administered a tumor antigen peptide). + At least two methods are provided for generating T lymphocytes: a population of CD8 T cells capable of recognizing multiple tumor antigen peptides using combinations or pools of tumor antigen peptides bound to MHC class I molecules; +Alternatively, tumor antigen-specific or targeted T lymphocytes can be generated by targeting the MHC class I molecule / tumor antigen peptide complex (i.e., engineered or recombinant CD8 + The tumor antigen peptide-specific TCRs of the present disclosure may be produced / generated in vitro or ex vivo by cloning one or more nucleic acids (genes) encoding a TCR (more specifically, an α chain and a β chain) that specifically binds to a tumor antigen peptide (T lymphocyte). Nucleic acids encoding the tumor antigen peptide-specific TCRs of the present disclosure may be obtained from T lymphocytes activated ex vivo against tumor antigen peptides (e.g., APCs loaded with tumor antigen peptides) or from individuals exhibiting an immune response to peptide / MHC molecule complexes, using methods known in the art. The tumor antigen peptide-specific TCRs of the present disclosure may be recombinantly expressed in host cells and / or host lymphocytes obtained from a graft recipient or a graft donor, and may be optionally differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). Nucleic acids (transgenes) encoding the TCR α and β chains may be introduced into T cells (e.g., from the subject to be treated or another individual) using any suitable method, such as transfection (e.g., electroporation) or transduction (e.g., using a viral vector). Engineered CD8 expressing TCR specific for tumor antigen peptide + T lymphocytes may be expanded in vitro using well-known culture methods.

[0072] The present disclosure relates to isolated CD8+ cells that are specifically induced, activated, and / or expanded (grown) by tumor antigen peptides (i.e., tumor antigen peptides bound to MHC class I molecules expressed on the cell surface) or combinations of tumor antigen peptides. + The present disclosure also provides a CD8 T lymphocyte capable of recognizing a tumor antigen peptide, or a combination thereof, according to the present disclosure. +In another aspect, the present disclosure provides a CD8 T-lymphocyte that specifically recognizes one or more MHC class I molecule / tumor antigen peptide complexes described herein, and a composition comprising the tumor antigen peptide. + Cell populations or cell cultures enriched for T lymphocytes (e.g., CD8 + Such enriched populations can be obtained by performing ex vivo expansion of specific T lymphocytes using cells such as APCs expressing MHC class I molecules loaded (e.g., presenting) with one or more of the tumor antigen peptides disclosed herein. As used herein, "enriched" refers to the number of tumor antigen-specific CD8 T lymphocytes in a population. + This means that the proportion of T lymphocytes is significantly higher compared to a natural population of cells, i.e., one that has not been subjected to the step of ex vivo expansion of specific T lymphocytes. In a further embodiment, the percentage of tumor antigen peptide-specific CD8 + The percentage of T lymphocytes is at least about 0.5%, e.g., at least about 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2% or 3%. In some embodiments, the percentage of tumor antigen peptide-specific CD8 + The percentage of T lymphocytes 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 populations or cultures enriched for CD8+ T lymphocytes that specifically recognize one or more MHC class I molecule / peptide (tumor antigen peptide) complexes of interest (e.g., CD8+ T lymphocyte populations) can be used in tumor antigen-based cancer immunotherapy, as described in more detail below. In some embodiments, tumor antigen peptide-specific CD8 + The population of T lymphocytes can be further enriched, for example, using multimers of MHC class I molecules loaded (covalently or non-covalently) with tumor antigen peptides as defined herein. Thus, the present disclosure provides, for example, a method for the detection of tumor antigen peptide-specific CD8+ A proportion of tumor antigen peptide-specific CD8 T lymphocytes is at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. + A purified or isolated population of T lymphocytes is provided.

[0073] The present disclosure further relates to the use of any tumor antigen peptide, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocyte, APC), and / or composition, or any combination thereof, according to the present disclosure as a medicament or in the manufacture of a medicament. In an embodiment, the medicament is for the treatment of cancer, e.g., a cancer vaccine. The present disclosure relates to any tumor antigen peptide, nucleic acid, expression vector, T cell receptor, cell (e.g., T lymphocyte, APC), and / or composition (e.g., vaccine composition), or any combination thereof, according to the present disclosure, for use in the treatment of cancer, e.g., as a cancer vaccine. The tumor antigen peptide sequences identified herein can be used for the production of synthetic peptides i) used for in vitro priming and expansion of tumor antigen-specific T cells to be injected into tumor patients, and / or ii) used as a vaccine to induce or enhance anti-tumor T cell responses in cancer patients.

[0074] In another aspect, the present disclosure provides for the use of the tumor antigen peptides described herein, or combinations thereof (e.g., peptide pools) as a vaccine for treating cancer in a subject. The present disclosure also provides for the use of the tumor antigen peptides described herein, or combinations thereof (e.g., peptide pools) as a vaccine for treating cancer in a subject. In an embodiment, the subject is a subject that has tumor antigen peptide-specific CD8 +Thus, in another aspect, the present disclosure provides a method of treating cancer (e.g., reducing the number of tumor cells, killing tumor cells), comprising administering to a subject an effective amount of a CD8 T cell that recognizes (i.e., expresses a TCR that binds to) one or more MHC class I molecule / tumor antigen peptide complexes (expressed on the surface of a cell, such as an APC). + In an embodiment, the method comprises administering (injecting) the CD8 T lymphocytes to a subject in need thereof. + After administration / infusion of T lymphocytes, the method further comprises administering to the subject an effective amount of tumor antigen peptides, or a combination thereof, and / or cells expressing MHC class I molecules loaded with tumor antigen peptides (e.g., APCs such as dendritic cells). In yet another embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of dendritic cells loaded with one or more tumor antigen peptides. In yet a further embodiment, the method comprises administering to a patient in need thereof an effective amount of allogeneic or autologous cells expressing a recombinant TCR that binds to tumor antigen peptides presented by MHC class I molecules.

