Novel tumor-specific antigens for ovarian cancer and uses thereof

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 filed on June 25, 2019, which is incorporated herein by reference. 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 an ovarian cancer-specific tumor antigen that is 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 more than 1,000 deaths in 2017. These 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. Moreover, 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 + TILs are It has been negatively associated with malignant cell diversity (5). Considering the therapeutic efficacy of immune checkpoint inhibitors in the type, Clinical trials with 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 immune responses to immunization (3, 8). (± immune checkpoint inhibitors) or as T cell receptor-based approaches ( These could be used as targets for therapeutic approaches (cell therapy, bispecific biologics) (9).

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

[0006] This disclosure provides the following items 1 to 61. 1. A tumor antigen peptide comprising one of the amino acid sequences set forth in SEQ ID NOs: 1 to 103. 2. The tumor according to item 1, comprising one of the amino acid sequences set forth in SEQ ID NOs: 19 to 103. Tumor antigen peptides. 3. The tumor antigen peptide binds to the HLA-A*01:01 molecule and is selected from SEQ ID NOs: 21, 28 40, 41, 66 or 88. Tumor antigen peptides. 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 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence according to 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. A tumor antigen peptide described in 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 according to item 43. 7. The tumor antigen peptide binds to the 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 the 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 described in item 1 or 2. 11. The tumor antigen peptide binds to the HLA-B*08:01 molecule and has SEQ ID NOs: 23, 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 an 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 of the amino acid sequence according to item 1 or 2. Chid. 16. The tumor antigen peptide binds to the HLA-B*40:01 molecule and is represented by SEQ ID NO: 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 the 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 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence according to item 94. 19. The tumor antigen peptide binds to the HLA-C*03:03 molecule and is selected from 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 according to item 72. 23. The tumor antigen peptide binds to the HLA-C*07:01 molecule and is selected from SEQ ID NOs: 38, 6 3. The tumor antigen peptide according to item 1 or 2, comprising the amino acid sequence according to item 1 or 93. 24. The tumor antigen peptide binds to the 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 represented by SEQ ID NO: 25, 5 7 or 79. The tumor antigen peptide according to item 1 or 2, comprising an amino acid sequence according to item 7 or 79. 27. A gene encoding a nucleic acid sequence located in a non-protein-coding region of the genome, comprising: , 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 a 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: 28. The method according to claim 27, comprising the amino acid sequence of any one of sequence numbers 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 NO: 16, 17, and 36 to 64, preferably any one of SEQ ID NO: 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 27. The method according to claim 27, further comprising the step of: The described tumor antigen peptide. 31. The non-protein coding region of the genome is an endogenous non-coding RNA transcript (ncRNA). 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 an 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. 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 the tumor antigen peptide according to item 33 or 35. A liposome comprising the nucleic acid according to claim 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. or the liposome according to item 33, and a pharma- ceutically acceptable carrier. , 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 claim 34, the liposome according to claim 35, or the composition according to claim 36, Vaccines, including Vaccin. 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 42. A tumor antigen peptide according to any one of items 1 to 32 in its peptide-binding groove. A single molecule that 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% of 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 to a subject 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 claims 33 to 34; (iii) the liposome according to item 35, (iv) the composition according to item 36, (v) (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 + or (viii) 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, an immunotherapy 52. The method according to 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, or (iv) the nucleic acid according to item 36. (v) the composition 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) a tumor antigen peptide according to item 33 or 34, (iii) the liposome according to item 35, or (iv) the nucleic acid according to item 36. (v) the composition 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 pharmaceutical 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 to 56, further comprising the use of at least one additional antitumor agent or therapy. The use according to any one of claims 1 to 5. 58. The at least one additional anti-tumor agent or therapy is a chemotherapeutic agent, an immunotherapy, an immunotherapy 58. The use according to item 57, wherein the treatment is a checkpoint inhibitor, radiation therapy or surgery. 59. (i) any one 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, (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; 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 lymphocyte, or cell population. 62. Tumor antigen peptide, nucleic acid, liposome, composition, vaccine, cell, CD8 + T.Rin The cell or cell population is combined with at least one additional anti-tumor agent or therapy. A tumor antigen peptide, a nucleic acid, or a 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, an immunotherapy 63. The method according to claim 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 population.

[0007] Further objects, advantages and features of the present invention are given by way of example only with reference to the accompanying drawings in which: These and other aspects will become apparent from a reading of the following non-limiting description of specific embodiments. [Brief description of the drawings]

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

[0009] The terms and symbols of genetics, molecular biology, biochemistry, and nucleic acids used herein are , standard articles 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 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 than one (i.e., at least one) of the grammatical objects of the article. As an example, "an element" is used herein to refer to at least one The term "ent" means one element or more than one element. Unless otherwise required by the context, the words "comprise" and "include" are used interchangeably. The terms "comprising" and "comprising" mean the steps or denotes the inclusion of an element, or a group of steps or elements, but does not include any other steps or elements. It will be understood that no exclusion of any group of steps or elements is implied by the inclusion of any of the following: .

[0011] The recitation of ranges of values ​​herein includes all values ​​within that range unless otherwise indicated herein. is intended to serve as a closed expression referring individually to each separate value that falls within Each separate value is incorporated herein as if it were individually recited herein. Also, every subset of values ​​within a range is included herein as if each was individually recited. No. 6,399,433, which is incorporated herein by reference in its entirety.

[0012] All methods described herein are intended to be illustrative, unless otherwise indicated herein or in the 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 indicative of

[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. or range of values).

[0016] In the study described herein, the inventors used a proteogenomics-based approach Using this method, 111 TSA candidates (103 novel candidates and The majority of these TSAs (93) were normal. 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) mainly consists of non-exon sequences, especially introns. The expression of thrombin-dependent markers 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 novel TSA candidate that inhibits T cell proliferation in ovarian cancer. These may be useful for immunotherapy based on

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

[0018] In an embodiment, the present disclosure relates to SEQ ID NOs: 1, 8, 10, 13, 15, and 19-27, preferably Preferably, the antibody 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 an embodiment, the tumor antigen peptide is are SEQ ID NOs: 1, 10, 13, 15, and 19 to 23, preferably SEQ ID NOs: 19 to 23 The amino acid sequence of the 5'-UTR region of the 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.

[0019] In an embodiment, the present disclosure relates to SEQ ID NOs: 16, 17, and 36 to 64, preferably SEQ ID NOs: In an intron, the amino acid sequence of the present invention is selected from the group consisting of 36 to 64 amino acids. The present invention relates to a tumor antigen peptide encoded by a sequence located within the

[0020] In embodiments, the present disclosure provides a method for the preparation of a nucleic acid sequence 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 relates to SEQ ID NOs: 4, and 85 to 92, preferably SEQ ID NO: 8 A non-coding RNA sequence that contains or consists of one of the following amino acid sequences: 5 to 92 amino acids. Tumor anti-cancer drugs encoded by 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 sequence comprising the steps of: or consisting of a tumor-specific antitumor agent encoded by a sequence that is an antisense sequence of a gene. Concerning the original peptide.

[0024] In another embodiment, the present disclosure provides a method for the preparation of a medicament comprising administering to a subject a medicament the invention, ... A tumor antigen peptide comprising or consisting of a sequence from a mucin gene. Concerning Petite.

