Novel tumor-specific antigens for ovarian cancer and their uses

Abstract

To provide novel tumor-specific antigens (TSAs), since ovarian cancer, notably high-grade serous ovarian cancer (HGSC), the principal cause of death from gynecological malignancies in the world, has not significantly benefited from recent progress in cancer immunotherapy and the nature of antigens that can elicit anti-HGSC immune responses is unknown while HGSC infiltration by lymphocytes correlates with superior survival.SOLUTION: Novel tumor-specific antigens (TSAs) shared by a large proportion of ovarian tumors are described herein. Most of the TSAs (>80%) described herein derives from aberrantly expressed unmutated genomic sequences, such as intronic and intergenic sequences, which 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 treatment of ovarian cancer is also described.SELECTED DRAWING: Figure 6A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is incorporated herein by reference in its entirety. The benefit of U.S. Provisional Patent Application No. 62 / 866,089 is claimed.

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

[0003] Ovarian cancer is the leading cause of death from gynecological malignancies worldwide, with 14,000 deaths annually in the United States. High-grade serous ovarian cancer (HGSC) is responsible for over 10,000 deaths (1). These causes account for 70-80% of deaths, and overall survival has not changed significantly over the decades. (2) A positive correlation between the abundance of tumor-infiltrating lymphocytes (TILs) and increased overall survival The relationship is that T cells can recognize biologically relevant tumor antigens in HGSC. Furthermore, strong evidence suggests that HGSCs adjacent to tumor epithelial cells These results suggest that TILs are actively involved in local immune editing. In a multimodality study of 212 HGSC samples from + TIL is It has been negatively associated with malignant cell diversity (5). Considering the therapeutic efficacy of immune checkpoint inhibitors in the type, Clinical trials using cross-linking inhibitors are currently underway in HGSC. However, early studies with anti-PD1 showed limited activity in HGSC. (6, 7).

[0004] Considering this, it is important to induce a therapeutic immune response in ovarian tumors such as HGSC. There is an urgent need to identify antigens that can stimulate the immune system (3, 8). (± immune checkpoint inhibitors) or as T cell receptor-based approaches (cell These proteins can be used as targets for cell therapy, bispecific biologics, and other therapeutics (9).

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

[0006] The present 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 according to item 1, comprising one of the amino acid sequences set forth in SEQ ID NOs: 19 to 103. Tumor antigen peptide. 3. The tumor antigen peptide binds to the HLA-A*01:01 molecule and is selected from SEQ ID NOs: 21 and 28. 40, 41, 66 or 88. Tumor antigen peptide. 4. The tumor antigen peptide binds to an HLA-A*02:01 molecule and is selected from SEQ ID NOs: 1, 19, 20, 22, 30, 31, 36, 50, 52, 60, 62, 73, 84, 85, 86 and 91. The tumor antigen peptide according to Item 1 or 2, comprising the amino acid sequence of Item 91. 5. The tumor antigen peptide binds to the HLA-A*11:01 molecule and is selected from SEQ ID NOs: 32, 54 55, 67, 69, 81, 87, 90 or 102. 3. The tumor antigen peptide according to 1 or 2. 6. The tumor antigen peptide binds to an HLA-A*24:02 molecule and is SEQ ID NO: 33 or 43. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence of 43. 7. The tumor antigen peptide binds to an HLA-A*25:01 molecule and is represented by SEQ ID NO: 24. 3. The tumor antigen peptide according to item 1 or 2, comprising one of the amino acid sequences: 8. The tumor antigen peptide binds to an HLA-A*29:02 molecule and is SEQ ID NO: 34 or 58. The tumor antigen peptide according to item 1 or 2, comprising one of the amino acid sequences according to item 58. Do. 9. The tumor antigen peptide binds to an HLA-A*32:01 molecule and is represented by SEQ ID NO: 16. 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence: 10. The tumor antigen peptide binds to an HLA-B*07:02 molecule and is selected from SEQ ID NOs: 4, 6, 8, 9, 26, 49, 78, 92, 97, or 101. A tumor antigen peptide according to item 1 or 2. 11. The tumor antigen peptide binds to the HLA-B*08:01 molecule and is selected from SEQ ID NOs: 23 and 35 , 42, 44, 46, 59, 63, 70, 74, 76, 83 or 103 3. The tumor antigen peptide according to item 1 or 2, comprising one of the following sequences: 12. The tumor antigen peptide binds to an HLA-B*14:01 molecule and is represented by SEQ ID NO: 53. 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence described above. 13. The tumor antigen peptide binds to an HLA-B*15:01 molecule and is SEQ ID NO: 2, 3 or 6. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence according to item 5. 14. The tumor antigen peptide binds to an HLA-B*18:01 molecule and is represented by SEQ ID NO: 89. 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence described above. 15. The tumor antigen peptide binds to the HLA-B*39:01 molecule and is selected from SEQ ID NOs: 47, 6 4, 96 or 99. Chid. 16. The tumor antigen peptide binds to the HLA-B*40:01 molecule and is selected from SEQ ID NOs: 11, 1 14. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence according to item 2 or 13. 17. The tumor antigen peptide binds to an HLA-B*44:02 molecule and is represented by SEQ ID NO: 65. 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence described above. 18. The tumor antigen peptide binds to an HLA-B*44:03 molecule and is SEQ ID NO: 37 or 94. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence of item 94. 19. The tumor antigen peptide binds to an HLA-C*03:03 molecule and is selected from the group consisting of SEQ ID NOs: 10, 2 9, 71 or 95. Chid. 20. The tumor antigen peptide binds to an HLA-C*04:01 molecule and is SEQ ID NO: 6 or 15. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence according to item 15. 21. The tumor antigen peptide binds to an HLA-C*05:01 molecule and is represented by SEQ ID NO: 27. 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence described above. 22. The tumor antigen peptide binds to an HLA-C*06:02 molecule and is SEQ ID NO: 18 or 72. The tumor antigen peptide according to Item 1 or 2, comprising the amino acid sequence of 72. 23. The tumor antigen peptide binds to an HLA-C*07:01 molecule and is selected from the group consisting of SEQ ID NOs: 38, 6 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence of 1 or 93. 24. The tumor antigen peptide binds to an HLA-C*07:02 molecule and is represented by SEQ ID NO: 7. 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence: 25. The tumor antigen peptide binds to an HLA-C*12:03 molecule and is represented by SEQ ID NO: 80. 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence described above. 26. The tumor antigen peptide binds to the HLA-C*14:02 molecule and is selected from SEQ ID NOs: 25, 5 7 or 79. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence of 27. Encoded by a sequence located in a non-protein-coding region of the genome, SEQ ID NO: 1 , 4, 6 to 8, 10, 13, 15 to 27 and 36 to 99, preferably SEQ ID NOs: 19 to 2 7 and 36 to 99. A tumor antigen peptide described in any one of claims 1 to 4. 28. The non-protein-coding region of the genome is an untranslated transcribed region (UTR), and the tumor The antigen peptide is selected from SEQ ID NOs: 1, 8, 10, 13, 15, and 19 to 27, preferably SEQ ID NOs: Item 27, which contains an amino acid sequence of any one of sequences 19 to 27. Tumor antigen peptides. 29. The non-protein coding region of the genome is an intron, and the tumor antigen peptide is , SEQ ID NOs: 16, 17, and 36 to 64, preferably any of SEQ ID NOs: 36 to 64 28. The tumor antigen peptide according to Item 27, comprising any one of the amino acid sequences described in Item 27. 30. The non-protein-coding region of the genome is an intergenic region, and the tumor antigen peptide Item 27, which comprises an amino acid sequence set forth in any one of SEQ ID NOs: 65 to 84. The described tumor antigen peptide. 31. The non-protein-coding region of the genome encodes non-coding RNA transcripts (ncRNAs). and the tumor antigen peptide is selected from SEQ ID NOs: 4 and 85 to 92, preferably SEQ ID NOs: 28. The tumor according to item 27, comprising an amino acid sequence according to any one of items 85 to 92. Antigenic peptides. 32. The non-protein-coding region of the genome is the antisense strand of a gene, and the tumor anti- The original peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 93 to 99. 28. The tumor antigen peptide according to Item 27. 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. The tumor antigen peptide according to any one of items 1 to 32, or item 33 or 34. A liposome comprising the nucleic acid according to 34. 36. The tumor antigen peptide according to any one of items 1 to 32, or the tumor antigen peptide according to item 31 or 32 34. The method of claim 33, further comprising administering to said patient a nucleic acid comprising: , composition. 37. The tumor antigen peptide according to any one of items 1 to 32, or the tumor antigen peptide according to item 33 or 34. 35. The nucleic acid according to Item 34, the liposome according to Item 35, or the composition according to Item 36, and azido Vaccines, including Influenza. 38. A tumor antigen peptide according to any one of items 1 to 32 in its peptide-binding groove. An isolated major histocompatibility complex (MHC) class I molecule comprising: 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. (i) A tumor antigen peptide according to any one of items 1 to 32, or (ii) A nucleotide sequence encoding the tumor antigen peptide according to any one of items 1 to 32. An isolated cell comprising a vector comprising the vector. 42. A tumor antigen peptide according to any one of items 1 to 32 in its peptide-binding groove. A single cell expresses major histocompatibility complex (MHC) class I molecules, including the mitochondrial endothelial cell line, on its surface. Isolated cells. 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. The isolated MHC class I molecule and / or or an MHC class I molecule expressed on the surface of the cell according to any one of items 42 to 44. T cell receptor (TCR) specifically recognizes 46. ​​An isolated CD8 expressing the TCR according to item 45 on its cell surface. + T lymphocytes. 47. At least 0.5% CD8 as defined in item 46 + A cell population that includes T lymphocytes . 48. A method for treating ovarian cancer in a subject, comprising administering an effective amount of (i) a compound according to any one of items 1 to 32. (ii) a tumor antigen peptide according to any one of items 33 and 34; and (iii) the liposome according to item 35, (iv) the composition according to item 36, (v) item (vi) a vaccine according to any one of items 41 to 45; (vii) a cell according to any one of items 41 to 45; ) CD8 as described in item 46 + (viii) a T lymphocyte, or a cell population according to item 47. to a subject. 49. The method according to item 48, wherein the ovarian cancer is serous carcinoma. 50. The method according to item 49, wherein the serous cancer is high-grade serous carcinoma (HGSC). . 51. The method further comprises administering to the subject at least one additional anti-tumor agent or therapy. 51. The method according to any one of Items 48 to 50. 52. The at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, an immunotherapy, or an immunotherapy. 52. The method of item 51, wherein the treatment is a checkpoint inhibitor, radiation therapy, or surgery. 53. (i) a tumor antigen peptide according to any one of items 1 to 32, (ii) item 33 or 34, (iii) the liposome according to Item 35, (iv) the nucleic acid according to Item 36 (v) the vaccine according to item 37; (vi) any one of items 41 to 45. (vii) the cell according to claim 46, + T lymphocytes, or (viii 48. Use of the cell population described in 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) item 33 or 34, (iii) the liposome according to Item 35, (iv) the nucleic acid according to Item 36 (v) the vaccine according to item 37; (vi) any one of items 41 to 45. (vii) the cell according to claim 46, + T lymphocytes, or (viii 48.) The cell population according to item 47, for the manufacture of a medicament for treating ovarian cancer in a subject. For, use. 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. Items 53-56 further including the use of at least one additional anti-tumor agent or therapy. The use according to any one of the preceding claims. 58. The at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, an immunotherapy, or an immunotherapy. 58. The use according to item 57, wherein the treatment is a checkpoint inhibitor, radiation therapy or surgery. 59. (i) any of items 1 to 32 for use in treating ovarian cancer in a subject. (ii) a tumor antigen peptide according to any one of items 33 and 34; and (iii) a nucleic acid according to item 33 or 34. (iii) the liposome according to item 35; (iv) the composition according to item 36; (v) (vi) a vaccine according to item 37, (vi) a cell according to any one of items 41 to 45, (vi i) CD8 as described in item 46 + (viii) a cell population according to item 47 Group. 60. The tumor antigen peptide for use according to item 59, wherein the ovarian cancer is serous carcinoma. Do, nucleic acid, liposome, composition, vaccine, cell, CD8 + T lymphocytes, or cell populations . 61. The use according to item 60, wherein the serous cancer is high-grade serous carcinoma (HGSC). tumor antigen peptides, nucleic acids, liposomes, compositions, vaccines, cells, CD8 + T lymphocytes, or cell populations. 62. Tumor antigen peptides, nucleic acids, liposomes, compositions, vaccines, cells, CD8 + T. Lin The cell or cell population is combined with at least one additional anti-tumor agent or therapy. A tumor antigen peptide, nucleic acid, or liposome for use according to any one of items 59 to 61. , composition, vaccine, cells, CD8 + T lymphocytes, or cell populations. 63. The at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, an immunotherapy, or an immunotherapy. 63. The method for treating tumors according to item 62, wherein the treatment is a checkpoint inhibitor, radiotherapy or surgery. Tumor antigen peptide, nucleic acid, liposome, composition, vaccine, cell, CD8 + T lymphocytes, or cell populations.

