Methods for selecting neoantigens from subject

Abstract

The purpose of the present invention is to provide a means for selecting a neoantigen from a subject. The above-mentioned technical problem is achieved by providing a method for selecting a neoantigen from a subject, and the like, the method comprising the steps of: acquiring sequence data of normal cells and cancer cells from the subject; identifying a gene having the cancer cell specific gene mutation; and identifying, from a major histocompatibility complex (MHC) presenting peptide database, a peptide based on a wild-type gene corresponding to a gene having the cancer cell-specific gene mutation, where the gene mutation is a missense mutation, and a peptide based on a wild-type gene corresponding to a gene having the gene mutation has a wild-type amino acid corresponding to the position of an amino acid mutation caused by the gene mutation.

Description

Technical Field

[0001] The present invention relates to a method for selecting neoantigens from a subject, etc. Background Art

[0002] As a treatment method for cancer, in addition to surgery, radiotherapy, and chemotherapy, immunotherapy can also be mentioned. Immunotherapy is a treatment method that utilizes the immune response against cancer that naturally exists in the organism, and controls the proliferation of cancer by enhancing this immune response, etc. Immunotherapy is expected to be a treatment method that acts on the whole body of the organism, has few side effects, and exhibits a continuous therapeutic effect.

[0003] As immune cells responsible for immunotherapy, T cells, particularly CD8-positive T cells (cytotoxic T cells: CTLs) and CD4-positive T cells can be mentioned. Cancer cells present peptides from specific proteins that are expressed at higher levels in cancer cells than in normal cells, i.e., peptides from cancer antigens, to CTLs via major histocompatibility complex (MHC) class I molecules on the cancer cell surface. CTLs recognize and activate this peptide through the T cell receptor (TCR), thereby eliminating cancer cells. That is, by inducing CTLs to recognize this peptide, the immune response against cancer can be enhanced, and thus immunotherapy can be achieved. In addition, CD4-positive T cells promote the immune response generated by CTLs by recognizing and activating peptides presented via MHC class II molecules. That is, by inducing CD4-positive T cells to recognize this peptide, the immune response against cancer can also be enhanced, and thus immunotherapy can be achieved.

[0004] In recent years, in addition to the above peptides from cancer antigens, it has been reported that peptides from specific proteins having mutations that do not exist in normal cells but are specifically present only in cancer cells (the peptides have mutations that are specifically present only in the cancer cells) can also be used as peptides from cancer cells for immunotherapy (Non-Patent Document 1). This peptide from a specific protein is called a new antigen from cancer cells, i.e., a "neoantigen". The gene mutations that bring about neoantigens account for only a small part of the large number of gene mutations in cancer cells. In addition, since these gene mutations vary among cancer patients, neoantigens also vary among cancer patients.

[0005] Cancer cells present neoantigens to CD8-positive T cells and CD4-positive T cells via major histocompatibility complex (MHC) class I molecules and MHC class II molecules on the cancer cell surface. CD8-positive T cells and CD4-positive T cells recognize and activate neoantigens through the T cell receptor (TCR), thereby distinguishing normal cells from cancer cells and specifically eliminating cancer cells. That is, by inducing CD8-positive T cells and CD4-positive T cells to recognize neoantigens through the TCR, the immune response against cancer can be specifically enhanced, thereby achieving highly specific immunotherapy. In particular, since neoantigens vary among individual cancer patients, by inducing CD8-positive T cells and CD4-positive T cells to recognize neoantigens through the TCR, the immune response against cancer can be specifically enhanced for each cancer patient, thereby achieving highly specific immunotherapy personalized for each patient. Moreover, neoantigens have mutations that do not exist in normal cells but are specifically present only in cancer cells. Since they do not naturally exist in the body, they can avoid immune tolerance in the thymus. Therefore, it is expected that a strong immune response can be induced by inducing CD8-positive T cells and CD4-positive T cells to recognize neoantigens through the TCR.

[0006] As methods for predicting neoantigens that vary among individual cancer patients, in silico and mass spectrometry (MS) methods can be cited. In the in silico method, using the highly developed next-generation sequencing technology in recent years, cancer cell-specific gene mutations are identified for each cancer patient, and then an MHC binding prediction algorithm is used to predict peptides with the gene mutations that can be presented by MHC, thereby predicting neoantigens that vary among individual cancer patients (Non-Patent Document 2). Since this method uses the above-mentioned next-generation sequencing technology and algorithm, it has the advantages of high sensitivity and simplicity. However, since it is not clear whether the predicted neoantigens are actually presented by MHC in the body, there are problems in terms of specificity. In the MS method, MHC-peptide complexes are enriched from the cancer cells of each cancer patient, the peptides are eluted from the MHC of the complex, and then the peptides are identified by MS, thereby predicting neoantigens that vary among individual cancer patients (Non-Patent Document 3). Since this method identifies peptides that are actually presented by MHC in the body by MS, it has the advantage of high specificity. However, it is limited to cases where the peptide is abundant in the body. In addition, this method involves complex experimental operations, so there are problems in terms of sensitivity and simplicity. Prior Art Documents Non-Patent Documents

[0007] Non-Patent Document 1: Nat. Biotechnol., 35, 97 (2017) Non-Patent Document 2: Nat. Rev. Drug Discov., 21, 261 - 282 (2022) Non - Patent Document 3: Nat.Rev.Clin.Oncol., 17, 595 - 610 (2020) Summary of the Invention Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a means for selecting neoantigens from a subject. Means for Solving the Problems

[0009] When studying methods for selecting neoantigens from a subject, the present inventors noticed that by combining existing in silico methods and MS methods, it is possible to simultaneously obtain the advantages of high sensitivity and simplicity of the former and the high specificity of the latter. And, to prove this possibility, the present inventors conducted in - depth research and found that by comparing the sequence data of normal cells and cancer cells with each other, it is possible to identify genes with cancer - specific gene mutations, and by identifying peptides based on the wild - type genes corresponding to these genes from the MHC - presented peptide database, it is possible to select neoantigens from a subject. Based on this discovery, the present inventors further conducted research and finally completed the present invention.

[0010] For some cancers, it is extremely difficult to collect a sufficient amount of tumor biopsy samples from an organ and isolate cells. The present inventors found that a MHC - presented peptide database obtained from cells other than the cancer cells of the subject itself can be used to select neoantigens. In this specification, this database can be denoted as a Surrogate immunopeptidome. That is, the cells used to produce the Surrogate immunopeptidome can be from any organ or tissue, and can be from the subject patient or a third party. In addition, cells from blood can be used in the Surrogate immunopeptidome. The collection and isolation of cells from blood are generally simple and rapid. In addition, even when it is not possible to obtain a sufficient amount of blood cells, it is possible to ensure a sufficient amount of cells for MS analysis by proliferating them using EBV. The present inventors achieved the identification of neoantigen peptides using the Surrogate immunopeptidome. For example, by comparing the sequence data of cells collected from a subject's cancer biopsy tissue and tissues other than the cancer tissue with each other, it was possible to identify genes with cancer - specific gene mutations, and by identifying peptides based on the wild - type genes corresponding to these genes from the Surrogate immunopeptidome, neoantigens from the subject were selected.

[0011] That is, the present invention relates to the following aspects. [1]A method for selecting neoantigens from a subject, comprising the steps of: (1) obtaining sequence data of normal cells and cancer cells from the subject; (2) identifying genes with cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; and (3) identifying peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database, wherein the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene with the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0012] [2]The method according to [1], wherein the sequence data is whole exome sequence data. [3]The method according to [2], wherein the MHC presented peptide database includes a list of wild-type MHC presented peptides according to each MHC class and / or each organ type. [4]The method according to [1], wherein the MHC presented peptide database includes a list of wild-type MHC presented peptides. [5]The method according to [4], wherein step (3) is a step of identifying peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations from the MHC presented peptide database according to each MHC class and / or each organ type. [6]The method according to [5], wherein the amino acid mutation caused by the gene mutation is a single amino acid mutation. [7]The method according to [6], wherein the peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations are scored based on their respective MHC presentation frequencies in the MHC presented peptide database.

[0013] [8]The method according to [7], wherein the peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations are identified based on the scoring. [9]The method according to [8], wherein the MHC presented peptide database is created by an MHC presented peptide analysis method, which includes the steps of: obtaining MHC-peptide complexes from cells; obtaining peptides from the complexes; and identifying the amino acid sequences of the peptides by mass spectrometry analysis.

[10] The method according to [9], wherein the cells are from a subject or a non-subject.

[11] The method according to [9], wherein the cells are collected from blood.

[12] The method according to [9], wherein the MHC is MHC class I and / or class II.

[0014]

[13] According to the method of

[12] , wherein MHC is human leukocyte antigen (HLA).

[14] According to the method of

[13] , which further comprises the following steps: introducing an amino acid mutation caused by the gene mutation into a peptide based on a wild-type gene corresponding to the gene having the cancer cell-specific gene mutation, wherein the peptide has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation; and / or determining the gene sequence encoding the peptide into which the amino acid mutation has been introduced.

[15] According to the method of

[14] , which further comprises the following steps: selecting a nucleic acid composed of a peptide into which the amino acid mutation has been introduced and / or a base sequence into which the gene mutation has been introduced as a neoantigen from a subject.

[0015]

[16] A method for producing a peptide containing a neoantigen from a subject, comprising the following steps: adding 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of the neoantigen from the subject selected by the method of

[15] , wherein the number of amino acids contained in the neoantigen to which the amino acid has been added is 40 or less.

[17] A peptide obtained by the method of

[16] .

[18] A method for producing a nucleic acid containing a neoantigen from a subject, comprising the following steps: adding 0 or more arbitrary bases to the 5'-end and / or 3'-end of the nucleic acid containing the neoantigen from the subject selected by the method of

[15] .

[19] A nucleic acid containing a neoantigen from a subject, wherein the neoantigen is selected by the method of

[15] .

[20] According to the method of

[18] , which further comprises the following steps: producing an mRNA containing the nucleic acid containing the neoantigen from the subject.

[21] A nucleic acid mRNA containing a neoantigen from a subject, wherein the neoantigen is selected by the method of

[15] .

[22] A vaccine composition comprising the peptide of

[17] , the nucleic acid of

[19] or the mRNA of

[21] .

[0016]

[23] A system for selecting neoantigens from a subject, comprising: (1) a sequence data acquisition unit that acquires sequence data of normal cells and cancer cells from the subject; (2) a gene identification unit that identifies genes having cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; and (3) a peptide identification unit that identifies peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database, wherein the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[24] The system according to

[23] , wherein the cells are from a subject or a non-subject.

[25] The system according to

[23] , wherein the cells are collected from blood.

[0017] In addition, the present invention also relates to the following aspects. [1]A method for selecting neoantigens from a subject, comprising the steps of: (1) acquiring sequence data of normal cells and cancer cells from the subject; (2) identifying genes having cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; and (3) identifying peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database, wherein the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0018] [2]The method according to [1], wherein the sequence data is whole exome sequence data. [3]The method according to [1] or [2], wherein the MHC presented peptide database includes a list of wild-type MHC presented peptides according to each MHC class and / or each organ type. [4]The method according to [1], wherein the MHC presented peptide database includes a list of wild-type MHC presented peptides. [5]The method according to any one of [1] to [4], wherein step (3) is a step of identifying peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from the MHC presented peptide database according to each MHC class and / or each organ type. [6] The method according to any one of [1] to [5], wherein the amino acid mutation caused by the gene mutation is a single amino acid mutation. [7] The method according to any one of [1] to [6], wherein the peptides based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation are scored based on their respective MHC presentation frequencies in the MHC-presented peptide database.

[0019] [8] The method according to [7], wherein the peptides based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation are identified based on the scoring. [9] The method according to any one of [1] to [8], wherein the MHC-presented peptide database is created by an MHC-presented peptide analysis method, which includes the following steps: obtaining MHC-peptide complexes from cells; obtaining peptides from the complexes; and identifying the amino acid sequences of the peptides by mass spectrometry.

[10] The method according to any one of [1] to [9], wherein the cells are from a subject or a non-subject.

[11] The method according to [1] to [9], wherein the cells are collected from blood.

[12] The method according to any one of [1] to [9], wherein the MHC is MHC class I and / or class II.

