Multi-gene mutation tumor neoantigen polypeptide algorithm

Abstract

The invention discloses a multi-gene mutation tumor new antigen polypeptide algorithm, the specific sites of the mutated antigen targets are genes APRT p.E141V, KCTD10 p.Y172N, PABPC1 p.L218V, ADAM28 p.C315G, ARF3 p.G6V, KLHL2 p.E88G, CNOT11 p.S148R, C5orf42 p.Q1002L, ABLIM1 p.C190Y, BCLAF1 p.N629S, PABPC1 p.K312RfsTer10 and KRAS p.G12D, the 12 gene mutations cover hotspot mutation, low-frequency mutation and rare mutation, and the antigen polypeptide can be synthesized on a large scale and is used for subsequent PABPC1 p.L218V and a more effective personalized treatment scheme can be established for the patient.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of immunology, and specifically discloses a polynucleotide algorithm for tumor neoantigens. BACKGROUND

[0002] Pancreatic cancer (PC) is a malignant tumor originating from pancreatic duct epithelial or acinar cells, with a global incidence ranking 11th and a mortality rate ranking 7th. In China, the mortality rate of pancreatic cancer has climbed to the 6th. Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of pancreatic cancer, which is a common clinical subtype with poor prognosis. Due to the insidious early symptoms of pancreatic cancer, lack of specific clinical manifestations and efficient early screening methods, about 80% of patients are in locally advanced or distant metastasis at the time of initial diagnosis, missing the opportunity for radical surgery. According to the data of the 2023 China Pancreatic Cancer Diagnosis and Treatment Guidelines, the 5-year survival rates of pancreatic cancer patients in the United States and Japan are about 12% and 10%, respectively, and the 5-year survival rate of pancreatic cancer patients in China is only 7.2%, which is much lower than that of other common malignant tumors.

[0003] The current standard first-line treatment for pancreatic ductal adenocarcinoma needs to be developed according to the disease stage: resectable pancreatic cancer is mainly treated with radical surgery such as pancreaticoduodenectomy and distal pancreatectomy, and postoperative adjuvant chemotherapy with gemcitabine or fluorouracil drugs; locally advanced pancreatic cancer is mainly treated with concurrent chemoradiotherapy or chemotherapy, and the possibility of surgery is evaluated after the tumor is reduced; metastatic pancreatic cancer is mainly treated with systemic chemotherapy, and common regimens include FOLFIRINOX (fluorouracil + calcium folinate + irinotecan + oxaliplatin), gemcitabine combined with albumin-bound paclitaxel, etc. Some patients with specific target mutations can use targeted drugs (such as larotrectinib for NTRK fusion), but the number of suitable people is very small. Due to the characteristics of pancreatic ductal adenocarcinoma such as "difficult early diagnosis, low resection rate, strong drug resistance, and fast recurrence", the existing treatment methods are difficult to significantly improve the prognosis of patients, and the development of new pancreatic ductal adenocarcinoma treatment technology has urgent clinical needs and important social significance.

[0004] Pancreatic ductal adenocarcinoma hotspot mutation neoantigens (such as neoantigens targeting KRAS G12D and PIK3CA E545K) are designed based on "high-frequency mutation sites" and are only suitable for patients carrying specific hotspot mutations. However, pancreatic ductal adenocarcinoma has significant mutational heterogeneity: on the one hand, the mutation frequency of major driver genes varies among individuals; on the other hand, patients may carry multiple low-frequency mutations, which may also produce immunologically active neoantigens, but are not covered by the "hotspot mutation library".

[0005] The individualized tumor neoantigen is captured by whole exome sequencing (WES) + transcriptome sequencing (mRNA), and can comprehensively capture the somatic mutations (including hotspot mutations, low-frequency mutations and rare mutations) unique to the tumor tissue of the patient, and screen specific antigens of "mutation-MHC binding adaptation" in combination with the HLA typing (such as HLA-A02:01, HLA-B15:01, etc.) of the patient. The individualized scheme can design a neoantigen polypeptide for any mutation, and the hotspot mutation neoantigen cannot provide a treatment scheme for the site because it does not cover the site. Clinical data shows that only about 30%-40% of pancreatic cancer patients carry core hotspot mutations such as KRAS and PIK3CA, and the individualized neoantigen can increase the coverage rate of the applicable population to more than 80%, significantly expanding the treatment benefit range. SUMMARY