[0075] In another aspect, the present disclosure provides a method for treating cancer in a subject by administering to the subject a CD8+ antigen that recognizes one or more MHC class I molecules that are loaded (presenting) with a tumor antigen peptide, or a combination thereof, for treating cancer in a subject (e.g., reducing the number of tumor cells, killing tumor cells). + In another aspect, the present disclosure provides a use of CD8 T lymphocytes that recognize one or more MHC class I molecules loaded (presenting) tumor antigen peptides, or a combination thereof, for the preparation / manufacture of a medicament for treating a cancer in a subject (e.g., reducing the number of tumor cells, killing tumor cells). + In another aspect, the present disclosure provides a method for the treatment of cancer in a subject, comprising administering to the subject a T-lymphocyte that recognizes one or more MHC class I molecules that are loaded with (present) a tumor antigen peptide, or a combination thereof, for use in treating the cancer in a subject (e.g., to reduce the number of tumor cells and kill tumor cells). +Provide T lymphocytes (cytotoxic T lymphocytes). In a further embodiment, the use further comprises the use of an effective amount of tumor antigen peptide (or a combination thereof) and / or a cell (e.g., APC) expressing one or more MHC class I molecules loaded (presenting) with the tumor antigen peptide, after the use of the tumor antigen peptide-specific CD8+ T lymphocytes.

[0076] The present disclosure also provides a method for generating an immune response in a subject against tumor cells expressing human class I MHC molecules loaded with any of the tumor antigen peptides disclosed herein or a combination thereof, the method comprising administering cytotoxic T lymphocytes that specifically recognize the class I MHC molecules loaded with the tumor antigen peptides or combinations of tumor antigen peptides. The present disclosure also provides the use of cytotoxic T lymphocytes that specifically recognize the class I MHC molecules loaded with any of the tumor antigen peptides or combinations of tumor antigen peptides disclosed herein to generate an immune response against tumor cells expressing human class I MHC molecules loaded with the tumor antigen peptides or combinations thereof.

[0077] In an embodiment, the method or use described herein further comprises determining the HLA class I alleles expressed by the patient before treatment / use, and administering or using a tumor antigen peptide that binds to one or more of the HLA class I alleles expressed by the patient.For example, if the patient is determined to express HLA-A2*01, HLA-B14*01 and HLA-C05*01, any combination of tumor antigen peptides of (i) SEQ ID NO: 14, 17, 45, 48, 51, 56, 75, 77, 82, 98 and / or 100 (binds to HLA-A2*01), (ii) SEQ ID NO: 53 (binds to HLA-B14*01), and / or (iii) SEQ ID NO: 27 (binds to HLA-C05*01) can be administered or used to the patient.

[0078] In an embodiment, the cancer is a solid cancer, preferably ovarian cancer. In an embodiment, the ovarian cancer is an ovarian carcinoma. In an embodiment, the ovarian cancer is an epithelial cancer, a serous carcinoma, a small cell carcinoma, a primary peritoneal carcinoma, a clear cell carcinoma or adenocarcinoma, an endometrial adenocarcinoma, a malignant mixed Mullerian tumor, a mucinous or cystadenocarcinoma, a malignant Brenner tumor, a transitional cell carcinoma, a sex cord-stromal tumor, a granular cell tumor, a Sertoli-Leydig tumor, a germ cell tumor, a dysgerminoma, a trophoblastic tumor, an immature (solid) or mature teratoma, a yolk sac tumor, a squamous cell carcinoma, or a secondary ovarian cancer. In an embodiment, the ovarian cancer is a type I ovarian carcinoma. In another embodiment, the ovarian cancer is a type II ovarian cancer. In an embodiment, the ovarian cancer is a serous carcinoma. In a further embodiment, the serous carcinoma is a high-grade serous carcinoma (HGSC). In an embodiment, the ovarian cancer is a stage I, II, III, or IV ovarian cancer.

[0079] In embodiments, the tumor antigen peptides, nucleic acids, expression vectors, T cell receptors, cells (e.g., T lymphocytes, APCs), and / or compositions according to the present disclosure, or any combination thereof, may be used in combination with chemotherapy (e.g., vinca alkaloids, agents that interfere with microtubule formation (e.g., colchicine and its derivatives), antiangiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents (e.g., tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (e.g., nucleoside analogs), alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (e.g., all trans retinoic acid or derivatives thereof), galdamin or its derivatives (17-AAG), immune checkpoint inhibitors (e.g., PD-1 / -PD-L1 inhibitors and CTLA-4 inhibitors, B7-1 / B7-2 inhibitors), antibodies, cell-based therapies (e.g., CAR inhibitors, CTLA-4 inhibitors, B7-1 / B7-2 inhibitors), ... In embodiments, the tumor antigen peptides, nucleic acids, expression vectors, T cell receptors, cells (e.g., T lymphocytes, APCs), and / or compositions according to the present disclosure are administered / used in combination with an immune checkpoint inhibitor. EXAMPLES

[0080] The present disclosure is illustrated in further detail by the following non-limiting examples.