[0025] In general, peptides such as tumor antigen peptides that are presented in the context of HLA class I are long 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 peptide sequence as defined herein. Longer peptides are artificially loaded into cells, such as antigen-presenting cells (APCs), and are then delivered to 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 ) can be loaded into APCs for presentation. The present invention relates to a method for treating tumors comprising administering to a patient a tumor antigen peptide comprising administering to said ... In some embodiments, the tumor antigen peptides defined herein are processed by The precursor peptide / polypeptide used to generate , 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 “top-down” after treatment with cells (APCs). The use of 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 to HLA class I molecules. In an embodiment, the tumor antigen peptide is small, having 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. A peptide is 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 the naturally occurring amino acids. To prepare both dimers and D-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 Natural amino acids include those that are not encoded by a nucleic acid sequence. 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. Examples of amino acids include β-alanine, 3-aminopropionic acid, 2,3-diamino Nopropionic acid, α-aminoisobutyric acid (Aib), 4-aminobutyric acid, N-methylglycine sarcosine, hydroxyproline, ornithine (e.g., L-ornithine), cytosine leucine, t-butylalanine, t-butylglycine, N-methylisoleucine, phenyl Glycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-naphthyl 3-benzothienylalanine, 4-chlorophenylalanine, pyridylalanine, 3-benzothienylalanine, 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 Nobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine Stains, homoserine (HoSer), cysteic acid, ε-aminohexanoic acid, δ-amino These 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 altered 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 (similar physicochemical properties) leads to alteration The substitution of an amino acid within a sequence can be performed by changing the amino acid to which it belongs. For example, positively charged (basic) amino acids and other members of the classes Examples 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 are serine, threonine, cysteine, and tyrosine. Negatively charged (acidic) amino acids include selenium, 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 amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions. The tumor antigen peptides described herein may be composed of all L-amino acids, all D-amino acids, or or a mixture of L- and D-amino acids. The tumor antigen peptides described herein contain all L-amino acids.

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

[0029] The tumor antigen peptide may also be modified to prevent degradation and increase stability, affinity and / or uptake. The N-terminus and / or C-terminus 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 SEQ ID NOs: 1 to 103, preferably SEQ ID NOs: 19 to 1 03 is a tumor antigen peptide that includes or consists of one of the amino acid sequences of (Table 3A, Table 3B).

[0030] In embodiments, the amino terminal residue of the tumor antigen peptide (i.e., the free amino group at the N-terminus) ) can be, for example, a 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, propionyl, butanyl, isopropionyl, or iso-butanyl), or aroyl In embodiments, the aryl group is an acyl group. is C1~C 16 Or C3~C 16 Acyl groups (linear or branched, saturated or unsaturated) In further embodiments, a saturated C1-C6 acyl group (linear or branched) or an unsaturated and C3-C6 acyl groups (straight-chain 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 (converted to an NH2 group), for example. This is because the OH groups of the hydroxyl ... 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, iso-butylamine, isobutylamine, Aliphatic amines of 1 to 10 carbons, such as sovalerylamine or cyclohexylamine , aniline, naphthylamine, benzylamine, cinnamylamine, or phenylethylamine Aromatic or arylalkylamines such as arylamine, 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, and more preferably SEQ ID NOs: 1 to 103. Preferably, the amino acid sequence is one 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 steps of: HLA-A, comprising or consisting of * 01:01 Tumor antigen peptide that binds to the molecule The present invention provides a method for the treatment of ovarian tumors comprising administering to the patient a therapeutically effective amount of a peptide (or tumor-specific peptide), preferably an ovarian tumor antigen peptide.

[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 a tumor-specific peptide), preferably an ovarian tumor antigen peptide, 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 the patient a therapeutically effective amount of a cytotoxic T cell, ...

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

[0035] In another embodiment, the disclosure provides a method for the preparation of a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO: 33 or 43. HLA-A * 24:02 A tumor antigen peptide (or tumor-specific peptide) that binds to a molecule tides), preferably ovarian tumor antigen peptides.

[0036] In another aspect, the present disclosure relates to 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, ovarian tumor antigen peptides are provided.

[0037] In another embodiment, the disclosure provides a method for the preparation of a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO: 34 or 58. HLA-A * 29:02 A tumor antigen peptide (or tumor-specific peptide) that binds to a molecule tides), preferably ovarian tumor antigen peptides.

[0038] In another aspect, the present disclosure relates to 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, ovarian tumor antigen peptides are provided.

[0039] In another aspect, the 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 Tumor antigen peptide (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 relates to an HLA antibody comprising or consisting of the sequence of SEQ ID NO:53. -B * 14:01 A tumor antigen peptide (or tumor-specific peptide) that binds to a molecule. Preferably, ovarian tumor antigen peptides are provided.

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

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

[0044] In another embodiment, 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 A tumor antigen peptide (or tumor The present invention provides a method for the preparation of a medicament for the treatment of cancer, comprising administering to a patient a medicament for the treatment of cancer, the method ...

[0045] In another embodiment, 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. Consisting of HLA-B * 40:01 binds to tumor antigen peptides (or tumor-specific The present invention provides a method for the preparation of a medicament for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of a medicament for the treatment of cancer, the method ...

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

[0047] In another embodiment, the disclosure provides a method for the preparation of a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO: 37 or 94. HLA-B * 44:03 A tumor antigen peptide (or tumor-specific peptide) that binds to the molecule tides), preferably ovarian tumor antigen peptides.

[0048] In another aspect, the present disclosure relates to a nucleic acid sequence similar to SEQ ID NO: 10, 29, 71 or 95, preferably SEQ ID NO: HLA-C comprising or consisting of sequences 29, 71 or 95 *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 embodiment, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising or consisting of the sequence of SEQ ID NO: 6 or 15. , HLA-C * 04:01 Tumor antigen peptide (or tumor-specific peptide) that binds to a molecule ), preferably an ovarian tumor antigen peptide.

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

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

[0052] In another embodiment, the disclosure provides a method for the preparation of a nucleic acid sequence comprising or including the sequence of SEQ ID NO: 38, 61 or 93. Consisting of HLA-C * 07:01 Tumor antigen peptide (or tumor specific peptide) that binds to a molecule The present invention provides a method for the preparation of a medicament for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of a medicament for the treatment of cancer, the method ...

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

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

[0055] In another embodiment, the disclosure provides a method for the preparation of a nucleic acid sequence comprising or including the sequence of SEQ ID NO: 25, 57 or 79. Consisting of HLA-C * 14:02 Tumor antigen peptides (or tumor-specific peptides) that bind to the molecule The present invention provides a method for the preparation of a medicament for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of a medicament for the treatment of cancer, the method ...

[0056] In embodiments, the tumor antigen peptide is a non-translated transcribed region (UTR), i.e., a 3′-U In another embodiment, the 5'-UTR region 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 administered in a host cell comprising a nucleic acid encoding the tumor antigen peptide. 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 Eppard, IRL, Oxford University Press, 19 Alternatively, the tumor antigen peptides can be prepared as described, for example, 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 Another method useful for synthesizing tumor antigen peptides is described in 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, the peptide comprising consisting of or consisting essentially of an amino acid sequence as defined herein and being pharma- ceutically acceptable. The present disclosure relates to synthetically produced (e.g., synthesized) peptides as salts. Salts of tumor antigen peptides are not salts, and therefore in vivo production of the peptides is not possible. The non-natural salt forms of peptides are very different from the peptides in their natural state. In the context of compositions, such as peptide vaccines, disclosed herein, the solubility of the peptides Preferably, the salt is a pharma- ceutically 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 which naturally accompany it. Typically The compound should be at least 60% by weight of the total material in the sample, more typically 75% % or 85% by weight, preferably more than 90% by weight, more preferably more than 95% by weight. Therefore, it is not acceptable for the product to be chemically synthesized, for example, by recombinant techniques, or to be substantially pure. 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 is derived. Not immediately contiguous with coding sequences that are normally contiguous in naturally occurring genomes A substantially pure compound is one in which the aryl, aryl, and aryl groups are not covalently bonded to one another. A substantially pure compound is, for example, For example, by extraction from a natural source and expression of a recombinant nucleic acid molecule encoding a peptide compound. The purity can be determined by column chromatography or by chemical synthesis. The amount of the ion exchange reaction can be measured using any suitable method, such as by 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., lyophilized.