[0006] To solve the above problems, the present application provides a multi-gene mutation tumor neoantigen polypeptide algorithm, which provides an effective solution to the problem of specific antibodies for genes APRT p.E141V, KCTD10 p.Y172N, PABPC1 p.L218V, ADAM28 p.C315G, ARF3 p.G6V, KLHL2 p.E88G, CNOT11 p.S148R, C5orf42 p.Q1002L, ABLIM1 p.C190Y, BCLAF1 p.N629S, PABPC1 p.K312RfsTer10, KRAS p.G12D, and designs polypeptides with antibody characteristics.

[0007] Specifically, the present application provides a multi-gene mutation tumor neoantigen polypeptide, which can promote the effective killing of tumor cells by CTL, and can use a tumor neoantigen prediction platform to predict and screen tumor neoantigens. The present application also relates to the design of antigen polypeptides for tumors.

[0008] The technical scheme adopted by the present application is as follows: A multi-gene mutation tumor neoantigen polypeptide algorithm, characterized in that the neoantigen polypeptide is detected by whole exome (WES) and transcriptome (mRNA) sequencing of tumor tissue and control blood, and the specific multi-gene mutations (12 mutations) of the tumor tissue are calculated to evaluate the antigen potential of the mutations.

[0009] 1. The neoantigen polypeptide of claim 1, wherein the specific site of the multi-gene mutated antigen target is APRT p.E141V, KCTD10 p.Y172N, PABPC1 p.L218V, ADAM28 p.C315G, ARF3 p.G6V, KLHL2 p.E88G, CNOT11 p.S148R, C5orf42 p.Q1002L, ABLIM1 p.C190Y, BCLAF1 p.N629S, PABPC1 p.K312RfsTer10, KRAS p.G12D.

[0010] The algorithm of the immune epitope database (IEDB) used by the multi-gene mutated antigen includes analysis of the protein structure of the neoantigen, polypeptide antigenicity, whether it can stimulate the production of IFN-γ, T cell epitope toxicity / adverse reactions, hydrophilicity / hydrophobicity, and other characteristics.

[0011] The neoantigen polypeptide is an amino acid sequence, and the design of the amino acid sequence of 12 polypeptide sequences includes the mRNA expression level of the antigen in tumor cells and the recognition ability of immune cells, and the epitope with potential immunogenicity is screened from the predicted neoantigen, and based on the determined antigenic recognition region, preferably, the functional same or similar polypeptide obtained by substituting and / or deleting at least one amino acid in the amino acid sequence of the neoantigen polypeptide.

[0012] The functional same or similar polypeptide refers to the tumor neoantigen polypeptide capable of activating HLA typing HLA-A 01:01, HLA-A 02:03, HLA-B 37:01, HLA-B 38:02, HLA-C 07:02, HLA-C 06:02, DRB1 10:01, DRB1 12:02.

[0013] The multi-gene mutation specific to the tumor tissue and expressed has 12 gene mutations, and the 12 gene mutations correspond to specific neoantigen T cells.

[0014] Compared with the prior art, the present application has the following beneficial effects: The antigen peptide of the present application can stimulate the activation of human specific T cells against gene mutations in vitro, and be amplified in large quantities for adoptive therapy of patients. The neoantigen polypeptide synthesized according to the PABPC1 gene p.L218V mutation can activate T lymphocytes specific to the PABPC1 gene p.L218V mutation peptide in vitro and release cytokine IFN-γ, indicating that the polypeptide has obvious immunogenicity and improves the killing ability of T cells to cancer cells of patients with pancreatic ductal adenocarcinoma with PABPC1 gene p.E141V mutation.

