An antigenic peptide and its application

Abstract

This application relates to an antigenic peptide, particularly for treating individuals suffering from ovarian cancer. The antigenic peptide is preferably determined based on the BRCA1 gene c.5470_5477del8 mutation, and is preferably selected from the amino acid sequences of SEQ ID NO 1 to SEQ ID NO 6 or the foregoing amino acid sequences with substitution and / or deletion and / or addition of at least one amino acid. The novel antigen polypeptide provided by the present invention can activate a large number of T lymphocytes specific to the BRCA1 gene c.5470_5477del8 mutation in vitro and release the cytokine IFN-γ, indicating that the polypeptide has obvious immunogenicity and improves the killing ability of T cells against cancer cells of ovarian cancer patients with the BRCA1 gene c.5470_5477del8 mutation. The antigenic peptide of the present invention can stimulate and activate human T cells specific to the BRCA1 gene c.5470_5477del8 mutation in vitro and amplify them in large numbers for adoptive transfer treatment of patients. The antigenic peptide of the present invention fills the blank in the treatment of ovarian cancer patients with BRCA1-c.5470_5477del8 somatic mutations with individualized antigenic peptides.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor immunotherapy, and particularly relates to an antigenic peptide, and more particularly to the design of a novel tumor neoantigen polypeptide based on the c.5470_5477del8 mutation of the BRCA1 gene and its application in the treatment of ovarian cancer. Background Art

[0002] Ovarian cancer (OC) is a malignant tumor that ranks third in incidence among malignant tumors of the female reproductive system and originates from ovarian or fallopian tube cells. It can be divided into three types, corresponding to the tumor development of three cell types: epithelial tumors, germ cell tumors, and stromal tumors, among which epithelial cancers are the most common. Currently, there is no clear cause of ovarian cancer, and there is a lack of specific early screening methods, resulting in about 70% of ovarian cancer patients being in the advanced stage at the time of initial diagnosis, thus missing the best treatment opportunity. Standard care for first-line treatment usually includes extensive tumor resection surgery, combined with platinum-based chemotherapy, the use of the angiogenesis inhibitor bevacizumab, and in some cases, maintenance treatment with poly ADP-ribose polymerase inhibitors (PARPi). Due to the difficulties in early detection and treatment of ovarian cancer, high recurrence rate, poor prognosis, etc., the development of new ovarian cancer treatment methods has important clinical and social significance.

[0003] BRCA1 (Breast Cancer gene 1) and BRCA2 (Breast Cancer gene 2) are the first two hereditary ovarian cancer susceptibility genes discovered, and they are also the most common mutated genes in ovarian cancer. Research has shown that individuals carrying BRCA1 or BRCA2 gene mutations have a significantly increased risk of developing breast or ovarian cancer. BRCA1 or BRCA2 genes belong to tumor suppressor genes, which repair DNA double-strand breaks through the homologous recombination repair pathway. Mutations in BRCA1 or BRCA2 genes lead to homologous recombination deficiency (HRD), which in turn causes the occurrence of malignant tumors. Among the currently known high-risk factors, pathogenic germline mutations in BRCA1 / 2 genes are one of the main inducing factors for ovarian cancer, accounting for 12% - 14% of all ovarian cancer patients. At the same time, the lifetime risk of ovarian cancer in women with positive mutations is 15 - 45% and 10 - 40% respectively. In contrast, the risk of ovarian cancer in ordinary women during their lifetime is 1.4%. Therefore, the National Comprehensive Cancer Network (NCCN) guidelines suggest that all patients with advanced ovarian cancer should undergo BRCA gene testing after diagnosis.

[0004] Cancer immunotherapy is a new type of cancer treatment method and is considered the next-generation treatment after surgery, radiotherapy, chemotherapy, and small molecule targeted therapy. The basic premise for the effectiveness of cancer immunotherapy is the presence of functional, antigen-specific T cells in the tumor. Neoantigens, as ideal targets for immunotherapy, are a promising treatment option. Tumor neoantigens are peptides presented on the surface of tumor cells. As nascent antigens encoded by mutated genes in tumor cells, they are mainly generated by point mutations, deletion mutations, gene fusions, etc. Therefore, they are new abnormal proteins different from the proteins expressed by normal cells. Tumor neoantigens combine with major histocompatibility complex (MHC) molecules and exist on the surface of tumor cells in the form of protein complexes, which can be specifically recognized by cytotoxic T cell receptors (TCRs), thus activating the immune response of T cells. Compared with traditional treatment methods, immunotherapy focuses on eliminating tumor cells by activating and enhancing the patient's own immune system. This treatment method has the following advantages: precise treatment, fewer side effects, and long-lasting effects. In addition, the body's immune system also has the characteristic of immune memory. Therefore, immunotherapy can help patients form memory-type immunity to prevent the recurrence and metastasis of tumors.