[0059] In another aspect, the present disclosure provides a method for the preparation of a tumor antigen peptide or tumor antigen precursor peptide described herein. Further provided herein is an isolated nucleic acid encoding a peptide. In an embodiment, 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 a molecule's natural environment or naturally occurring sources. Peptides separated from the source macromolecules (e.g., other nucleic acids, proteins, lipids, sugars, etc.) "Synthetic" as used herein refers to a peptide or nucleic acid molecule that is produced by, e.g., recombinant techniques. A peptide that has not been isolated from its natural source, produced through artifical techniques or using chemical synthesis. The nucleic acids of the present disclosure refer to peptides or nucleic acid molecules that are capable of 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 or plasmid may be included in the present disclosure. The cloning, expression, or viral vectors containing nucleic acid sequences encoding the tumor antigen peptides of the present disclosure. Alternatively, the tumor antigen peptide of the present disclosure may be encoded by a vector or plasmid. 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 The term host cell refers to the progeny or potential progeny of a host cell that is capable of expressing the tumor antigen peptides described herein. 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 the transcription and translation of the inserted coding sequence and , cloning sites, and other elements. A sequence encoding a peptide or polypeptide and an appropriate In one embodiment, an expression vector containing the appropriate 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 Ausubel 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 capable of expressing 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 a coding nucleic acid. Thus, the term regulatory region refers to the promoter sequence, regulatory protein binding site, upstream activation site, and In an embodiment, the nucleic acid (DNA) encoding the tumor antigen peptide of the present disclosure includes a nucleic acid sequence. A, RNA) may be contained within a liposome or any other suitable vehicle or 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 HLA-A*01:01 molecule. In one embodiment, the MHC class I molecule is an HLA-A2 molecule, and in a further embodiment, 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 HLA-A*25:01. 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. In a further embodiment, the HLA-B*07:02 molecule. In another embodiment, the MHC class I molecule is an HLA-B08 molecule, and In one embodiment, the HLA-B*08:01 molecule. The gene 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 yet another 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 In one embodiment, the HLA-C*03:03 molecule. In another embodiment, the 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 class I molecule is an HLA-C06 molecule, and in a further embodiment, the class I molecule is 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, the HLA-C*12:03 molecule. The gene is an HLA-C14 molecule, and in a further embodiment, an HLA-C*14:02 molecule. It is.

[0061] In embodiments, the tumor antigen peptide is non-covalently bound to the MHC class I molecule (i.e. 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 a construct, 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 fused to an MHC class I molecule (α chain). Nucleic acids encoding fusion proteins comprising tumor antigen peptides as 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 the preparation of a tumor antigen peptide as described herein (covalently linked). The present invention provides multimers of MHC class I molecules that are covalently or non-covalently loaded. 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. (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 the preparation of a method for the preparation of a tumor antigen peptide specific for the tumor antigen peptide 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 30-well platelet-transferase (30-well platelet-transferase) containing ... contacting the MHC class I molecule with a multimer and binding the MHC class I multimer to the CD8 + and detecting or isolating T lymphocytes. CD8 bound by rasI multimers + T lymphocytes may be isolated by known methods, e.g., fluorescent activity assay. Cells were isolated using fluorescent cell sorting (FACS) or magnetic activated cell sorting (MACS). This is also fine.

[0062] In yet another aspect, the present disclosure provides a method for the preparation of a nucleic acid, vector or plasmid of the present disclosure described herein. The peptides include a nucleic acid or vector encoding one or more tumor antigen peptides. A cell (e.g., a host cell), in embodiments an isolated cell, is provided. The disclosure also provides an MHC class I antigen binding or presenting tumor antigen peptide according to the disclosure. A cell 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 the 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 lipofectamine. injection, electroporation, microinjection and viral-mediated transfection Transforming a host cell refers to any technique for introducing foreign nucleic acid into a host cell, including recombinant gene transfer. Alternatively, suitable methods for transfection can be found, 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 vectors or vectors of the present disclosure for gene therapy. can be used to deliver the plasmid to a subject.

[0063] Using a variety of methods known in the art, cells, such as APCs, can be transfected with one or more 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 It means that the cell is transformed with a nucleic acid encoding an 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 cell proliferation and proliferation. The tumor antigen peptide may also be provided with 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 relates to a tumor antigen peptide as defined herein (or The present invention relates to a composition or a method for the preparation of a nucleic acid encoding a nucleic acid comprising the steps of: In an embodiment, the composition is 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 peptide encoding the tumor antigen peptide Any combination of tumor antigen peptides as defined herein. Compositions that include the combination / subcombination are encompassed by the present disclosure. The 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 one of the selected alleles of an MHC class I molecule. 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 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 incubated with tumor antigens. The APC can be obtained by contacting (stimulating) the APC with a peptide. 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 APC" and "stimulating APC" refer to the induction of tumor antigen receptors. The cells are then cultured in one or more tumor-associated media such 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 used to prepare tumor antigen-containing tumor cells. The peptide may be, for example, a longer peptide that includes the sequence of a tumor antigen peptide (including a naturally occurring protein). The peptide / polypeptide may then be indirectly loaded with the tumor antigen peptide / polypeptide. are processed internally within the APC to generate MHC class I complexes at the cell surface (e.g. The APC is loaded with tumor antigen peptides, and the APC then binds 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 can include the following steps: (a) obtaining APCs from a first subject; and (b) contacting / loading the APCs of step (a) with a tumor antigen peptide to form a surface of the APCs. and (c) forming an MHC class I / tumor antigen peptide complex in the tumor. 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., an allogeneic vaccine). The present invention relates to 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. Thus, 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 a tumor antigen peptide, or a combination thereof, to a patient. The tumor anti-tumor agent as defined herein may be mixed or formulated with a pharmaceutical acceptable carrier. MHC class I loaded with any one of the original peptides or any combination 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 are CD8 + T lymphocytes, e.g., autologous CD8 + Stimulates / expands T lymphocytes Thus, in another aspect, the present disclosure provides a method for the preparation of a method for the preparation of a compound according to the present invention, Any of the tumor antigen peptides (or nucleic acids or vectors encoding same) MHC class I molecules and T lymphocytes, more specifically CD8 + Cells expressing T lymphocytes (e.g., CD8 + A population of cells that contains T lymphocytes (a) a composition comprising:

[0067] In embodiments, the composition may comprise a buffer, excipient, carrier, diluent, and / or medium (e.g., In further embodiments, the composition further comprises a buffer, excipient, carrier, diluent, or and / or the medium may contain pharma- ceutically acceptable buffers, excipients, carriers, diluents and / or As used herein, "a medicament containing pharma- ceutically acceptable buffers, excipients, The carrier, diluent and / or medium are physiologically compatible and are capable of promoting the biological activity of the active ingredient. Any and all solvents, buffers, binders, etc. 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 pharma- ceutical 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 Armaceutical 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, the buffer, excipient, carrier, and / or A medium is a non-naturally occurring buffer, excipient, carrier, and / or medium. In one embodiment, 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 relates to a tumor antigen peptide as defined herein (or any one or any combination of the nucleic acids encoding the , an excipient, carrier, diluent and / or medium. For compositions that include cells (e.g., APCs, T lymphocytes), the compositions may be used to maintain viable cells. Representative examples of such media include physiological saline, Earl's Balanced Salt Solution (Life Technologies®), PlasmaLyte® (registered trademark) (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 is present therein when administered to a subject. A composition capable of inducing an immune response against one or more tumor antigen peptides Vaccination methods for inducing an immune response in mammals include the use of vaccine formulations. By any conventional route known in the art, for example, mucous membranes (e.g., ophthalmic, intranasal, pulmonary, Orally, through the gastric, intestinal, rectal, vaginal, or urinary surfaces; parenterally (e.g., subcutaneously, intradermally, intramuscularly); via routes of administration (intramuscular, intravenous, or intraperitoneal) or via topical administration (e.g., via a patch or similar) The use of a vaccine or vaccine vector administered by a dermal delivery system 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 The present invention relates to a method for producing a tumor antigen peptide comprising administering to a subject the tumor antigen peptide or a combination thereof, and a nucleic acid encoding the 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. ll-like receptor (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. An "adjuvant" refers to an antigen (a tumor antigen peptide, nucleic acid and / or cell according to the present disclosure) ), it suppresses the immune response to the agent in the host upon exposure to the mixture. This refers to a substance that specifically enhances or enhances. Ajuva, 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 (Oil emulsions and surfactant-based formulations, etc.), e.g. MF59 (Micro Fluid 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 ([SBAS4] aluminum salt containing MPL), ISCOMS (saponin (4) structural complex of lactic acid and lipids, polylactide-co-glycolide (PLG), and (5) microbial derived Body (natural and synthetic), e.g., Monophosphoryl lipid A (MPL), Detox (MP L+M. Phlei cell wall skeleton), AGP[RC-529](synthetic acylated monosaccharide), D C_Chol (a lipoid immunostimulant that can self-assemble into liposomes), OM -174 (Lipid A derivative), CpG motif (synthetic compound containing immune-stimulating 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, Immudatan (C3d tandem array), and and / or (6) an inert vehicle such as gold particles.

[0069] In an embodiment, the tumor antigen peptide or a composition comprising the same is in a lyophilized form. In another embodiment, the tumor antigen peptide or the composition comprising 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: 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 or any combination of MHC class I antigens. Cells such as APCs expressing the molecule can be used to recruit CD8+ T lymphocytes in vivo or ex vivo. Thus, in another aspect, the present disclosure provides The present invention relates to a method for the preparation of a tumor antigen peptide complex comprising administering to said tumor antigen peptide complex a therapeutically effective amount of said tumor antigen peptide complex. and nucleic acid molecules encoding such TCR molecules. and vectors comprising such nucleic acid molecules. The TCR according to the present disclosure is 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. The TCR, particularly the nucleic acid encoding the TCR 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. Generate 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 the 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 a T lymphocyte, e.g., a vector encoding a tumor antigen peptide-specific TCR. Vector or plasmid transformed / transfected CD8 + T lymphocytes In a further embodiment, the present disclosure provides a method for the preparation of a tumor antigen peptide-specific TCR-transduced tumor antigen peptide-specific TCR. Methods of treating patients with the transformed autologous or allogeneic cells are provided. In this study, we aimed to develop a method to target tumor antigen-specific TCRs 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 Treatment 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) The present disclosure relates to a tumor antigen peptide that can be recognized by a T-lymphocyte cluster. The method for providing a loan includes administering to a subject (e.g., a graft recipient), e.g., an ASC In T and / or donor lymphocyte infusion (DLI) recipients, tumor antigen peptides The antibodies may be generated for and specifically target tumor cells expressing the IL-1 domain. Therefore, the present disclosure provides a method for specifically recognizing a tumor antigen peptide / MHC class I molecule complex. 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 naturally selected T lymphocytes. This can be done in vivo (i.e., APCs are loaded with tumor antigen peptides) The study was performed in patients who received IFN-γ-γ or in patients who received tumor antigen peptides. The undifferentiated lymphocytes are cultured under conditions favorable for inducing T cell activation and proliferation. Tumor antigen peptide / MHC class I molecule complex (typically on the surface of a cell such as an APC) The method of the present disclosure includes contacting a CD8 + A small number of cells are required to produce T lymphocytes. The present invention provides at least two methods for detecting tumor antigen peptides bound to MHC class I molecules. Using combined or pooled C populations capable of recognizing multiple tumor antigen peptides D8 + Alternatively, tumor antigen-specific or targeted T lymphocytes can be generated. T lymphocytes react with MHC class I molecule / tumor antigen peptide complexes (i.e., engineered or recombinant CD8 + TCR (more specifically, the α chain and By cloning one or more nucleic acids (genes) encoding the The tumor antigen peptide-specific T cells of the present disclosure can be produced / generated in vitro or ex vivo. The nucleic acid encoding the CR can be synthesized by synthesis of the tumor antigen peptide using methods known in the art. 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 be optionally differentiated in vitro to provide cytotoxic T lymphocytes (CTLs). Nucleic acids (transgenes) encoding the α and β chains can be introduced into the cells by transfection (e.g., electroporation). Any suitable method, such as perforation (poration) or transduction (e.g., using a viral vector), may be used. 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 peptide + T lymphocytes are well known They can be grown in vitro using culture methods.

[0072] The present disclosure relates to tumor antigen peptides (i.e., those that bind to MHC class I molecules expressed on the cell surface). The tumor antigen peptides bound to the tumor antigens are specifically induced by the tumor antigens. Isolated CD8 cells that are induced, activated, and / or expanded (proliferated) + T.Rin The present disclosure also provides a method for the preparation of a tumor antigen peptide, or a combination thereof, according to the present disclosure. CD8 that can recognize the match + T lymphocytes (i.e., cells that bind to MHC class I molecules) and compositions comprising the tumor antigen peptides. In another aspect, the present disclosure provides a method for the preparation of a tumor antigen comprising administering to a subject ... CD8 specifically recognizes peptide complexes + T lymphocyte enriched cell population or cell culture Nutrients (e.g., CD8 + Such enriched populations are provided herein. and MH loaded with (e.g., presenting) one or more of the tumor antigen peptides disclosed herein. Ex vivo transduction of specific T lymphocytes using cells such as APCs expressing C class I molecules As used herein, "enriched" means , tumor antigen-specific CD8 + The proportion of T lymphocytes compared to the natural population of cells , i.e., more significantly than those that have not been subjected to the step of ex vivo expansion of specific T lymphocytes. In a further embodiment, the tumor antigen peptide-specific target CD8 + The percentage 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 an embodiment, the tumor antigen peptide-specific CD8 + The proportion of T lymphocytes is approximately 0 .5 to 10%, 0.5 to 8%, 0.5 to 5%, 0.5 to 4%, 0.5 to About 3%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, or about 3% to about 4%. CD40 specifically recognizes the MHC class I molecule / peptide (tumor antigen peptide) complex. Such cell populations or cultures enriched for 8+ T lymphocytes (e.g., CD8+ T lymphocyte populations) The antibody fragments may be used in tumor antigen-based cancer immunotherapy, as described in more detail below. In some embodiments, tumor antigen peptide-specific CD8 + A population of T lymphocytes, e.g. , which is (covalently or non-covalently) loaded with a tumor antigen peptide as defined herein. The multimers of MHC class I molecules are then used to further enrich the 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 The present invention provides prepared or isolated populations.

[0073] The present disclosure further relates to a method for the preparation of a pharmaceutical composition comprising administering to a patient a pharmaceutical composition comprising any of the methods disclosed herein, as a medicament or in the manufacture of a medicament. 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: i) administered to tumor patients by injection; 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 may be used for the production of synthetic peptides to be used as cutins.

[0074] In another aspect, the disclosure provides a method for the treatment of cancer in a subject, comprising administering to a subject a composition comprising the compound 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 the use of a vaccine for treating cancer in a subject. , a tumor antigen peptide described herein, or a combination thereof (e.g., peptide In an embodiment, the subject is a subject having 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 includes administering an effective amount of one or more MHC class I molecule / tumor antigen peptide complexes (AP C) expresses a TCR that recognizes (i.e. binds to) (CD8) + and 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 is administered. peptides, or combinations thereof, and / or M loaded with tumor antigen peptides. 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 to a subject. 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 then administered to the recipient. administering to a patient.