[0015] The antigen peptide of the present application fills the blank of individualized antigen peptide in the treatment of pancreatic ductal adenocarcinoma patients with PABPC1 p.L218V somatic mutation. At the same time, the antigen polypeptide of the present application can be synthesized in large quantities and used in subsequent standardized and individualized immunotherapy of PABPC1 p.L218V mutation tumor patients. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure is the evaluation of immune infiltration level of four molecular markers of pancreatic ductal adenocarcinoma patients (part) of the present application by immunohistochemical staining; Figure 2 Figure is the immune infiltration score results of 12 cases (Pt01-Pt12) of pancreatic ductal adenocarcinoma patients in Example 2 of the present application; Figure 3 Figure is the in vitro cell evaluation experiment results of the effectiveness evaluation of the synthetic polypeptide sequences of No. 3 and No. 8 in Example 3 of the present application; Figure 4 Figure is the ELISPOT results statistical chart of the synthetic polypeptide sequences of No. 3 and No. 8 in Example 3 of the present application; Figure 5 Figure is the CTL results statistical chart of the synthetic polypeptide sequences of No. 3 and No. 8 in Example 3 of the present application; Figure 6 Figure is the flow chart of tumor neoantigen polypeptide design of the present application. DETAILED DESCRIPTION

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only one embodiment of the present application, and other embodiments can also be obtained by those skilled in the art according to these drawings. DEFINITIONS As used herein, the terms "a" and "an" and "the" and similar referents are intended to indicate the singular as well as the plural, unless the context clearly indicates otherwise or it is obvious from the circumstances that the ind icated number is not intended to be singular. As used herein, the terms "about," "substantially," and "approximately" mean within an acceptable range of error for the particular value as determined by one of ordinary skill in the art to which the value pertains, which will depend in part on how the value is measured or determined, or on the limitation of the measurement system. The term "polypeptide" as used herein refers to a linear chain of three or more amino acids linked by peptide bonds multimer. The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the terms "nucleic acid" or "polynucleotide" also include nucleic acids comprising analogues of natural nucleotides, which have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated. Unless otherwise noted, modifications to DNA sequences are intended to apply mutatis mutandis to the corresponding RNA sequences. A "construct" refers to any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, linear or circular, single- or double-stranded DNA or RNA polynucleotide molecule) that can be derived from any source, capable of genomic integration or autonomous replication, which can operably link one or more polynucleotide molecules. In the present application, a construct typically comprises a polynucleotide molecule of the present application operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide molecule of the present application in a host cell. Expression of the nucleic acids of the present application can be directed using a heterologous promoter or an endogenous promoter. A "vector" refers to any recombinant nucleic acid construct that can be used for the purpose of transformation (i.e., introduction of heterologous DNA into a host cell). A vector can contain a resistance gene for growth in an organism and a promoter for expression of a protein of interest in the organism. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., vectors having a replication origin that is functional in the host cell). Other vectors can be integrated into the genome of a host cell and are thus replicated with the host genome upon introduction into the host cell. Additionally, certain preferred vectors are capable of directing expression of genes to which they are operatively linked. One type of vector is a "plasmid," which generally refers to a circular double- stranded DNA loop that is used as a vehicle to carry additional DNA segments (foreign genes) but can also include linear double-stranded molecules, such as linear double-stranded molecules obtained from amplification by polymerase chain reaction (PCR) or from linearizing circular plasmids with restriction enzymes. Plasmid vectors include the vector backbone (i.e., the empty vector) and the expression framework. The term "expression cassette" refers to a nucleic acid construct that encodes a gene of interest and is capable of being expressed in a host cell. The expression cassette can include a promoter, a gene of interest, and a terminator. The expression cassette can also include a selection marker. The expression cassette can be incorporated into a vector. The term "gene" as used herein refers to a hereditary unit occupying a specific locus on a chromosome. The term "fragment" means a polypeptide with one or more (several) amino acids deleted from the amino and / or carboxyl terminus of a polypeptide. The term "variant" means a polypeptide comprising an alteration, i.e., a substitution, an insertion, and / or a deletion of one or more (several) amino acids at one or more (several) positions of a parent or wild-type polypeptide, which has the activity of the parent or wild-type polypeptide. The term "variant" means a polypeptide comprising an alteration, i.e., a substitution, an insertion, and / or a deletion of one or more (several) amino acids at one or more (several) positions of a parent or wild-type polypeptide, which has the activity of the parent or wild-type polypeptide.

[0018] "Substitution" means replacing the amino acid occupying a certain position with a different amino acid; "Deletion" means removing the amino acid occupying a certain position; and "insertion" means adding one or several amino acids next to the amino acid occupying a certain position. As used herein, the term "host cell" refers to a cell into which a vector is introduced, including a number of cell types such as prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as HEK 293T cells, MDAMB231 cells, 4T1 cells, etc.; in the present application, the "host cell" refers especially to a mammalian cell, more particularly a human-derived cell.