[0020]

[13] The method according to any one of [1] to

[12] , wherein the MHC is human leukocyte antigen (HLA).

[14] The method according to any one of [1] to

[13] , further comprising the following steps: introducing the amino acid mutation caused by the gene mutation into the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation, wherein the peptide has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation; and / or determining the gene sequence encoding the peptide into which the amino acid mutation has been introduced.

[15] The method according to

[14] , further comprising the following steps: selecting a nucleic acid composed of the peptide into which the amino acid mutation has been introduced and / or the base sequence into which the gene mutation has been introduced as a neoantigen from a subject.

[0021]

[16] A method for producing a peptide containing a neoantigen from a subject, comprising the following steps: adding 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of the neoantigen from a subject selected by the method of [1] to

[15] , wherein the number of amino acids contained in the neoantigen to which the amino acid has been added is 40 or less.

[17] A peptide obtained by the method of

[16] .

[18] A method for producing a nucleic acid containing a neoantigen from a subject, comprising the step of adding more than 0 arbitrary bases to the 5'-end and / or 3'-end of a nucleic acid containing a neoantigen from a subject selected by the method of [1] to

[15] .

[19] A nucleic acid containing a neoantigen from a subject, wherein the neoantigen is selected by the method of

[15] .

[20] According to the method of

[18] , further comprising the step of producing an mRNA containing a nucleic acid containing a neoantigen from a subject.

[21] An mRNA of a nucleic acid containing a neoantigen from a subject, wherein the neoantigen is selected by the method of

[15] .

[22] A vaccine composition comprising the peptide of

[17] , the nucleic acid of

[19] or the mRNA of

[21] .

[0022]

[23] A system for selecting a neoantigen from a subject, comprising: (1) a sequence data acquisition unit that acquires sequence data of normal cells and cancer cells from a subject; (2) a gene identification unit that identifies a gene having a cancer cell-specific gene mutation by comparing the sequence data of the normal cells and cancer cells with each other; and (3) a peptide identification unit that identifies a peptide based on a wild-type gene corresponding to the gene having the cancer cell-specific gene mutation from a major histocompatibility complex (MHC) presented peptide database, wherein the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[24] The system according to

[23] , wherein the cells are from a subject or a non-subject.

[25] The system according to

[23] or

[24] , wherein the cells are collected from blood. Effects of the Invention

[0023] According to the present invention, it is possible to select a neoantigen from a subject. In particular, according to the present invention, by combining the existing in silico method and the MS method, it is possible to simultaneously obtain the advantages of high sensitivity and simplicity of the former and the high specificity of the latter. That is, according to the present invention, it is possible to select a neoantigen from a subject with high sensitivity, high specificity and simplicity. In addition, in other words, according to the present invention, it is possible to select a neoantigen from a subject efficiently, at low cost and quickly. In addition, in other words, according to the present invention, it is possible to select a neoantigen from a subject accurately, simply and quickly.

[0024] For some cancers, it is extremely difficult to collect tumor biopsy samples from organs and isolate cells. According to the present invention, for sequence analysis for identifying cancer cell-specific gene mutations, only a small number of cells need to be obtained from cancer and control normal tissues. The cells for MS analysis, i.e., for creating a surrogate immunopeptidome, can be cells obtained from tissues other than the cancer tissue of the subject. For example, they can be cells from blood, and the collection and isolation of such cells are generally simple and rapid. That is, the surrogate immunopeptidome can be created from the subject's own blood cells, third-party blood cells, third-party cancer cells, etc., but is not limited thereto. In the present invention, by comparing the sequence data of cells collected from the cancer biopsy tissue and tissues other than the cancer tissue of the subject, genes with cancer cell-specific gene mutations are identified, and the wild-type gene corresponding to the gene is compared with the surrogate immunopeptidome, MHC-presented peptides based on the gene are screened, and the gene mutation is introduced into the peptide, thereby achieving high-sensitivity, high-specificity, and rapid and simple selection of neoantigens from the subject. This method is sometimes denoted as NESSIE (Neoantigen Selection using Surrogate Immunopeptidome) in this specification. Generally, MS analysis, including the creation of an immunopeptidome, requires a relatively large number of cells, and cells from blood can be collected in large quantities. This is a great advantage, for example, in cases where it is very necessary to obtain normal cells from the subject patient, such as when dealing with minor histocompatibility antigens or HLA class II. In addition, even if an insufficient number of cells cannot be collected, a large amount of samples can be obtained from a small number of cells by amplifying the cells from blood with EBV virus. In addition, as long as a relatively large number of cells can be collected for MS analysis, including the creation of an immunopeptidome, the collected cells are not limited to cells from blood. According to the present invention, as long as the MHC matches, any surrogate immunopeptidome can be used to determine neoantigens of MHC class I and class II. For example, when using a surrogate immunopeptidome from the subject's own blood cells (which have been immortalized), not only can neoantigens of HLA class II be determined, but also neoantigens of both HLA class I and class II can be determined. Even in the case of using a surrogate immunopeptidome from the tumor or normal tissue of another person, if the HLA matches, neoantigens of both HLA class I and class II can also be determined.

[0025] In addition, according to the present invention, based on the selected neoantigens from a subject, peptides and / or nucleic acids containing the neoantigens can be produced. Moreover, according to the present invention, based on the selected neoantigens from a subject, peptides and / or nucleic acids containing the neoantigens can be produced and used for personalized medicine of the subject. Furthermore, according to the present invention, based on the selected neoantigens from a subject, mRNA containing the neoantigens can be produced and used for personalized medicine of the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Concept of the neoantigen selection method of the present invention is shown. Figure 2 Principle of the neoantigen selection method of the present invention is shown. Figure 3 Features of the neoantigen selection method of the present invention are shown. Figure 4 Neoantigens from a mouse colorectal cancer cell line selected by the neoantigen selection method of the present invention are shown. Figure 5 Antitumor effects brought about by administering neoantigens from a mouse colorectal cancer cell line selected by the neoantigen selection method of the present invention are shown.

[0027] Figure 6 Antitumor effects brought about by combining neoantigens from a mouse colorectal cancer cell line selected by the neoantigen selection method of the present invention and anti-PD-1 antibody are shown. Figure 7 Induction of CD8-positive T cells against neoantigens from a mouse colorectal cancer cell line selected by the neoantigen selection method of the present invention is shown. Figure 8 Neoantigens from a human colorectal cancer tissue (CRC) panel selected by the neoantigen selection method of the present invention are shown. Figure 8 A shows the number of neoantigens (upper) and the number of missense mutations carried by the patient (lower) in each CRC case of HLA-A24. Figure 8 B shows the number of neoantigens (upper) and the number of missense mutations carried by the patient (lower) in each CRC case of HLA-A02.

[0028] Figure 9 Tumor infiltrating lymphocyte (TIL) responses against neoantigens from a human colorectal cancer tissue (CRC) panel selected by the neoantigen selection method of the present invention are shown. Figure 9 A shows the TIL response against the TUBB-RAF9 neoantigen in the CRC111 case (HLA-A24). Figure 9 B shows the TIL response against the STT3A-KRV9 neoantigen in the CRC135 case (HLA-A02). Figure 10 Shows the reaction of HLA class II-presented neoantigens selected by the neoantigen selection method of the present invention with CD4-positive T cells. Figure 10 A shows the reaction of the SCO1 neoantigen of the CRC66 case (HLA-DR ＊ 04:06) with CD4-positive T cell clones from the patient. Figure 10 B shows the reaction of the CD4-positive T cell clone with the neoantigens presented by DRB1 ＊ 04:06. Figure 11 Shows an increase in neoantigen-reactive CD8+ cells in the spleen by combining a vaccine against neoantigens selected by the neoantigen selection method of the present invention and ICB. Figure 12 Shows an overview of the neoantigen selection method of the present invention using NESSIE. Figure 13 Shows an overview of the selection of HLA class II neoantigens using autologous peripheral blood. Figure 14 Shows that patient peripheral blood CD4+ T cells strongly react with the detected neoantigens.

[0029] Unless otherwise defined in this specification, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, and other publications and information cited in this specification are hereby incorporated by reference in their entirety. Detailed Description

[0030] [Method for Selecting Neoantigens from a Subject] One aspect of the present invention relates to a method for selecting neoantigens from a subject (sometimes denoted as "the neoantigen selection method of the present invention"), comprising the following steps: (1) obtaining sequence data of normal cells and cancer cells from the subject; (2) identifying genes having cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; and (3) identifying peptides based on wild-type genes corresponding to the genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database, wherein the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0031] Another aspect of the present invention relates to a method for selecting neoantigens from a subject (sometimes also denoted as NESSIE (Neoantigen Selection using Surrogate Immunopeptidome)), comprising the following steps: (1) obtaining sequence data of cells from a subject; (2) using an MHC-presented peptide database to compare the sequence data of cells from the subject or cells other than the subject with each other, thereby identifying genes with cancer cell-specific gene mutations; and (3) identifying peptides based on wild-type genes corresponding to genes with cancer cell-specific gene mutations from a major histocompatibility complex (MHC class) presented peptide database, wherein MHC is MHC class I and II, the cells can be from blood, cancer cells expressing neoantigens can be from an organ different from the organ from which the cells are collected, the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene with the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0032] In the present invention, a "subject" refers to any animal having a major histocompatibility complex (MHC) in its body cells and having cancer cells in its body. For example, the subject is a vertebrate such as a mammal, a bird, a reptile, etc. For example, the subject is a mammal such as a human, a mouse, a rat, etc. Preferably, the subject is a human. In the present invention, "cancer" refers to any tumor that has an adverse effect on a subject by means of infiltration, metastasis to surrounding tissues, etc. For example, cancers include adrenocortical carcinoma, anal cancer, cholangiocarcinoma, bladder cancer, breast cancer, ovarian cancer, cervical cancer, leukemia (acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, etc.), myeloproliferative neoplasms (chronic myeloid leukemia, chronic neutrophilic leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic eosinophilic leukemia, etc.), colorectal cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, Hodgkin's disease, head and neck cancer, lip and oral cavity cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, malignant lymphoma (non-Hodgkin lymphoma, etc.), melanoma, mesothelioma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, gastric cancer, testicular cancer, brain tumor, neuroendocrine tumor, neuroendocrine carcinoma (small cell carcinoma, etc.), etc. In the present invention, "cancer" includes blood cancer. For example, blood cancer includes leukemia (acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, etc.), myeloproliferative neoplasms (chronic myeloid leukemia, chronic neutrophilic leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic eosinophilic leukemia, etc.). In the present invention, a "tumor" refers to a cell mass formed by cells that autonomously proliferate excessively in violation of the regulation in the body.

[0033] In the present invention, a "neoantigen" refers to any mutant protein that does not exist in normal cells but specifically exists only in cancer cells, a peptide derived from the protein (the peptide has a mutation that specifically exists only in cancer cells), or a nucleic acid composed of the base sequence encoding the protein or the peptide, and particularly refers to a peptide derived from the protein or a nucleic acid composed of the base sequence encoding the peptide. The gene mutations that bring about neoantigens only account for a small part of the large number of gene mutations in cancer cells. In addition, since these gene mutations vary among each subject, the neoantigens also vary among each subject.

[0034] Cancer cells present neoantigens to CD8-positive T cells via MHC class I molecules on the cancer cell surface. CD8-positive T cells recognize and activate neoantigens through TCR, thereby distinguishing normal cells from cancer cells and specifically eliminating cancer cells. That is, by inducing CD8-positive T cells to recognize neoantigens through TCR, the immune response against cancer can be specifically enhanced, thus achieving highly specific immunotherapy. In particular, since neoantigens vary among each subject, by inducing CD8-positive T cells to recognize neoantigens through TCR, the immune response against cancer can be specifically enhanced for each subject, thereby achieving highly specific immunotherapy personalized for each subject. Moreover, neoantigens have mutations that do not exist in normal cells but specifically exist only in cancer cells. Since they do not naturally exist in the body, immune tolerance in the thymus can be avoided. Therefore, it is expected that a strong immune response can be induced by inducing CD8-positive T cells to recognize neoantigens through TCR.