[0081] Example 1: Materials and Methods Human HGSC samples. Tumor fragments for HGSC1–6 and matched normal adjacent tissues for HGSC1–3 were obtained from Tissue Solutions (Glasgow, GB). Tumor tissue (OV606) or ascites (OV633 and OV642) were obtained from the Princess Margaret Cancer Registry (Toronto, ON, Canada). Snap frozen samples were used for RNA extraction and MHCI-associated peptide isolation. RNA sequencing data for OvCa48–114 were downloaded from the National Center for Biotechnology Information Sequence Read Archive under project PRJNA398141, converted to fastq files, and processed similarly to other samples. MS raw data for samples within this cohort were downloaded from the ProteomeXchange consortium via the PRIDE partner with identifier PXD007635. HLA typing for each sample was obtained from RNA sequencing (RNA-Seq) data using OptiType™ v1.0 with default parameters (24). Sample information is presented in Table 1. [Table 1]

[0082] RNA extraction and sequencing. For HGSC1-6, total RNA was isolated using the AllPrep® DNA / RNA / miRNA Universal Kit (Qiagen) according to the manufacturer's recommendations. For OV606, OV633, and OV642, total RNA was isolated using TRIzol® (Invitrogen). RNA from each sample was assessed on a 2100 Bioanalyzer® (Agilent Genomics) to ensure it had a RIN>6 and was used to run one replicate of RNA-Seq per sample. cDNA libraries were prepared from polyA-rich mRNA using the KAPA Standard mRNA-Seq Kit. Libraries were further amplified and used for paired-end RNA-Seq on a HiSeq® 2000 or Illumina NextSeq® 500, yielding 150-300 million reads per sample.

[0083] Generation of customized reference databases for MS analysis. For each sample, a customized “global cancer database” was generated by concatenating two modules, “canonical cancer proteome” and “cancer-specific proteome,” as previously described (15 and U.S. Provisional Application No. 62 / 724,760). Briefly, RNA-Seq reads were trimmed of adapters and low-quality 3′ bases using Trimmomatic v0.35 (25). To generate the canonical cancer proteome, trimmed reads were aligned to the reference human genome version GRCh38.88 using STAR v2.5.1b. Transcript expression was quantified in transcripts per million (tpm) using kallisto v0.43.0 using default parameters. Nucleotide variants were identified using FreeBayes (26) and converted to an agnostic single nucleotide polymorphism file format as input for pyGeno (27). Sample-specific proteomes for each sample were then constructed in pyGeno by inserting single base variants (FreeBayes quality >20) into the reference genome. Sample-specific sequences of expressed proteins (tpm>0) were added to the canonical cancer proteome in fasta format.

[0084] To generate cancer-specific proteomes, trimmed R1 reads were reverse-complemented using FASTX-Toolkit version 0.0.14 and used together with trimmed R2 reads to generate 33- and 24-nucleotide-long k-mer databases. To exclude sequencing errors and limit database size, sample-specific thresholds of minimal k-mer occurrences were applied for 33 nucleotides as follows: 7 for HGSC1-3, 8 for OV642, 10 for OV633, 4 for HGSC4 and OV606, 6 for HGSC5, 5 for HGSC6, and 3 for OvCa48-114. Cancer-specific k-mers were obtained by subtracting k-mers expressed in human thymic epithelial cells (TECs), and then assembled into longer sequences (contigs) by the kmer_assembly tool in NEKTAR (in-house developed software, https: / / github.com / iric-soft / nektar). Contigs >34 nucleotides in length were translated in three frames into amino acid sequences and split at internal stop codons. Resulting stretches of at least 8 amino acids in length were included in the associated cancer-specific proteome.

[0085] Isolation of MAP. Tumor and tissue samples were cut into small pieces (cubes, size approximately 3 mm) and 5 ml of ice-cold PBS containing protein inhibitor cocktail (Sigma, Cat. No. P8340-5ml) was added. They were homogenized twice, first for 20 seconds using an Ultra Turrax™ T25 homogenizer (IKA-Labortechnik) set at a speed of 20000 rpm, then for 20 seconds using an Ultra Turrax™ T8 homogenizer (IKA-Labortechnik) set at a speed of 25,000 rpm. Then, 550 μl of ice-cold 10x lysis buffer (5% w / v CHAPS) was added to each sample. After 60 minutes of incubation with tumbling at 4° C., the samples were centrifuged at 10000 g for 30 minutes at 4° C. The supernatant was transferred to a new tube containing 1 mg of W6 / 32 antibody covalently linked protein A magnetic beads, and MAPs were immunoprecipitated as previously described ( 28 ). The MAP extract was then dried using a Speed-Vac and kept frozen prior to MS analysis.

[0086] MS analysis. The dried peptide extract was resuspended in 0.2% formic acid. The peptide extract was loaded onto a home-made C18 analytical column (15 cm x 150 μm i.d. packed with C18 Jupiter Phenomenex™) with a 56 min gradient from 0-30% acetonitrile (0.2% formic acid) and 600 nl.min on an Easy-nLC II system. -1 The samples were analyzed on a Q-Exactive™ HF mass spectrometer (Thermo Fisher Scientific). Each full MS spectrum was acquired at 60,000 resolution, followed by 20 MS / MS spectra, at 30,000 resolution, 5×10 4 The most abundant multiply charged ions were selected for MS / MS sequencing with an automatic gain control target of 100 ms, an injection time of 100 ms, and a collision energy of 25%. For HGSC4-6, each full MS spectrum acquired at a resolution of 60,000 was followed by 20 MS / MS spectra, one each at a resolution of 30,000, 2 × 10 4Select the most abundant multiply charged ions for MS / MS sequencing with an automatic gain control target of 100 ms, an injection time of 800 ms, and a collision energy of 25%.

[0087] MAP Identification. Peptides were identified using PEAKS 8.5 or Peaks X (Bioinformatics Solution Inc.) to search peptide sequences against the Global Cancer Database. For peptide identification, the precursor and fragment ion tolerances were set to 10 ppm and 0.01 Da, respectively. For samples from Schuster et al. (13), the precursor and fragment ion tolerances were set to 5 ppm and 0.5 Da, respectively. The occurrence of oxidation (M) and deamidation (NQ) were considered as post-translational modifications. A sample-specific threshold was applied to the PEAKS score to ensure that the list of MAPs included only 5% of the decoy specifics. Peptides that exceeded this threshold were further filtered according to the following criteria: peptide length 8–11 amino acids, and MHC allele affinity rank ≤ 2% based on NetMHC4.0 predictions (29).