[0019] As used herein, the term "antigenic epitope," also known as an antigenic determinant, refers to a specific chemical group with a certain composition and structure on the surface or other sites of an antigen molecule, capable of specifically binding to corresponding antibodies or sensitized lymphocytes. During the immune response, the epitopes recognized by the antigen receptors TCR (T cell receptor) and B cell receptors BCR have different characteristics and are respectively called T-cell epitopes and B-cell epitopes. T-cell epitopes are generally not located on the surface of antigen molecules; they must be processed into small polypeptide molecules by antigen-presenting cells and bound to MHC molecules before they can be recognized by the TCR. T cells can only recognize processed epitopes. B-cell epitopes, on the other hand, can exist on the surface of antigen molecules and can be directly recognized by B cells without processing. In this application, it also refers to one or more peptide segments that are predicted or screened to specifically bind to antibodies. The term "peptide carrier protein conjugate" refers to the conjugate formed by the coupling of the peptide and the carrier protein of this application.

[0020] In this embodiment, a carrier protein can couple one or more polypeptides. When multiple polypeptides are coupled, the polypeptides have the same amino acid sequence. The number of polypeptides coupled to each carrier protein varies depending on the physicochemical properties of the specific coupled polypeptide sequences, the type of carrier protein, and the coupling method. In this application, 2 to 50 polypeptides are preferred; more preferably, 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, or 15 to 30; or, even more preferably, 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, or 10 to 24. Any one of Articles 18, 10-16, and 10-15. As used herein, the term "antigen" refers to any substance capable of inducing an immune response in the body. Specifically, it refers to a substance that can specifically bind to antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, producing immune response products (sensitized lymphocytes or antibodies), and that can specifically bind to these products in vivo and in vitro. Therefore, antigens possess two important characteristics: immunogenicity and immunoreactivity. In this application, antigen also refers to a complete antigen with immunogenicity formed by the conjugation of a polypeptide hapten with a carrier protein. This can be a polypeptide-carrier-protein conjugate formed by conjugating a polypeptide with a single amino acid sequence with a carrier protein; or a composition of polypeptide-carrier-protein conjugates formed by conjugating polypeptides with multiple different amino acid sequences with a carrier protein.

[0021] As used herein, the term "vaccine" generally refers to the ability of a substance to have both immunogenic and reactogenic properties. Immunogenicity refers to the ability to stimulate the body to produce an immune response, i.e., the ability to stimulate the body to produce specific immune cells, to activate, proliferate, differentiate, and ultimately produce immune effector substances, such as specific antibodies or sensitized lymphocytes. Reactogenicity refers to the ability to specifically bind to the antibodies or sensitized lymphocytes that it induces. As used herein, the term "preventing" refers to an approach taken to stop or delay the occurrence of a disease or condition or symptoms (e.g., HBV infection or a disease associated with HBV infection) in a subject.

[0022] As used herein, the term "treating" refers to an approach taken to obtain a beneficial or desired clinical result. For the purposes of the present application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. In the present application, the antibodies of the present application have the ability to neutralize HBV and thus can be used to prevent / stop HBV infection in a subject or cells thereof who is not yet diseased. In addition, the antibodies of the present application have the ability to clear HBV (i.e., are capable of clearing HBV DNA and / or HBsAg in the body and HBV and cells infected with HBV in the body) and thus can be used to treat HBV infection or a disease associated with HBV infection in a subject who is already diseased. As used herein, the term "subject" refers to a mammal, such as a primate, e.g., a human. As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., HBV infection or a disease associated with HBV infection) refers to an amount that is sufficient to prevent, stop, or delay the occurrence of the disease (e.g., HBV infection or a disease associated with HBV infection); an effective amount for treating a disease refers to an amount that is sufficient to cure or at least partially arrest the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight, and gender, the mode of administration of a drug, and other therapies being administered concurrently, etc. As used herein, the term "pharmaceuticalally acceptable excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween 80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin.