[0005] Since mutant proteins generate antigenic epitopes that are not present in wild-type proteins, finding somatic mutation sites specific to the mutant genes of cancer patients and designing and screening effective neoantigen polypeptides based on this have become a popular research direction. The patent document with publication number CN104962612A discloses a frameshift mutation of BRCA1 gene g.41256139delT and its application in preparing a breast cancer auxiliary diagnosis kit, which screens out highly specific mutation sites related to breast cancer and provides support for the screening and diagnosis of breast cancer. Earlier studies by ARuangapirom L et al. (Vaccines. 2022 Sep 22; 10(10): 1597) showed that the antigen potential of top recurrent somatic mutations was evaluated by calculating the binding affinity between mutant epitopes and MHC class I, and it was found that most recurrent mutations were predicted to be antigenic. The researchers designed neoantigen polypeptides IKILCATYVK and KILCATYVK for the N345K site mutation of the PIK3CA gene (in vitro studies showed it was antigenic), and found that the neoantigen polypeptides had a strong affinity with HLA-A*11:01 and HLA-A*31:01 molecules and could effectively activate T cells in patients and kill tumors. The Human Mutation article showed that the detection methods used in previous studies mainly covered the exon regions of genes and a small part of intron positions, and the reported BRCA gene variations were widely distributed at various positions of the genes and did not show a concentrated distribution. Among them, the c.5470_5477del ATTGGGCA variation of the BRCA1 gene and the c.3109C>T variation of the BRCA2 gene were reported the most times (Comprehensive Profiling of BRCA1 and BRCA2 Variants in Breast and Ovarian Cancer in Chinese Patients. Hum Mutat. 2019 Dec 11. doi: 10.1002 / humu.23965). Further, among BRCA1 mutations, the c.5470_5477del8 mutation site had the highest occurrence frequency.

[0006] Effective neoantigens need to meet the following conditions: they must be tumor-specific to prevent off-target effects from affecting the healthy tissues of patients; they can be presented on the surface of tumor cells so that immune cells can recognize and kill tumor cells; they must be able to activate an immune response (be immunogenic). Screening neoantigens from a vast number of tumor peptides is not easy and requires considering various factors. There has been no report on effective tumor neoantigens targeting the c.5470_5477del8 mutation in BRCA1.

[0007] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, when the applicant made this invention, a large number of literatures and patents were studied, but due to space limitations, all details and contents were not listed in detail. However, this does not mean that this invention does not possess the features of these prior arts. On the contrary, this invention already possesses all the features of the prior arts, and the applicant reserves the right to add relevant prior arts in the background art. SUMMARY OF THE INVENTION

[0008] In a first aspect of the present invention, there is provided an antigenic peptide, particularly for treating an individual suffering from ovarian cancer, which is an amino acid sequence selected from SEQ ID NO 1 to SEQ ID NO 6 or the foregoing amino acid sequence with at least one amino acid substituted and / or deleted and / or added.

[0009] Specifically, SEQ ID NO 1 is MVRWAAASK.

[0010] Specifically, SEQ ID NO 2 is MPVSTTLVA.

[0011] Specifically, SEQ ID NO 3 is ILSVMNVHDF.

[0012] Specifically, SEQ ID NO 4 is SSAKRPLWLK.

[0013] Specifically, SEQ ID NO 5 is RVAQDTPHL.

[0014] Specifically, SEQ ID NO 6 is SLPPLPFWV.

[0015] It has been reported that the frequency of large rearrangements of BRCA1 and BRCA2 is less than 10% of all BRCA1 / 2 mutations in the Asian population. One possible reason is the heterogeneity of the population. Therefore, it is necessary to conduct genetic analysis by stratifying populations in different regions. By consulting a large number of literatures, the present invention finds that Shi T et al. (Int J Cancer. 2017 May 1; 140(9): 2051 - 2059) studied the mutation distribution of ovarian cancer in the Chinese population and reported a high - frequency pathogenic mutation c.5470_5477del8 on BRCA1, and the positive mutation is highly associated with an increased risk of ovarian cancer. Selecting this locus is in line with the current situation of ovarian cancer in China and can establish a more effective personalized treatment plan for patients.