[0035] In the present invention, a "normal cell" refers to any cell that proliferates according to the regulation in the body and thus does not cause cancer in the subject. In the present invention, a "cancer cell" refers to any cell that autonomously proliferates excessively in violation of the regulation in the body and thus causes cancer in the subject. Normal cells and cancer cells can be derived from any tissue and organ of a subject. For example, normal cells and cancer cells are derived from the digestive system (esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, etc.), circulatory system (blood, blood vessels, lymphatic vessels, lymph nodes, spleen, thymus, etc.), respiratory system (trachea, bronchi, lungs, etc.), urinary system (kidneys, ureters, bladder, urethra, etc.), reproductive system (testes, prostate, ovaries, uterus, etc.), endocrine system (pituitary gland, thyroid gland, adrenal gland, pancreas, etc.), nervous system (brain, spinal cord, etc.) or musculoskeletal system (bones, cartilage, skeletal muscles, etc.). Normal cells can be derived from organs other than the cancerous organ of the subject. In one embodiment of the present invention, normal cells and / or cancer cells can be derived from blood.

[0036] In the present invention, "sequence data" refers to the base sequence of nucleotides that make up any nucleic acid. In the present invention, "comparing sequence data with each other" means comparing two or more types of sequence data so as to be able to determine regions of similarity in the base sequences of nucleotides. For example, the base sequences of nucleotides can be represented as rows of a matrix, with gaps inserted so that sequences of the same or similar properties are aligned in the same column.

[0037] In the present invention, "cancer cell-specific gene mutation" refers to any gene mutation that occurs only in the cancer cells of a subject and does not occur in the normal cells of the subject. In the present invention, "wild-type gene corresponding to a gene having a cancer cell-specific gene mutation" refers to the gene before the cancer cell-specific gene mutation occurs. In the present invention, "peptide based on the wild-type gene corresponding to a gene having a cancer cell-specific gene mutation" refers to a peptide encoded by the base sequence in the wild-type gene corresponding to the gene having a cancer cell-specific gene mutation.

[0038] In the present invention, "major histocompatibility complex (MHC)" refers to a cell transmembrane glycoprotein that is present on the cell surface and presents peptides of proteins from inside and outside the cell. MHC is mainly divided into class I and class II. Most nucleated cells in the body have MHC class I molecules, and peptides of proteins from inside the cell can be presented to CD8-positive T cells via MHC class I molecules. On the other hand, antigen-presenting cells have MHC class II molecules, and peptides of proteins from outside the cell can be presented to CD4-positive T cells via MHC class II molecules.

[0039] In the present invention, the "major histocompatibility complex (MHC)-presented peptide database" refers to a database containing the amino acid sequences of each MHC-presented peptide created by an MHC-presented peptide analysis method, and the MHC-presented peptide analysis method includes the following steps: obtaining an MHC-peptide complex from any cell; obtaining a peptide from the complex; and identifying the amino acid sequence of the peptide by mass spectrometry. The MHC-presented peptide database includes the amino acid sequences of peptides that are indeed presented by MHC in vivo. In addition, the content of this peptide is abundant in vivo. Thus, CD8-positive T cells have a tendency to be easily induced to recognize this peptide in vivo through TCR. In the present invention, "missense mutation" refers to the substitution of a base within a codon resulting in the replacement of the original amino acid (wild-type amino acid) with a different amino acid (mutant amino acid).

[0040] In the present invention, the "position of the amino acid mutation caused by a cancer cell-specific gene mutation" refers to the position of the amino acid encoded by the base sequence present at the position where the gene mutation has occurred in the gene having the cancer cell-specific gene mutation. In the present invention, the "wild-type amino acid corresponding to the position of the amino acid mutation caused by a cancer cell-specific gene mutation" refers to the amino acid encoded by the base sequence present at the position where the gene mutation has occurred in the gene before the occurrence of the cancer cell-specific gene mutation. In the present invention, "the peptide based on the wild-type gene corresponding to the gene having a cancer cell-specific gene mutation has the wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation" means that the peptide encoded by the base sequence in the wild-type gene corresponding to the gene having a cancer cell-specific gene mutation has the amino acid encoded by the base sequence present at the position where the gene mutation has occurred in the gene before the occurrence of the cancer cell-specific gene mutation.

[0041] In the present invention, the "peptide based on the gene having a cancer cell-specific gene mutation (mutant peptide)" and the "peptide based on the wild-type gene corresponding to the gene having a cancer cell-specific gene mutation (wild-type peptide)" can be represented as follows, respectively. Mutant peptide: (X) m -Mt-(X) n Wild-type peptide: (X) m -Wt-(X) n wherein, Mt is the mutant amino acid, "Wt" is the wild-type amino acid corresponding to the mutant amino acid, X is independently any wild-type amino acid, m and n are independently any integers of 0 or more. In addition, (X) m is located on the N-terminal side, (X) nLocated on the C-terminal side. For example, m and n are each independently any integer from 0 to 20, 0 to 15, 0 to 10, or 0 to 5, and the sum of m and n is any integer of 40 or less, 30 or less, 20 or less, or 10 or less.

[0042] In one embodiment, the sequence data is whole exome sequence data. In the present invention, "whole exome sequence data" refers to information on base sequences obtained by selectively obtaining exon regions encoding proteins in the genome of any cell and determining the sequences of these regions using a next-generation sequencer. By using whole exome sequencing data, it is possible to efficiently explore gene mutations related to cancer.

[0043] In one embodiment, the MHC-presented peptide database includes a list of wild-type MHC-presented peptides for each MHC class and / or each organ type. In the present invention, "including a list of wild-type MHC-presented peptides for each MHC class and / or each organ type" means classifying wild-type MHC-presented peptides, i.e., peptides actually presented by MHC in vivo, according to the MHC class and / or organ type of various peptide sources and including them in list form. In another embodiment, the MHC-presented peptide database includes a list of wild-type MHC-presented peptides for both MHC class I and MHC class II.

[0044] In one embodiment, step (3) is a step of identifying, from the MHC-presented peptide database, a peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation for each MHC class and / or each organ type. In the present invention, "identifying from the MHC-presented peptide database for each MHC class and / or each organ type" means, after determining the MHC class of the subject and / or the type of the cancerous organ, identifying a peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation from the MHC-presented peptide database so that the MHC class and / or organ type of the subject is consistent with the MHC class and / or organ type in the MHC-presented peptide database. To determine the MHC class of a subject, any cell of the subject can be used. For example, the cell can be from an organ other than the cancerous organ of the subject. In another embodiment, step (3) is a step of identifying a peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation from a database of MHC-presented peptide databases including MHC class I and MHC class II, wherein the cell can be from blood.

[0045] In one embodiment, the amino acid mutation caused by the gene mutation is a single amino acid mutation. In one embodiment, peptides based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation are scored based on their respective MHC presentation frequencies in the MHC-presented peptide database. In the present invention, "scoring based on their respective MHC presentation frequencies in the MHC-presented peptide database" means that for peptides based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation (i.e., the above-mentioned "wild-type peptides") and the amino acid sequences on the N-terminal side and C-terminal side thereof (i.e., the above-mentioned "(X) m " and "(X) n "), it is investigated whether they are registered in the MHC-presented peptide database. If the peptide is registered in the MHC-presented peptide database, it is regarded as a "hit". If the peptide has been registered in the MHC-presented peptide database once, it is regarded as a "single hit". For each hit, at most one point is added to the score of the peptide. It should be noted that, conversely, if the peptide is not registered in the MHC-presented peptide database at all, the score of the peptide is 0 points.

[0046] In one embodiment, peptides based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation are identified based on the scoring. In the present invention, "identifying based on the scoring" means that when the score is a value greater than 0 points, for peptides registered in the MHC-presented peptide database, each amino acid sequence and the calculated score are listed. From the perspective of the in vivo effectiveness of neoantigens, the score is preferably 1 point or more, more preferably 2 points or more, and further preferably 3 points or more.

[0047] In one embodiment, the MHC-presented peptide database is created by an MHC-presented peptide analysis method, which includes the following steps: obtaining an MHC-peptide complex from cells; obtaining a peptide from the complex; and identifying the amino acid sequence of the peptide by mass spectrometry. In the present invention, "MHC-peptide complex" refers to an MHC that presents peptides of proteins from inside and outside cells. In one embodiment, the MHC is MHC class I and / or class II.

[0048] In one embodiment, the MHC is human leukocyte antigen (HLA). In the present invention, "human leukocyte antigen (HLA)" refers to the MHC of humans. HLA is mainly divided into class I and class II. Class I is further divided into HLA-A, HLA-B, HLA-C, etc., and class II is further divided into HLA-DR, HLA-DQ, HLA-DP, etc.

[0049] In one embodiment, the method for selecting neoantigens of the present invention further comprises the following steps: identifying a base sequence encoding a peptide based on a wild-type gene corresponding to a gene having a cancer cell-specific gene mutation. In one embodiment, the method for selecting neoantigens of the present invention further comprises the following steps: introducing an amino acid mutation caused by the gene mutation into a peptide based on a wild-type gene corresponding to a gene having the cancer cell-specific gene mutation; and / or introducing the gene mutation into the base sequence encoding the peptide.

[0050] In the present invention, "introducing an amino acid mutation caused by a cancer cell-specific gene mutation" means replacing "Wt" with "Mt" in a wild-type peptide already registered in the MHC-presented peptide database. In the present invention, "introducing a cancer cell-specific gene mutation" means replacing the base sequence corresponding to "Wt" with the base sequence corresponding to "Mt" in the base sequence encoding a wild-type peptide already registered in the MHC-presented peptide database. In one embodiment, the method for selecting neoantigens of the present invention further comprises the following steps: selecting a nucleic acid composed of a peptide having the introduced amino acid mutation and / or a base sequence having the introduced gene mutation as a neoantigen from a subject.

[0051] Neoantigens from a subject selected by the method for selecting neoantigens of the present invention can be applied to immunotherapies such as cancer vaccine therapy and genetically modified T cell therapy. In cancer vaccine therapy, the immune response of the subject against cancer is activated by administering neoantigens to the subject. In genetically modified T cell therapy, T cells collected from the subject's body are genetically modified to express a TCR gene for recognizing a neoantigen presented by MHC or a gene of a fusion protein of an antigen-binding site of an antibody for recognizing a neoantigen presented by MHC and an activation domain of TCR, and then cultured and returned to the subject, thereby activating the immune response of the subject against cancer. Any one of genetically modified T cell therapy (TCR-T cell therapy) and genetically modified T cell therapy (CAR-T cell therapy) can be used. Among them, genetically modified T cell therapy (TCR-T cell therapy) uses a cell (TCR-T cell) that has been genetically modified to express a TCR gene for recognizing a neoantigen presented by MHC; genetically modified T cell therapy (CAR-T cell therapy) uses a cell (CAR-T cell) that has been genetically modified to express a gene of a fusion protein of an antigen-binding site of an antibody for recognizing a neoantigen presented by MHC and an activation domain of TCR.

[0052] In addition, neoantigens from a subject selected by the neoantigen selection method of the present invention can also be used in combination with immune checkpoint therapy. In immune checkpoint therapy, an immune checkpoint inhibitor is bound to a specific molecule on the surface of cancer cells that inhibits the immune response to block the inhibition of the immune response, thereby activating the subject's immune response against cancer. When applied to this immunotherapy, the neoantigens from a subject selected by the neoantigen selection method of the present invention can be in either the form of a peptide or a nucleic acid consisting of a base sequence encoding the peptide. In addition, amino acids can be added to the N-terminus and / or C-terminus of the neoantigens from a subject selected by the neoantigen selection method of the present invention. In addition, nucleic acids can be added to the 5'-end and / or 3'-end of the neoantigens from a subject selected by the neoantigen selection method of the present invention. For example, in cancer vaccine therapy, the neoantigens from a subject selected by the neoantigen selection method of the present invention can be used in various forms, such as peptide vaccines, mRNA vaccines, DNA vaccines, viral vector vaccines, virus-like particle vaccines, plasmid DNA vaccines, bacterial vector vaccines, dendritic cell vaccines, and the like.