[0075] In another aspect, the disclosure provides a method for treating cancer in a subject (e.g., reducing the number of tumor cells, and loading the tumor antigen peptides, or combinations thereof, to kill tumor cells. CD8 recognizes one or more MHC class I molecules that present + Proposes the use of T lymphocytes In another aspect, the present disclosure provides a method for treating cancer in a subject (e.g., reducing the number of tumor cells). and killing tumor cells, or a combination thereof. CD8 + In another aspect, the present disclosure provides a method for treating cancer in a subject, comprising administering to a subject a therapeutically effective amount of a T lymphocyte. For use in (e.g., to reduce the number of tumor cells and kill tumor cells) ), 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., This further includes the use of APCs.

[0076] The present disclosure also relates to any of the tumor antigen peptides disclosed herein or combinations thereof. Targeting immune responses 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 comprising the steps of: Cytotoxic T lymphocytes that specifically recognize class I MHC molecules loaded with the combination were generated. The present disclosure also relates to a method for the preparation of a tumor antigen peptide or a combination thereof. To generate 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 Provide.

[0077] In embodiments, the methods or uses described herein include administering to the patient a therapeutically effective amount of To determine the HLA class I alleles expressed by the patient and and administering or using a tumor antigen peptide that binds 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 an embodiment, 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, and 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 agents and CTLA-4 inhibitors, B7-1 / B7-2 inhibitors) antibodies, cell-based therapies (e.g. The present invention provides a method for treating cancer by administering one or more additional active agents or In embodiments, the 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. EXAMPLES

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

[0081] Example 1: Materials and Methods Human HGSC samples. Tumor fragments 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 obtained from Princeps s Margaret Cancer Registry(Toronto, ON, Ca Snap frozen samples were obtained from the University of California, San Diego, for RNA extraction and storage. and MHCI-associated peptides. The data was collected by the National Ce ter for Biotechnology Information Seque Download the data 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 OV606, OV633, and OV642. For each sample, 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 to use it for the sample RNA-Seq was performed once per replicate using the KAPA Standard mRNA-Seq Kit. A cDNA library was prepared from polyA-rich mRNA using Further amplification and sequencing were performed using HiSeq® 2000 or Illumina NextSe Used for paired-end RNA-Seq on the q®500, 150 million samples We got 0-300 million leads.

[0083] Generation of a customized reference database for MS analysis. The customized Global Cancer Database was developed as described above (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 The RNA-Seq reads were adapted using rimmomatic v0.35(25). The 3′-peptide and low-quality 3′ bases were trimmed. To achieve this, the trimmed reads were analyzed using STAR v2.5.1b to identify the reference human genome. The transcript expression was measured using default parameters. The number of transcripts per million ( Nucleotide variants were quantified using FreeBayes (26). We identified and ran agnostic ) was converted to single nucleotide polymorphism file format. Then, the sample-specific proteomic sequence of each sample was 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] To generate cancer-specific proteomes, trimmed R1 reads were FASTXed -Reverse complemented and trimmed using Toolkit version 0.0.14 R 33-nt and 24-nt 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 for 33 nucleotides as follows: The following were applied: HGSC1-3 (7), OV642 (8), and OV633 (9). 10 for HGSC4 and OV606, 4 for HGSC5, 6 for HGS 5 for C6 and 3 for OvCa48~114. The expressed k-mers were subtracted to obtain cancer-specific k-mers, which were then analyzed using 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 assembled into amino acid sequences. The resulting stretches of at least 8 amino acids were It was included in the cancer-specific proteome.

[0085] Isolation of MAPs. Tumor and tissue samples were cut into small pieces (cubes, size approximately 3 mm) and Ice-cold 1000-mL IgG1-containing protein inhibitor cocktail (Sigma, Cat. No. P8340-5ml) 5 ml of PBS was added. First, the Ultra Tuner was 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 s, 2 Then, 550 μl of ice-cold 10x lysis buffer (5% w / v CHAPS) was added. was added to each sample. After 60 minutes of incubation with tumbling at 4°C, The samples were centrifuged at 10000g for 30 min at 4° C. The supernatant was diluted with 1 mg of W6 / 32 antibody. Transfer the mixture to a new tube containing covalently bound Protein A magnetic beads and 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 15 cm × 150 μm inner diameter column filled with 0.2% acetonitrile was added to the column. A 56-minute gradient from 0.01% HCl (formic acid) and 600 nl.min on an Easy-nLC II system. - 1 The samples were loaded at a flow rate of 1000 rpm on a Q-Exactive™ HF mass spectrometer (Ther The data were analyzed using a 3D microscope (Fisher Scientific) at 60,000 resolution. Each complete MS spectrum was followed by 20 MS / MS spectra, resulting in 30,000 Resolution: 5×104 with an automatic gain control target of 100 ms, an injection time of 25 Select the most abundant multiply charged ions for MS / MS sequencing with a collision energy of 0.5%. 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 controlled 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] Identification of MAPs. PEAKS 8.5 or Peaks X (Bioinformatics Peptides were identified using the Global Cancer Data Sheet (GCD) Peptide sequences were searched against a database of 1000 nucleotides. Peptide identification was based on precursor ions and The tolerances for the S and fragment ions were set at 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 scores to ensure inclusion of Peptides that exceeded 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 ≦2%(29).

[0088] Identifying and validating TSA candidates. To identify TSA candidates, each MAP and its code The sequences were then compared to the associated cancer and normal canonical proteomes, or the cancer and normal canonical proteomes, respectively. The 24-nucleotide k-mer database was queried against the normal 24-nucleotide k-mer database. d) Normal canonical proteome and normal 24-nucleotide k-mer data. The database was generated using RNA-Se from purified TECs from six human thymes. The construct was constructed using the reads of MAq (15 and US Provisional Application No. 62 / 724,760). P can be expressed in two cases: i) in the normal canonical proteome of the sample, That is, if the peptide sequence was not detected in the k-mer, or i) The peptide is absent from both the cancer and normal canonical proteomes, RNA coding sequences are overexpressed at least 10-fold in cancer cells compared to TECs. If the MAP corresponds to some RNA sequence, it is labeled as a TSA candidate. If so, it will be considered a TSA candidate only if all sequences are consistent with TSA candidate status. All TSA candidates were considered as TSA candidates. MS / MS spectra were manually verified. This eliminated any false positives. Supported by RNA data that were distinguishable by MS. For TSA candidates with I / L variants that were retained, the most expressed variant was the TSA candidate. If so, then 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. We removed TSA candidates from reads that matched the MAP code (CR gene). The sequences contained variants that did not match known germline polymorphisms (reported in dbSNP v149). If the mutation was present, it was classified as an mTSA. If the mutation was not present, it was classified as an aeTSA. 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 are available at the Genotype-Tissue expression (GTEx) portal. Downloaded from (phs000424.v7 accessed on 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 covered the MAP coding sequence was significantly higher than that of the RNA-Seq reads for each tissue. The 24-mer of the MAP coding sequence in the database of 24-mers transformed from the The read counts were estimated by the minimum occurrence of the r set. The results were normalized to the number of reads (rphm) and then log-transformed (log 10 (rphm+1) Averaged across all available RNA-Seq experiments for each tissue. MHC 低 Peripheral expression was observed in tissues other than the brain cortex, nerves, and testis at rphm>10. aeTSA candidates that did not have a genomic identity were considered as bona fide aeTSA. [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 expression of the TSA coding region on the TSA coding strand. Count all reads overlapping the A coding region with parameter orientation = "same" The qCount function from the R package QuasR (30) was used to map the Counts were calculated as 100 million mapped reads per 100 million reads. The aeTSA expression analysis was performed using the context of the surrounding sequences. Since the aeTSA expression may be affected by the t, the individual aeTSAs were mapped to 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 immune cell populations, is calculated based on RNA-Seq data. For each tumor, a score was estimated according to 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 presentation. To estimate the number of aeTSA presented by individual patients To estimate the variance, we performed a bioinformatics simulation. First, the likelihood of aeTSA expression was based on the TCGA-OV cohort. The results were based on the proportion of tumors expressing the corresponding RNA containing the germline SNP. For eTSA, the expression likelihood was calculated as follows: SNP frequency is calculated by the Genome Agg Second, the European American population (n=1,242,890), African-American (n=416,581), and Chinese ( HLA allele frequencies were obtained from the National Marrow Donor Program (NMDP) for the 14 patients (n=99,672). The patient's HLA genotype was then determined based on reported frequencies in a given population. , six HLA class I alleles were simulated. Six HLA alleles were independently 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, The expression of each aeTSA was considered to be the same for the same overlapping group. The expression state was simulated only once for each of the 14 independent events, except for the duplicate aeTSA. 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 tumor plots were performed using the gplots package in R. 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. Comparisons of the cloths were performed using ANOVA, and pairwise comparisons between groups were performed using the Ulcoxon The rank sum test was used. The log-rank P value of the survival analysis was calculated by the “survival” Calculated in case.