[0088] Identification and validation of TSA candidates. To identify TSA candidates, each MAP and its coding sequence was queried against the associated cancer and normal canonical proteomes or cancer and normal 24-nucleotide k-mer databases, respectively. The normal canonical proteome and normal 24-nucleotide k-mer databases were constructed using RNA-Seq reads from purified TECs from six human thymes (15 and US Provisional Application No. 62 / 724,760). MAPs were labeled as TSA candidates in two cases: i) if the peptide sequence was not detected in either the normal canonical proteome or normal (i.e., TEC) k-mers of the sample, or ii) if the peptide was absent from both the cancer and normal canonical proteomes and their RNA coding sequences were overexpressed at least 10-fold in cancer cells compared to TECs. If a MAP corresponded to several RNA sequences, it was considered as a TSA candidate only if all sequences were consistent with TSA candidate status. MS / MS spectra of all TSA candidates were manually verified to remove any false identifications. For TSA candidates with I / L variants supported by RNA data that were distinguishable by MS, both variants were examined further if the most expressed variant was a TSA candidate.

[0089] Finally, genomic locations were assigned to all MS-validated TSA candidates by mapping the mapping reads containing the MAP coding sequence on the reference genome (GRCh38) using BLAT (UCSC Genome Browser). TSA candidates whose reads matched to hypervariable regions (HLA, Ig or TCR genes) were excluded. TSA candidates were classified as mTSA if they contained variants in the MAP coding sequence that did not match known germline polymorphisms (reported in dbSNP v149). Non-mutated candidates were classified as aeTSA candidates and subjected to further evaluation of their expression in normal tissues and organs.

[0090] Tissue expression of sequences encoding aeTSA candidates. RNA-Seq data from 27 different tissues were downloaded from the Genotype-Tissue expression (GTEx) portal (phs000424.v7.p2 accessed on April 16, 2018) and used to assess the expression of coding sequences of aeTSA candidates, as previously described (15). RNA-Seq data were obtained from 50 donors, excluding cervix (n = 6), fallopian tube (n = 7), adipose tissue (n = 49), bladder (n = 12), and kidney (n = 38). The accession numbers of the GTEx datasets used in this study are listed in Table 2. Briefly, the number of reads with complete coverage of the MAP coding sequence was estimated by the minimum occurrence of the 24-mer set of the MAP coding sequence in the database of 24-mers transformed from the RNA-Seq reads of each tissue. Read counts were normalized to reads per 100 million sequenced (rphm) and further log-transformed (log 10 (rphm+1)), averaged across all available RNA-Seq experiments for each tissue. MHC 低 Candidate aeTSAs that showed no peripheral expression with rphm>10 in tissues other than those (brain cortex, nerve, and testis) were considered as bona fide aeTSAs. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0091] Expression of TSA coding regions. RNA expression of TSA coding regions was quantified from mapped BAM files using the qCount function in the R package QuasR (30) with parameter orientation = "same" as the count of all reads overlapping the TSA coding region on the TSA coding strand. Counts were normalized to reads per 100 million mapped reads. For aeTSA expression analysis, we specifically analyzed the unique regions to which individual aeTSAs were mapped, as the context of the surrounding sequences can affect aeTSA expression.

[0092] Clinical and genomic data from TCGA. Level 3 data processed and normalized with hg38 for HM27 methylation, DNA copy number variation, RNA-Seq gene expression, and clinical data were downloaded from the TCGA open access database using the R package TCGAbiolinks (31). Arm-level DNA copy number alterations were downloaded from the Broad Institute TCGA Genomic Data Analysis Center (doi:10.7908 / C1P84B9Q).

[0093] Immune cell score. An immune cell score, representing the immune cell population, was estimated based on the RNA-Seq data. For each tumor, the score was estimated as the average of the log-transformed FPKM values ​​of their marker genes, as described by Danaher et al. (32).

[0094] Frequency of aeTSA presentation. Bioinformatics simulations were performed to estimate the number of aeTSAs presented by individual patients. This estimation was based on two parameters. First, the likelihood of aeTSA expression was based on the proportion of tumors expressing the corresponding RNA in the TCGA-OV cohort. For aeTSAs containing germline SNPs, the likelihood of expression was calculated as follows: (Proportion of TCGA tumors expressing aeTSA) × (SNP frequency in a given population). SNP frequencies were obtained from the Genome Aggregation Database. Second, HLA allele frequencies were obtained from the National Marrow Donor Program (NMDP) for European American populations (n ​​= 1,242,890), African Americans (n ​​= 416,581) and Chinese (n = 99,672). The HLA genotypes of patients were then simulated with six HLA class I alleles based on their reported frequencies in the given population. Six HLA alleles were assumed to be independent events, so some HLA loci were homozygous in the simulated patients. An aeTSA was considered to be presented in a simulated patient when both the aeTSA and the associated HLA allele were expressed. Expression of each aeTSA was considered to be an independent event, except for duplicate aeTSAs, whose expression status was simulated only once for the same duplicate population. One million simulated patients and their aeTSA-presenting status were generated for each of the three populations and used to plot the distributions.

[0095] Statistical analysis and data visualization. Analyses and figures were performed using R v3.5.1 or Python v2.7.6. Heat maps of TSA coding region expression in tumors were generated using the 'gplots' package in R. Correlation tests were performed with the R function 'cor.test' using Spearman's method unless otherwise indicated. Tests involving comparison of distributions were performed with ANOVA tests, and pairwise comparisons between groups were performed by Wolcoxon rank sum tests. Log-rank P values ​​for survival analyses were calculated with the 'survival' package.