[0007] Other objects, advantages and features of the present invention are given by way of example only with reference to the accompanying drawings in which: This will become apparent from a reading of the following non-limiting description of specific embodiments. [Brief explanation 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. MAPs were identified by searching for matches in customized individual global cancer databases constructed from RNA-Seq data. Peptide sequences were identified using MS analysis. CDS: coding sequence. [Figure 2] Figures 2A-B show the expression of RNAs encoding aeTSA candidates in normal tissues. Heatmaps depict the 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 with above-threshold RNA expression (average rphm > 10). Numbers next to each peptide sequence indicate the number of tissues with above-threshold expression of the corresponding RNA. [Figure 3]Figures 3A-3D 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 the 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), and their reporting status (outer circle) of aeTSAs. [Figure 4] Expression of aeTSA coding regions across ovarian cancer samples. Heat maps show RNA expression of each aeTSA coding region in the nine samples reported in this study (left) and 378 samples from the TCGA-OV cohort, with color intensity indicating RNA levels in reads mapped to the region per million reads. [Figure 5] Figures 5A-5B show that copy number changes correlate with the expression of several aeTSAs. Figure 5A: Heatmap shows Spearman correlations between aeTSA RNA expression levels and DNA copy number, promoter methylation, or gene expression for aeTSAs located intragenicly (left) or extragenicly (right). Unavailable data are displayed as light gray. Figure 5B: Number of aeTSAs identified from each chromosome arm (top) with arm-level amplification scores (bottom). Asterisks indicate amplifications considered significant (Q-value < 0.25). [Figure 6]Figures 6A-6E show that presentation of three aeTSAs can induce spontaneous antitumor immune responses. Figures 6A-6C: Kaplan-Meier curves show the 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-encoding RNA in the absence of relevant HLA allotypes; ND: No expression of aeTSA-encoding RNA in the absence of relevant HLA allotypes; EP: Expression of aeTSA-encoding RNA in the presence of relevant HLA allotypes; NP: No expression of aeTSA-encoding RNA in the presence of relevant HLA allotypes. Color shades represent 95% confidence intervals. Log-rank P values ​​are shown. Figures 6D and 6E: Abundance of T and cytotoxic cells in tumors from the four groups shown in Figure 6C. [Figure 7] Figure 1 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 if 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 dashed red lines indicate the median number of aeTSAs per tumor in each population. [Figure 8] Figures 8A-8B show that dbSNP-based mTSA validation is also supported by sequencing data from 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 aesTSAs if both their coding sequences and the corresponding reference sequences restricted RNA expression in peripheral tissues. Figure 2 shows a heatmap showing the average RNA expression of aesTSA 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 indicates tissues / organs with an average rphm value greater than 10. The number next to each peptide sequence indicates the number of tissues with significant RNA expression for the given peptide. Red asterisks indicate peptides that were retained as aesTSAs. [Figure 10] Graph showing the correlation between the number of MAPs and tumor size. The scatter plot shows the 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] Figures 11A-11B are graphs showing the relationship between aeTSA expression and DNA copy number variation (CNV). Figure 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). Figure 11B: Correlation between aeTSA counts and chromosome arm amplifications. Scatter plots show the Pearson correlation between aeTSA counts and chromosome arm amplifications (x-axis) identified from each sample arm (y-axis). DETAILED DESCRIPTION OF THE INVENTION

[0009] The genetic, molecular biology, biochemistry, and nucleic acid terms and symbols used herein are , standard papers and texts in the field, e.g., Kornberg and Baker,DNA Replication,Second Edition(WH Freeman, New York, 1992), Lehninger, Bioche mistry,Second Edition(Worth Publishers,N ew York, 1975), Strachan and Read, Human Mo. lecular Genetics,Second Edition(Wiley-Li ss, New York, 1999), Eckstein, editor, Oligon. ucleotides and analogs: A Practical Appro ach(Oxford University Press, New York, 199 1), Gait, editor, Oligonucleotide Synthesis :A Practical Approach(IRL Press,Oxford,1 984) etc. All terms are based on their typical meanings established in the relevant technical field. must be understood in a meaningful way.

[0010] The articles "a" and "an" refer to one or more (i.e., at least) of the grammatical objects of the article. As an example, "an element" is used herein to refer to at least one element. "ent") means one element or more than one element. Unless the context requires otherwise, we use the words "comprise" and "comprise The terms "(s)" and "comprising" mean the specified steps or denotes the inclusion of an element, or group of steps or elements, but does not include any other step or element. It will be understood that no exclusion of steps or groups of elements is implied. .

[0011] The recitation of ranges of values ​​herein includes all values ​​within that range unless otherwise indicated herein. It is intended to serve as a complete expression that individually refers to each distinct value that falls within it. and each separate value is incorporated herein as if it were individually recited herein. Also, all subsets of values ​​within ranges are included herein as if individually enumerated. and is incorporated herein by reference in its entirety.

[0012] All methods described herein are intended to be illustrative unless otherwise indicated herein or in context. The steps may be performed in any suitable order unless clearly contradicted by the

[0013] Any and all examples or exemplary language provided herein (e.g., "etc.") ") is intended only to better illustrate the invention and, unless otherwise stated, It is not intended to limit the scope of the present invention.

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

[0015] As used herein, the term "about" has its ordinary meaning. includes the inherent variation of error for the device or method used to determine the value used to indicate that a value is an enumeration or is close to an enumerated value, e.g., values ​​within 10% or 5% of the

[0016] In the work described herein, we used a proteogenomics-based approach Using this method, 111 TSA candidates (103 novel candidates and We identified 93 TSAs (93 of which were previously reported candidates). These aberrant genes originate from aberrantly expressed, non-mutated genomic sequences that are not expressed in normal tissues. The current TSA (herein referred to as aeTSA) is composed primarily of non-exon sequences, especially introns. The expression of thrombin-like proteins was shown to be derived from tron ​​(31%) and intergenic (22%) sequences. was regulated at the transcriptional level by variations in gene copy number and DNA methylation. aeTSA is shared by the majority of HGSCs, and the frequency and H Taking into account LA allele frequency, the median number of aeTSAs per tumor in Caucasians was The novel TSA candidate identified herein is a candidate for ovarian cancer T cell This may be useful for immunotherapy based on

[0017] Thus, in an embodiment, the present disclosure provides the nucleic acid sequences of SEQ ID NOs: 1 to 103, preferably SEQ ID NOs: 19 to 1 Tumors containing or consisting of one of the amino acid sequences of Table 3A, Table 3B Concerning antigenic peptides (or tumor-specific peptides).

[0018] In embodiments, the present disclosure provides SEQ ID NOs: 1, 8, 10, 13, 15, and 19-27, preferably Preferably, the amino acid sequence comprises or consists of one of the amino acid sequences of SEQ ID NOs: 19 to 27. , located in the untranslated transcribed region (UTR), i.e., the 3'-UTR or 5'-UTR region In embodiments, the tumor antigen peptide is encoded by a sequence are SEQ ID NOs: 1, 10, 13, 15, and 19 to 23, preferably SEQ ID NOs: 19 to 23 a 5'-UTR region comprising or consisting of one of the amino acid sequences In embodiments, the tumor antigen peptide is encoded by a sequence represented by SEQ ID NO: 8, and 24 to 27, preferably one of the amino acid sequences of SEQ ID NOs: 24 to 27, or is encoded by a sequence located in the 3'-UTR region, which consists of:

[0019] In embodiments, the present disclosure provides SEQ ID NOs: 16, 17, and 36-64, preferably SEQ ID NOs: Introns containing or consisting of one of the amino acid sequences of Nos. 36 to 64 The present invention relates to a tumor antigen peptide encoded by a sequence located within the present invention.

[0020] In embodiments, the present disclosure provides a method for the production of a polypeptide comprising one of the amino acid sequences of SEQ ID NOs: 65-84; or a tumor antigen peptide encoded by a sequence located in an intergenic region, Regarding do.

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

[0022] In another embodiment, the present disclosure provides SEQ ID NOs: 4, and 85-92, preferably SEQ ID NO: 8 Non-coding RNA sequences containing or consisting of one of 5 to 92 amino acid sequences. Tumor anti-cancer drugs encoded by ncRNAs (ncRNAs) located in exons of non-coding transcripts Concerning the original peptide.

[0023] In another embodiment, the present disclosure provides a method for the preparation of a nucleic acid comprising one of the amino acid sequences of SEQ ID NOs: 93-99. or consisting of a tumor anti-cancer agent encoded by a sequence that is an antisense of a gene. Concerning the original peptide.

[0024] In another embodiment, the present disclosure provides a method for the preparation of a medicament comprising one of the amino acid sequences of SEQ ID NOs: 100-103. A tumor antigen peptide comprising or consisting of a sequence from a mucin gene. Regarding Petite.

[0025] Generally, peptides such as tumor antigen peptides presented in the context of HLA class I are long peptides. The length is about 7 or 8 to about 15 amino acid residues, or preferably 8 to 14 amino acid residues. In some embodiments of the methods described herein, the method further comprises administering to the subject a tumor antigen comprising a tumor antigen peptide sequence as defined herein. Longer peptides are artificially loaded onto cells such as antigen-presenting cells (APCs) and are then absorbed by the cells. The tumor antigen peptides are then processed by MHC class I molecules on the surface of APCs. In this method, a 15 amino acid residue (i.e., tumor antigen precursor peptide) is synthesized. ) can be loaded onto APCs for presentation. The tumor antigen peptides are then delivered to a protease in the APC cytoplasm, which then delivers the corresponding tumor antigen peptides as defined in the specification. In some embodiments, the tumor antigen peptides defined herein are processed by The precursor peptide / polypeptide used to generate, e.g., 1000, 500 , 400, 300, 200, 150, 100, 75, 50, 45, 40, 35, 30, 2 5, 20, or 15 amino acids or less. Thus, the tumor antigens described herein All methods and processes using peptides are based on the "top-up" method after treatment with cells (APCs). Longer peptides or peptides to induce the final presentation of 8-14 tumor antigen peptides The present invention relates to polypeptides (including natural proteins), i.e., tumor antigen precursor peptides / polypeptides. In some embodiments, the tumor antigen peptides described herein include the use of: Approximately 8-14, 8-13, or 8-12 amino acids in length (e.g., 8, 9, 10, 11, 12 or 13 amino acids long) and are sufficient to directly match with HLA class I molecules. In embodiments, tumor antigen peptides are small, consisting of 20 amino acids or less, preferably 15 amino acids or less. In an embodiment, the tumor antigen comprises 14 or fewer amino acids, more preferably 14 or fewer amino acids. Peptides are at least 7 amino acids, preferably at least 8 amino acids, more preferably Preferably it contains at least 9 amino acids.

[0026] As used herein, the term "amino acid" includes the L-variants of naturally occurring amino acids. to prepare both the D- and C-isomers, as well as synthetic analogs of tumor antigen peptides. Other amino acids used in peptide chemistry (e.g., naturally occurring amino acids, naturally occurring These include amino acids that are not naturally occurring, amino acids that are not encoded by nucleic acid sequences, etc. Examples of amino acids present in Other amino acids include, for example, non-genetically encoded forms of Conservative substitutions of L-amino acids, as well as L-amino acids, are included. Examples of amino acids include β-alanine, 3-aminopropionic acid, 2,3-diamino α-aminoisobutyric acid (Aib), 4-aminobutyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), cytochrome c leucine, t-butylalanine, t-butylglycine, N-methylisoleucine, phenyl Glycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-naphthyl thialanine, pyridylalanine, 3-benzothienylalanine, 4-chlorophenylalanine 2-Fluorophenylalanine, 3-Fluorophenylalanine, 4-Fluorophenylalanine Phenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylate Carboxylic acid, β-2-thienylalanine, methionine sulfoxide, L-homoarginine ( Hoarg), N-acetyl lysine, 2-aminobutyric acid, 2-aminobutyric acid, 2,4-diamino N-butyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine Stains, homoserine (HoSer), cysteic acid, ε-aminohexanoic acid, δ-amino Examples include valeric acid and 2,3-diaminobutyric acid (D- or L-). The amino acids are well known in the field of biochemistry / peptide chemistry. The original peptide contains only naturally occurring amino acids.