[0023] Example 1 like Figures 1-6 As shown, an algorithm for detecting neoantigen peptides in tumors with multiple gene mutations is characterized by the following: The neoantigen peptides are detected by whole-exome sequencing (WES) and mRNA sequencing of tumor tissue and control blood to identify 12 tumor-specific and expressed multi-gene mutations. The antigenic potential of the mutations is assessed by calculating the binding affinity between the mutated epitopes and major histocompatibility complex (MHC) classes I and II. The specific antigenic target sites of the multi-gene mutations are the genes APRT p.E141V, KCTD10 p.Y172N, PABPC1 p.L218V, ADAM28 p.C315G, ARF3 p.G6V, KLHL2 p.E88G, CNOT11 p.S148R, C5orf42 p.Q1002L, ABLIM1 p.C190Y, BCLAF1 p.N629S, and PABPC1. p.K312RfsTer10, KRAS p.G12D.

[0024] The algorithm of the immune epitope database (IEDB) used for the multi-gene mutated antigen includes: analysis of the protein structure, peptide antigenicity, whether it will stimulate the production of IFN-γ, T cell epitope toxicity / adverse reactions, hydrophilicity, hydrophobicity, and other properties of the neoantigen.

[0025] The new antigen polypeptide is an amino acid sequence, and the amino acid sequence is a polypeptide sequence designed by screening epitopes with potential immunogenicity from predicted neoantigens based on mRNA expression levels of antigens in tumor cells and recognition ability of immune cells, a determined antigenic recognition region, preferably, a polypeptide with the same or similar function obtained by substituting and / or deleting at least one amino acid in the amino acid sequence of the new antigen polypeptide.

[0026] The polypeptide with the same or similar function refers to the tumor neoantigen polypeptide capable of activating HLA typing HLA-A 01:01, HLA-A 02:03, HLA-B 37:01, HLA-B 38:02, HLA-C 07:02, HLA-C 06:02, DRB1 10:01, DRB1 12:02.

[0027] The tumor tissue-specific and expressed multi-gene mutation has 12 gene mutations, and the 12 gene mutations correspond to specific T cells of neoantigens.

[0028] The sample of the tumor tissue and the corresponding blood have 3 cases, respectively, and the genomic DNA and the transcriptome mRNA are extracted and subjected to whole exome sequencing and transcriptome sequencing, and the prediction and screening include the following steps: S1: through paired analysis of whole exome and blood (normal tissue), specific mutations (such as INDEL) of tumor tissue can be obtained, mutation frequency VAF>1%, mRNA expression amount FPKM>1 of corresponding mutation gene, and polypeptides of these specific mutations form a first-stage tumor neoantigen database; S2: the HLA typing of the sample and the first-stage tumor neoantigen database are subjected to antigen affinity prediction, and the tumor neoantigen polypeptide with high affinity is selected, and the tumor neoantigen polypeptide with high affinity forms a second-stage tumor neoantigen database; S3: according to the polypeptide in the second-stage tumor neoantigen database, the polypeptide is elongated before and after, and the TAP transport efficiency after elongation, the immunogenicity score, the flanking enzyme cutting site, and the hydrophilic and hydrophobic are calculated to form a final tumor neoantigen database.

[0029] The sample of the tumor tissue and the corresponding blood are subjected to tumor microenvironment immune infiltration evaluation, and the immune infiltration evaluation includes immunohistochemical staining of four molecular markers CD3, CD8, CD45RO and FOXP3 Figure 1(Only staining results from some patients are listed); Immune Score analysis was performed using tumor tissue samples and corresponding blood samples (e.g.) Figure 2 As shown in the figure, the analysis of the infiltration levels of 10 types of cells in tumor tissue was completed.

[0030] Immunostaining evaluation revealed: 1) The overall immune infiltration rate is not high in patients with pancreatic ductal adenocarcinoma; 2) The immune levels vary among patients with pancreatic ductal adenocarcinoma.

[0031] The above results demonstrate that the peptides containing the 12 key antigenic information presented in this application can effectively target DC-CTL cell immunotherapy, activating immune cells and giving them specific killing ability against hepatitis B-infected cells. This result was observed both in vitro and in vivo in mice. Therefore, these peptides can be used as therapeutic vaccines to directly activate the cellular immune system in the human body, or they can be loaded onto antigen-presenting cells in vitro and further activated to activate cytotoxic T cells, ultimately being autologously reinfused as a cell-based therapeutic vaccine to achieve the effects of preventing and / or treating hepatitis B.