[0096] Example 2: Proteogenomic analysis identifies 111 TSAs in 23 HGSC. To obtain a systems-level characterization of the TSA landscape, Direct MAP identification was performed using deconvolutional mass spectrometry (MS / MS) analysis (34-36). The search engine searches for a peptide sequence to match each acquired MS / MS spectrum. , 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 be used to identify All genome sequences are free of specific mutations, out-of-frame translation events, and non-exonic sequences. The search to capture TSAs encoded by tumor-specific regions was performed for each tumor. 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 include (TEC 3-frame translation of the cancer-specific RNA sequences after subtraction of normal RNA sequences (from 1) TECs have been implicated in 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 promiscuously expressed ( 38 ); MAPs from nine primary HGSC samples were obtained by immunoprecipitation of MHC I molecules and then Analyses were performed by liquid chromatography-MS / MS (15, 28). Immunopeptideemia data from an additional cohort of 14 HGSCs reported by R. et al. (13) The data were also included for samples where matching RNA-Seq and MS data were available, as described herein. 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 may represent germline polymorphisms These classifications were consistent across the 23 analyzed Eighteen mTSAs were obtained from the samples (Table 3A). Of the 18 mTSAs, 7 result from in-frame exon translation and 4 1 arose from out-of-frame exon translation and 8 from non-coding sequences. Therefore, matched normal tissues were available and RNA-Seq analysis demonstrated that the mTSA variant was reproductively It was confirmed that this was not due to cell lineage polymorphism (Figures 8A to 8B). In this case, some mTSAs may correspond to rare polymorphisms not present in dbSNP. It cannot be formally excluded that the number of mTSAs may be slightly overestimated. The efficacy was higher in patients with less than one mTSA per tumor (18 mTSA / 23 tumors). We conclude that A is rare in HGSC and therefore represents a less attractive target. Moreover, the classical TSA discovery method does not detect exons in-frame or 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 were applied to identify pure aeTSAs, i.e. We identified cancer-specific expression of 27 genes across the human body. Their RNA expression in peripheral tissues was analyzed. MHC 低 Other than tissues (brain, nerves, testes) All candidates whose coding RNA was 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 (SNP), and was therefore removed from the aeTSA list. If the candidate contains the SNP-containing sequence (as reported in dbSNP), the candidate is and labeled as a valid aeTSA only if the reference sequence met the above criteria. (Figure 9). Overall, 93 aeTSA candidates met these stringent criteria (Figure 2). , Tables 3B and 3C), of which 85 have never been reported to our knowledge ( Interestingly, 5 of the 93 aeTSAs were expressed in the testis, Although some cancer bacterial antigens (CGAs) are aeTSAs, most aeTSAs are CGAs. CGA is a canonical exon normally expressed only by germ cells. The abnormal expression of these proteins in cancer cells is mainly mediated by epigenetic mechanisms. However, some CGAs are expressed by adult mTECs. (16) and CGA expressed in mTECs (or other somatic tissues) are used as TAAs and and those that are not expressed by any normal tissue (including mTECs) are bona fide aeTS is regarded as A.

[0099] A genomic location was assigned to each aeTSA. When multiple locations were possible, the matching RNA The characteristics of all TSAs are shown in Tables 3B and 3C. The stringent approach is based on the use of non-standard translation (5'UTR, 3'UTR, It is possible that the total number of aeTSAs arising from gene-specific mutations (e.g., intergenic, frameshift) may be formally underestimated. Indeed, the open reading frame used to generate MAP in tumors is It is known that their coding RNA is expressed in some normal tissues, which 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 of the TSA coding sequence to the ORFs, and marks the area outside the ORF as "non-translational." "Code", "ORF overlap but frame shifted" is "Code out", and "ORF matrix" is "ORF The code that was switched in was called the "code-in." 2 The "Genome Origin" column further annotates aeTSA according to the biotype from Ensembl. For example, "antisense" refers to the opposite side of the gene to which the sequence is annotated. "Canonical" means that it is in the stand and is a canonical / annotated ORF. "ncRNA" refers to RNA that is annotated without ORF according to Ensembl. It is NA. 3 "ncRNA" refers to a transcript whose coding sequence aligns with an exon of a non-coding transcript. Refers to... 4 "Non-coding antisense" refers to a sequence that is a TSA coding sequence that 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 It 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 favorably with the number of MAPs (Figure 3A). 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 noncoding sequences are the limiting factor for 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 be expected not to be shared by a large number of 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 samples treated with This is because the proteogenomic approach described here is generally similar to RNA-Seq and This suggests that the method can be applied to MS data and is seemingly immune to inter-laboratory variability. In both cohorts, approximately 83% of TSAs were unmutated, and the majority of TSAs The majority of the genomes arose from atypical translation: mainly from non-coding regions and, to a lesser extent, from frame Two features of aeTSA are noteworthy: i) 80% are derived from non-coding sequences, specifically introns (31%) and intergenic (22%) and ii) 90% are novel MAPs (Figure 3D). The corresponding protein isoforms were identified in the UniProt database (13, 39–43 and and U.S. Patent Publication No. 2012 / 0077696A1). Except for those that match the 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 arises from repeated transcriptional abnormalities, we used the results of this study and TCGA eggs. 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 between Expression of this set of TSA-encoding transcripts is not a rare or random event, but a common 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 used vectors from the TCGA-OV dataset. Using the localized data, we correlated aeTSA RNA expression with local genes or epigenetic We explored the relationship between genetic abnormalities, regional DNA copy number alterations, DNA methylation levels on gene promoter regions and RNA methylation levels for each aeTSA We tested the correlation between expression and genomic regions that are part of genes (exons, introns, etc.) (Figure 5A). When aeTSA derived from a genomic DNA sequence (e.g., chromosome 1 or UTR) was used, the expression of the relevant gene was significantly improved. We also analyzed the correlation between the expression of aeTSA. In the latter situation, the correlation between genes and aeTSA expression was A significant correlation was observed between the aeT For SA, the regulation of aeTSA expression generally affects the entire gene. Furthermore, the DNA copy number changes were positively correlated with the RNA expression levels of aeTSA. This was due to the fact that intragenic and extragenic aeTSA (antisense and genomic This suggests that DNA copy number changes have a substantial effect on aeTSA expression. Notably, this correlation was observed in a larger proportion of tumors. The increase in the aeTSA coding region was particularly strong for the intragenic aeTSA (Fig. 11A). Examination of the chromatid distribution revealed that some chromosome arms frequently amplified in HGSC were associated with many ae For example, the TSA gene was found to be amplified in ovarian cancer. The long arm of chromosome 3, which is known to contain a genomic DNA fragment (44), was the source of eight aeTSAs. One, MECOM, located at 3q26.2(44), is a triple-exon duplication. However, amplification of the chromosome arm did not necessarily result in the generation of an out-of-frame aeTSA (Table 3B). was neither necessary (e.g., 15q) nor sufficient to generate aeTSA (e.g., For example, 8q) (Figure 5B, Figure 11B).