[0027] In embodiments, the tumor antigen peptides described herein have a sequence similar to that described herein. and peptides having modified sequences containing substitutions of functionally equivalent amino acid residues. For example, one or more amino acid residues within the sequence may act as functional equivalents and silence Similar polarity (having similar physicochemical properties) leads to alteration The substitution of an amino acid within a sequence can be performed by replacing the amino acid with another amino acid of the same sequence. For example, positively charged (basic) amino acids and other members of the same class may be selected. These include arginine, lysine, and histidine (as well as homoarginine and ornithidine). Nonpolar (hydrophobic) amino acids include leucine, isoleucine, and These include lanine, phenylalanine, valine, proline, tryptophan, and methionine. The uncharged polar amino acids include serine, threonine, cysteine, and tyrosine. Negatively charged (acidic) amino acids include riboflavin, asparagine, and glutamine. Examples include glutamic acid and aspartic acid. The amino acid glycine is a non-polar It can be included in either the polar or uncharged (neutral) 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 contain all L-amino acids, all D-amino acids, or In embodiments, the present invention may include a mixture of L- and D-amino acids. The tumor antigen peptides described herein contain all L-amino acids.

[0028] In embodiments, the antibody comprises or is derived from one of the sequences disclosed in Tables 3A-3B. The sequence of the tumor antigen peptide does not substantially contribute to the interaction with the T cell receptor. The amino acid residues, the incorporation of which does not substantially affect T cell reactivity, are It may be modified by substitution with other amino acids that do not eliminate binding to the amino acid.

[0029] The tumor antigen peptide may also be modified to prevent degradation, increase stability, affinity, and / or uptake. The N- and / or C-termini may be capped or modified to enhance Thus, in another aspect, the present disclosure provides a compound of formula Z 1 -XZ 2 modified tumors The present invention provides an antigenic peptide, wherein X is selected from the group consisting of SEQ ID NOs: 1 to 103, preferably SEQ ID NOs: 19 to 1 03 amino acid sequence. (Table 3A, Table 3B).

[0030] In embodiments, the amino terminal residue (i.e., the free amino group at the N-terminus) of the tumor antigen peptide ) can be used to refer to, for example, the moiety / chemical group (Z 1 ) by covalent bonding (e.g., for protection from degradation) Z 1 is a straight or branched chain alkyl group of 1 to 8 carbons, or It may be a silyl group (R—CO—), where R is a hydrophobic moiety (e.g., acetyl, propanol, propionyl, butanyl, isopropionyl, or isobutanyl), or aroyl In embodiments, the acyl group is an (Ar—CO—) group, where Ar is an aryl group. is C1~C 16 or C3~C 16 Acyl groups (straight-chain or branched, saturated or unsaturated) In a further embodiment, a saturated C1-C6 acyl group (linear or branched) or an unsaturated and C3-C6 acyl groups (linear or branched), such as acetyl group (CH3-CO-Ac). In an embodiment, Z 1 The carboxy-terminal residue of the tumor antigen peptide (i.e., That is, the free carboxyl group at the C-terminus of the tumor antigen peptide can be amidated (transferred to the NH2 group), for example. can be modified (e.g., for protection from degradation) by substitution of OH groups by OH groups. In such a case, Z 2 is an NH group. In embodiments, Z 2 is a hydroxamate group, tolyl group, amide (primary, secondary or tertiary) group, methylamine, isobutylamine, isobutylamine, Aliphatic amines of 1 to 10 carbons, such as sovalerylamine or cyclohexylamine , aniline, naphthylamine, benzylamine, cinnamylamine, or phenylethylamine aromatic or arylalkylamines such as methylamine, alcohols, or CHOH In an embodiment, Z 2 In embodiments, the tumor antigen peptide is , the amino acid sequences of SEQ ID NOs: 1 to 103, preferably SEQ ID NOs: 19 to 103 (Table 3A, Table 3 In an embodiment, the tumor antigen peptide comprises one of SEQ ID NOs: 1 to 103, preferably SEQ ID NOs: 1 to 103. Preferably, it consists of one of the amino acid sequences of 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 a method for the preparation of a nucleic acid comprising the sequence of SEQ ID NO: 21, 28, 40, 41, 66 or 88. HLA-A, comprising or consisting of * 01:01 binds to tumor antigen peptides The present invention provides a method for the treatment of ovarian tumors comprising administering to a mammalian subject the present invention ....

[0032] In another aspect, the present disclosure provides SEQ ID NOs: 14, 17, 45, 48, 51, 56, 75, 77 , 82, 98 or 100, preferably 45, 48, 51, 56, 75, 77, 82, 9 HLA-A comprising or consisting of 8 or 100 sequences * 02:01 Molecular A tumor antigen peptide (or tumor-specific peptide), preferably an ovarian tumor antigen peptide, is combined with the Provide chid.

[0033] In another aspect, the present disclosure provides SEQ ID NOs: 1, 19, 20, 22, 30, 31, 36, 50, 52, 60, 62, 73, 84, 85, 86 or 91, preferably 19, 20, 22, Sequences of 30, 31, 36, 50, 52, 60, 62, 73, 84, 85, 86 or 91 HLA-A, comprising or consisting of * 03:01 Tumor antigen peptide binding to a molecule The present invention provides a method for the treatment of ovarian tumors comprising administering to a mammalian subject the present invention ...

[0034] In another aspect, the present disclosure provides SEQ ID NOs: 32, 54, 55, 67, 69, 81, 87, 90 or 102 sequences, or HLA-A * 11:01 Binding to molecules a tumor antigen peptide (or tumor-specific peptide), preferably an ovarian tumor antigen peptide, Provide the

[0035] In another aspect, the disclosure provides a method for the detection of a mutated or mutated nucleotide sequence comprising or consisting of the sequence of SEQ ID NO: 33 or 43. HLA-A * 24:02 molecule, binding to tumor antigen peptides (or tumor-specific peptides) tides), preferably ovarian tumor antigen peptides.

[0036] In another aspect, the present disclosure provides an HLA antibody comprising or consisting of the sequence of SEQ ID NO: 24. -A * 25:01 molecule, tumor antigen peptide (or tumor-specific peptide), Preferably, an ovarian tumor antigen peptide is provided.

[0037] In another aspect, the disclosure provides a method for the detection of a mutated or mutated nucleotide sequence comprising or consisting of the sequence of SEQ ID NO: 34 or 58. HLA-A * 29:02 molecule, binding to tumor antigen peptides (or tumor-specific peptides) tides), preferably ovarian tumor antigen peptides.

[0038] In another aspect, the present disclosure provides an HLA antibody comprising or consisting of the sequence of SEQ ID NO: 16. -A * 32:01 molecule, tumor antigen peptide (or tumor-specific peptide), Preferably, an ovarian tumor antigen peptide is provided.

[0039] In another aspect, the present disclosure provides SEQ ID NOs: 4, 6, 8, 9, 26, 49, 78, 92, 97 or or 101, preferably 26, 49, 78, 92, 97 or 101 sequences or consisting of HLA-B * 07:02 Binding to tumor antigen peptides (or tumor-specific peptides), preferably ovarian tumor antigen peptides.

[0040] In another aspect, the present disclosure provides SEQ ID NOs: 23, 35, 42, 44, 46, 59, 63, 70 HLA-B comprising or consisting of the sequence of * 0 A tumor antigen peptide (or tumor-specific peptide) that binds to the 8:01 molecule, preferably Ovarian tumor antigen peptides are provided.

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

[0042] In another aspect, the disclosure provides a method for the detection of a nucleotide sequence comprising or consisting of the sequence of SEQ ID NO: 2, 3 or 5. HLA-B * 15:01 molecule binds to tumor antigen peptides (or tumor-specific peptides) tides), preferably ovarian tumor antigen peptides.

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

[0044] In another aspect, the disclosure provides a method for the preparation of a nucleic acid comprising the sequence of SEQ ID NO: 47, 64, 96 or 99; consists of HLA-B * 39:01 molecule, which binds to tumor antigen peptides (or tumor tumor-specific peptides, preferably ovarian tumor antigen peptides.

[0045] In another aspect, the disclosure provides a method for the preparation of a nucleic acid sequence comprising or comprising the sequence of SEQ ID NO: 11, 12 or 13. HLA-B * 40:01 molecule binds to tumor antigen peptides (or tumor-specific peptides) The present invention provides a peptide, preferably an ovarian tumor antigen peptide.

[0046] In another aspect, the present disclosure provides an HLA antibody comprising or consisting of the sequence of SEQ ID NO: 65. -B * 44:02 molecule, tumor antigen peptide (or tumor-specific peptide), Preferably, an ovarian tumor antigen peptide is provided.

[0047] In another aspect, the disclosure provides a method for the detection of a mutated or mutated nucleotide sequence comprising or consisting of the sequence of SEQ ID NO: 37 or 94. HLA-B * 44:03 molecule, which binds to tumor antigen peptides (or tumor-specific peptides) tides), preferably ovarian tumor antigen peptides.

[0048] In another aspect, the present disclosure provides a nucleic acid sequence similar to SEQ ID NO: 10, 29, 71 or 95, preferably SEQ ID NO: HLA-C comprising or consisting of 29, 71 or 95 sequences *03:03 minutes A tumor antigen peptide (or tumor-specific peptide), preferably an ovarian tumor antigen, that binds to the The raw peptide is provided.

[0049] In another aspect, the disclosure provides a method for the detection of a nucleotide sequence comprising or consisting of the sequence of SEQ ID NO: 6 or 15. , HLA-C * 04:01 molecule, binding to tumor antigen peptides (or tumor-specific peptides) ), preferably ovarian tumor antigen peptides.

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

[0051] In another aspect, the present disclosure provides a method for the preparation of a nucleic acid sequence of SEQ ID NO: 18 or 72, preferably SEQ ID NO: 18 or 72. HLA-C comprising or consisting of the sequence * 06:02 Binding to tumor antigen molecules Peptides (or tumor-specific peptides), preferably ovarian tumor antigen peptides, are provided.

[0052] In another aspect, the disclosure provides a method for the preparation of a nucleic acid comprising or containing the sequence of SEQ ID NO: 38, 61 or 93. Consisting of HLA-C * 07:01 Tumor antigen peptide (or tumor-specific peptide) binding to the molecule The present invention provides a peptide, preferably an ovarian tumor antigen peptide.

[0053] In another aspect, the present disclosure provides an HLA- C * 07:02 A tumor antigen peptide (or tumor-specific peptide), preferably The present invention also provides ovarian tumor antigen peptides.

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

[0055] In another aspect, the disclosure provides a method for the preparation of a nucleic acid comprising or containing the sequence of SEQ ID NO: 25, 57 or 79. Consisting of HLA-C * 14:02 binding to tumor antigen peptides (or tumor-specific The present invention provides a peptide, preferably an ovarian tumor antigen peptide.

[0056] In embodiments, the tumor antigen peptide is located in the untranslated transcribed region (UTR), i.e., the 3′-U In another embodiment, the nucleic acid sequence is encoded by a sequence located in the TR or 5'-UTR region. 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 this embodiment, the tumor antigen peptide is located within an exon and has a sequence derived from a frameshift. Coded by columns.

[0057] The tumor antigen peptides of the present disclosure can be expressed by infecting host cells containing nucleic acids encoding the tumor antigen peptides. Produced by expression (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis) Peptides can be produced using manual and / or automated solid-phase procedures well known in the art. Suitable syntheses include, for example, "T-boc" or " This can be done by utilizing the "Fmoc" procedure. The procedure is, for example, Solid Phase Peptide Synthesis: Practical Approach(E.Atherton and RCSh by Eppard, IRL, Oxford University Press, 19 Alternatively, tumor antigen peptides can be prepared using methods such as those described in Liu et al. t al., Tetrahedron Lett. 37:933-936, 1996, B aca et al.,J.Am.Chem.Soc.117:1881-1887,1 995, Tam et al., Int. J. Peptide Protein Res .45:209-216,1995, Schnolzer and Kent,Scie. nce 256:221-225, 1992, Liu and Tam, J.Am.Ch em.Soc.116:4149-4153, 1994, Liu and Tam, Pr. Natl.Acad.Sci.USA 91:6584-6588,1994, and Yamashiro and Li, Int. J. Peptide Protein Res. 31:322-334, 1988), segment condensation Other methods useful for synthesizing tumor antigen peptides include those described by Nakagawa et al. et al., J.Am.Chem.Soc.107:7087-7092,1985 In embodiments, the tumor antigen peptides are chemically synthesized (synthetic peptides). Another embodiment of the present disclosure is a non-naturally occurring peptide, wherein the peptide is consisting of or consisting essentially of an amino acid sequence as defined herein and being pharmaceutically acceptable The present disclosure relates to synthetically produced (e.g., synthesized) peptides as salts. Salts of tumor antigen peptides are not salts, so in vivo production of the peptides is not a The non-natural salt form of peptides is very different from the peptides in the state. In the context of compositions, e.g., peptide vaccines disclosed herein, the solubility of the peptides Preferably, the salt is a pharmaceutically acceptable salt of the peptide.