[0096] Example 2: Proteogenomic analysis identifies 111 TSAs in 23 HGSC. To obtain a systems-level characterization of the TSA landscape, we performed direct MAP identification using high-throughput tandem MS (MS / MS) analysis (34-36). Current search engines rely on user-defined protein databases to match each acquired MS / MS spectrum with peptide sequences (37). Thus, peptides in a test sample can only be identified by the search engine if their sequence is included in the reference database. Because general reference protein databases such as UniProt do not contain sample-specific mutations, out-of-frame translation events, and non-exonic sequences, the search to capture TSAs encoded by all genomic regions requires the construction of a customized database containing tumor-specific translation products for each tumor. Therefore, we constructed a customized search database from the RNA-Seq reads of each analyzed sample using a recently described proteogenomic approach (15). Such a customized database contains two modules: the canonical proteome (in-frame translation of exons) and the cancer-specific proteome, which is a three-frame translation of the cancer-specific RNA sequences after subtraction of the normal RNA sequences (from TECs) (Figure 1). TECs were used as normal controls for two reasons: i) their important role in establishing immune tolerance during immature T cell development (i.e., mid-aqueous tolerance), and ii) their remarkable ability to promiscuously express more transcripts than other types of somatic cells (38). MAPs from nine primary HGSC samples were obtained by immunoprecipitation of MHC I molecules and then analyzed by liquid chromatography-MS / MS (15, 28). Immunopeptidememia data of an additional cohort of 14 HGSCs reported by Schuster et al. (13) were also reanalyzed by applying the proteogenomic approach described herein to samples for which matching RNA-Seq and MS data were available. Each of the TSA candidates was confirmed by manual validation of the spectra and genomic locations.

[0097] Candidate overlapping genomic variants not present in dbSNP were unlikely to represent germline polymorphisms and therefore labeled as mTSAs. Such classification yielded 18 mTSAs from the 23 samples analyzed (Table 3A). None of these mTSAs have been reported previously. Of the 18 mTSAs, 7 arose from in-frame exon translation, 4 from out-of-frame exon translation, and 8 from non-coding sequences. For three tumors, matched normal tissue was available, and RNA-Seq analysis confirmed that the mTSA variants were not germline polymorphisms (Figure 8A-B). For tumors with unmatched normal tissue, it cannot be formally excluded that some mTSAs may correspond to rare polymorphisms not present in dbSNP. The possibility that the number of mTSAs may be slightly overestimated only strengthens the conclusion that with less than one mTSA per tumor (18 mTSAs / 23 tumors), mTSAs are rare in HGSC and therefore represent less attractive targets. Moreover, because classical TSA discovery methods strictly focus on mTSAs resulting from in-frame exon translation, they would only reveal 7 of the 111 TSAs reported herein. [Table 3]

[0098] For non-mutated TSA candidates, we applied strict criteria to identify pure aeTSAs, i.e., those whose expression is cancer-specific. We then analyzed their RNA expression in 27 peripheral tissues across the human body. 低All candidates whose coding RNA was expressed (rphm>10) in any peripheral tissue other than the tissues (brain, nerve, testis) were removed from the aeTSA list. If the coding sequence of an aeTSA candidate contained a germline single nucleotide polymorphism (reported in dbSNP), this candidate was labeled as a valid aeTSA only if the SNP-containing sequence and the reference sequence met the above criteria (Figure 9). Overall, 93 aeTSA candidates met these stringent criteria (Figure 2, Table 3B and Table 3C), of which, 85 have never been reported to the best of our knowledge (Table 2B). Interestingly, 5 of the 93 aeTSAs were expressed in testis, indicating that although some cancer germline antigens (CGAs) are aeTSAs, most aeTSAs are not CGAs. CGAs are encoded by canonical exons that are normally expressed only by germ cells, and their aberrant expression in cancer cells is mainly driven by epigenetic alterations. However, several CGAs are expressed by adult mTECs ( 16 ), and CGAs expressed in mTECs (or other somatic tissues) are considered as TAAs, and those not expressed by any normal tissues (including mTECs) are considered as bona fide aeTSAs.

[0099] A genomic location was assigned to each aeTSA. When multiple locations were possible, the one with the highest occurrence of matching RNA reads was selected. The characteristics of all TSAs are reported in Table 3B and Table 3C. A stringent approach is formally capable of underestimating the total number of aeTSAs resulting from atypical translation (5'UTR, 3'UTR, intergenic, frameshift). Indeed, although the reading frames used to generate MAPs in tumors are known, it is not possible to guess which reading frames may be translated when their coding RNAs are expressed in some normal tissues. Therefore, such aeTSA candidates were excluded to avoid false positives in the TSA list. [Table 4-1] [Table 4-2] 1 The “translation event” column summarizes the relationship between the TSA coding sequence and the ORF, with those outside the ORF designated as “non-coding,” those overlapping but frameshifted ORFs designated as “coding-out,” and those matching the ORFs designated as “coding-in.” 2 The "Genomic Origin" column further annotates the aeTSA according to the biotype from Ensembl, e.g., "antisense" means that the sequence is on the opposite stand as an annotated gene, "canonical" refers to the canonical / annotated ORF, and "ncRNA" is an annotated RNA that does not contain an ORF according to Ensembl. 3 "ncRNA" refers to when the TSA coding sequence aligns with exons of a non-coding transcript. 4 By "non-coding antisense" it is meant that the TSA coding sequence is antisense to a gene in the Ensembl annotation and thus it is a non-coding region. [Table 5]

[0100] Example 3: Most TSAs in HGSC are non-mutated MAPs resulting from non-canonical translation. With an average of 2200 unique MAPs identified per sample, a total of 111 unique TSAs were found (Figure 3A). The number of TSAs identified per sample correlated significantly with the number of MAPs (Figure 3B). Furthermore, there was a moderate correlation between the number of MAPs per HLA allele and tumor sample size (Figure 10). This is consistent with the idea that tumor sample size is a limiting factor in MS analysis (28). In principle, TSAs derived from mutated noncoding sequences can be designated as both mTSAs or aeTSAs. We decided to label them as mTSAs accordingly. The rationale was that mTSAs, regardless of genomic origin (exonic or not), are expected to be “private TSAs”, i.e., not shared by a large number of tumors. In contrast, nonmutated aeTSAs could theoretically be shared by a significant proportion of HGSCs.