[0104] For the technology that TCGA uses 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 for 17 However, the DNA sequence of six aeTSAs was not significantly different from that of the control. A significant correlation was found between chilling and aeTSA expression (Figure 5A). In both cases, the results were negative and positive, suggesting that promoter demethylation frequently leads to increased transcription. In particular, the two genes with the highest negative correlations were 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 involved in several cancer types, including HGSC. aeTSA is a CGA that is overexpressed in at least one type of encephalomyelitis (3). 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. We next assessed whether some aeTSAs could induce spontaneous protective immune responses. Expression of aeTSA at the tid level, in addition to expression of aeTSA RNA, was 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 randomized 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 size of each group. This significantly reduced the power of the analysis, and therefore the statistical power of the three aeTs. The log-rank p values ​​for SA ranged from 0.013 to 0.076 (Figure 6A-C). 6C). Nevertheless, two observations suggest that these correlations are biologically meaningful. First, the “protective effect” of these aeTSAs is 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 cytoplasmic vesicles (Figure 6D, Figure 6E; ANOVA, p<0.05). .

[0106] Example 7: Median number of aeTSA presented by individual tumors Using the list of 93 aeTSAs, we will finally determine how this study contributes to TSA-targeted immunotherapy. Therefore, the study estimated that 93% of 1 million patients would benefit from the treatment. The presentation status of each aeTSA was randomly simulated. To determine this, we used the three largest data sets from US bone marrow banks: European American Humans, African Americans, and Chinese (45) were used. Alleles in a given population The frequency, percentage of expression in TCGA-OV tumors, and SNP frequency, if applicable, were used. Six HLA alleles and aeTSA expression status were generated independently 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 whites and 98% of African-Americans determined that at least one aeTSA could be found in 74% of Japanese and 78% of Chinese. However, the median number of aeTSAs per tumor was 5 in European Caucasians and 4 in African-Americans. The differences between these populations were due to HLA allele inheritance. This is due to the change in offspring frequency and the fact that the tumor samples were primarily from Caucasian Europeans. These calculations underestimate the number of aeTSAs per tumor for three main reasons. First, more than 50% of MAPs bound 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 have generated AP. This cautious strategy was adopted because the current 93 a Vaccines containing the eTSA set are available for nearly all Caucasian and African It was concluded that the study covered a significant proportion of Canadian Americans and Asians (e.g., Chinese). It can be done.

[0107] Although the present technology has been described above with reference to specific embodiments thereof, the present invention is not limited to the above embodiments and may be modified in any manner without departing from the spirit and scope of the present invention. The scope of the present invention is not to be construed as limiting 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 phrase "commercial use" The singular forms "a," "an," and "the" are used unless otherwise clearly indicated by the context. Unless otherwise indicated, corresponding plural references are included.

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Claims

1. A tumor antigen peptide comprising one of the amino acid sequences set forth in SEQ ID NOs: 1 to 103.

2. The tumor marker according to claim 1, comprising one of the amino acid sequences set forth in SEQ ID NOs: 19 to 103. Tumor antigen peptides.

3. The tumor antigen peptide binds to an HLA-A*01:01 molecule and is selected from SEQ ID NOs: 21, 28, 40, 41, 66 or 88. Tumor antigen peptides.

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 The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence set forth in claim 91.

5. The tumor antigen peptide binds to an HLA-A*11:01 molecule and is selected from SEQ ID NOs: 32, 54 55, 67, 69, 81, 87, 90 or 102. Item 3. The tumor antigen peptide according to item 1 or 2.

6. The tumor antigen peptide binds to an HLA-A*24:02 molecule and has SEQ ID NO: 33 or 43. The tumor antigen peptide of claim 1 or 2, comprising the amino acid sequence of claim 43.

7. The tumor antigen peptide binds to an HLA-A*25:01 molecule and is represented by SEQ ID NO:

24. The tumor antigen peptide according to claim 1 or 2, comprising one of the amino acid sequences:

8. The tumor antigen peptide binds to an HLA-A*29:02 molecule and has SEQ ID NO: 34 or 58. The tumor antigen peptide according to claim 1 or 2, comprising one of the amino acid sequences set forth in claim 58. Chid.

9. The tumor antigen peptide binds to an HLA-A*32:01 molecule and is represented by SEQ ID NO:

16. The tumor antigen peptide according to claim 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. Item 3. The tumor antigen peptide according to item 1 or 2.

11. The tumor antigen peptide binds to an HLA-B*08:01 molecule, and is selected from SEQ ID NOs: 23, 35, Amino acid according to claim 42, 44, 46, 59, 63, 70, 74, 76, 83 or 103 The tumor antigen peptide of claim 1 or 2, comprising one of the sequences:

12. The tumor antigen peptide binds to an HLA-B*14:01 molecule and is represented by SEQ ID NO:

53. The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence:

13. The tumor antigen peptide binds to an HLA-B*15:01 molecule and is SEQ ID NO: 2, 3 or The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence of 5.

14. The tumor antigen peptide binds to an HLA-B*18:01 molecule and is represented by SEQ ID NO:

89. The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence:

15. The tumor antigen peptide binds to an HLA-B*39:01 molecule and is selected from SEQ ID NOs: 47, 64 3. The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence according to claim 1, 2 or 3. Chid.

16. The tumor antigen peptide binds to an HLA-B*40:01 molecule and is represented by SEQ ID NOs: 11 and 12.

14. The tumor antigen peptide of claim 1 or 2, comprising the amino acid sequence of claim 13.

17. The tumor antigen peptide binds to an HLA-B*44:02 molecule and is represented by SEQ ID NO:

65. The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence:

18. The tumor antigen peptide binds to an HLA-B*44:03 molecule and has SEQ ID NO: 37 or 3. The tumor antigen peptide of claim 1 or 2, comprising the amino acid sequence set forth in claim 94.

19. The tumor antigen peptide binds to an HLA-C*03:03 molecule and is represented by SEQ ID NO: 10, 29 3. The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence according to claim 2, 71 or 95. Chid.

20. The tumor antigen peptide binds to an HLA-C*04:01 molecule and is SEQ ID NO: 6 or 1 The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence described in claim 5.

21. The tumor antigen peptide binds to an HLA-C*05:01 molecule and is represented by SEQ ID NO:

27. The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence:

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 of claim 1 or 2, comprising the amino acid sequence of claim 72.

23. The tumor antigen peptide binds to an HLA-C*07:01 molecule and is represented by SEQ ID NOs: 38, 61 3. The tumor antigen peptide of claim 1 or 2, comprising an amino acid sequence as set forth in claim 93.

24. The tumor antigen peptide binds to an HLA-C*07:02 molecule and is represented by SEQ ID NO:

7. The tumor antigen peptide of claim 1 or 2, comprising an amino acid sequence.

25. The tumor antigen peptide binds to an HLA-C*12:03 molecule and is represented by SEQ ID NO:

80. The tumor antigen peptide according to claim 1 or 2, comprising the amino acid sequence:

26. The tumor antigen peptide binds to an HLA-C*14:02 molecule and is selected from SEQ ID NOs: 25, 57, 3. The tumor antigen peptide of claim 1 or 2, comprising an amino acid sequence as set forth in claim 79.