[0058] In embodiments, the tumor antigen peptides described herein are substantially pure. , is "substantially pure" when it is separated from the components that naturally accompany it. The compound should be at least 60% by weight of the total material in the sample, more typically 75% by weight, 80% by weight, or more typically 90% by weight. % or 85% by weight, preferably more than 90% by weight, more preferably more than 95% by weight Therefore, it is not suitable for use in a pharmaceutical composition that is chemically synthesized, for example, by recombinant technology. The polypeptide to be produced will generally be free of its naturally associated components, e.g., its source. The nucleic acid molecule will be substantially free of components of the macromolecular source from which it was derived. Not immediately contiguous with coding sequences that are normally contiguous in naturally occurring genomes (e.g., A substantially pure compound is one in which the hydroxyl groups are not covalently bonded (i.e., not covalently bonded). For example, by extraction from a natural source, followed by expression of a recombinant nucleic acid molecule encoding a peptide compound. Purity can be determined by column chromatography or by chemical synthesis. This can be measured using any suitable method, such as gel electrophoresis, HPLC, etc. In one embodiment, the tumor antigen peptide is in solution. In another embodiment, the tumor antigen peptide is , in solid form, e.g., freeze-dried.

[0059] In another aspect, the present disclosure provides a method for the production of a tumor antigen peptide or tumor antigen precursor peptide described herein. Further provided is an isolated nucleic acid encoding a peptide. In embodiments, the nucleic acid is 1 nucleotide to about 45 nucleotides, about 24 to about 45 nucleotides, for example, 24, 2 "Isolated" means that the nucleic acid sequence contains 7, 30, 33, 36, 39, 42, or 45 nucleotides. As used herein, other components present in the molecule's natural environment or naturally occurring sources Peptides separated from the macromolecules of their 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 produced by, for example, recombinant techniques. A peptide that has not been isolated from its natural source, produced through artifical techniques or using chemical synthesis. Nucleic acids of the present disclosure refer to peptides or nucleic acid molecules for recombinant expression of the tumor antigen peptides of the present disclosure. a cloning vector or expression vector that can be used to transfect a host cell The vector may be contained in a vector or plasmid, such as a current vector. The cloning, expression, or viral vectors containing nucleic acid sequences encoding the tumor antigen peptides of the present disclosure. Alternatively, a vector or plasmid containing the tumor antigen peptide of the present disclosure is provided. The loading nucleic acid may be integrated into the genome of the host cell. The cells express the tumor antigen peptide or protein encoded by the nucleic acid. The term "host cell" as used herein refers not only to a particular target cell, but also to any such Host cells refer to the progeny or potential progeny of such cells. Any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., For example, insect cells, yeast cells or mammalian cells. The plasmid contains the elements necessary for transcription and translation of the inserted coding sequence and encodes the resistance gene. , cloning sites, and other elements. A sequence encoding a peptide or polypeptide and an appropriate polypeptide operably linked thereto. Expression vectors containing suitable transcriptional and translational control / regulatory elements may be constructed. These include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al. (1989) Molecular Biology. ar Cloning,A Laboratory Manual,Cold Spri ng Harbor Press, Plainview, NY, and Ausube l,FMet al.(1989)Current Protocols in M Olecular Biology,John Wiley & Sons,New Y "Operably linked" means that the components, particularly the nucleic acid, It refers to the juxtaposition of components that enable a nucleotide sequence to perform its normal function. Thus, a coding sequence that is operably linked to a regulatory sequence is subject to the regulatory control of the regulatory sequence, i.e. That is, a nucleotide sequence that can express a coding sequence under transcriptional and / or translational control. The term "regulatory / control region" or "regulatory / control sequence" as used herein refers to the organization of a gene sequence. When used herein, it refers to a non-coding nucleotide sequence that is involved in regulating the expression of an encoding nucleic acid. Therefore, the term regulatory region refers to the promoter sequence, regulatory protein binding site, upstream activation site, and In an embodiment, the nucleic acid (DN) encoding the tumor antigen peptide of the present disclosure includes a factor sequence. A, RNA) may be contained within a liposome or any other suitable vehicle. It is bound to

[0060] In another aspect, the present disclosure provides a method for the preparation of a tumor antigen peptide comprising the steps of: In an embodiment, the MHC class I molecule is an HLA- In a further embodiment, the molecule is an HLA-A*01:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A2 molecule, and in a further embodiment, an HLA-A3 molecule. In another embodiment, the MHC class I molecule is an HLA-A*02:01 molecule. A3 molecule, and in a further embodiment, an HLA-A*03:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A11 molecule, and in a further embodiment, In another embodiment, the MHC class I molecule is an HLA-A*11:01 molecule. In a further embodiment, the molecule is an HLA-A*24:02 molecule. In an embodiment, the MHC class I molecule is an HLA-A25 molecule, and in a further embodiment In another embodiment, the MHC class I molecule is H In a further embodiment, it 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. In another embodiment, the MHC class I molecule is an HLA-A*32:02 molecule. , HLA-B07 molecule, and in a further embodiment, HLA-B*07:02 molecule. In another embodiment, the MHC class I molecule is an HLA-B08 molecule, and In one embodiment, it is an HLA-B*08:01 molecule. In another embodiment, it is an MHC class I molecule. The 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 one embodiment, the MHC class is HLA-B*15:01 molecule. The I molecule is an HLA-B18 molecule, and in a further embodiment, HLA-B*18:01 In another embodiment, the MHC class I molecule is an HLA-B39 molecule, and In a further embodiment, the MHC class is an HLA-B*39:01 molecule. The ras I molecule is an HLA-B40 molecule, and in a further embodiment, HLA-B*40: In another embodiment, the MHC class I molecule is an HLA-B44 molecule. In a further embodiment, the HLA-B*44:02 molecule or the HLA-B*44:03 molecule In another embodiment, the MHC class I molecule is an HLA-C03 molecule, and further In one embodiment, the molecule is an HLA-C*03:03 molecule. In another embodiment, the molecule is an MHC class I The 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 one embodiment, the MHC class is an HLA-C*05:01 molecule. In a further embodiment, the antigen binding molecule is an HLA-C*06 molecule, and in a further embodiment, an HLA-C*06:0 In another embodiment, the MHC class I molecule is an HLA-C07 molecule, In a further embodiment, the antibody is an HLA-C*07:01 or HLA-C*07:02 molecule. In another embodiment, the MHC class I molecule is an HLA-C12 molecule, and In one embodiment, it is an HLA-C*12:03 molecule. The molecule is an HLA-C14 molecule, and in a further embodiment, an HLA-C*14:02 molecule. is.

[0061] In embodiments, the tumor antigen peptide is non-covalently bound to the MHC class I molecule (i.e., That is, tumor antigen peptides are loaded into the peptide-binding groove / pocket of MHC class I molecules. In another embodiment, the tumor antigen peptide is covalently or non-covalently linked to the MHC class It is covalently attached / bound to the I molecule (α chain). In such constructs, the tumor antigen peptide and the MHC class I molecule (α chain) are typically , short (e.g., 5-20 residues, preferably about 8-12, e.g., 10), flexible Synthetic fusion proteins with linkers or spacers (e.g., polyglycine linkers) In another aspect, the present disclosure provides a method for producing a fusion protein of an MHC class I molecule (α chain) fused to the fusion protein. Nucleic acids encoding fusion proteins comprising the tumor antigen peptides defined herein are provided. In embodiments, the MHC class I molecule (α chain)-peptide complex is multimerized. Thus, in another aspect, the present disclosure provides a method for preparing a tumor antigen peptide as described herein, comprising (covalently binding) The present invention provides multimers of MHC class I molecules loaded (directly or non-covalently). Such multimers may be linked to a tag, e.g., a fluorescent tag, that allows for detection of the multimer. Multiple strategies for the production of MHC multimers, including C dimers, tetramers, pentamers, octamers, etc. has been developed (Bakker and Schumacher, Current O Pinion in Immunology 2005, 17:428-433 MHC multimers are useful, for example, for the detection and purification of antigen-specific T cells. Thus, in another aspect, the present disclosure provides a method for producing a tumor antigen peptide specific for the tumor antigen peptides defined herein. Typical CD8 + 1. A method for detecting or purifying (isolating, enriching) T lymphocytes, comprising: The cell population was subjected to immunoprecipitation using a cytoplasmic immunoglobulin (C1)-containing IgG1-associated ... contacting the cells with a multimer of MHC class I molecules and binding the cells to the MHC class I multimer; CD8 + and detecting or isolating T lymphocytes. CD8 bound by rasI multimers + T lymphocytes can be isolated by known methods, e.g., fluorescent activity isolated using fluorescently activated cell sorting (FACS) or magnetic activated cell sorting (MACS) That's fine.

[0062] In yet another aspect, the present disclosure provides a method for the production of a nucleic acid, vector, or plasmid of the present disclosure as described herein. a smid, i.e., a nucleic acid or vector encoding one or more tumor antigen peptides Cells (e.g., host cells), in embodiments, isolated cells, are provided. The disclosure also provides an MHC class I antigen binding to or presenting a tumor antigen peptide according to the disclosure. Cells expressing a molecule (e.g., an MHC class I molecule of one of the alleles disclosed above) In one embodiment, the host cell is a eukaryotic cell, e.g., a mammalian cell, preferably In another embodiment, the cell is an antigen-presenting cell. In one embodiment, the host cell is a primary cell, a cell line, or an immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC). The nucleic acid and vector are , can be introduced into cells via conventional transformation or transfection techniques The terms "transformation" and "transfection" refer to the use of calcium phosphate or The transfection was carried out by calcium chloride coprecipitation, DEAE-dextran mediated transfection, and lipofection. cytochemistry, electroporation, microinjection and viral-mediated transfection Transforming a host cell refers to techniques for introducing foreign nucleic acid into a host cell, including transformation. Alternatively, suitable methods for transfection are described, for example, in Sambrook et al. al. (supra), and other laboratory manuals. Methods for introducing nucleic acids into animal cells are also known, and include the use of the vectors or vectors of the present disclosure for gene therapy. can be used to deliver the plasmid to a subject.

[0063] Cells, such as APCs, can be transfected with one or more markers using a variety of methods known in the art. As used herein, a tumor antigen peptide can be loaded. "Loading" a cell with a tumor antigen peptide or a RNA encoding a tumor antigen peptide NA or DNA is transfected into cells, or alternatively, APCs are used to It means that the cell is transformed with nucleic acid encoding the antigenic peptide. It can directly bind to MHC class I molecules present on cells (e.g., peptide-pulsed cells). Tumor antigens can be loaded by contacting the cells with exogenous tumor antigen peptides that can be used to stimulate tumor growth. The tumor antigen peptide also contains a domain or domains that facilitate its presentation by MHC class I molecules. motifs, e.g., endoplasmic reticulum (ER) retrieval signals, C-terminal Lys-Asp-Glu-Le u sequence (Wang et al. Eur J Immunol. See 2004 Dec:34(12):3582-94).

[0064] In another aspect, the present disclosure provides a tumor antigen peptide as defined herein (or the peptide a nucleic acid encoding a nucleic acid encoding a nucleic acid encoding a nucleic acid sequence; In embodiments, the composition comprises a peptide combination / pool as defined herein. Any combination of tumor antigen peptides (2, 3, 4, 5, 6, 7, 8, 9, 10 any combination of the above tumor antigen peptides), or a compound encoding the tumor antigen peptide any combination of tumor antigen peptides as defined herein. Compositions containing the combination / subcombination are encompassed by the present disclosure. The antibody or pool may contain one or more known tumor antigens.

[0065] Thus, in another aspect, the present disclosure provides a tumor antigen peptide as defined herein. or any combination thereof, and an MHC class I molecule (e.g., and a cell expressing an MHC class I molecule of one of the selected alleles. APCs for use in the present disclosure are not limited to a particular type of cell, CD8 + Presents protein antigens on its cell surface for recognition by T lymphocytes dendritic cells (DCs), Langerhans cells, macrophages, and For example, APCs can be expressed in vitro, in vivo, or in vivo. DCs are derived from peripheral blood mononuclear cells either ex vivo or in vivo, and then transfected with tumor antigens. APCs can also be obtained by contacting (stimulating) them with peptides. One or more of the tumor antigen peptides shown are administered to a subject to present the tumor antigen peptide. APCs are induced in the subject's body and activated to present tumor antigen peptides in vivo. The phrases "inducing APCs" and "stimulating APCs" refer to the induction of tumor antigen receptors. The cells are then transfected with one or more tumor-associated proteins so that the peptides are presented on their surface by MHC class I molecules. contacting the tumor antigen peptide or a nucleic acid encoding the tumor antigen peptide, or As described herein, in accordance with the present disclosure, tumor antigen peptides can be loaded onto the tumor cells. The peptide may be, for example, a longer peptide containing the sequence of a tumor antigen peptide (including a naturally occurring protein). The antigen may be indirectly loaded using a peptide / polypeptide, followed by the addition of a tumor antigen peptide / polypeptide. are processed internally within the APC to generate MHC class I complexes at the cell surface (e.g., APCs are loaded with tumor antigen peptides, and the APCs then bind to the tumor antigen peptides. After allowing the APCs to present the vaccine, the APCs can be administered to a subject as a vaccine. For example, ex vivo administration may involve the following steps: (a) collecting APCs from a first subject; and (b) contacting / loading the APCs of step (a) with tumor antigen peptides to form tumor antigen peptides on the surface of the APCs. (c) forming an MHC class I / tumor antigen peptide complex in the tumor; and administering the peptide-loaded APCs to a second subject.