[0053] In peptide vaccines, the immune response of the subject against cancer is activated by administering a peptide containing the neoantigen to the subject. In mRNA vaccines, an artificial copy of mRNA containing a base sequence encoding the neoantigen is administered to the subject, so that the neoantigen is expressed in vivo, thereby activating the subject's immune response against cancer. In DNA vaccines, an artificial copy of DNA containing a base sequence encoding the neoantigen is administered to the subject, so that the neoantigen is expressed in vivo, thereby activating the subject's immune response against cancer. In viral vector vaccines, a virus containing a nucleic acid consisting of a base sequence encoding the neoantigen is administered to the subject, so that the neoantigen is expressed in vivo, thereby activating the subject's immune response against cancer. In virus-like particle vaccines, virus-like particles (having an external structure similar to that of a virus but not having genetic information) containing a nucleic acid consisting of a base sequence encoding the neoantigen are administered to the subject, so that the neoantigen is expressed in vivo, thereby activating the subject's immune response against cancer. In plasmid DNA vaccines, plasmid DNA containing a base sequence encoding the neoantigen is administered to the subject, so that the neoantigen is expressed in vivo, thereby activating the subject's immune response against cancer. In bacterial vector vaccines, bacteria containing a nucleic acid consisting of a base sequence encoding the neoantigen are administered to the subject, so that the neoantigen is expressed in vivo, thereby activating the subject's immune response against cancer. In dendritic cell vaccines, a peptide containing the neoantigen is bound to dendritic cells in vitro, and the dendritic cells are administered to the subject to activate T cells under conditions closer to the physiological environment, thereby activating the subject's immune response against cancer.

[0054] In addition, the present invention may also be a nucleic acid containing neoantigens from a subject selected by the neoantigen selection method of the present invention. The nucleic acid preferably contains a tumor-specific framework, tumor-related genes, and / or tumor-specific missense mutations. Further, in another aspect, the present invention may be an mRNA containing neoantigens from a subject selected by the neoantigen selection method of the present invention. The mRNA preferably contains a tumor-specific framework, tumor-related genes, and / or tumor-specific missense mutations. Preferably, the neoantigens from a subject selected by the neoantigen selection method of the present invention are used in the form of an mRNA vaccine. Additionally, from the perspective of in vivo stability, it is preferred to introduce modified nucleosides such as pseudouridine and 2'-O-methylated nucleosides into the mRNA. The above features related to the neoantigens from a subject selected by the neoantigen selection method of the present invention are equally applicable to substances produced by the following neoantigen production method of the present invention, the neoantigen peptide production method of the present invention, the neoantigen nucleic acid production method of the present invention, the method for producing a substance containing a neoantigen peptide of the present invention, and the method for producing a substance containing a neoantigen nucleic acid of the present invention.

[0055] Method for producing neoantigens from a subject Another aspect of the present invention relates to a method for producing neoantigens from a subject (sometimes also denoted as "the neoantigen production method of the present invention"), comprising the following steps: (1) obtaining sequence data of normal cells and cancer cells from a subject; (2) identifying genes having cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; (3) identifying peptides based on the wild-type genes corresponding to the genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presentation peptide database; (4) identifying the base sequences encoding the peptides based on the wild-type genes corresponding to the genes having cancer cell-specific gene mutations; and (5) introducing amino acid mutations caused by the gene mutations into the peptides based on the wild-type genes corresponding to the genes having cancer cell-specific gene mutations; and / or introducing the gene mutations into the base sequences encoding the peptides, wherein the cancer cell-specific gene mutations are missense mutations, and the peptides based on the wild-type genes corresponding to the genes having cancer cell-specific gene mutations have wild-type amino acids corresponding to the positions of the amino acid mutations caused by the gene mutations.

[0056] Another aspect of the present invention relates to a method for producing neoantigens from a subject (sometimes referred to as the method for producing neoantigens using NESSIE of the present invention), comprising the following steps: (1) obtaining sequence data of cells from a subject; (2) identifying genes with cancer cell-specific gene mutations by comparing the sequence data of the cells with the sequence data of cells from the subject or from outside the subject; (3) identifying peptides based on the wild-type genes corresponding to the genes with the cancer cell-specific gene mutations from a database including a major histocompatibility complex presentation peptide database; (4) identifying the base sequences encoding the peptides based on the wild-type genes corresponding to the genes with the cancer cell-specific gene mutations; and (5) introducing amino acid mutations caused by the gene mutations into the peptides based on the wild-type genes corresponding to the genes with the cancer cell-specific gene mutations; and / or introducing the gene mutations into the base sequences encoding the peptides, wherein the cells can be from blood, the cancer cells expressing the neoantigens can be from an organ different from the organ from which the cells are derived, the MHC can be MHC class I and MHC class II, the cancer cell-specific gene mutations are missense mutations, and the peptides based on the wild-type genes corresponding to the genes with the cancer cell-specific gene mutations have wild-type amino acids corresponding to the positions of the amino acid mutations caused by the gene mutations.

[0057] In one embodiment, the method for producing neoantigens of the present invention further comprises the following step: selecting a nucleic acid composed of the peptide into which the amino acid mutations are introduced and / or the base sequence into which the gene mutations are introduced as a neoantigen from the subject. In one embodiment, the method for producing neoantigens of the present invention comprises the following step: adding 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of the neoantigen from the subject, wherein the number of amino acids contained in the neoantigen to which the amino acids are added is 40 or less.

[0058] In the present invention, "adding 0 or more arbitrary amino acids to the N-terminus and / or C-terminus" means, for example, adding 0 to 40, 0 to 25, 0 to 20, 0 to 15, 0 to 10, or 0 to 5 arbitrary amino acids to the N-terminus and / or C-terminus. The arbitrary amino acids can be amino acids based on the wild-type genes corresponding to the genes with cancer cell-specific gene mutations, or can be arbitrarily selected amino acids. In the present invention, "the number of amino acids contained in the neoantigen to which the amino acids are added is 40 or less" means, for example, the number of amino acids contained in the neoantigen to which the amino acids are added is 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, or 15 or less.

[0059] For a neoantigen presented by MHC class I, one or more arbitrary amino acids can be added only to the N-terminus or C-terminus of the neoantigen from the subject. For a neoantigen presented by MHC class II, one or more arbitrary amino acids can be added only to the N-terminus or C-terminus of the neoantigen from the subject. In one embodiment, the method for producing a neoantigen of the present invention comprises the following steps: adding zero or more arbitrary bases to the 5'-end and / or 3'-end of the neoantigen from the subject. Additionally, the number of bases contained in the neoantigen to which the base has been added can be 900 or less.

[0060] In the present invention, "adding zero or more arbitrary bases to the 5'-end and / or 3'-end" means, for example, adding 0 to 900, 0 to 750, 0 to 600, 0 to 450, 0 to 30, or 0 to 150 arbitrary bases to the 5'-end and / or 3'-end. The arbitrary bases can be bases based on the wild-type gene corresponding to the gene having a cancer cell-specific gene mutation, or can be arbitrarily selected bases. In the present invention, "the number of bases contained in the neoantigen to which the base has been added is 900 or less" means, for example, the number of bases contained in the neoantigen to which the base has been added is 900 or less, 300 or less, 150 or less, 75 or less, 60 or less, or 45 or less.

[0061] Regarding the neoantigen presented by MHC class I, three or more arbitrary bases can be added only to the 5'-end or 3'-end of the neoantigen from the subject. Regarding the neoantigen presented by MHC class II, three or more arbitrary bases can be added only to the 5'-end or 3'-end of the neoantigen from the subject. From the perspective of the in vivo effectiveness of the neoantigen, preferably, in the neoantigen to which amino acids have been added, 10 to 14 amino acids are present on the N-terminal side and C-terminal side of "Mt", and the number of amino acids contained in the neoantigen to which amino acids have been added is 23 to 27. From the perspective of the in vivo effectiveness of the neoantigen, preferably, in the neoantigen to which bases have been added, 30 to 42 bases are present on the 5'-end side and 3'-end side of the base sequence corresponding to "Mt", and the number of bases contained in the neoantigen to which bases have been added is 69 to 81.

[0062] Method for producing a neoantigen peptide from a subject Another aspect of the present invention relates to a method for producing neoantigens from a subject (sometimes denoted as "the method for producing neoantigen peptides of the present invention"), comprising the following steps: (1) obtaining sequence data of normal cells and cancer cells from the subject; (2) identifying genes with cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; (3) identifying peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database; and (4) introducing amino acid mutations caused by the gene mutations into the peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations, wherein the cancer cell-specific gene mutations are missense mutations, and the peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations have wild-type amino acids corresponding to the positions of the amino acid mutations caused by the gene mutations.

[0063] Yet another aspect of the present invention relates to a method for producing neoantigens from a subject (sometimes referred to as "the method for producing neoantigen peptides using NESSIE of the present invention"), comprising the following steps: (1) obtaining sequence data of cells from the subject; (2) identifying genes with cancer cell-specific gene mutations by comparing the sequence data of the cells with sequence data of cells from the subject or from outside the subject; (3) identifying peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations from an MHC presented peptide database; and (4) introducing amino acid mutations caused by the gene mutations into the peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations, wherein the cells can be from blood, the cancer cells expressing neoantigens can be from an organ different from the organ from which the cells are derived, the MHC can be MHC class I and MHC class II, the cancer cell-specific gene mutations are missense mutations, and the peptides based on wild-type genes corresponding to the genes with cancer cell-specific gene mutations have wild-type amino acids corresponding to the positions of the amino acid mutations caused by the gene mutations.

[0064] In one embodiment, the method for producing neoantigen peptides of the present invention further comprises the following step: selecting the peptides introduced with the amino acid mutations as neoantigens from the subject. In one embodiment, the method for producing neoantigen peptides of the present invention comprises the following steps: adding 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of the neoantigens from the subject produced by the method for producing neoantigen peptides of the present invention, wherein the number of amino acids contained in the neoantigens added with the amino acids is 40 or less.

[0065] Method for producing neoantigen nucleic acids from a subject Another aspect of the present invention relates to a method for producing neoantigens from a subject (sometimes denoted as "the method for producing neoantigen nucleic acids of the present invention"), comprising the following steps: (1) obtaining sequence data of normal cells and cancer cells from the subject; (2) identifying genes with cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; (3) identifying peptides based on the wild-type genes corresponding to the genes with cancer cell-specific gene mutations from the major histocompatibility complex (MHC) presented peptide database; (4) identifying the base sequences encoding the peptides based on the wild-type genes corresponding to the genes with cancer cell-specific gene mutations; and (5) introducing the gene mutation into the base sequences encoding the peptides based on the wild-type genes corresponding to the genes with cancer cell-specific gene mutations, wherein the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene with cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0066] Yet another aspect of the present invention relates to a method for producing neoantigens from a subject (sometimes denoted as the method for producing neoantigen nucleic acids using NESSIE of the present invention), comprising the following steps: (1) obtaining sequence data of cells from the subject; (2) identifying genes with cancer cell-specific gene mutations by comparing the sequence data of the cells with the sequence data of cells from the subject or from outside the subject; (3) identifying peptides based on the wild-type genes corresponding to the genes with cancer cell-specific gene mutations from the major histocompatibility complex (MHC) presented peptide database; (4) identifying the base sequences encoding the peptides based on the wild-type genes corresponding to the genes with cancer cell-specific gene mutations; and (5) introducing the gene mutation into the base sequences encoding the peptides based on the wild-type genes corresponding to the genes with cancer cell-specific gene mutations, wherein the cells can be from blood, the cancer cells expressing neoantigens can be from different organs, the MHC can be MHC class I and MHC class II, the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene with cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0067] In one embodiment, the method for producing neoantigen nucleic acids of the present invention further comprises the following step: selecting the nucleic acid composed of the base sequences introduced with the gene mutation as the neoantigen from the subject. In one embodiment, the method for producing a neoantigen nucleic acid of the present invention comprises the following steps: adding 0 or more arbitrary bases to the 5'-end and / or 3'-end of a neoantigen from a subject produced by the method for producing a neoantigen nucleic acid of the present invention. Additionally, the number of bases contained in the neoantigen to which the bases have been added may be 900 or less.