27. Encoded by a sequence located in a non-protein coding region of the genome, SEQ ID NO: 1, 4 , 6-8, 10, 13, 15-27 and 36-99, preferably SEQ ID NOs: 19-27 and 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 a untranslated transcribed region (UTR), The tumor antigen peptide is selected from SEQ ID NOs: 1, 8, 10, 13, 15, and 19-27, preferably The present invention relates to a method for the preparation of a nucleic acid sequence comprising the amino acid sequence of any one of SEQ ID NOs: 19 to 27. The described tumor antigen peptide.

29. The non-protein coding region of the genome is an intron, and the tumor antigen peptide The nucleic acid sequence of SEQ ID NO: 16, 17, and 36 to 64, preferably SEQ ID NO: 36 to 64, The tumor antigen peptide of claim 27, comprising any one of the amino acid sequences described above.

30. The non-protein coding region of the genome is an intergenic region, The method of claim 1, wherein the tide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 65 to 84.

28. A tumor antigen peptide according to 27.

31. The non-protein coding region of the genome is a non-coding RNA transcript (ncRNA). exon, and the tumor antigen peptide is selected from SEQ ID NOs: 4 and 85-92, preferably The method according to claim 27, comprising the amino acid sequence set forth in any one of sequence numbers 85 to 92. Tumor antigen peptide.

32. the non-protein coding region of the genome is an antisense strand of a gene; The tumor antigen peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 93 to 99. The tumor antigen peptide of claim 27.

33. A nucleic acid encoding a tumor antigen peptide according to any one of claims 1 to 32.

34. 34. The nucleic acid of claim 33 which is an mRNA or a viral vector.

35. A tumor antigen peptide according to any one of claims 1 to 32, or claim 33 or A liposome comprising the nucleic acid according to claim 34.

36. A tumor antigen peptide according to any one of claims 1 to 32, or a tumor antigen peptide according to claim 33 or 34. or the liposome of claim 35, and a pharma- ceutically acceptable carrier. Hmm, composition.

37. A tumor antigen peptide according to any one of claims 1 to 32, or a tumor antigen peptide according to claim 33 or 34.

37. The nucleic acid according to claim 35, the liposome according to claim 35, or the composition according to claim 36, A vaccine comprising an adjuvant.

38. A method for treating a tumor comprising administering to a subject a tumor antigen peptide according to any one of claims 1 to 32 within its peptide-binding groove. , an isolated major histocompatibility complex (MHC) class I molecule.

39. 39. The isolated MHC class I molecule of claim 38 in the form of a multimer.

40. 40. The isolated MHC class I molecule of claim 39, wherein the multimer is a tetramer.

41. (i) a tumor antigen peptide according to any one of claims 1 to 32, or (ii) A method for the preparation of a tumor antigen peptide comprising the steps of: An isolated cell comprising a vector comprising the

42. Tumor antigen peptides according to any one of claims 1 to 32 in their peptide binding groove 1. An isolated mouse model of an ... The cells were

43. The cell of claim 42, which is an antigen-presenting cell (APC).

44. The cell of claim 43 , wherein the APC is a dendritic cell.

45. An isolated MHC class I molecule according to any one of claims 38 to 40 and / or is an MHC class I antigen expressed on the surface of a cell according to any one of claims 42 to 44. A T cell receptor (TCR) that specifically recognizes a molecule.

46. An isolated CD8 antigen expressing the TCR of claim 45 on its cell surface. ＋ T Lymphocytes.

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

48. 1. A method of treating ovarian cancer in a subject, comprising administering to a subject an effective amount of: (i) Any of claims 1 to 32 (ii) a tumor antigen peptide according to any one of claims 33 and 34, (iii) the liposome according to claim 35; (iv) the composition according to claim 36; (v) (vi) a vaccine according to claim 37; (vi) a cell according to any one of claims 41 to 45; (vii) CD8 according to claim 46 ＋ or (viii) a T lymphocyte as defined in claim 47. administering to said subject the cell population described above.

49. 49. The method of claim 48, wherein the ovarian cancer is a serous carcinoma.

50. 50. The method of claim 49, wherein the serous cancer is high-grade serous carcinoma (HGSC). 。

51. and further comprising administering to said subject at least one additional anti-tumor agent or therapy. The method according to any one of claims 48 to 50.

52. The at least one additional anti-tumor agent or therapy may be a chemotherapeutic agent, an immunotherapy, an immune chemotherapeutic agent, or a combination thereof.

52. The method of claim 51, wherein the therapeutic agent is a checkpoint inhibitor, radiation therapy or surgery.

53. (i) a tumor antigen peptide according to any one of claims 1 to 32, (ii) claim 33 or 34, (iii) the liposome according to claim 35, (iv) the (v) the composition according to claim 37; (vi) the vaccine according to claims 41 to 45. The cell according to any one of claims 1 to 46, (vii) the CD8 ＋ T lymphocytes, or (viii) The cell population of claim 47 for treating ovarian cancer in a subject. use.

54. (i) a tumor antigen peptide according to any one of claims 1 to 32, (ii) claim 33 or 34, (iii) the liposome according to claim 35, (iv) the (v) the composition according to claim 37; (vi) the vaccine according to claims 41 to 45. The cell according to any one of claims 1 to 46, (vii) the CD8 ＋ T lymphocytes, or (viii) A method for treating ovarian cancer in a subject, comprising administering to said subject the cell population of claim 47. Use for the manufacture of medicines.

55. 55. The use of claim 53 or 54, wherein the ovarian cancer is serous carcinoma.

56. 56. The use according to claim 55, wherein the serous cancer is high-grade serous carcinoma (HGSC). 。

57. The method of any one of claims 53 to 56, further comprising the use of at least one additional anti-tumor agent or therapy.

2. The use according to any one of claims 1 to 11.

58. The at least one additional anti-tumor agent or therapy may be a chemotherapeutic agent, an immunotherapy, an immune chemotherapeutic agent, or a combination thereof.

58. The use according to claim 57, wherein the therapeutic agent is a checkpoint inhibitor, radiation therapy or surgery.

59. (i) a compound according to any one of claims 1 to 32 for use in treating ovarian cancer in a subject; A tumor antigen peptide according to any one of claims 1 to 3, (ii) a nucleic acid according to claim 33 or 34. (iii) the liposome according to claim 35; (iv) the composition according to claim 36; (v) (vi) a vaccine according to claim 37, (vi) a cell according to any one of claims 41 to 45. (vii) the CD8 of claim 46 ＋ or (viii) a T lymphocyte according to claim 47. The cell populations described.

60. The tumor antigen peptide for use according to claim 59, wherein the ovarian cancer is a serous cancer. do, nucleic acid, liposome, composition, vaccine, cell, CD8 ＋ T lymphocytes, or cell populations 。

61. 61. The use according to claim 60, wherein the serous cancer is high-grade serous carcinoma (HGSC). Tumor antigen peptides, nucleic acids, liposomes, compositions, vaccines, cells, and CD8 ＋ T lymphocyte, or cell population.

62. The tumor antigen peptide, the nucleic acid, the liposome, the composition, the vaccine, the cell, and the CD8 ＋ T.Rin The cell or cell population is combined with at least one additional anti-tumor agent or therapy. A tumor antigen peptide, a nucleic acid, a liposome, or the like for use according to any one of claims 59 to 61. Composition, vaccine, cell, CD8 ＋ T lymphocytes, or cell populations.

63. The at least one additional anti-tumor agent or therapy may be a chemotherapeutic agent, an immunotherapy, an immune chemotherapeutic agent, or a combination thereof.

63. The method of claim 62, wherein the therapeutic agent is a checkpoint inhibitor, radiotherapy or surgery. Tumor antigen peptide, nucleic acid, liposome, composition, vaccine, cell, CD8 ＋ T lymphocytes, or cell population.

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