[0066] The first subject and the second subject may be the same subject (e.g., an autologous vaccine); Or it may be a different subject (e.g., allogeneic vaccine). The present invention provides a method for producing a composition (e.g., a pharmaceutical composition) for inducing antigen-presenting cells. Use of the tumor antigen peptides (or combinations thereof) described herein is provided. Therefore, the present disclosure relates to a method or process for producing a pharmaceutical composition for inducing antigen-presenting cells. The present invention provides a method or process for administering tumor antigen peptides, or a combination thereof, to a patient. The anti-tumor agent as defined herein may be mixed or formulated with a pharmaceutical acceptable carrier. MHC class I loaded with any one or any combination of the original peptides 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- APs expressing HLA-C06, HLA-C07, HLA-C12, or HLA-C14 molecules Cells such as C, CD8 + T lymphocytes, e.g., autologous CD8 + Stimulates / expands T lymphocytes Thus, in another aspect, the present disclosure provides a method for detecting a cellular component, as defined herein, Any of the tumor antigen peptides (or nucleic acids or vectors encoding same) MHC class I molecules and T lymphocytes, more specifically CD8 + T lymphocyte-expressing cells (e.g., CD8 + A population of cells that includes T lymphocytes )

[0067] In embodiments, the composition may contain a buffer, excipient, carrier, diluent, and / or medium (e.g., In further embodiments, the composition further comprises a buffer, excipient, carrier, diluent, and The medium and / or the medium may contain pharmaceutically acceptable buffers, excipients, carriers, diluents and / or As used herein, "a pharmaceutically acceptable buffer, excipient, , carriers, diluents and / or media" are physiologically compatible and capable of supporting the biological activity of the active ingredient. Any and all solvents, buffers, binders that do not interfere with the efficacy of the , lubricants, fillers, thickeners, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants , stabilizers, release retardants, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, etc. The use of such media and agents for pharmaceutically active substances is within the skill of the art. It is well known in the field (Rowe et al., Handbook of Pharmacology aceutical excipients,2003,4 th edition,Ph (Arachnetical Press, London, UK). Any conventional medium or In the compositions of the present disclosure, unless the agent is incompatible with the active compound (peptide, cell), In embodiments, buffers, excipients, carriers, and / or A medium is a non-naturally occurring buffer, excipient, carrier, and / or medium. In some embodiments, a tumor antigen peptide as defined herein, or one or more of said tumor antigen peptides. One or more of the nucleic acids (e.g., mRNA) encoding the Contained within or complexed to liposomes (e.g., Vit or MT et al.,Recent Pat Drug Deliv Formu l.2013 Aug;7(2):99-110).

[0068] In another aspect, the present disclosure provides a tumor antigen peptide as defined herein (or the peptide any one or any combination of the nucleic acids encoding the , an excipient, a carrier, a diluent and / or a medium. For compositions containing cells (e.g., APCs, T lymphocytes), the composition may be used to maintain viable cells. Representative examples of such media include physiological saline, Earl's Balanced Salt Solution (Life Technologies®), and is a registered trademark of PlasmaLyte® (Baxter International In embodiments, the compositions (e.g., pharmaceutical compositions) include "immunogens." "immunogenic composition," "vaccine composition," or "vaccine." The term "vaccine composition" or "vaccine" as used herein refers to one or more The tumor antigen peptide or vaccine vector contains the A composition capable of inducing an immune response against one or more tumor antigen peptides Vaccination methods for inducing an immune response in mammals involve the administration of vaccine compositions. By any conventional route known in the art, for example, mucosal membranes (e.g., ophthalmic, intranasal, pulmonary, Oral (gastric, intestinal, rectal, vaginal, or urinary tract) surfaces; parenteral (e.g., subcutaneous, intradermal, intramuscular) via intramuscular, intravenous, or intraperitoneal routes, or via topical administration (e.g., via a patch) the use of vaccines or vaccine vectors administered by transdermal delivery systems In embodiments, the tumor antigen peptide (or a combination thereof) is coupled to a carrier protein. Conjugation to (conjugate vaccine) increases the immunogenicity of tumor antigen peptides Thus, the present disclosure provides a method for the preparation of a tumor antigen peptide (or a combination thereof), or A nucleic acid encoding a tumor antigen peptide or a combination thereof, and a carrier protein. For example, a tumor antigen peptide or nucleic acid is provided as a conjugate. TLR ligands (e.g., Zom et al., Adv Immun ol.2012,114:177-201) or polymers / dendrimers (e.g., L iu et al., Biomacromolecules.2013 Aug 12; 14(8):2798-806) In embodiments, the immunogenic composition or vaccine further comprises an adjuvant. "Adjuvant" refers to an antigen (tumor antigen peptide, nucleic acid and / or cell according to the present disclosure) ), it suppresses the immune response to the drug in the host upon exposure to the mixture. It refers to a substance that specifically improves or enhances. Ajuba, which is currently used in the field of vaccines, Examples of salts include: (1) mineral salts (such as aluminum phosphate and aluminum hydroxide); Aluminum salts, calcium phosphate gel), squalene, (2) oil-based adjuvants (such as oil emulsions and surfactant-based formulations), e.g., MF59 (Micro Fluidized detergent stabilized oil-in-water emulsion), QS21 (purified saponin), AS02 [SB AS2] (oil-in-water emulsion + MPL + QS-21), (3) particulate adjuvant, For example, virosomes (unilamellar liposome vehicles incorporating influenza hemagglutinin) AS04 (aluminum salt containing MPL [SBAS4]), ISCOMS (saponins) (4) Microbial derived structural complexes of lactic acid bacteria and lipids, polylactide-co-glycolide (PLG), Lipids (natural and synthetic), such as monophosphoryl lipid A (MPL), Detox (MP L+M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), D C_Chol (a lipoid immunostimulatory substance that can self-assemble into liposomes), OM -174 (lipid A derivative), CpG motif (synthetic compound containing immunostimulatory CpG motif) synthetic oligonucleotides), modified LT and CT (to provide a non-toxic adjuvant effect) (5) endogenous human immunomodulators, e.g., human G M-CSF or hIL-12 (either protein or encoding plasmid) cytokines that can be administered in combination with other drugs), Immudaptin (C3d tandem array), and and / or (6) an inert vehicle such as gold particles.

[0069] In embodiments, the tumor antigen peptide or a composition comprising the same is in a lyophilized form. In another embodiment, the tumor antigen peptide or a composition comprising the same is a liquid composition. In a further embodiment, the tumor antigen peptide is present in the composition at a concentration of about 0.01 μg / mL to about 1 In a further embodiment, the tumor antigen peptide is present in the composition at a concentration of 00 μg / mL. Approx. 0.2 μg / mL ~ approx. 50 μg / mL, approx. 0.5 μg / mL ~ approx. 10, 20, 30, 4 0, or 50 μg / mL, about 1 μg / mL to about 10 μg / mL, or about 2 μg / mL is the concentration.

[0070] As described herein, any of the tumor antigen peptides defined herein MHC class I antigens that are loaded with or bind to one or any combination of MHC class I antigens. Cells such as APCs expressing the molecule can be used to induce CD8+ T lymphocytes in vivo or ex vivo. Thus, in another aspect, the present disclosure provides The present invention relates to a method for treating tumors comprising administering to a subject a therapeutic agent capable of interacting with or binding to the MHC class I molecule / tumor antigen peptide complex described herein. T cell receptors (TCRs) capable of binding to the TCR molecule, and nucleic acid molecules encoding such TCR molecules, and vectors comprising such nucleic acid molecules. The TCRs according to the present disclosure are preferably loaded onto MHC class I molecules on the surface of living cells in vitro or in vivo; or specifically interact with tumor antigen peptides presented by MHC class I molecules. Nucleic acids encoding TCRs, particularly TCRs of the present disclosure, can bind to, for example, M We isolated a new T lymphocyte clone that specifically recognizes HC class I / tumor antigen peptide complexes. Generates T lymphocytes (e.g., CD8 + T lymphocytes) or other types of lymphocytes In certain embodiments, the method may be applied to genetically transform / modify a patient-derived T lymphocytes (e.g., CD8 + T lymphocytes) express one or more antigens that recognize tumor antigen peptides. The cells are transformed to express a TCR and the transformed cells are administered to the patient (autologous In certain embodiments, T lymphocytes (e.g., CD8 + T lymphocytes) are transformed to express one or more TCRs that recognize tumor antigen peptides. The transformed cells are then administered to the recipient (allogeneic cell transfusion). In one embodiment, the present disclosure provides a method for the production of T lymphocytes, e.g., vectors encoding tumor antigen peptide-specific TCRs. CD8 transformed / transfected by vector or plasmid + T lymphocytes In a further embodiment, the present disclosure provides a method for the preparation of a tumor antigen peptide-specific TCR-transfected cell line. Methods of treating patients with the transformed autologous or allogeneic cells are provided. In this study, tumor antigen-specific TCRs were used in the production of autologous or allogeneic cells for the treatment of cancer. Use is provided.

[0071] In some embodiments, a patient treated with a composition (e.g., a pharmaceutical composition) of the present disclosure is treated with allogeneic stem cell transplantation (ASCL), allogeneic lymphocyte infusion, or autologous lymphocyte infusion. The compositions of the present disclosure react ex vivo with tumor antigen peptides. Activated allogeneic T lymphocytes (e.g., CD8 + T lymphocytes), loaded with tumor antigen peptides Allogeneic or autologous APC vaccines, tumor antigen peptide vaccines, and tumor antigen-specific TCR-transformed allogeneic or autologous T lymphocytes (e.g., CD8 + T lymphocytes) and The present disclosure relates to a T lymphocyte cluster that can recognize tumor antigen peptides. The method for providing a loan involves administering to a subject (e.g., a transplant recipient), e.g., an ASC In T and / or donor lymphocyte infusion (DLI) recipients, tumor antigen peptides The IL-14 receptor may be generated for and specifically target tumor cells that express the IL-14 receptor. Therefore, the present disclosure provides a method for specifically recognizing tumor antigen peptide / MHC class I molecule complexes. CD8, which encodes and expresses a T cell receptor that can recognize or bind to + T lymphocytes The T lymphocytes (e.g., CD8 + T lymphocytes) are recombinant (engineered) or or naturally selected T lymphocytes. This can be done in vivo (i.e., by using APCs loaded with tumor antigen peptides). The study was conducted in patients receiving IFN-γ-γ-α or IFN-γ-γ-α or in patients receiving tumor antigen peptides. The undifferentiated lymphocytes are cultured under conditions favorable for inducing (potentially) T cell activation and proliferation. Tumor antigen peptide / MHC class I molecule complex (typically on the surface of cells such as APCs) The method of the present disclosure, comprising contacting a CD8 + A small number of cells are needed to produce T lymphocytes. The present invention provides at least two methods for combining tumor antigen peptides bound to MHC class I molecules. Using combined or pooled populations capable of recognizing multiple tumor antigen peptides D8 + Alternatively, tumor antigen-specific or targeted T lymphocytes can be generated. T lymphocytes express MHC class I molecule / tumor antigen peptide complexes (i.e., engineered or recombinant CD8 + TCR (more specifically, α chain and By cloning one or more nucleic acids (genes) encoding the β-chains, The tumor antigen peptide-specific T cells of the present disclosure can be produced / generated in vitro or ex vivo. Nucleic acids encoding CRs can be synthesized using methods known in the art to encode tumor antigen peptides. T lymphocytes activated ex vivo against the target (e.g., loaded with tumor antigen peptides) APC), or from an individual that exhibits an immune response to the peptide / MHC molecule complex. The tumor antigen peptide-specific TCR of the present disclosure can bind to host cells and / or graft tissue. recombinantly expressed in host lymphocytes obtained from the recipient or graft donor , which can optionally be differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). Nucleic acids (transgenes) encoding the α and β chains are introduced into the cells by transfection (e.g., electroporation). Any suitable method, such as perforation) or transduction (e.g., using a viral vector) can be used. The antibody may be used to introduce T cells (e.g., from the subject to be treated or from another individual). Engineered CD8 expressing TCR specific for tumor antigen peptides + T lymphocytes are well known They can be grown in vitro using culture methods.