[0068] Method for producing a peptide comprising a neoantigen from a subject Another aspect of the present invention relates to a method for producing a peptide comprising a neoantigen from a subject (sometimes referred to as "the method for producing a neoantigen-containing peptide of the present invention"), which comprises the following steps: adding 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of a neoantigen from a subject selected by the neoantigen selection method of the present invention, wherein the number of amino acids contained in the neoantigen to which the amino acids have been added is 40 or less. In the present invention, a "nucleic acid peptide comprising a neoantigen from a subject" means a neoantigen from a subject to which 0 or more arbitrary amino acids have been added to the N-terminus and / or C-terminus, wherein the number of amino acids contained in the neoantigen to which the amino acids have been added is 40 or less.

[0069] Method for producing a nucleic acid comprising a neoantigen from a subject Another aspect of the present invention relates to a method for producing a nucleic acid comprising a neoantigen from a subject (sometimes referred to as "the method for producing a neoantigen-containing nucleic acid of the present invention"), which comprises the following steps: adding 0 or more arbitrary bases to the 5'-end and / or 3'-end of a neoantigen from a subject selected by the neoantigen selection method of the present invention. Additionally, the number of bases contained in the neoantigen to which the bases have been added may be 900 or less.

[0070] In the present invention, a "nucleic acid comprising a neoantigen from a subject" means a neoantigen from a subject to which 0 or more arbitrary bases have been added to the 5'-end and / or 3'-end. In one embodiment, the method for producing a neoantigen-containing nucleic acid of the present invention further comprises the following step: producing an mRNA comprising a nucleic acid containing a neoantigen from a subject.

[0071] System for selecting a neoantigen from a subject Another aspect of the present invention relates to a system for selecting neoantigens from a subject (sometimes denoted as "the neoantigen selection system of the present invention"), comprising: (1) a sequence data acquisition unit that acquires sequence data of normal cells and cancer cells from the subject; (2) a gene identification unit that identifies genes having cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; and (3) a peptide identification unit that identifies peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database, wherein the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0072] Another aspect of the present invention relates to a system for selecting neoantigens from a subject (sometimes denoted as "the neoantigen selection system using NESSIE of the present invention"), comprising: (1) a sequence data acquisition unit that acquires sequence data of cells from the subject; (2) a gene identification unit that identifies genes having cancer cell-specific gene mutations by comparing the sequence data of the cells with each other; and (3) a peptide identification unit that identifies peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database, wherein the cells can be from blood, the cancer cells expressing neoantigens can be from different organs, the MHC can be MHC class I and MHC class II, the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0073] In the present invention, the "sequence data acquisition unit" refers to the part that acquires sequence data of normal cells and cancer cells from the subject. The sequence data acquisition unit may include a sequencer. For example, the sequencer is a next-generation sequencer. In the present invention, the "gene identification unit" refers to the part that identifies genes having cancer cell-specific gene mutations by comparing the sequence data of normal cells and cancer cells with each other. The gene identification unit may include software for aligning (comparing) two or more sequence data to determine similar regions of the nucleotide base sequences. In the present invention, the "peptide identification unit" refers to the part that identifies peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from the MHC presented peptide database. The peptide identification unit may include software for aligning (comparing) the amino acid sequences of two or more peptides to determine similar regions of the peptide amino acid sequences.

[0074] System for producing neoantigens from a subject Another aspect of the present invention relates to a system for producing neoantigens from a subject (sometimes referred to as "the neoantigen production system of the present invention"), comprising: (1) a sequence data acquisition unit that acquires sequence data of normal cells and cancer cells from a subject; (2) a gene identification unit that identifies genes having cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; (3) a peptide identification unit that identifies peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database; (4) a base sequence identification unit that identifies base sequences encoding peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations; and (5) an amino acid mutation introduction unit that introduces amino acid mutations caused by the gene mutations into peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations; and / or introduces the gene mutations into the base sequences encoding the peptides, wherein the cancer cell-specific gene mutations are missense mutations, and the peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations have wild-type amino acids corresponding to the positions of the amino acid mutations caused by the gene mutations.

[0075] Yet another aspect of the present invention relates to a system for producing neoantigens from a subject (sometimes referred to as "the neoantigen production system using NESSIE of the present invention"), comprising: (1) a sequence data acquisition unit that acquires sequence data of cells from a subject; (2) a gene identification unit that identifies genes having cancer cell-specific gene mutations by comparing the sequence data of the cells with sequence data of cells from the subject or from outside the subject; (3) a peptide identification unit that identifies peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database; (4) a base sequence identification unit that identifies base sequences encoding peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations; and (5) an amino acid mutation introduction unit that introduces amino acid mutations caused by the gene mutations into peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations; and / or introduces the gene mutations into the base sequences encoding the peptides, wherein the cells can be from blood, the cancer cells expressing the neoantigens can be from different organs, the MHC can be MHC class I and MHC class II, the cancer cell-specific gene mutations are missense mutations, and the peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations have wild-type amino acids corresponding to the positions of the amino acid mutations caused by the gene mutations.

[0076] In the present invention, the "base sequence identification unit" refers to a part that identifies a base sequence encoding a peptide based on a wild-type gene corresponding to a gene having a cancer cell-specific gene mutation. The base sequence identification unit may include a sequencer. In the present invention, the "amino acid mutation introduction unit" refers to a part that introduces an amino acid mutation caused by the gene mutation into a peptide based on a wild-type gene corresponding to a gene having a cancer cell-specific gene mutation. The amino acid mutation introduction unit may include software that replaces "Wt" with "Mt" in a wild-type peptide registered in the MHC-presented peptide database. In the present invention, the "gene mutation introduction unit" refers to a part that introduces the gene mutation into a base sequence encoding a peptide based on a wild-type gene corresponding to a gene having a cancer cell-specific gene mutation. The gene mutation introduction unit refers to software that replaces a base sequence corresponding to "Wt" with a base sequence corresponding to "Mt" in a base sequence encoding a wild-type peptide registered in the MHC-presented peptide database.

[0077] In one aspect, the neoantigen production system of the present invention further includes: a neoantigen selection unit that selects a nucleic acid composed of a peptide into which the amino acid mutation has been introduced and / or a base sequence into which the gene mutation has been introduced as a neoantigen from a subject. In the present invention, the "neoantigen selection unit" refers to a part that selects a nucleic acid composed of a peptide into which the amino acid mutation has been introduced and / or a base sequence into which the gene mutation has been introduced as a neoantigen from a subject. The neoantigen selection unit may include software that selects a nucleic acid composed of a peptide into which the amino acid mutation has been introduced and / or a base sequence into which the gene mutation has been introduced as a neoantigen from a subject.

[0078] In one aspect, the neoantigen production system of the present invention includes: an amino acid addition unit that adds 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of a neoantigen from a subject produced by the neoantigen production system of the present invention, wherein the number of amino acids contained in the neoantigen to which the amino acid has been added is 40 or less. In the present invention, the "amino acid addition unit" refers to a part that adds 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of a neoantigen from a subject produced by the neoantigen production system of the present invention. The amino acid addition unit may include a peptide synthesis device.

[0079] In one aspect, the neoantigen production system of the present invention includes: a base addition unit that adds 0 or more arbitrary bases to the 5'-end and / or 3'-end of a neoantigen from a subject produced by the neoantigen production system of the present invention. In the present invention, the "base addition part" refers to a part that adds 0 or more arbitrary bases to the 5'-end and / or 3'-end of a neoantigen from a subject produced by the neoantigen production system of the present invention. The base addition part may include a nucleic acid synthesis device.

[0080] System for producing neoantigen peptides from a subject Another aspect of the present invention relates to a system for producing neoantigens from a subject (sometimes referred to as the "neoantigen peptide production system of the present invention"), comprising: (1) a sequence data acquisition part that acquires sequence data of normal cells and cancer cells from a subject; (2) a gene identification part that identifies genes having cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; (3) a peptide identification part that identifies peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database; and (4) an amino acid mutation introduction part that introduces amino acid mutations caused by the gene mutations into peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations, wherein the cancer cell-specific gene mutations are missense mutations, and the peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations have wild-type amino acids corresponding to the positions of the amino acid mutations caused by the gene mutations.

[0081] Another aspect of the present invention relates to a system for producing neoantigens from a subject (sometimes referred to as the "neoantigen peptide production system using NESSIE of the present invention"), comprising: (1) a sequence data acquisition part that acquires sequence data of cells from a subject; (2) a gene identification part that identifies genes having cancer cell-specific gene mutations by comparing the sequence data of the cells with sequence data of cells from a subject or other than the subject; (3) a peptide identification part that identifies peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database; and (4) an amino acid mutation introduction part that introduces amino acid mutations caused by the gene mutations into peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations, wherein the cells may be from blood, the cancer cells expressing neoantigens may be from different organs, MHC may be MHC class I and MHC class II, the cancer cell-specific gene mutations are missense mutations, and the peptides based on wild-type genes corresponding to genes having cancer cell-specific gene mutations have wild-type amino acids corresponding to the positions of the amino acid mutations caused by the gene mutations.

[0082] In one embodiment, the neoantigen peptide production system of the present invention further includes: a neoantigen selection unit that selects a peptide into which the amino acid mutation has been introduced as a neoantigen from a subject. In one embodiment, the neoantigen peptide production system of the present invention includes: an amino acid addition unit that adds 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of a neoantigen from a subject produced by the neoantigen peptide production system of the present invention, wherein the number of amino acids contained in the neoantigen to which the amino acid has been added is 40 or less.

[0083] System for producing a neoantigen nucleic acid from a subject Another aspect of the present invention relates to a method for producing a neoantigen from a subject (sometimes referred to as "the neoantigen nucleic acid production system of the present invention"), including: (1) a sequence data acquisition unit that acquires sequence data of normal cells and cancer cells from a subject; (2) a gene identification unit that identifies a gene having the cancer cell-specific gene mutation by comparing the sequence data of the normal cells and cancer cells with each other; (3) a peptide identification unit that identifies a peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation from a major histocompatibility complex (MHC) presented peptide database; (4) a base sequence identification unit that identifies a base sequence encoding the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation; and (5) a gene mutation introduction unit that introduces the gene mutation into the base sequence encoding the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation, wherein the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0084] Another aspect of the present invention relates to a method for producing neoantigens from a subject (sometimes denoted as "neoantigen nucleic acid production system using NESSIE of the present invention"), including: (1) a sequence data acquisition unit that acquires sequence data of cells from a subject; (2) a gene identification unit that identifies a gene having a cancer cell-specific gene mutation by comparing the sequence data of the cells with sequence data of cells from the subject or other than the subject; (3) a peptide identification unit that identifies a peptide based on a wild-type gene corresponding to the gene having the cancer cell-specific gene mutation from a major histocompatibility complex (MHC) presented peptide database; (4) a base sequence identification unit that identifies a base sequence encoding the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation; and (5) a gene mutation introduction unit that introduces the gene mutation into the base sequence encoding the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation, wherein the cells can be from blood, the cancer cells expressing the neoantigen can be from different organs, the MHC can be MHC class I and MHC class II, the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0085] In one embodiment, the neoantigen nucleic acid production system of the present invention further includes: a neoantigen selection unit that selects a nucleic acid composed of the base sequence introduced with the gene mutation as a neoantigen from a subject. In one embodiment, the neoantigen nucleic acid production system of the present invention includes: a base addition unit that adds 0 or more arbitrary bases to the 5'-end and / or 3'-end of a neoantigen from a subject produced by the neoantigen nucleic acid production system of the present invention. Additionally, the number of bases contained in the neoantigen to which the bases are added can be 900 or less.

[0086] System for producing a peptide containing a neoantigen from a subject Another aspect of the present invention relates to a system for producing a peptide containing a neoantigen from a subject (sometimes denoted as "neoantigen-containing peptide production system of the present invention"), including the following steps: an amino acid addition unit that adds 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of a neoantigen from a subject selected by the neoantigen selection system of the present invention, wherein the number of amino acids contained in the neoantigen to which the amino acids are added is 40 or less.