[0016] The present invention provides a design of a polypeptide sequence for an antigenic recognition region determined based on the c.5470_5477del8 mutation of the BRCA1 gene. First, the antigenic potential of top recurrent somatic mutations is evaluated by calculating the binding affinity between mutant epitopes and major histocompatibility complex (MHC) class I. The present invention uses the ImmuneEpitope Database (IEDB) algorithm to analyze the characteristics of antigens such as protein structure, surface accessibility, and hydrophilicity. At the same time, considering the expression level of antigens on tumor cells and the recognition ability of immune cells to them, epitopes with potential immunogenicity are screened from the predicted neoantigens, and polypeptides with specific amino acid sequences are designed based on the determined antigenic recognition region. Based on the above path, the amino acid sequence of the neoantigen polypeptide screened by the present invention is MVRWAAASK (SEQ ID NO.6) corresponding to No. 6, or a polypeptide with the same or similar function obtained by substituting and / or deleting and / or adding at least one amino acid in the amino acid sequences of the above polypeptides. The above polypeptides with the same or similar function (tumor antigen polypeptides) can stimulate patients with HLA typing of HLA-A*03:01 and accompanied by the c.5470_5477del8 mutation of BRCA1 to produce specific antibodies. The highly immunogenic and tumor-specific neoantigens provided by the present invention against the c.5470_5477del8 mutation of BRCA1 provide an effective treatment method for individualized ovarian cancer immunotherapy.

[0017] The present invention also provides a method for verifying the in vitro immunogenicity of the above tumor neoantigen polypeptide, which specifically includes the following three steps: in vitro isolation of peripheral blood mononuclear cells (PBMC) from tumor patients, T cell incubation, and detection of T cell IFN-γ secretion by enzyme-linked immunospot assay (ELISPOT). Through scientific design and combination, a better scheme for isolating and culturing PBMC is provided, and the specific T lymphocytes activated by the polypeptide and the cytokine IFN-γ released by them are verified in vitro based on the color reaction, so as to evaluate the immunological activity of the synthesized tumor neoantigen, providing a theoretical basis for the subsequent development of tumor neoantigen vaccines. The tumor antigen peptide provided by the present invention can stimulate and activate human T cells specific for the c.5470_5477del8 mutation of the BRCA1 gene in vitro, and cause the activated T cells to expand in large numbers. This tumor antigen peptide can be used for adoptive transfer therapy of patients.

[0018] According to a preferred embodiment, the antigenic peptide is determined based on the c.5470_5477del8 mutation of the BRCA1 gene. More preferably, the antigenic peptide is SEQ ID NO 1 or the amino acid sequence of SEQ ID NO 1 with at least one amino acid substitution and / or deletion and / or addition.

[0019] According to a preferred embodiment, the antigenic peptide is an HLA tumor antigen peptide corresponding to the major histocompatibility complex class I complex.

[0020] According to a preferred embodiment, the HLA tumor antigen peptide provided in the first aspect of the present invention has one or more mutations relative to the wild-type HLA tumor antigen peptide. Compared with the wild-type HLA tumor antigen peptide, when the body is stimulated with the HLA tumor antigen peptide provided in the first aspect of the present invention, the affinity of the body for the specific binding of the T cell receptor increases.

[0021] According to a preferred embodiment, the antigenic peptide activates and releases the cytokine IFN-γ by stimulating T lymphocytes to improve the body's immune response ability.

[0022] According to a preferred embodiment, the IC50 of the affinity between the antigenic peptide and the major histocompatibility complex class I complex is less than 500 nM. Preferably, the IC50 of the affinity between the antigenic peptide and the major histocompatibility complex class I complex is less than 50 nM.

[0023] The present invention also relates to the use of the antigenic peptide provided in the first aspect of the present invention in the early diagnosis and prognosis evaluation of ovarian cancer. This application is especially the application of preparing reagents, kits or substances used in the early diagnosis and prognosis evaluation of ovarian cancer.

[0024] The second aspect of the present invention provides a polynucleotide comprising a base sequence encoding the antigenic peptide provided in the first aspect of the present invention.

[0025] The third aspect of the present invention provides a population of antigen-presenting cells obtained by pulsing with the antigenic peptide provided in the first aspect of the present invention or transfected with the polynucleotide provided in the second aspect of the present invention.

[0026] The fourth aspect of the present invention provides a vaccine or immunogenic composition capable of eliciting a T cell response, comprising: the antigenic peptide provided in the first aspect of the present invention, optionally having a physiologically acceptable buffer, carrier or excipient, and / or optionally having an adjuvant or immunostimulant; and / or the polynucleotide provided in the second aspect of the present invention; and / or the population of antigen-presenting cells provided in the third aspect of the present invention.