[0101] Of note, the features of TSAs identified in samples originally processed in this study or by Schuster et al. were remarkably similar (Figure 3C). This suggests that the proteogenomic approach described herein can be generally applied to RNA-Seq and MS data and is ostensibly not subject to inter-laboratory variability. In both cohorts, approximately 83% of TSAs were not mutated, and the majority of TSAs arose from atypical translation: primarily from non-coding regions and, to a lesser extent, from out-of-frame exon translation (Figure 3C). Two features of aeTSAs are noteworthy: i) 80% originate from non-coding sequences, especially introns (31%) and intergenics (22%), and ii) 90% are novel MAPs (Figure 3D). Previously reported MAPs were derived from in-frame exon translation, except for those that match processed transcripts (biotypes annotated by the Ensembl database) whose corresponding protein isoforms are included in the UniProt database ( 13 , 39 - 43 and U.S. Patent Publication No. 2012 / 0077696A1 ).

[0102] Example 4: Expression of aeTSA-encoding transcripts in ovarian cancer samples. To determine whether cancer-specific expression of aeTSA-encoding transcripts arises from random transcriptional noise or from recurrent transcriptional aberrations, we analyzed RNA expression of 93 aeTSA-encoding genomic regions identified in samples from this study and the TCGA ovarian cancer cohort. Regions encoding aeTSAs were expressed in a significant proportion of ovarian cancers; 72 (77%) were expressed in at least 10% of samples and 16 (17%) were expressed in at least 80% of samples (Figure 4). These commonly expressed regions likely generate shared TSAs between patients. Thus, it may be concluded that expression of this set of 93 aeTSA-encoding transcripts in HGSC is not a rare or random event, but rather a common feature of HGSC.

[0103] Example 5: Genomic correlation of aeTSA expression. To understand the mechanism of aeTSA expression, we used multi-omic data from the TCGA-OV dataset to explore the relationship between aeTSA RNA expression and local genetic or epigenetic abnormalities. Where applicable, we tested the correlation between local DNA copy number changes, DNA methylation levels on gene promoter regions, and RNA expression for each aeTSA (Figure 5A). In cases where aeTSAs derived from genomic regions that are part of a gene (exon, intron or UTR) were used, we also analyzed the correlation between the expression of the associated gene and the expression of the aeTSA. In the latter situation, a remarkable correlation was observed between the gene and aeTSA expression (Figure 5A). This suggests that for aeTSAs whose coding regions are intragenic, the regulation of aeTSA expression generally affects the entire gene. Furthermore, DNA copy number changes were positively correlated with the RNA expression levels of aeTSA. This was similar for both intragenic and extragenic aeTSAs (antisense and intergenic). This suggests that DNA copy number changes have a substantial effect on aeTSA expression. Notably, this correlation was particularly strong for intragenic aeTSAs expressed in a larger proportion of tumors (Figure S11A). When we examined the chromosomal distribution of aeTSA coding regions, we found that several chromosomal arms frequently amplified in HGSC produced many aeTSAs (Figure 5B). For example, the long arm of chromosome 3 (44), which is commonly amplified in ovarian cancer, was the source of eight aeTSAs. As one of the top amplified regions, MECOM, located at 3q26.2 (44), generated three duplicated exonic out-of-frame aeTSAs (Table 3B). However, amplification of a chromosomal arm was neither necessary (e.g., 15q) nor sufficient (e.g., 8q) to generate aeTSAs (Figure 5B, Figure S11B).

[0104] Due to the technology used by TCGA to analyze DNA methylation (HM27 array), no methylation data were available for the promoters of extragenic aeTSAs and several aeTSA source genes. Thus, the analysis of promoter methylation was limited to a subset of 17 aeTSAs. Nevertheless, for six aeTSAs, a significant correlation between DNA methylation and aeTSA expression was found (Figure 5A). The correlation was negative in five cases and positive in one case. This is consistent with the idea that promoter demethylation frequently leads to enhanced transcription. Notably, the two genes showing the highest negative correlation were MAGEC1 (ρ = -0.53, P adj =1.6x10 -26 ) and MAGEA4 (ρ = −0.51, P adj =6.7x10 -25 ), which are represented by the dark bars with arrows in Figure 5A. The MAGE family of genes are CGAs that are overexpressed in several cancer types, including HGSC (3). Overall, it can be concluded that aeTSA expression is regulated at the transcriptional level, at least in part, by variations in gene copy number and DNA methylation.

[0105] Example 6: Expression of three aeTSAs correlates with improved survival. We next evaluated whether some aeTSAs could induce spontaneous protective immune responses. Addressing this question is complicated by the fact that expression of aeTSAs at the peptide level requires, in addition to expression of aeTSA RNA, the presence of associated HLA allotypes. Therefore, we subdivided patients from the TCGA cohort into four subgroups based (or not) on the expression of individual aeTSA RNAs and based (or not) on the presence of associated HLA allotypes. Presentation of three aeTSAs correlated with a more favorable clinical outcome (Figure 6A-6C). Polymorphism of HLA alleles considerably reduced the size of each group and therefore the power of this analysis. Thus, the log-rank p-values ​​for the three aeTSAs ranged from 0.013 to 0.076 (Figure 6A-6C). Nevertheless, two observations provide supporting evidence that these correlations are biologically meaningful. First, the "protective effect" of these aeTSAs appeared to be HLA-restricted: patients expressing aeTSA RNA had superior survival if they also expressed the relevant HLA allele. Second, expression of the RTHQMNTFQR aeTSA and its relevant HLA allotypes positively correlated with tumor infiltration by T cells and cytotoxic T cells (Figure ​(Figure6D,E); ANOVA, p<0.05).