[0087] System for producing a nucleic acid containing a neoantigen from a subject Another aspect of the present invention relates to a system for producing nucleic acids containing neoantigens from a subject (sometimes denoted as "the neoantigen-containing nucleic acid production system of the present invention"), comprising: a base addition unit that adds zero or more arbitrary bases to the 5'-end and / or 3'-end of a neoantigen from a subject selected by the neoantigen selection system of the present invention. Additionally, the number of bases contained in the neoantigen to which bases have been added may be 900 or less. In one embodiment, the neoantigen-containing nucleic acid production system of the present invention further comprises: an mRNA production unit that produces mRNA containing a nucleic acid containing a neoantigen from a subject. In the present invention, the "mRNA production unit" refers to the part that produces mRNA containing a nucleic acid containing a neoantigen from a subject. The mRNA production unit may include an RNA synthesis device.

[0088] The present invention will be described in more detail with reference to the following examples, which show specific embodiments of the present invention, and the present invention is not limited thereto. Examples

[0089] Concept of the neoantigen selection method of the present invention The concept of the neoantigen selection method of the present invention is shown in Figure 1 . It should be noted that Figure 1 the various embodiments shown show specific embodiments of the present invention, but the present invention is not limited thereto. Additionally, "HLA" may be used interchangeably with "MHC". First, users such as medical staff identify peptides based on wild-type genes corresponding to genes with cancer cell-specific gene mutations from the HLA-presented peptide database according to each HLA class and each organ type based on information about a subject such as a patient (HLA class, type of cancerous organ, and cancer cell-specific gene mutations). Among them, the cancer cell-specific gene mutation is a missense mutation, and the peptide based on the wild-type gene corresponding to the gene with the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

[0090] The gene with the cancer cell-specific gene mutation is identified by comparing the whole exome sequence (WES) data of normal cells and cancer cells. Additionally, the HLA-presented peptide database is created by an HLA-presented peptide analysis method and includes a list of wild-type HLA-presented peptides according to each HLA class and each organ type, and the method includes the following steps: obtaining an HLA-peptide complex from cells; obtaining a peptide from the complex; and identifying the amino acid sequence of the peptide by mass spectrometry.

[0091] Next, peptides based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation are scored based on their respective HLA presentation frequencies in the HLA-presented peptide database, and identification is performed based on this score. Peptides produced by introducing amino acid mutations caused by the gene mutation into the peptides based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation are selected as neoantigens from the subject.

[0092] More specifically, the HLA-presented peptide database can be created by combining proteomic HLA ligandome analysis techniques of HLA immunoprecipitation, mass spectrometry, and whole exome transcriptome analysis. This analysis technique can reveal a complete picture of the HLA ligandome of a sample from human tissue.

[0093] The present inventors have used proteomic HLA ligandome analysis techniques to identify more than 100,000 natural HLA-presented peptides (i.e., peptides actually presented to HLA in vivo) from clinical samples to date. In addition, a panel of cancer antigens was created simultaneously to investigate the tumor-infiltrating lymphocyte (TIL) response of patients, and an interesting finding was obtained: the TIL response was more common in peptides identified by proteomic HLA ligandome analysis techniques than in peptides predicted by existing in silico methods. Since the peptides identified by proteomic HLA ligandome analysis techniques are natural HLA-presented peptides and are abundant in vivo, CD8-positive T cells tend to be easily induced to recognize the peptide in vivo through TCR.

[0094] In addition, the same tendency was also observed for neoantigens. That is, the neoantigens identified by proteomic HLA ligandome analysis techniques are natural HLA-presented peptides and are abundant in vivo, so CD8-positive T cells tend to be easily induced to recognize the neoantigen in vivo through TCR. Moreover, neoantigens have mutations that do not exist in normal cells but are specifically present only in cancer cells. Since they do not naturally exist in vivo, immune tolerance in the thymus can be avoided. Thus, it is expected that a strong immune response can be induced by inducing CD8-positive T cells to recognize neoantigens through TCR.

[0095] That is, neoantigens identified by proteomic HLA ligandome analysis techniques are considered to be able to effectively induce the TIL response of patients, and thus have high immunogenicity. In addition, it is considered that this neoantigen can effectively induce the TIL response of patients, thereby converting a tumor (cold tumor) to which TIL has no response into a tumor (hot tumor) to which TIL has a response.

[0096] In order to effectively utilize the above phenomenon, an HLA-presented peptide database was created from the data obtained by proteomic HLA ligandome analysis technology. Based on this database, a neoantigen selection method of the present invention was constructed. The neoantigen selection method of the present invention utilizes an existing cumulative database of HLA-presented peptides, and thus can select neoantigens from a subject with high sensitivity, high specificity, and simply from the WES data of normal cells and cancer cells.

[0097] Principle of the neoantigen selection method of the present invention The principle of the neoantigen selection method of the present invention is as Figure 2 shown. It should be noted that Figure 2 The various schemes shown show specific specific examples of the present invention, but the present invention is not limited thereto. In addition, "HLA" can be used interchangeably with "MHC". The principle of the neoantigen selection method of the present invention is to compare a peptide based on a wild-type gene corresponding to a gene having a cancer cell-specific gene mutation of a subject such as a patient with a natural HLA-presented peptide in an existing cumulative database of HLA-presented peptides, thereby selecting a neoantigen. At this time, for each HLA class and each organ type, a peptide based on a wild-type gene corresponding to a gene having a cancer cell-specific gene mutation is identified from the HLA-presented peptide database. This is based on the fact that when the HLA class and organ type are the same among different individuals, the same peptide is presented to HLA at a high frequency.

[0098] First, by comparing the WES data of normal cells and cancer cells with each other, a gene having a cancer cell-specific gene mutation of a subject such as a patient is identified. Then, only genes having missense mutations are extracted from the genes having the cancer cell-specific gene mutation. Then, the amino acid sequence based on the base sequence of the gene having the missense mutation is listed, and the mutated amino acid in the amino acid sequence is designated as "Mt", and the wild-type amino acid corresponding to the mutated amino acid is designated as "Wt". That is, a peptide based on a gene having the cancer cell-specific gene mutation (mutant peptide) and a peptide based on a wild-type gene corresponding to the gene having the cancer cell-specific gene mutation (wild-type peptide) are respectively represented as follows. Mutant peptide: (X) m -Mt-(X) n Wild-type peptide: (X) m -Wt-(X) n wherein X is independently any wild-type amino acid, and m and n are independently any integers of 0 or more. In addition, (X) m is located on the N-terminal side, and (X) nLocated on the C-terminal side. For example, m and n are each independently any integer from 0 to 20, 0 to 15, 0 to 10, or 0 to 5, and the sum of m and n is any integer of 40 or less, 30 or less, 20 or less, or 10 or less.

[0099] Next, after determining the HLA class of a subject such as a patient and the type of cancerous organ, a peptide based on the wild-type gene corresponding to the gene having a cancer cell-specific gene mutation (i.e., the above-mentioned "wild-type peptide") is identified from the HLA-presented peptide database so that the HLA class and organ type of the subject are consistent with the HLA class and organ type in the HLA-presented peptide database. That is, for each HLA class and each organ type, a peptide based on the wild-type gene corresponding to the gene having a cancer cell-specific gene mutation is identified from the HLA-presented peptide database.

[0100] More specifically, for the "Wt" of the peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation (i.e., the above-mentioned "wild-type peptide") and the amino acid sequences on its N-terminal side and C-terminal side (i.e., the above-mentioned "(X) m " and "(X) n "), it is investigated whether it has been registered in the HLA-presented peptide database. In the case where this peptide has been registered in the HLA-presented peptide database, it is regarded as a "hit". In the case where this peptide has been registered in the HLA-presented peptide database once, it is regarded as a "single hit". Each time there is a hit, at most one point is added to the score of this peptide. Conversely, in the case where this peptide has not been registered in the HLA-presented peptide database at all, the score of this peptide is 0 points. That is, the peptides based on the wild-type genes corresponding to the genes having the cancer cell-specific gene mutations are scored based on their respective HLA presentation frequencies in the HLA-presented peptide database.

[0101] In the case where the score is a value greater than 0 points, for the peptides that have been registered in the HLA-presented peptide database, the amino acid sequence and the calculated score of each peptide are listed. That is, peptides based on the wild-type genes corresponding to the genes having cancer cell-specific gene mutations are identified based on the scoring. It should be noted that at this time, the peptides that have been registered in the HLA-presented peptide database are also given a score between 0 and 1 according to the reliability of the MS method (i.e., the confidence level (FDR) of the amino acid sequence of the HLA-presented peptide), so there may be scores containing decimal points. For example, in the case where the FDR of the peptide that has been registered in the HLA-presented peptide database is 0.01, then each time there is a hit, one point is added to the score of this peptide; in the case where the FDR of the peptide that has been registered in the HLA-presented peptide database is 0.05, then each time there is a hit, 0.5 points are added to the score of this peptide. Finally, for the peptides already registered in the HLA-presented peptide database, "Wt" was replaced with "Mt". The peptides generated by this replacement were selected as neoantigens.

[0102] Features of the neoantigen selection method of the present invention The features of the neoantigen selection method of the present invention are as Figure 3 shown. It should be noted that Figure 3 The various schemes shown represent specific examples of the present invention, but the present invention is not limited thereto. In addition, "HLA" and "MHC" can be used interchangeably. According to the neoantigen selection method of the present invention, neoantigens from a subject can be selected. In particular, according to the neoantigen selection method of the present invention, by combining the existing in silico method and the MS method, the advantages of high sensitivity and simplicity of the former and the high specificity of the latter can be obtained simultaneously. That is, according to the neoantigen selection method of the present invention, neoantigens from a subject can be selected with high sensitivity, high specificity and simplicity. In addition, in other words, according to the neoantigen selection method of the present invention, neoantigens from a subject can be selected efficiently, at low cost and quickly. In addition, in other words, according to the neoantigen selection method of the present invention, neoantigens from a subject can be selected accurately, simply and quickly.

[0103] Example 1. Creation of MHC-presented peptide database (MHC ligandome analysis) 0.5 - 2.0 g of tissues (14 cases of human colorectal cancer tissues and 11 cases of human normal colorectal mucosa tissues with HLA-A24, 3 cases of human colorectal cancer tissues and 2 cases of human normal colorectal mucosa tissues with HLA-A02, and colorectal tissues of 21 5-week-old male and 5 6-week-old female Balb / c mice with H-2K d and H-2D d ) that were quickly frozen in liquid nitrogen were respectively cryo-ground using a Mixer Mill MM400 (Retsch), and then dissolved in 30 mL of lysis buffer (0.25% sodium deoxycholate (Wako), 0.2 mmol / L iodoacetamide (Wako), 1 mmol / L EDTA (Dojindo), 200× protease inhibitor mixture (Sigma), 1 mmol / L PMSF (Sigma), 1% octyl-β-D-glucopyranoside (Dojindo) / DPBS (Gibco)) to prepare a lysate containing MHC-peptide complexes. The lysate was added to a column immobilized with an MHC-specific antibody to concentrate and purify the MHC-peptide complexes. MHC-presented peptides were eluted from the MHC-peptide complexes with 0.2% trifluoroacetic acid (TFA), and then further concentrated and purified by Sep-Pak tC18 (Waters) and ZipTip U-C18 (Millipore).

[0104] Dissolve the MHC-presented peptides in 5% acetonitrile / 0.1% TFA, and then use them for nano-LC coupled with an Orbitrap mass spectrometer (Easy-nLC 1000 system (Thermo) and Q Exactive Plus (Thermo)) to obtain a peak list. The peptide separation conditions for LC are as follows: 3% - 30% solution B (100% ACN / 0.1% FA) 80-minute gradient, flow rate 300 nL / min, 3 μm C18 nano-HPLC capillary column (75 mm × 20 cm, Nikkyo Technos). The measurement conditions for mass spectrometry are as follows: In Full MS, resolution 70000 (m / z = 200), AGC target value = 3e6, maximum Injection Time = 100 ms, scan range 350 - 2,000 m / z; in MS / MS, resolution 17500 (m / z = 200), AGC target value = 1e5, maximum Injection Time = 120 ms. The measurement is performed by the Data-dependent MS / MS, Top 10 method, which selects the 10 precursor ions with high signal intensity in each scan as the objects for MS / MS measurement.