[0027] A vaccine composition refers to a composition used to generate immunity for preventing and / or treating diseases. Specifically, a vaccine is a drug containing or generating antigens, which is used for humans or animals to produce specific antibodies and protective substances through the vaccine.

[0028] An immunogenic composition refers to a composition that contains or generates antigens and is capable of eliciting an antigen-specific humoral or cellular immune response (such as a T cell response).

[0029] A vaccine or immunogenic composition can be a pharmaceutical composition, and the pharmaceutical composition also includes pharmaceutically acceptable adjuvants, immunostimulants, stabilizers, carriers, diluents, and / or excipients, etc. Such materials are non-toxic and do not interfere with the efficacy of the active ingredients.

[0030] The fifth aspect of the present invention provides an antibody or its antigen-binding fragment, a T cell receptor, or a chimeric antigen receptor (CAR) that specifically binds to the antigen peptide provided in the first aspect of the present invention.

[0031] According to a preferred embodiment, the antibody can be a T cell receptor-like antibody.

[0032] Preferably, the antibody includes monoclonal antibodies and polyclonal antibodies, which include intact antibodies and functional antibody fragments (i.e., antigen-binding fragments).

[0033] Preferably, the antibody includes a light chain variable region and a heavy chain variable region, for example, in the form of scFv.

[0034] Preferably, the antibody includes variant polypeptide species having one or more amino acid substitutions, insertions, or deletions in the natural amino acid sequence. The antibody retains or substantially retains its specific binding function.

[0035] According to a preferred embodiment, the chimeric antigen receptor (CAR) is a chimeric antigen receptor based on a T cell receptor-like antibody.

[0036] Preferably, the chimeric antigen receptor includes: an extracellular region, a transmembrane region, one or more co-stimulatory domains, and an intracellular signaling domain.

[0037] Preferably, the chimeric antigen receptor can be designed to separately recognize tumor neoantigen peptides, or to recognize tumor neoantigen peptides in combination with HLA molecules or MHC molecules.

[0038] The sixth aspect of the present invention provides a method for producing the antibody or its antigen-binding fragment, T cell receptor or chimeric antigen receptor provided by the fifth aspect of the present invention, which includes: the step of selecting an antibody or its antigen-binding fragment, T cell receptor or chimeric antigen receptor that binds to the antigen peptide provided by the first aspect of the present invention, wherein the antibody or its antigen-binding fragment, T cell receptor or chimeric antigen receptor optionally binds to a major histocompatibility complex or HLA molecule, or is optionally expressed on the cell surface.

[0039] The seventh aspect of the present invention provides a polynucleotide that encodes the antibody or its antigen-binding fragment, T cell receptor or chimeric antigen receptor provided by the fifth aspect of the present invention.

[0040] The eighth aspect of the present invention provides a vector that contains the polynucleotide provided by the seventh aspect of the present invention.

[0041] The ninth aspect of the present invention provides an immune cell that specifically binds to the antigen peptide provided by the first aspect of the present invention. Preferably, the immune cell can be a T cell, NK cell or dendritic cell.

[0042] The present invention also relates to the use of the antigen peptide provided by the first aspect of the present invention, the polynucleotide provided by the second aspect, the population of antigen-presenting cells provided by the third aspect, the vaccine or immunogenic composition provided by the fourth aspect that can elicit a T cell response, the antibody or its antigen-binding fragment, T cell receptor or chimeric antigen receptor provided by the fifth aspect, the method for producing an antibody or its antigen-binding fragment, T cell receptor or chimeric antigen receptor provided by the sixth aspect, the polynucleotide provided by the seventh aspect, the vector provided by the eighth aspect, and the immune cell provided by the ninth aspect in the treatment of ovarian cancer. Preferably, this use is particularly the use for preparing substances used in the treatment of ovarian cancer. Preferably, this use includes directly or indirectly treating ovarian cancer using the aforementioned substances.

[0043] In addition, the present invention also relates to the use of the antigen peptide provided by the first aspect of the present invention, the polynucleotide provided by the second aspect, the population of antigen-presenting cells provided by the third aspect, the vaccine or immunogenic composition provided by the fourth aspect that can elicit a T cell response, the antibody or its antigen-binding fragment, T cell receptor or chimeric antigen receptor provided by the fifth aspect, the method for producing an antibody or its antigen-binding fragment, T cell receptor or chimeric antigen receptor provided by the sixth aspect, the polynucleotide provided by the seventh aspect, the vector provided by the eighth aspect, and the immune cell provided by the ninth aspect in adoptive immunotherapy for ovarian cancer. Preferably, this use is particularly the use for preparing substances used in adoptive immunotherapy for ovarian cancer.