[0106] Example 7: Median number of aeTSA presented by individual tumors With the list of 93 aeTSAs, we finally estimated how much this study could benefit TSA-targeted immunotherapy. Therefore, we randomly simulated the presentation status of the 93 aeTSAs in 1 million patients. To estimate the frequency of HLA alleles, we used the three largest datasets from the United States Bone Marrow Bank: European Americans, African Americans, and Chinese (45). Using the allele frequency in the given population, the expression percentage in TCGA-OV tumors, and, if applicable, the SNP frequency, we independently generated the six HLA alleles and aeTSA expression status. We calculated the number of aeTSAs per individual tumor as the sum of the expressed HLA-aeTSA pairs. Based on these simulations, we determined that at least one aeTSA could be found in 98% of European Caucasians, 74% of African Americans, and 78% of Chinese, and the median number of aeTSAs per tumor was 5 in European Caucasians, 2 in African Americans, and 4 in Chinese (Figure 7). These differences between populations arose from the variation in HLA allele frequencies and the fact that the tumor samples were mainly from Caucasians of European descent. These calculations are suspected to underestimate the number of aeTSAs per tumor for three main reasons. First, the fact that >50% of MAPs binding to more than one HLA allotype often bind across supertypes or loci (46) is not taken into account. Second, genomic regions encoding a given MAP frequently generate overlapping MAPs presented by different HLA allotypes (23). Third, for the five aeTSAs containing nonsynonymous SNPs listed in dbSNP, we assumed that only SNP variants that generated MAPs in the samples were active, while other SNP variants did not generate MAPs. This cautious strategy was adopted because a single amino acid change may be sufficient to abrogate MAP presentation (47). It can be concluded that the vaccine, which contains the current set of 93 aeTSAs, covers almost all Caucasians with HGSC, as well as a significant proportion of African-Americans and Asians (e.g., Chinese).

[0107] Although the present technology has been described above with reference to specific embodiments thereof, modifications can be made 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

1. A tumor antigen peptide of 15 amino acids or less encoded by a sequence located in a non-protein coding region of the genome, A tumor antigen peptide comprising one of the amino acid sequences set forth in SEQ ID NOs: 1, 4, 8, 10, 13, 15-17, 19-27 and 36-99.

2. The tumor antigen peptide according to claim 1, which consists of one of the amino acid sequences set forth in SEQ ID NOs: 19 to 27 and 36 to 99.

3. The tumor antigen peptide of claim 1, wherein the tumor antigen peptide binds to an HLA-A*01:01 molecule and comprises or consists of an amino acid sequence set forth in SEQ ID NO: 21, 40, 41, 66 or 88.

4. The tumor antigen peptide described in claim 1, wherein the tumor antigen peptide binds to an HLA-A*02:01 molecule and comprises or consists of an amino acid sequence set forth in SEQ ID NO: 17, 45, 48, 51, 56, 75, 77, 82 or 98.

5. The tumor antigen peptide of claim 1, wherein the tumor antigen peptide binds to an HLA-A*03:01 molecule and comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1, 19, 20, 22, 36, 50, 52, 60, 62, 73, 84, 85, 86 or 91.

6. The tumor antigen peptide of claim 1, which binds to an HLA-A*11:01 molecule and comprises or consists of an amino acid sequence set forth in SEQ ID NO: 54, 55, 67, 69, 81, 87 or 90.

7. The tumor antigen peptide of claim 1, which binds to an HLA-A*24:02 molecule and comprises or consists of the amino acid sequence set forth in SEQ ID NO:

43.

8. The tumor antigen peptide of claim 1, wherein the tumor antigen peptide binds to an HLA-A*25:01 molecule and comprises or consists of one of the amino acid sequences set forth in SEQ ID NO:

24.

9. The tumor antigen peptide of claim 1, wherein the tumor antigen peptide binds to an HLA-A*29:02 molecule and comprises or consists of one of the amino acid sequences set forth in SEQ ID NO:

58.

10. The tumor antigen peptide of claim 1, which binds to an HLA-A*32:01 molecule and comprises or consists of the amino acid sequence set forth in SEQ ID NO:

16.

11. The tumor antigen peptide of claim 1, wherein the tumor antigen peptide binds to an HLA-B*07:02 molecule and comprises or consists of an amino acid sequence set forth in SEQ ID NO: 4, 8, 26, 49, 78, 92 or 97.

12. The tumor antigen peptide of claim 1, wherein the tumor antigen peptide binds to an HLA-B*08:01 molecule and comprises or consists of one of the amino acid sequences set forth in SEQ ID NOs: 23, 42, 44, 46, 59, 63, 70, 74, 76 or 83.

13. The tumor antigen peptide of claim 1, which binds to an HLA-B*14:01 molecule and comprises or consists of the amino acid sequence set forth in SEQ ID NO:

53.

14. The tumor antigen peptide of claim 1, wherein the tumor antigen peptide binds to an HLA-B*18:01 molecule and comprises the amino acid sequence set forth in SEQ ID NO:

89.

15. The tumor antigen peptide of claim 1, wherein the tumor antigen peptide binds to an HLA-B*39:01 molecule and comprises an amino acid sequence set forth in SEQ ID NO: 47, 64, 96 or 99.