[0105] Use Proteome Discoverer v2.3 (Thermo) software to compare the obtained peak list with an MS reference database (protein sequences obtained from GENCODE, v31 and vM23) to interpret the amino acid sequences of the MHC-presented peptides. For the matching algorithm, use Sequest HT, and for the false discovery rate (FDR), use Percolator. The MHC-presented peptides after interpreting the amino acid sequences are detected with FDR 0.05, and peptides 8 - 14 amino acids long are identified from these peptides. Integrate the MHC-presented peptides identified from each sample to create an MHC-presented peptide database. The following information is registered in the MHC-presented peptide database: (1) the amino acid sequences of the MHC-presented peptides, (2) the confidence level (FDR) of the amino acid sequences of the MHC-presented peptides, (3) the MHC class of the samples from which the amino acid sequences of the MHC-presented peptides are derived, and (4) the types of organs of the samples from which the amino acid sequences of the MHC-presented peptides are derived.

[0106] Example 2. Identify cancer cell-specific Foreign gene Mutated gene DNA was extracted from human colorectal cancer tissues, human normal colorectal mucosa tissues, mouse colorectal cancer cell line CT26 (ATCC), and spleens of Balb / c mice (5 six-week-old females) using the Allprep DNA / RNA / Protein Kit (Qiagen) or DNeasy Blood&Tissue Kit (Qiagen). Libraries were created using SureSelect Human All Exon V6 (Agilent) or SureSelect Mouse (Agilent), and data were obtained through a NovaSeq 6000 (Illumina) sequencer to achieve 150bp paired-end short reads with an average coverage of 150× (data volume obtained: 18 Gb / sample). The obtained short reads were aligned with the reference genomes hg38 or mm10 using BWA (v0.7.17, v0.7.10), PCR duplicates were removed using Picard tools (2.18.2, 1.118), variant calling was performed using GATK (v4.0.5.1, v4), the detected variants were annotated using SnpEff (v4.3t, v4.1), SNPs were filtered using dbSNP and the 1000Genome Project (www.internationalgenome.org), and the sequence data of normal and cancer cells were compared with each other using Mutect2 to identify genes with cancer cell-specific gene mutations. It should be noted that the gene mutations are SNV, Indel, etc.

[0107] Example 3. Identification of wild-type peptides from the MHC-presented peptide database For the genes with missense mutations among the genes with cancer cell-specific gene mutations identified in Example 2, referring to the CDS nucleotide sequences and protein information registered in NCBI (GRCh38.p13, GRCm38.p6), the full-length amino acid sequence of the protein translated by the gene and the amino acid mutation positions were determined. Then, it was investigated whether the peptides based on the wild-type genes corresponding to the genes had been registered in the MHC-presented peptide database created in Example 1. That is, as described in the "Principle of the method for selecting neoantigens of the present invention" above, for each MHC class and each organ type, peptides based on the wild-type genes corresponding to the genes with the cancer cell-specific gene mutations were identified from the MHC-presented peptide database. Specifically, the respective MHC presentation frequencies in the MHC-presented peptide database were used to score the peptides, and the identification was performed based on the scores. It should be noted that when a 0.01 FDR was assigned to the peptides registered in the MHC-presented peptide database, the score of the peptide was increased by one point for each hit, and when a 0.05 FDR was assigned to the peptides registered in the MHC-presented peptide database, the score of the peptide was increased by 0.5 points for each hit.

[0108] Example 4. Selection of neoantigens from a mouse colorectal cancer cell line (1) Selection of neoantigens As described in Example 3, for each type of MHC class and each type of organ, peptides were identified from the MHC-presented peptide database of Balb / c mouse large intestine tissue based on the wild-type genes corresponding to the genes with specific gene mutations in the mouse large intestine cancer cell line CT26. Specifically, these peptides were scored based on their respective MHC presentation frequencies in the MHC-presented peptide database, and identification was performed based on this score. Then, amino acid mutations caused by the gene mutations were introduced into these peptides, and the peptides into which the amino acid mutations were introduced were selected as neoantigens from the mouse large intestine cancer cell line.

[0109] As a result, six neoantigens presented by H-2K d and H-2D d could be selected, namely KYLSVQ S QL (SEQ ID NO: 1), KYS N ASEAI (SEQ ID NO: 2), KGPKRDEQ C (SEQ ID NO: 3), QPV S SLRF (SEQ ID NO: 4), A R PTVVSHL (SEQ ID NO: 5), and HRQ E RRERPY (SEQ ID NO: 6) (the underlined part indicates mutant amino acids) ( Figure 4 ). The score based on the MHC presentation frequency was at most 2 points.

[0110] (2) Antitumor effects of the selected neoantigens For the six selected neoantigens, peptides were synthesized respectively (Genscript), and the antitumor effects brought about by administering the peptides to syngeneic mice were investigated. For 6-week-old Balb / c mice (male, Hokudo), 10 days and 3 days before transplanting tumors into the mice, a mixture of peptides (or an equal amount of DMSO) each containing 50 μg of the six neoantigens and 100 μg of Poly(I:C) HMW (InvivoGen) (adjuvant) was administered subcutaneously. A mixture of 5×10 6 mouse large intestine cancer cell line CT26 and Matrigel (Corning) was subcutaneously transplanted into the above mice. The tumors in the mice were measured with vernier calipers every 2 - 3 days, and the tumor volume was calculated by the following formula: Tumor volume = (long diameter of the tumor) × (short diameter of the tumor) 2 ÷2. For the control groups (i.e., the untreated group without any administration and the group administered DMSO and Poly(I:C) HMW (adjuvant)), n = 5 was used, and for the group administered the peptide mixture and Poly(I:C) HMW (adjuvant), n = 6 was used.

[0111] As a result, in the group administered with the peptide mixture and Poly(I:C)HMW (adjuvant), significant antitumor effects were found in 2 out of 6 Figure 5 . From this, it was found that antitumor effects were obtained by administering neoantigens selected from a mouse colorectal cancer cell line by the neoantigen selection method of the present invention.

[0112] (3) Antitumor effect of the selected neoantigen in combination with an anti-PD-1 antibody Then, the antitumor effect of the above peptide mixture in combination with an immune checkpoint inhibitor (anti-PD-1 antibody) was investigated. For 4-week-old Balb / c mice (male, Hokudo), 10 days and 3 days before transplanting tumors into the mice, a mixture containing 50 μg each of six neoantigens and 100 μg of Poly(I:C)HMW (InvivoGen) (adjuvant) was administered subcutaneously. 7 days and 14 days after tumor transplantation, 18 mg / kg and 10 mg / kg of anti-mouse PD-1 antibody (clone 29F.1A12, Bio XCell) were administered intraperitoneally to the above mice, respectively. The tumors in the mice were measured with vernier calipers every 2 - 3 days, and the tumor volume was calculated by the following formula: Tumor volume = (long diameter of the tumor) × (short diameter of the tumor) 2 ÷2. For the control group (not administered with the peptide mixture and Poly(I:C)HMW (adjuvant), but administered with the anti-PD-1 antibody alone), n = 9 was used, and for the group administered with the peptide mixture and Poly(I:C)HMW (adjuvant) and then the anti-PD-1 antibody, n = 10 was used.

[0113] As a result, in the control group (not administered with the peptide mixture and Poly(I:C)HMW (adjuvant), but administered with the anti-PD-1 antibody alone), antitumor effects were found in 3 out of 9, but in the group administered with the peptide mixture and Poly(I:C)HMW (adjuvant) and then the anti-PD-1 antibody, antitumor effects were found at a ratio of 7 out of 10 Figure 6 . From this, it was found that the neoantigens selected by the neoantigen selection method of the present invention can enhance the antitumor effect of immune checkpoint inhibitors.

[0114] (4) Confirmation of induction of CD8-positive T cells against the selected neoantigen In the group in which the anti-PD-1 antibody was administered after the administration of the above peptide mixture and Poly(I:C)HMW (adjuvant), individuals with high anti-tumor effects were specifically used, and the induction of CD8-positive T cells against the peptide mixture was confirmed. The spleen was removed from the mice euthanized by cervical dislocation, and the spleen was passed through a 70-μm cell strainer twice to disperse the cells contained in the spleen. Then, the cells were treated with ammonium chloride hemolysis buffer for 5 minutes to remove red blood cells, and splenocytes were collected. To 2.5×10 6 splenocytes, 1 μM of each peptide (peptides of the above six neoantigens), anti-mouse CD16 / CD32 antibody (FcBlock, BD), GolgiPlug (BD), Monensin (BioLegend), and anti-mouse CD107a-FITC antibody (BioLegend) were added, and the cells were incubated at 37 °C in a 5% CO2 environment for 4 hours. Then, the splenocytes were washed with 2% FBS / 0.04% NaN3 / PBS, anti-mouse CD3-PE antibody (BioLenegnd) and anti-mouse CD8-APC antibody (BioLegend) were added to the splenocytes, and the cells were incubated at 4 °C for 20 minutes. The splenocytes were fixed with Cytofix / Cytoperm (BD), and after permeabilization, anti-mouse IFN-γ-PE-Cy7 antibody (BioLegend) was added to the splenocytes, and the cells were incubated at 4 °C for 20 minutes. The antibody-stained splenocytes were measured using a flow cytometer (FACS Canto II, BD) and FACSDiva analysis software (BD). The expression levels of CD107a and the production levels of IFNγ were measured in the cell population expressing CD3 and CD8 on the cell surface in the cell population excluding doublet and autofluorescent cells, and the induction of CD8-positive T cells against each of the above six neoantigen peptides was evaluated.

[0115] As a result, the induction of CD8-positive T cells was significantly found, especially in the Mtch1-KSL9 neoantigen, and the induction of CD8-positive T cells was detected in 0.24% of the CD8-positive T cell population ( Figure 7 ). Thus, it was confirmed that CD8-positive T cells were induced against the neoantigens selected by the neoantigen selection method of the present invention, and it was found that the CD8-positive T cells induced in this way would bring anti-tumor effects.

[0116] Example 5. Selection of neoantigens from a human colorectal cancer tissue (CRC) panel (1) Selection of neoantigens As shown in Example 3, peptides corresponding to wild-type genes based on genes with specific gene mutations in human colorectal cancer tissues (15 cases of HLA-A24 human colorectal cancer tissues and 5 cases of HLA-A02 human colorectal cancer tissues) were identified from the HLA-presented peptide database of human large intestine tissues according to each HLA class and each organ type. Specifically, the peptides were scored based on their respective HLA presentation frequencies in the HLA-presented peptide database, and identification was performed based on this score. Then, amino acid mutations caused by the gene mutations were introduced into the peptides, and the peptides into which the amino acid mutations were introduced were selected as neoantigens from the human colorectal cancer tissue (CRC) group. As a result, neoantigens could be selected in several cases among CRC cases of HLA-A24 and HLA-A02 ( Figure 8 ). In addition, after examining the number of missense mutations carried by patients in each CRC case of HLA-A24 and HLA-A02, a tendency was found that the number of selected neoantigens increased in direct proportion to the number of missense mutations. For example, in the CRC111 case among CRC cases of HLA-A24, the patient carried the largest number of missense mutations and the largest number of selected neoantigens.

[0117] (2) Confirmation of TIL response against the selected neoantigens To confirm whether the selected neoantigens actually induce an immune response in patients, tumor-infiltrating lymphocytes (TIL) from human colorectal cancer tissues were cultured to investigate the TIL response against the selected neoantigens. This investigation used the TUBB-RAF9 neoantigen (RYL A VAAVF (SEQ ID NO: 7)) of the CRC111 case (HLA-A24) and the STT3A-KRV9 neoantigen (KLNPQ R FEV (SEQ ID NO: 8)) of the CRC135 case (HLA-A02) (the underlined part indicates mutant amino acids).

[0118] Human colorectal cancer tissues cut into slices 1 - 2 mm thick were cultured in AIM-V medium (Gibco) supplemented with 10% human serum (1% penicillin / streptomycin, 1% GlutaMAX, 10 mmol / L HEPES, 1 mmol / L sodium pyruvate, 55 mmol / L 2-mercaptoethanol, 2.5 μg / mL amphotericin B, 6000 U / mL rhIL2) for 2 - 4 weeks to isolate and proliferate TIL. The culture medium was changed every 3 days. Then, 30 ng / mL anti-human CD3 antibody (clone OKT3, BioLegend) was added to the above culture medium to proliferate TIL, and then TIL was co-cultured with irradiated (100 Gy) PBMC from healthy humans for 2 weeks.