[0072] The present disclosure provides a method for the preparation of tumor antigen peptides (i.e., antigens that bind to MHC class I molecules expressed on the cell surface). Specifically induced by tumor antigen peptides (or combinations of tumor antigen peptides) Isolated CD8 that is induced, activated, and / or expanded (grown) + T. Lin The present disclosure also provides tumor antigen peptides, or combinations thereof, according to the present disclosure. CD8 can recognize the match + T lymphocytes (i.e., cells that bind to MHC class I molecules) and a composition comprising the tumor antigen peptide. In another aspect, the present disclosure provides a method for the treatment of cancer by administering to a patient a therapeutically effective amount of one or more MHC class I molecules / tumor antigens described herein. CD8 specifically recognizes peptide complexes + T lymphocyte-enriched cell populations or cell cultures Nutrients (e.g., CD8 + Such enriched populations are provided by the methods described herein. and MH loaded with (e.g., presenting) one or more of the tumor antigen peptides disclosed herein. Ex vivo transfection of specific T lymphocytes using cells such as APCs expressing C class I molecules As used herein, "enriched" means , tumor antigen-specific CD8 in the population + The proportion of T lymphocytes compared to the natural population of cells , i.e., more significantly than those not subjected to the process of ex vivo expansion of specific T lymphocytes. In a further embodiment, the tumor antigen peptide-specific target CD8 + The proportion of T lymphocytes is at least about 0.5%, for example, at least about 0.6% , 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2% or 3%. In embodiments, tumor antigen peptide-specific CD8 + The proportion of T lymphocytes is approximately 0 0.5 to approximately 10%, approximately 0.5 to approximately 8%, approximately 0.5 to approximately 5%, approximately 0.5 to approximately 4%, approximately 0.5 to Approximately 3%, approximately 1% to approximately 5%, approximately 1% to approximately 4%, approximately 1% to approximately 3%, approximately 2% to approximately 5%, approximately 2% or more about 4%, about 2% to about 3%, about 3% to about 5%, or about 3% to about 4%. CD specifically recognizes the MHC class I molecule / peptide (tumor antigen peptide) complex. Such cell populations or cultures enriched for CD8+ T lymphocytes (e.g., CD8+ T lymphocyte populations) The antibody fragments can be used in tumor antigen-based cancer immunotherapy, as described in more detail below. In some embodiments, tumor antigen peptide-specific CD8 + T lymphocyte populations, e.g. , loaded (covalently or non-covalently) with a tumor antigen peptide as defined herein. The present disclosure provides a method for the preparation of MHC class I molecules for further enrichment using multimers of MHC class I molecules. , e.g., tumor antigen peptide-specific CD8 + The proportion of T lymphocytes is at least about 30% 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, Tumor antigen peptide-specific CD8 of 98%, 99%, or 100% + T lymphocyte essence A prepared or isolated population is provided.

[0073] The present disclosure further provides a method for the preparation of a pharmaceutical composition comprising administering to a subject in need thereof ... a tumor antigen peptide, a nucleic acid, an expression vector, a T cell receptor, a cell (e.g., a T lymphocyte , APC), and / or compositions, or any combination thereof. In embodiments, the medicament is for the treatment of cancer, e.g., a cancer vaccine. The present disclosure also provides a method for the treatment of cancer, for example, as a cancer vaccine, using any of the methods disclosed herein. tumor antigen peptides, nucleic acids, expression vectors, T cell receptors, cells (e.g., T lymphocytes, APC), and / or composition (e.g., vaccine composition), or any combination thereof The tumor antigen peptide sequences identified herein are used in combination with: i) a tumor patient injected with used for in vitro priming and expansion of tumor antigen-specific T cells, and / or ii) vaccines for inducing or enhancing anti-tumor T cell responses in cancer patients. It can be used for the production of synthetic peptides, which are used as cutins.

[0074] In another aspect, the present disclosure provides a method for treating cancer in a subject, comprising administering to a subject a compound of the invention as described herein as a vaccine for treating cancer in a subject. or a combination thereof (e.g., a peptide pool) The present disclosure also provides for the use of a vaccine for treating cancer in a subject. , the tumor antigen peptides described herein, or combinations thereof (e.g., peptides In embodiments, the subject is administered a tumor antigen peptide-specific CD8 + Thus, in another aspect, the present disclosure provides a method for treating cancer. (e.g., reducing the number of tumor cells, killing tumor cells) The method comprises administering an effective amount of one or more MHC class I molecule / tumor antigen peptide complexes (APs). expressing TCRs that recognize (i.e., bind to) T cells expressed on the surface of cells such as C (Do)CD8 + administering (infusing) T lymphocytes to a subject in need thereof In embodiments, the method comprises: + After administration / infusion of T lymphocytes, an effective amount of tumor antigen peptides, or combinations thereof, and / or tumor antigen peptide-loaded M Administering cells (e.g., APCs such as dendritic cells) that express HC class I molecules to the subject. In yet another embodiment, the method further comprises administering a therapeutically effective amount of one or more tumor anti-cancer drugs. The method includes administering dendritic cells loaded with the original peptide to a subject in need thereof. In a further embodiment, the method comprises administering an effective amount of a tumor antigen presented by an MHC class I molecule. Allogeneic or autologous cells expressing recombinant TCRs that bind to the original peptide are used. administering the compound to a patient.

[0075] In another aspect, the present disclosure provides a method for treating cancer in a subject (e.g., by reducing the number of tumor cells, tumor antigen peptides, or combinations thereof, for killing tumor cells. CD8 recognizes one or more MHC class I molecules that present + The use of T lymphocytes is suggested In another aspect, the present disclosure provides a method for treating cancer in a subject (e.g., reducing the number of tumor cells). tumor antigen peptides for the preparation / manufacture of pharmaceuticals for (inducing the proliferation and killing of tumor cells), or a combination thereof. CD8 + In another aspect, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to a subject a therapeutically effective amount of T lymphocytes. for use in (e.g., reducing tumor cell numbers and killing tumor cells) one or more antigen-loaded (presenting) tumor antigen peptides, or a combination thereof CD8 recognizes MHC class I molecules + Provides T lymphocytes (cytotoxic T lymphocytes) In a further embodiment, the use comprises the use of tumor antigen peptide-specific CD8+ T lymphocytes. Then, an effective amount of a tumor antigen peptide (or a combination thereof), and / or a tumor antigen peptide is administered. Cells expressing one or more MHC class I molecules (e.g., IgG) loaded with (presenting) tumor antigen peptides This further includes the use of APCs (e.g., APCs).

[0076] The present disclosure also provides any of the tumor antigen peptides disclosed herein or combinations thereof. Targeting the immune response against tumor cells expressing human class I MHC molecules loaded with IgG The present invention provides a method for producing a tumor antigen peptide or a tumor antigen peptide. Cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with the combination The present disclosure also relates to a method for administering a tumor antigen peptide or a combination thereof to a patient. for generating an immune response against tumor cells expressing selected human class I MHC molecules. , any of the tumor antigen peptides or combinations of tumor antigen peptides disclosed herein The use of cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with β-glucan has also been proposed. Provide.

[0077] In embodiments, the methods or uses described herein may be administered by the patient prior to treatment / use. To determine the HLA class I alleles expressed by the patient and administration or use of tumor antigen peptides that bind to one or more of the ras I alleles For example, if the patient has HLA-A2*01, HLA-B14*01 and If determined to express HLA-C05*01, (i) SEQ ID NOs: 14, 17, 45, 48, 51, 56, 75, 77, 82, 98 and / or 100 (HLA-A2*01 (ii) SEQ ID NO: 53 (binds to HLA-B14*01), and / or or (iii) a tumor antigen peptide of SEQ ID NO: 27 (which binds to HLA-C05*01) Any combination may be administered to or used by a patient.

[0078] In embodiments, the cancer is a solid cancer, preferably ovarian cancer. is an ovarian carcinoma. In embodiments, the ovarian cancer is epithelial carcinoma, serous carcinoma, small cell carcinoma, , primary peritoneal cancer, clear cell carcinoma or adenocarcinoma, endometrial adenocarcinoma, malignant mixed Mullerian tumor , mucinous adenocarcinoma or cystadenocarcinoma, malignant Brenner tumor, transitional cell carcinoma, sex cord stromal tumor, condylar Granular cell tumor, Sertoli-Leydig tumor, germ cell tumor, dysgerminoma, trophoblastic tumor, Immature (solid) teratoma or mature teratoma, yolk sac tumor, squamous cell carcinoma, or secondary In one embodiment, the ovarian cancer is type I ovarian carcinoma. In some embodiments, the ovarian cancer is type II ovarian cancer. In some embodiments, the ovarian cancer is serous carcinoma. In a further embodiment, the serous cancer is high-grade serous carcinoma (HGSC). In some embodiments, the ovarian cancer is 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, or any combination thereof Combinations include chemotherapy (e.g., vinca alkaloids), drugs 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, inhibitors, antimetabolites (e.g., nucleoside analogues), alkylating agents, platinum-based agents, Anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (e.g. For example, all-trans retinoic acid or its derivatives), Galdamin or its derivatives (17-AAG), immune checkpoint inhibitors (e.g., PD-1 / PD-L1 inhibitors) anti-cancer drugs and CTLA-4 inhibitors, B7-1 / B7-2 inhibitors), antibodies, cell-based therapies (e.g. and one or more additional active agents or chemotherapeutic agents, such as CAR T cells, to treat cancer. In embodiments, a tumor antigen peptide according to the present disclosure, Nucleic acids, expression vectors, T cell receptors, cells (e.g., T lymphocytes, APCs), and / or or the composition is administered / used in combination with an immune checkpoint inhibitor. [Example]

[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 of HGSC1-6 and matched normal neighbors of HGSC1-3. The explant tissue was obtained from Tissue Solutions (Glasgow, GB). Tumor tissue (OV606) or ascites fluid (OV633 and OV642) were collected from Prince s Margaret Cancer Registry(Toronto, ON, Ca Snap frozen samples were obtained from the University of California, San Diego, for RNA extraction and storage. RNA sequencing of OvCa48-114 was performed using the MHCI-associated peptides. The data was collected by the National Ce under project PRJNA398141. ter for Biotechnology Information Seque Download it from the nce Read Archive, convert it to a fastq file, The raw MS data for samples in this cohort were processed in the same way as the other samples. ProteomeXchange Consortium via 7635 PRIDE Partners HLA typing of each sample was performed using the default parameters (24). RNA sequencing (RNA-S) using OptiType™ v1.0 eq) data. Sample information is presented in Table 1. [Table 1]

[0082] RNA extraction and sequencing. For HGSC1-6, AllPrep® Using a DNA / RNA / miRNA universal kit (Qiagen), manufacturer Total RNA was isolated according to the recommendations of [1]. For the 100-kDa genomic DNA fragments, total RNA was isolated using TRIzol® (Invitrogen). The RNA from each sample was analyzed using a 2100 Bioanalyzer (registered trademark) (Agilent Genomics) to confirm that the RIN was >6, and then used to RNA-Seq was performed once per replicate using the KAPA Standard mRNA-Seq Kit. A cDNA library was prepared from polyA-rich mRNA using the following method. Further amplification and sequencing were performed using HiSeq® 2000 or Illumina NextSe Used for paired-end RNA-Seq on the q®500, 150 million per sample We got 0-300 million leads.

[0083] Generation of a customized reference database for MS analysis. The customized Global Cancer Database, as previously mentioned (15 and US Provisional Patent No. Application No. 62 / 724,760), "canonical cancer proteome" and "cancer-specific proteome" The roteome was generated by concatenating two modules of the T RNA-Seq reads were adapted using rimmomatic v0.35(25). The nucleotides and low-quality 3' bases were trimmed. To do this, trimmed reads were analyzed using STAR v2.5.1b to identify the reference human genome. The transcript expression was measured using the default parameters. The number of transcripts per million ( Nucleotide variants were quantified using FreeBayes (26). Identify and run agnostic The sample-specific proteomic sequences for each sample were then converted to a single nucleotide polymorphism file format. Insert single base variants (FreeBayes quality >20) into the reference genome. The sample-specific sequences of the expressed proteins (tpm>0 ) were added to the canonical cancer proteome in fasta format.

[0084] FASTX the trimmed R1 reads to generate cancer-specific proteomes -Reverse complementary and trimmed R using Toolkit version 0.0.14 33-nucleotide and 24-nucleotide k-mer databases with 2 reads To exclude sequencing errors and limit the database size, To achieve this, we set a sample-specific threshold for the occurrence of the minimum k-mer, as follows: The following were applied: HGSC1-3: 7, OV642: 8, OV633: 10 for HGSC4 and OV606, 4 for HGSC5, 6 for HGS 5 for C6 and 3 for OvCa48~114. After subtracting the expressed k-mers to obtain cancer-specific k-mers, we used NEKTAR (in-house) Developed software, https: / / github.com / iric-soft / nektar's kmer_assembly tool can be used to create longer sequences (continuous Contigs >34 nucleotides in length were translated in three frames and the amino acid sequence was The resulting sequences of at least eight amino acids in length were considered relevant. These were included in the cancer-specific proteome.