16. The tumor antigen peptide of claim 1, which binds to an HLA-B*40:01 molecule and comprises or consists of the amino acid sequence set forth in SEQ ID NO:

13.

17. The tumor antigen peptide of claim 1, which binds to an HLA-B*44:02 molecule and comprises or consists of the amino acid sequence set forth in SEQ ID NO:

65.

18. The tumor antigen peptide of claim 1, which binds to an HLA-B*44:03 molecule and comprises or consists of an amino acid sequence set forth in SEQ ID NO: 37 or 94.

19. The tumor antigen peptide of claim 1, which binds to an HLA-C*03:03 molecule and comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10, 71 or 95.

20. The tumor antigen peptide of claim 1, which binds to an HLA-C*04:01 molecule and comprises or consists of the amino acid sequence set forth in SEQ ID NO:

15.

21. The tumor antigen peptide of claim 1, which binds to an HLA-C*05:01 molecule and comprises or consists of the amino acid sequence set forth in SEQ ID NO:

27.

22. The tumor antigen peptide of claim 1, which binds to an HLA-C*06:02 molecule and comprises or consists of the amino acid sequence set forth in SEQ ID NO:

72.

23. The tumor antigen peptide of claim 1, which binds to an HLA-C*07:01 molecule and comprises or consists of an amino acid sequence set forth in SEQ ID NO: 38, 61 or 93.

24. The tumor antigen peptide of claim 1, which binds to an HLA-C*12:03 molecule and comprises or consists of the amino acid sequence set forth in SEQ ID NO:

80.

25. The tumor antigen peptide of claim 1, which binds to an HLA-C*14:02 molecule and comprises or consists of an amino acid sequence set forth in SEQ ID NO: 25, 57 or 79.

26. The tumor antigen peptide of claim 1, wherein the non-protein coding region of the genome is a non-translated transcribed region (UTR), and the tumor antigen peptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 1, 8, 10, 13, 15, and 19-27.

27. The tumor antigen peptide of claim 1, wherein the non-protein coding region of the genome is an intron, and the tumor antigen peptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 16, 17, and 36-64.

28. The tumor antigen peptide of claim 1, wherein the non-protein coding region of the genome is an intergenic region, and the tumor antigen peptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 65 to 84.

29. The tumor antigen peptide of claim 1, wherein the non-protein coding region of the genome is an exon of a non-coding RNA transcript (ncRNA), and the tumor antigen peptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 4 and 85-92.

30. The tumor antigen peptide of claim 1, wherein the non-protein coding region of the genome is an antisense strand of a gene, and the tumor antigen peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 93 to 99.

31. A nucleic acid encoding one or more of the tumor antigen peptides of claim 1.

32. 32. The nucleic acid of claim 31 which is an mRNA or a viral vector.

33. A composition comprising a tumor antigen peptide of claim 1 or a nucleic acid of claim 31 or 32, and a pharma- ceutically acceptable carrier.

34. A vaccine comprising a tumor antigen peptide according to claim 1, a nucleic acid according to claim 31 or 32, or a composition according to claim 33, and an adjuvant.

35. A T cell receptor (TCR) that specifically recognizes an MHC class I molecule expressed on the surface of a cell and containing the tumor antigen peptide described in claim 1 within its peptide binding groove.

36. An isolated CD8 antigen expressing the TCR of claim 35 on its cell surface. ＋ T lymphocytes.

37. At least 0.5% of the CD8 ＋ A population of cells comprising T lymphocytes.

38. (i) a tumor antigen peptide according to any one of claims 1 to 30, (ii) a nucleic acid according to claim 31 or 32, (iii) a composition according to claim 33, (iv) claim 34 (v) the TCR of claim 35; and (vi) the CD8 of claim 36. ＋ 40. Use of a T lymphocyte, or (vii) the cell population of claim 37, for the manufacture of a medicament for treating ovarian cancer in a subject.

39. 39. The use of claim 38, wherein the ovarian cancer is serous carcinoma.

40. 39. The use of claim 38, wherein the serous cancer is high-grade serous carcinoma (HGSC).

41. The use described in claim 38, wherein the pharmaceutical agent is for use in combination with at least one additional anti-tumor agent or therapy.

42. 42. The use of claim 41, wherein the at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy or surgery.

43. (i) a tumor antigen peptide according to any one of claims 1 to 30, (ii) a nucleic acid according to claim 31 or 32, (iii) a composition according to claim 33, (iv) a vaccine according to claim 34, (v) a TCR according to claim 35, or (vi) a CD8 according to claim 36, for use in treating ovarian cancer in a subject. ＋ or (vii) a T lymphocyte, or the cell population of claim 37.

44. The tumor antigen peptide, nucleic acid, composition, vaccine, TCR, CD8 for use according to claim 43, wherein the ovarian cancer is a serous carcinoma. ＋ T lymphocytes, or cell populations.

45. The tumor antigen peptide, nucleic acid, composition, vaccine, TCR, CD8 for use according to claim 44, wherein the serous cancer is high-grade serous carcinoma (HGSC). ＋ T lymphocytes, or cell populations.

46. The tumor antigen peptide, the nucleic acid, the composition, the vaccine, the TCR, the CD8 ＋ 44. The tumor antigen peptide, nucleic acid, composition, vaccine, TCR, CD8, or T lymphocyte or cell population for use according to claim 43, for use in combination with at least one additional anti-tumor agent or therapy. ＋ T lymphocytes, or cell populations.

47. The tumor antigen peptide, nucleic acid, composition, vaccine, TCR, CD8 for use according to claim 46, wherein the at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, an immunotherapy, an immune checkpoint inhibitor, radiation therapy or surgery. ＋ T lymphocytes, or cell populations.

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