[0119] 1~5×10 5 TIL, 1 μM neoantigen (TUBB-RAF9 neoantigen or STT3A-KRV9 neoantigen) were intermittently mixed with an equal amount of T2 or T2-A24 cells presented to HLA, Human FcR blocking reagent (MBL), GolgiPlug (BD), Monensin (BioLegend) and anti-human CD107a-PE antibody (BioLegend) and incubated for 4 hours at 37°C and 5% CO2. Then, the cells were washed with 2% FBS / 0.04% NaN3 / PBS, and anti-human CD3-PE-Cy7 (BD) and anti-human CD8-APC (BioLegend) were added to the cells and incubated at 4°C for 20 minutes. The cells were fixed with Cytofix / Cytoperm (BD), permeabilized, and anti-human IFN-γ-FITC antibody (BioLegend) was added to the cells and incubated at 4°C for 20 minutes. The cells stained with such antibodies were measured using a flow cytometer (FACS Canto II, BD) and FACSDiva analysis software (BD). The CD107a expression and IFNγ production of cell populations expressing CD3 and CD8 on the cell surface in cell populations other than bimodal and autofluorescent cells were measured to evaluate the TIL response to each of the two neoantigens mentioned above. In order to determine the nonspecific response level of TIL, HIV peptides (HLA-A24: RYLRDQQLL (sequence number 9) and HLA-A02: SLYNTVATL (sequence number 10)) were used as negative controls.

[0120] As a result, TIL responses were found in both the TUBB-RAF9 neoantigen of CRC111 case (HLA-A24) and the STT3A-KRV9 neoantigen of CRC135 case (HLA-A02). Figure 9 ). It can be seen that in human colorectal cancer tissue, CD8-positive T cells that recognize the selected neoantigens and react to the neoantigens infiltrate and accumulate in the tumor. That is, it can be seen that the selected neoantigens do induce immune responses in the patient. In addition, it can be seen that the neoantigen selection method of the present invention is indeed presented to HLA in tumor tissue, and can select immunogenic neoantigens that are the attack targets of CD8-positive T cells after tumor infiltration with high sensitivity, high specificity and simplicity.

[0121] (3) Application of presenting new antigens to HLA class I According to the new antigen selection method of the present invention, not only HLA class I presenting new antigens but also HLA class I presenting new antigens can be selected. As described in Example 3, peptides corresponding to wild-type genes based on genes with tissue-specific gene mutations in human colorectal cancer tissue were identified from the HLA-presented peptide database derived from human colorectal tissue, for each HLA class and each organ type. Specifically, the peptides were scored based on their respective HLA presentation frequencies in the HLA-presented peptide database, and identification was performed based on this score. Then, amino acid mutations caused by the gene mutations were introduced into the peptides, and the peptides into which the amino acid mutations were introduced were selected as neoantigens from human colorectal cancer tissue.

[0122] As a result, the SCO1 neoantigen (KGEIAASITHMRPY (SEQ ID NO: 11)) (the underlined part indicates mutant amino acids) of the CRC66 case (HLA-DR ＊ 04:06) was selected. V ). Reactive CD4-positive T cells were cloned from the peripheral blood of the patient, and the reaction of the CD4-positive T cell clone with the SCO1 neoantigen was investigated. As a result, SCO1 neoantigen-specific cytokines (TNFα and IFNγ) were produced. For the wild-type peptide corresponding to the SCO1 neoantigen, production of this cytokine was not observed. In addition, investigation was performed using different antigen-presenting cells of the HLA class, and as a result, presentation of the SCO1 neoantigen on HLA-DR Figure 10 04:06 was confirmed. ＊

[0123] Example 6: Efficacy of combination of vaccine and immune checkpoint inhibitor (ICB) For CT26 tumor-implanted Balb / c mice, using SEQ ID NO: 1 (KYLSVQ S QL) as the neoantigen, spleens were collected from individuals who had not been treated, had received vaccine administration, had received anti-PD-1 antibody administration, or had received combined vaccine and anti-PD-1 antibody administration, and the reaction of CD8+ cells to the neoantigen (CD107a expression level and IFNγ production level) was investigated. As a result, it was confirmed that the combination of vaccine and immune checkpoint inhibitor (ICB) increased the neoantigen-reactive CD8+ cells in the spleen ( Figure 11 ).

[0124] Example 7. Selection of neoantigens from the genes identified in Example 2 (using HLA from multiple organs) (Presented peptide) Similarly to Example 1, an MHC-presented peptide database was prepared from a sample containing peripheral blood tissue. Then, similarly to Example 2, genes with cancer cell-specific gene mutations were identified. Similarly to Example 3, regardless of the HLA class and organ type, peptides based on the wild-type genes corresponding to the genes with tumor-specific gene mutations were identified from a database that integrated HLA-presented peptides from various HLA classes and organs. Specifically, the peptides were scored based on their respective HLA presentation frequencies in the HLA-presented peptide database, and identification was performed based on this score. Then, amino acid mutations caused by the gene mutations were introduced into the peptides, and the peptides into which the amino acid mutations were introduced were selected as neoantigens ( Figure 12 ).

[0125] Example 8. Selection of neoantigens from the genes identified in Example 2 (using (HLA-presented peptide) Similarly to Example 3, neoantigens presented to all HLA classes, particularly HLA class II, were targeted, and peptides based on the wild-type genes corresponding to the genes with tumor-specific gene mutations were identified from the HLA-presented peptide database of B-LCLs from autologous PBMCs. Specifically, a database was prepared from the HLA-presented peptide sets of B-LCLs obtained in the same manner as in Example 1, and the peptides were identified based on this HLA-presented peptide database. Then, amino acid mutations caused by the gene mutations were introduced into the peptides, and the peptides into which the amino acid mutations were introduced were selected as neoantigens from tumor cells ( Figure 13 ).

[0126] As a result, the SCO1 neoantigen (KGEIAASIVTHMRPY (SEQ ID NO: 11)) (the underlined part indicates mutant amino acids) of the CRC66 case (HLA-DR ＊ 04:06) was selected ( Figure 10 ). Reactive CD4-positive T cells were cloned from the patient's peripheral blood, and the reaction of this CD4-positive T cell clone with the SCO1 neoantigen was investigated. As a result, SCO1 neoantigen-specific cytokines (TNFα and IFNγ) were found ( Figure 14 ). No cytokine production was observed for the wild-type peptide corresponding to the SCO1 neoantigen. In addition, investigations were performed using different antigen-presenting cells of the HLA class, and SCO1 neoantigen presentation on HLA-DR ＊ 04:06 was confirmed.

[0127] As can be seen from the above results, according to the present invention, it is possible to select neoantigens from a subject. In particular, according to the present invention, by combining the existing in silico method and the MS method, it is possible to simultaneously obtain the advantages of high sensitivity and simplicity of the former and the high specificity of the latter. That is, according to the present invention, it is possible to select neoantigens from a subject with high sensitivity, high specificity, and simplicity. In other words, according to the present invention, it is possible to select neoantigens from a subject efficiently, at low cost, and rapidly. In other words, according to the present invention, it is possible to select neoantigens from a subject accurately, simply, and rapidly.

[0128] In addition, according to the present invention, based on the selected neoantigens from a subject, it is possible to produce a peptide and / or nucleic acid containing the neoantigen. And, according to the present invention, based on the selected neoantigens from a subject, it is possible to produce a peptide and / or nucleic acid containing the neoantigen and apply it to personalized medicine for the subject. And, according to the present invention, based on the selected neoantigens from a subject, it is possible to produce mRNA containing the neoantigen and apply it to personalized medicine for the subject.

Claims

1. A method for selecting neoantigens from a subject, comprising the following steps: (1) Obtaining sequence data of normal cells and cancer cells from the subject; (2) Identifying genes with cancer cell-specific gene mutations by comparing the sequence data of the normal cells and cancer cells with each other; And (3) Identifying peptides based on wild-type genes corresponding to genes with cancer cell-specific gene mutations from a major histocompatibility complex (MHC) presented peptide database, Wherein, The cancer cell-specific gene mutation is a missense mutation, and The peptide based on the wild-type gene corresponding to the gene with the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

2. The method according to claim 1, wherein The sequence data is whole exome sequence data.

3. The method according to claim 2, wherein The MHC presented peptide database includes a list of wild-type MHC presented peptides according to each type of MHC class and / or each type of organ.

4. The method according to claim 1, wherein, The MHC presented peptide database includes a list of wild-type MHC presented peptides.

5. The method according to claim 4, wherein, Step (3) is a step of identifying peptides based on wild-type genes corresponding to genes with cancer cell-specific gene mutations from the MHC presented peptide database according to each type of MHC class and / or each type of organ.

6. The method according to claim 5, wherein The amino acid mutation caused by the gene mutation is a single amino acid mutation.

7. The method according to claim 6, wherein, The peptides based on the wild-type genes corresponding to the genes with cancer cell-specific gene mutations are scored based on their respective MHC presentation frequencies in the MHC presented peptide database.

8. The method according to claim 7, wherein The peptides based on the wild-type genes corresponding to the genes with cancer cell-specific gene mutations are identified based on the scoring.

9. The method according to claim 8, wherein, The MHC presented peptide database is created by an MHC presented peptide analysis method, which includes the following steps: obtaining MHC-peptide complexes from cells; obtaining peptides from the complexes; and identifying the amino acid sequences of the peptides by mass spectrometry analysis.

10. The method according to claim 9, wherein, The cells are from a subject or a non-subject.

11. The method according to claim 9, wherein, The cells are collected from blood.

12. The method according to claim 9, wherein, The MHC is MHC class I and / or II.

13. The method according to claim 12, wherein, The MHC is human leukocyte antigen (HLA).

14. The method according to any one of claims 1 to 13, wherein, Further comprising the following steps: Introducing the amino acid mutation caused by the gene mutation into the peptide based on the wild-type gene corresponding to the gene with the cancer cell-specific gene mutation, wherein the peptide has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation; and / or determining the gene sequence encoding the peptide into which the amino acid mutation has been introduced.

15. The method according to claim 14, wherein Further comprising the following steps: Selecting a nucleic acid composed of the peptide into which the amino acid mutation has been introduced and / or the base sequence into which the gene mutation has been introduced as a neoantigen from the subject.

16. A method for making a peptide comprising neoantigens from a subject, comprising the steps of: Adding 0 or more arbitrary amino acids to the N-terminus and / or C-terminus of the neoantigen from the subject selected by the method according to claim 15, wherein the number of amino acids contained in the neoantigen to which the amino acids have been added is 40 or less.

17. A peptide obtained by the method according to claim 16.

18. A method for making a nucleic acid comprising neoantigens from a subject, comprising the steps of: Adding 0 or more arbitrary bases to the 5'-end and / or 3'-end of the nucleic acid containing the neoantigen from the subject selected by the method according to claim 15.

19. A nucleic acid comprising a neoantigen from a subject, the neoantigen being selected by the method according to claim 15.

20. The method according to claim 18, wherein Further comprising the following steps: Producing an mRNA comprising a nucleic acid containing a neoantigen from a subject.

21. An mRNA nucleic acid comprising a neoantigen from a subject, the neoantigen being selected by the method according to claim 15.

22. A vaccine composition comprising the peptide according to claim 17, the nucleic acid according to claim 19 or the mRNA according to claim 21.

23. A system for selecting neoantigens from a subject, comprising: (1) A sequence data acquisition unit that acquires sequence data of normal cells and cancer cells from a subject; (2) A gene identification unit that identifies a gene having a cancer cell-specific gene mutation by comparing the sequence data of the normal cells and cancer cells with each other; And (3) A peptide identification unit that identifies a peptide based on a wild-type gene corresponding to a gene having a cancer cell-specific gene mutation from a major histocompatibility complex (MHC) presented peptide database, wherein The cancer cell-specific gene mutation is a missense mutation, and The peptide based on the wild-type gene corresponding to the gene having the cancer cell-specific gene mutation has a wild-type amino acid corresponding to the position of the amino acid mutation caused by the gene mutation.

24. The system according to claim 23, wherein, The cells are from a subject or a non-subject.

25. The system according to claim 23, wherein, The cells are collected from blood.

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