[0085] Isolation of MAPs. Tumor and tissue samples were cut into small pieces (cubes, approximately 3 mm in size) and Ice-cold protein inhibitor cocktail (Sigma, Cat. No. P8340-5ml) 5 ml of PBS was added. First, the Ultra Tu set at a speed of 20,000 rpm A rrax™ T25 homogenizer (IKA-Labortechnik) was used. for 20 seconds, then the Ultra Turrax (commercially available) was set at a speed of 25,000 rpm. (Target) Using a T8 homogenizer (IKA-Labortechnik), 20 seconds, 2 Then, add 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 10,000 g for 30 minutes at 4° C. The supernatant was diluted with 1 mg of W6 / 32 antibody. Transfer the beads to a new tube containing covalently bound Protein A magnetic beads and analyze the MAP as previously described. The MAP extract was then immunoprecipitated using a Speed-Vac. They were dried and kept frozen prior to MS analysis.

[0086] MS analysis. The dried peptide extract was resuspended in 0.2% formic acid. , a homemade C18 analytical column (C18 Jupiter Phenomenex™) A 15cm x 150µm inner diameter tube filled with 0-30% acetonitrile (0.2% A 56-minute gradient from HCl (formic acid) and 600 nL min on the Easy-nLC II system. - 1 The samples were loaded at a flow rate of 1000 sq. m. The samples were analyzed on a Q-Exactive™ HF mass spectrometer (Ther The data were analyzed using a microscope (Fisher Scientific). Each complete MS spectrum obtained was followed by 20 MS / MS spectra, resulting in 30,000 Resolution: 5 x 104 Automatic gain control target of 100ms, injection time of 100ms, and Select the most abundant multiply charged ions for MS / MS sequencing with a collision energy of 100%. For HGSC4-6, each complete MS spectrum was acquired at 60,000 resolution. followed by 20 MS / MS spectra, with a resolution of 30,000 and 2 × 10 4 of with a dynamic gain control target, an injection time of 800 ms, and a collision energy of 25% The most abundant multiply charged ions were selected for MS / MS sequencing.

[0087] Identifying MAPs. PEAKS 8.5 or Peaks X (Bioinformatics) Peptides were identified using the Global Cancer Data Peptide sequences were searched against a database. Peptide identification was based on precursor ions and The tolerances for the S and fragment ions were set to 10 ppm and 0.01 Da, respectively. For the sample from Chuster et al. (13), precursor and fragment ions The tolerances for oxidation (M) and deamidation ( The occurrence of NQ was considered as a post-translational modification. Only 5% of the MAP list was decoy specific. A sample-specific threshold was applied to the PEAKS score to ensure inclusion of Peptides exceeding the values ​​were further filtered according to the following criteria: peptide length 8–1 MHC allele affinity ranking based on single amino acid and NetMHC4.0 predictions nk≦2%(29).

[0088] Identifying and validating TSA candidates. To identify TSA candidates, each MAP and its code The sequence of the nucleotides was compared with the relevant cancer and normal canonical proteomes, or the cancer and normal canonical proteomes, respectively. A database of normal and normal 24-nucleotide k-mers was queried. d) Conventional canonical proteome and conventional 24-nucleotide k-mer data The database was generated using RNA-Seq from purified TECs collected from six human thymuses. The construct was constructed using the reads of q (15 and U.S. Provisional Application No. 62 / 724,760). MA P can be expressed in two cases: i) in the normal canonical proteome of the sample, and in the normal (i.e. That is, if the peptide sequence was not detected in the TEC k-mer, or i) The peptide is absent from both the cancer and normal canonical proteomes, and RNA coding sequences are overexpressed at least 10-fold in cancer cells compared to TECs. If the MAP corresponds to some RNA sequences, the sequence is labeled as a TSA candidate. If so, it is only possible to select a TSA candidate if all sequences are consistent with TSA candidate status. All TSA candidates were considered as candidates for SA. MS / MS spectra of all TSA candidates were manually verified. This eliminated any false identifications. Supported by RNA data that were distinguishable by MS. For TSA candidates with harboring I / L variants, the most expressed variant was the TSA candidate. If so, both mutants were further examined.

[0089] Finally, the mapping reads containing the MAP coding sequence on the reference genome (GRCh38) were Genomic location was determined by mapping using BLAT (UCSC genome browser). The positions were assigned to all MS-validated TSA candidates. TSA candidates were excluded from the reads that matched the MAP code. The sequence contained variants that did not match known germline polymorphisms (reported in dbSNP v149). If the mutation was present, it was classified as an mTSA. Non-mutated candidates were classified as aesthesia-associated TSA candidates, and positive candidates were classified as These were 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. The data can be accessed through the Genotype-Tissue expression (GTEx) portal Downloaded from (phs000424.v7 accessed April 16, 2018) .p2), to assess the expression of the coding sequences of candidate aeTSAs, as previously described ( 15 ). RNA-Seq data were collected from the cervix (n=6), fallopian tube (n=7), and adipose tissue ( n = 49), bladders (n = 12), and kidneys (n = 38) from 50 donors. The accession numbers of the GTEx datasets used in this study are listed in Table 2. Therefore, the number of reads that completely cover the MAP coding sequence is significantly higher than the number of RNA-Seq reads for each tissue. The 24-mers of MAP coding sequences in the database of 24-mers transformed from the The read count was estimated by the minimum occurrence of the r set. The data were normalized to the number of reads (rphm) and then logarithmically transformed (log 10 (rphm+1)), Averaged across all available RNA-Seq experiments for each tissue. 低 Peripheral expression was observed in tissues other than the brain cortex, nerves, and testis at rphm>10. aeTSA candidates without a TSA were considered as genuine aeTSAs. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0091] Expression of the TSA coding region. RNA expression of the TSA coding region is determined by the TSA coding strand. As a count of all reads overlapping the coding region, with parameter orientation = "same" The “qCount” function in the R package “QuasR” (30) was used to map Counts were calculated as 100 million mapped reads per 100 million reads. The aeTSA expression analysis was normalized to the number of reads. Since the t) may affect aeTSA expression, the units where individual aeTSAs are mapped The key areas were specifically analyzed.

[0092] Clinical and genomic data from TCGA. hg38 for HM27 methylation, DNA Processed and normalized using copy number variation, RNA-Seq gene expression, and clinical data The level 3 data were analyzed using the R package TCGAbiolinks (31). Arm-level DNA copies were downloaded from the TCGA open access database. The number of changes was analyzed by the Broad Institute TCGA Center for Genomic Data Analysis (DCDA). i:10.7908 / C1P84B9Q).

[0093] Immune cell score. Immune cell score, which represents the immune cell population, is calculated based on RNA-Seq data. For each tumor, a score was estimated using the The FPKM values ​​were estimated as the average of the log-transformed FPKM values ​​of those marker genes, as shown in Fig. 1.

[0094] Frequency of aeTSA presentations. To estimate the number of aeTSAs presented by individual patients. We performed bioinformatics simulations to estimate the two parameters. First, the likelihood of developing aesthesia was based on the data in the TCGA-OV cohort. The data were based on the proportion of tumors expressing the corresponding RNA containing the germline SNP. For eTSA, the likelihood of expression was calculated as follows: (TCGA expressing eTSA) (proportion of tumors) × (SNP frequency in a given population). SNP frequency is calculated using the Genome Agg Second, the European American collection (n=1,242,890), African Americans (n=416,581), and Chinese ( HLA allele frequencies were obtained from the National Marrow Donor Program (NMDP) for the 2016 NIH ... The patient's HLA genotype was then determined based on reported frequencies in a given population. , simulated with six HLA class I alleles. Six HLA alleles are independent. Because it was assumed that this was a random event, some HLA loci were homozygous in the simulated patients. When both aeTSA and the relevant HLA allele were expressed, TSAs were considered to be presented in simulated patients. The expression of each aeTSA was compared for the same overlapping group. These are independent events, except for duplicate aeTSA, where the expression state was simulated only once. One million simulated patients and their aeTSA presentation status were divided into three groups. and used to plot the distribution.

[0095] Statistical analysis and data visualization. Analyses and figures were performed using R v3.5.1 or Python. The data were analyzed using R v2.7.6. The gplots package in R was used to plot the tumor data. A heat map of TSA coding region expression in tumors was generated. Correlation studies were performed unless otherwise indicated. Unless otherwise specified, the Spearman method was used and the R function "cor.test" was used. Tests involving comparison of the distributions were performed using the ANOVA test, and pairwise comparisons between groups were performed using the Ulcoxon The log-rank P value of the survival analysis was calculated by the rank sum test. Calculated in cases.

[0096] Example 2: Proteogenomic analysis identifies 111 TSAs in 23 HGSCs. To obtain a system-level characterization of the TSA landscape, high-throughput tanks Direct MAP identification was performed using deconvoluted MS (MS / MS) analysis (34-36). The search engine uses a sequence matching algorithm to match each acquired MS / MS spectrum with a peptide sequence. , which depends on a user-defined protein database (37). A peptide is identified by the search engine only if its sequence is included in the reference database. Public reference protein databases such as UniProt can identify All genomes are free of specific mutations, out-of-frame translation events, and non-exon sequences. The search to capture TSAs encoded by the genomic region was performed using tumor-specific This requires the construction of a customized database containing the alternative translation products. Therefore, each analyzed sample was analyzed using a recently described proteogenomic approach (15). We constructed a customized search database from the RNA-Seq reads of This customized database consists of two modules: the canonical proteome ( In-frame translation of exons) and cancer-specific proteomes, which are Figure 1 shows the three-frame translation of the cancer-specific RNA sequences after subtraction of the normal RNA sequences (from the normal RNA sequences). 1) TECs are important for i) their importance in establishing immune tolerance during immature T cell development; role (i.e., neutral water tolerance), and ii) more transcripts than other types of somatic cells. was used as a normal control for two reasons: its remarkable ability to be promiscuous ( 38 ); MAPs from nine primary HGSC samples were obtained by immunoprecipitation of MHC I molecules and then Analysis was performed by liquid chromatography-MS / MS (15, 28). Immunopeptidemic data from an additional cohort of 14 HGSCs reported by r et al. (13) The data also includes the data described herein for samples where matching RNA-Seq and MS data are available. Each of the TSA candidates was reanalyzed by applying a proteogenomic approach. Each was confirmed by manual verification of the spectra and genomic locations.

[0097] Candidate overlapping genomic variants not present in dbSNP likely represent germline polymorphisms. These were classified as mTSA. Eighteen mTSAs were obtained from the samples (Table 3A). Of the 18 mTSAs, 7 result from in-frame exon translation and 4 result from in-frame exon translation. 1 arose from out-of-frame exon translation and 8 from non-coding sequences. Therefore, matched normal tissues are available, and RNA-Seq analysis demonstrates that mTSA variants are reproductively It was confirmed that this was not due to cell lineage polymorphism (Figures 8A-8B). In this case, some mTSAs may correspond to rare polymorphisms not present in dbSNP. It cannot be officially ruled out that the number of mTSAs may be slightly overestimated. The incidence of mTSA was <1 per tumor (18 mTSA / 23 tumors). We conclude that A is rare in HGSC and therefore represents a less attractive target. Furthermore, classical TSA discovery methods rely on in-frame exon translation. Since the focus is strictly on mTSAs arising from It would reveal only 7 of the 111 TSAs. [Table 3]

[0098] For non-mutating TSA candidates, strict criteria are applied to identify pure aeTSAs, i.e., We identified cancer-specific genes whose expression is specific to cancer. Their RNA expression in peripheral tissues was analyzed. 低 Other than tissues (brain, nerves, testes) All candidates whose coding RNAs were expressed in any peripheral tissue (rphm>10) were included. The coding sequence of the aeTSA candidate was found to be a germline single nucleotide polymorphism. If the candidate contains a SNP-containing sequence (as reported in dbSNP), the candidate is and the reference sequence was labeled as a valid aeTSA only if it 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 our knowledge ( Interestingly, 5 of the 93 aeTSAs were expressed in the testis, and Although some cancer bacterial antigens (CGAs) are aesthesia-associated steroidal antisera (aeTSAs), most aesthesia-associated steroidal antisera (aeTSAs) are CGAs. CGA is a canonical expression vector normally expressed only by germ cells. Their abnormal expression in cancer cells is mainly due to epigenetic mechanisms. However, some CGAs are expressed by adult mTECs. (16) and CGAs expressed in mTECs (or other somatic tissues) are expressed as TAAs and and those not expressed by any normal tissue (including mTECs) are bona fide aeTS. is considered as A.