[0032] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A multi-gene mutation tumor neoantigen polypeptide algorithm, characterized in that: The novel antigenic peptide was developed by detecting tumor tissue-specific and expressed multi-gene mutations (12 mutations) through whole-exome (WES) and transcriptome (mRNA) sequencing of tumor tissue and control blood. The antigenic potential of the mutations was assessed by calculating the binding affinity between the mutated epitopes and major histocompatibility complex (MHC) class I and (MHC) class II.

2. The multi-gene mutation tumor neoantigen polypeptide algorithm according to claim 1, characterized in that, The specific sites of the antigenic targets of the multi-gene mutations are the genes APRT p.E141V, KCTD10 p.Y172N, PABPC1 p.L218V, ADAM28 p.C315G, ARF3 p.G6V, KLHL2 p.E88G, CNOT11 p.S148R, C5orf42 p.Q1002L, ABLIM1 p.C190Y, BCLAF1 p.N629S, PABPC1 p.K312RfsTer10, and KRAS p.G12D.

3. The multi-gene mutation tumor neoantigen polypeptide algorithm according to claim 1, characterized in that, The algorithm of the immune epitope database (IEDB) used for the multi-gene mutated antigen includes: analysis of the protein structure, peptide antigenicity, whether it will stimulate the production of IFN-γ, T cell epitope toxicity / adverse reactions, hydrophilicity, hydrophobicity, and other properties of the neoantigen.

4. The multi-gene mutation tumor neoantigen polypeptide algorithm according to claim 1, characterized in that, The neoantigen polypeptide is an amino acid sequence. The design of the 12-peptide sequence includes the antigen's mRNA expression level in tumor cells and the recognition ability of immune cells, screening epitopes with potential immunogenicity from the predicted neoantigens, and, based on the determined antigenic recognition region, preferably, the neoantigen polypeptide undergoes substitution and / or deletion, or the addition of at least one amino acid to obtain a polypeptide with the same or similar function.

5. The multi-gene mutation tumor neoantigen polypeptide algorithm according to claim 4, characterized in that, The polypeptides with the same or similar functions refer to tumor neoantigen polypeptides that can activate HLA typing for HLA-A. 01:01, HLA-A 02:03, HLA-B 37:01, HLA-B 38:02, HLA-C 07:02, HLA-C 06:02, DRB1 10:01, DRB1 12:

02.

6. The multi-gene mutation tumor neoantigen polypeptide algorithm according to claim 1, characterized in that, The tumor tissue is characterized by 12 gene mutations that are expressed and are specific to the tumor tissue. These 12 gene mutations correspond to T cells with specific neoantigens.

7. The multi-gene mutation tumor neoantigen polypeptide algorithm according to claim 6, characterized in that: Three tumor tissue samples and three corresponding blood samples were collected. Genomic DNA and transcriptome mRNA were extracted from each sample and subjected to whole-exome sequencing and transcriptome sequencing, respectively. The prediction and screening included the following steps: S1: By pairwise analysis of whole exome and blood (normal tissue), tumor-specific mutations (such as INDEL) can be obtained. The mutation frequency VAF>1% and the mRNA expression level of the corresponding mutated gene FPKM>1 are used to form the first-stage tumor neoantigen database. S2: Perform antigen affinity prediction on the HLA typing of the sample and the first-stage tumor neoantigen database, and select tumor neoantigen peptides with high affinity. The tumor neoantigen peptides with high affinity constitute the second-stage tumor neoantigen database. S3: Based on the second-stage tumor neoantigen database, the peptides are elongated before and after the elongation and the elongated TAP is calculated to have high transport efficiency, high immunogenicity score, flanking enzyme cleavage sites, and hydrophilicity / hydrophobicity to form the final tumor neoantigen.

8. The multi-gene mutation tumor neoantigen polypeptide algorithm according to claim 7, characterized in that: The tumor tissue samples and corresponding blood samples were used to assess the immune infiltration of the tumor microenvironment. The immune infiltration assessment included: immunohistochemical staining of four molecular markers, CD3, CD8, CD45RO, and FOXP3 (Figure 1, only showing the staining results of some patients); Immune Score analysis was performed on the tumor tissue samples and corresponding blood samples (as shown in Figure 2), and the infiltration levels of 10 types of cells in the tumor tissue were analyzed.

Images