[0099] Each aeTSA was assigned a genomic location. If multiple locations were possible, the matching RNA The highest incidence of lead was selected. The characteristics of all TSAs are listed in Table 3B and Table 3C. The stringent approach is based on the detection of atypical translation (5'UTR, 3'UTR, formally underestimate the total number of aeTSA arising from gene-specific mutations (e.g., intergenic, frameshift). Indeed, the open reading frame used to generate MAP in tumors is It is known that their coding RNAs are expressed in several normal tissues. It is not possible to predict whether the reading frame of a TS can be translated. To avoid false positives in the A list, such aeTSA candidates were excluded. . [Table 4-1] [Table 4-2] 1 The "Translation Event" column summarizes the relationship between the TSA coding sequence and the ORF, and refers to the area outside the ORF as "non-translated." "Code", ORF overlapping but frameshifted is "Code-out", ORF matrix is ​​" The code that was switched on was called "code-in." 2 The "Genome Origin" column further annotates aeTSA according to biotype from Ensembl. For example, "antisense" indicates that the sequence is opposite to the annotated gene. "Canonical" means that the ORF is in the canonical / annotated format. "ncRNA" refers to annotated RNAs that do not contain ORFs according to Ensembl. It is NA. 3 "ncRNA" refers to a transcript whose coding sequence aligns with the exons of a non-coding transcript. Refers to... 4 "Non-coding antisense" refers to a sequence in which the TSA coding sequence is It is meant to be the antisense of the gene and therefore it is a non-coding region. [Table 5]

[0100] Example 3: Most HGSC TSAs are non-mutated MAPs derived from non-canonical translation is. An average of 2200 unique MAPs were identified per sample, resulting in a total of 111 unique The number of TSAs identified per sample correlated with the number of MAPs. Furthermore, the number of MAPs per HLA allele correlated significantly with the tumor sample size (Figure 3B). There was a moderate correlation between tumor sample size and MS analysis (Figure 10). This is consistent with the idea that mutated non-coding sequences are the limiting factor for transcription (28). A TSA derived from a sequence can be designated as both an mTSA or an aeTSA. We decided to label them as mTSAs accordingly. The rationale is that they are of genomic origin ( mTSA, whether exonic or not, is a "private TSA" that is, they would not be expected to be shared by multiple tumors. Mutant aeTSA could theoretically be shared by a significant proportion of HGSC.

[0101] Of note, the first treatment in this study or the one described by Schuster et al. The TSA signatures identified in the samples treated with HCl were remarkably similar (Fig. 3C). This is because the proteogenomic approach described here is generally similar to RNA-Seq and This suggests that it can be applied to MS data and is superficially immune to inter-laboratory variability. In both cohorts, approximately 83% of TSAs were unmutated, suggesting that the majority of TSAs The fragments arose from atypical translation: mainly from non-coding regions and, to a lesser extent, from fragments Two features of aeTSA are noteworthy: i) 80% come from non-coding sequences, especially introns (31%) and intergenic sequences (22%) ii) 90% are novel MAPs (Fig. 3D). The corresponding protein isoforms are listed in the UniProt database (13, 39-43 and and U.S. Patent Publication No. 2012 / 0077696A1). except those that match biotypes annotated by the nsembl database). Derived from in-frame exon translation.

[0102] Example 4: Expression of aeTSA-encoding transcripts in ovarian cancer samples. Does cancer-specific expression of aeTSA-encoding transcripts arise from random transcriptional noise? To determine whether this is due to a recurrent transcriptional abnormality, we investigated the effects of this gene on the TCGA egg. Genomic regions encoding 93 aeTSAs identified in samples from the ductal cancer cohort RNA expression was analyzed. The region encoding aeTSA was found to be involved in a significant proportion of ovarian cancers. Seventy-two (77%) were expressed in at least 10% of the samples, and Sixteen (17%) were expressed in 80% of patients (Figure 4). Therefore, the 93 ae in HGSC are likely to generate shared TSAs. The expression of this set of TSA-encoding transcripts is not a rare or random event, but rather It can be concluded that this is a common feature of HGSC.

[0103] Example 5: Genomic correlation of aeTSA expression. To understand the mechanism of aeTSA expression, we investigated the mechanisms of aeTSA expression in mice from the TCGA-OV dataset. Using the chromosomal data, we correlated aeTSA RNA expression with local genes or epigenetic We explored the relationship between genetic abnormalities, regional DNA copy number alterations, if applicable, DNA methylation levels on gene promoter regions and RNA for each aeTSA The correlation between expression and genomic regions (exons, introns) that are part of genes was examined (Figure 5A). When aeTSA derived from a transgene (e.g., a transgene or UTR) was used, the expression of the relevant gene was significantly improved. The correlation between the expression of aeTSA and the genes involved in aeTSA expression was also analyzed. A significant correlation was observed between the aeT gene and the aeT gene (Fig. 5A). For SA, the regulation of aeTSA expression generally affects the entire gene. Furthermore, changes in DNA copy number were positively correlated with the RNA expression level of aeTSA. This was due to the presence of intragenic and extragenic aeTSA (antisense and genomic). This suggests that changes in DNA copy number have a substantial effect on aeTSA expression. Notably, this correlation was observed in a larger proportion of tumors. The staining of the aeTSA coding region was particularly strong for the intragenic aeTSA (Fig. 11A). Examination of chromatid distribution revealed that some chromosome arms frequently amplified in HGSC were associated with many ae For example, the TSA gene, which is commonly amplified in ovarian cancer, was found to produce TSA (Figure 5B). The long arm of chromosome 3 (44) was the source of eight aeTSAs. One, MECOM, located at 3q26.2(44), contains three duplicated exons. However, amplification of the chromosome arm did not necessarily result in the generation of an out-of-frame aeTSA (Table 3B). Neither was necessary (e.g., 15q) nor sufficient to generate aeTSA (e.g., For example, 8q) (Figure 5B, Figure 11B).

[0104] Due to the technology used by TCGA to analyze DNA methylation (HM27 array), The promoters of exogenous aeTSA and some aeTSA source genes were No promoter methylation data were available. Therefore, promoter methylation analysis was performed on 17 However, for six aeTSAs, DNA A significant correlation was found between thrombosis and aeTSA expression (Figure 5A). In most cases, the results were negative, and in one case the results were positive. This suggests that promoter demethylation frequently enhances transcription. In particular, the two genes with the highest negative correlations are MAGEC1 (ρ = −0.53, P adj =1.6x10 -26 ) and MAGEA4( ρ=-0.51, P adj =6.7x10 -25 ), which are indicated by arrows in Figure 5A. The MAGE family of genes is expressed in several cancer types, including HGSC. aeTSA is a CGA that is overexpressed in the thyroid gland type (3). Overall, aeTSA expression is at least Regulated at the transcriptional level, in part, by variations in gene copy number and DNA methylation It can be concluded that

[0105] Example 6: Expression of three aeTSAs correlates with improved survival. Next, we evaluated whether some aeTSAs could induce spontaneous protective immune responses. Expression of aeTSA at the thymocyte level, in addition to expression of aeTSA RNA, is associated with HL Addressing this issue is complicated by the fact that it requires the presence of the A allotype. Therefore, patients from the TCGA cohort were selected based on their individual aeTSA RNA expression. Based on (or not based on) the presence of relevant HLA allotypes The three aeTSA presentations were more favorable. The HLA allele polymorphisms correlated with the clinical outcomes (Fig. 6A-6C). This significantly reduced the statistical power of this analysis. The log-rank p values ​​for SA ranged from 0.013 to 0.076 (Figure 6A-B). 6C). Nevertheless, two observations suggest that these correlations are biologically meaningful. First, the "protective effect" of these aeTSAs is unclear. In patients expressing aeTSA RNA, the associated H Survival was superior when the LA allele was also expressed. Expression of aeTSA and its associated HLA allotypes is associated with T cell and cytotoxic T cell proliferation. The results showed a positive correlation with tumor invasion by vesicles (Figures 6D and 6E; ANOVA, p<0.05). .

[0106] Example 7: Median number of aeTSA presented by individual tumors Using the list of 93 aeTSAs, we will ultimately determine how this study will impact TSA-targeted immunotherapy. Therefore, the estimated benefit is 93% of the 1 million patients. The presentation status of each aeTSA was randomly simulated. To determine this, we used the three largest datasets from US bone marrow banks: European Americans; Humans, African Americans, and Chinese (45) were used. Alleles in a given population Frequency, percentage of expression in TCGA-OV tumors, and SNP frequency, if applicable, were used. Six HLA alleles and aeTSA expression statuses were independently generated for each tumor. The number of aeTSAs per IgG was calculated as the sum of the expressed HLA-aeTSA pairs. Based on these simulations, 98% of European Caucasians and 98% of African Americans determined that at least one aesa could be found in 74% of Japanese and 78% of Chinese. However, the median number of aesthesia-associated steroids (aeTSA) per tumor was 5 in European Caucasians and 4 in African Americans. The difference between these populations was due to HLA allele inheritance. due to the varying frequencies of tumors and the fact that tumor samples were primarily from Caucasians of European descent. These calculations underestimate the number of aeTSAs per tumor for three main reasons. First, more than 50% of MAPs bind to two or more HLA allotypes. The fact that, in many cases, binding occurs across supertypes or loci ( 46 ). Second, the genomic region encoding a given MAP is This is because they frequently generate overlapping MAPs presented by different HLA allotypes (23). Third, we investigated five aeTSAs containing nonsynonymous SNPs listed in dbSNP. In this case, only the SNP variants that produce MAP in the sample are effective, and other SNP variants are not effective. We hypothesized that a single amino acid change would not produce AP. This cautious strategy was adopted because the current 93 a The vaccine containing the eTSA suite is available for nearly all Caucasians and African Americans with HGSC. It was concluded that the study covered a significant proportion of African Americans and Asians (e.g., Chinese). It can be done.

[0107] While the present technology has been described above with reference to specific embodiments thereof, the appended claims and the scope of the present invention. In the claims, the word "comprising" means "including but not limited to" , including, but not limited to It is used as an open-ended term that is substantially equivalent to the expression " The singular forms "a," "an," and "the" are used unless the context clearly indicates otherwise. Unless otherwise indicated, corresponding plural references are included.

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Claims

1. A tumor antigen peptide of 15 amino acids or less, comprising the amino acid sequence set forth in SEQ ID NO:

103.

2. The tumor antigen peptide of claim 1, consisting of the amino acid sequence set forth in SEQ ID NO:

103.

3. A nucleic acid encoding the tumor antigen peptide of claim 1.

4. The nucleic acid of claim 3, which is mRNA.

5. The nucleic acid described in claim 3, present in a viral vector.

6. A composition comprising the tumor antigen peptide of claim 1 or 2, or the nucleic acid of any one of claims 3 to 5, and a pharmaceutically acceptable carrier.

7. A vaccine comprising the tumor antigen peptide of claim 1 or 2, or the nucleic acid of any one of claims 3 to 5, and an adjuvant.

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

9. An isolated CD8 expressing the TCR of claim 8 on its cell surface. ＋ T lymphocytes.

10. at least 0.5% of CD8 as defined in claim 9 ＋ A cell population that includes T lymphocytes.

11. (i) the tumor antigen peptide according to claim 1 or 2; (ii) (iii) the composition according to claim 6; (iv) the vaccine according to claim 7; (v) the TCR according to claim 8; (vi) the CD8 according to claim 9. ＋ 11. Use of T lymphocytes, or (vii) the cell population of claim 10, for the manufacture of a medicament for treating ovarian cancer in a subject.

12. The use of claim 11, wherein the ovarian cancer is serous carcinoma.

13. The use according to claim 12, wherein the serous cancer is high-grade serous carcinoma (HGSC).

14. The use of claim 11, wherein the pharmaceutical agent is for use in combination with at least one additional anti-tumor agent or therapy.

15. 15. The use of claim 14, wherein the at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy or surgery.

16. (i) a tumor antigen peptide according to claim 1 or 2; (ii) for use in treating ovarian cancer in a subject. (iii) the composition according to claim 6; (iv) the vaccine according to claim 7; (v) the TCR according to claim 8; (vi) the CD8 according to claim 9. ＋ or (vii) a cell population according to claim 10.

17. The tumor antigen peptide, nucleic acid, composition, vaccine, TCR, CD8 for use according to claim 16, wherein the ovarian cancer is serous carcinoma. ＋ T lymphocytes, or cell populations.

18. The tumor antigen peptide, nucleic acid, composition, vaccine, TCR, CD8 for use according to claim 17, wherein the serous cancer is high-grade serous carcinoma (HGSC). ＋ T lymphocytes, or cell populations.

19. The tumor antigen peptide, nucleic acid, composition, vaccine, TCR, CD8 ＋ 17. The tumor antigen peptide, nucleic acid, composition, vaccine, TCR, CD8 for use according to claim 16, wherein the T lymphocyte or cell population is for use in combination with at least one additional anti-tumor agent or therapy. ＋ T lymphocytes, or cell populations.

20. 20. The tumor antigen peptide, nucleic acid, composition, vaccine, TCR, CD8 for use according to claim 19, wherein the at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, immunotherapy, immune checkpoint inhibitor, radiation therapy or surgery. ＋ T lymphocytes, or cell populations.

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