[0044] Technical effects:

[0045] The neoantigen polypeptides provided by the present invention can serve as targets for tumor immunotherapy, attacking tumor cells by activating the patient's own immune system or enhancing the patient's immune response ability. The neoantigen polypeptides provided by the present invention can be polypeptides with the same or similar functions obtained by substituting and / or deleting and / or adding at least one amino acid in the amino acid sequences of the polypeptides of SEQ ID NO 1 to SEQ ID NO 6. Among them, the above-mentioned polypeptides with the same or similar functions refer to that the tumor antigen polypeptides can activate patients with HLA typing of HLA-A*03:01 and accompanied by a c.5470_5477del8 mutation in BRCA1 to generate specific T cells against tumors with a c.5470_5477del8 mutation in BRCA1. In particular, the neoantigen polypeptides provided by the present invention can massively activate T lymphocytes specifically targeting the c.5470_5477del8 mutation in the BRCA1 gene in vitro and release the cytokine IFN-γ, indicating that the polypeptide has obvious immunogenicity and enhances the killing ability of T cells against cancer cells of ovarian cancer patients with a c.5470_5477del8 mutation in the BRCA1 gene. The antigen peptides of the present invention fill the gap in the treatment of ovarian cancer patients with somatic mutations of BRCA1-c.5470_5477del8 in individualized antigen peptides. The expression of the tumor neoantigens of the present invention has individual differences, and individualized treatment plans can be formulated for patients according to the expression of the tumor neoantigens of the patients, so as to achieve the purpose of improving the treatment effect. The antigen polypeptides of the present invention can be synthesized on a large scale and used in the standardized and individualized immunotherapy of patients with BRCA1-c.5470_5477del8 mutant tumors in the future.

[0046] Furthermore, the present invention uses antigen peptide-specific CTL cells (Antigen Peptide-Specific Cytotoxic T Lymphocytes) as effector cells and human ovarian cancer cell line A2780 as target cells to explore the ability of neoantigen-specific CTL to kill tumor cells in vitro under the conditions of effector-to-target ratios of 1:1 and 4:1. The results show that when the effector-to-target ratio is 4:1, the tumor killing rate of the neoantigen peptide (MVRWAAASK) reaches 18.8%, and it can promote the effective killing of tumor cells by CTL cells.

[0047] The tumor neoantigens of the present invention can serve as tumor markers for ovarian cancer and be used for the early diagnosis and prognosis evaluation of ovarian cancer, and the presence and development of tumors can be judged by detecting the level of tumor neoantigens in the body fluids of patients.

[0048] In terms of drug research and development, the novel tumor antigens provided by the present invention can serve as targets for drug research and development, and play an important role in the development of antibody drugs or small molecule targeted drugs against the novel tumor antigens of ovarian cancer. These drugs can selectively target tumor cells and inhibit their growth and spread. At the same time, these drugs do not harm the normal healthy tissues of the individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a diagram for evaluating the immune infiltration level by immunohistochemical staining of four molecular markers in (partial) ovarian cancer patients of the present invention;

[0050] Figure 2 It is the result of immune infiltration scores of 38 ovarian cancer patients (Pt01 - Pt38) in Example 2 of the present invention;

[0051] Figure 3 It is a diagram of the experimental results of in vitro cell evaluation for evaluating the effectiveness of the synthetic polypeptide sequences numbered 6 and numbered 2 in Example 3 of the present invention;

[0052] Figure 4 It is a statistical chart of the ELISPOT results of the synthetic polypeptide sequences numbered 6 and numbered 2 in Example 3 of the present invention, where SFU / 10 6 cells are spot forming units per million cells, obtained by multiplying the number of plated cells 2×10 5 by 5 (i.e., multiplying the average number of spots by 5);

[0053] Figure 5 It is a statistical chart of the killing rate of neoantigen - specific CTL cells against tumor cells in Example 4 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0054] The following is a detailed description with reference to the accompanying drawings.

[0055] BRCA1 refers to the Breast cancer 1 gene.

[0056] In the present invention, antigenic peptides are equivalent to neoantigens, neoantigenic peptides, neoantigenic polypeptides, and novel tumor antigens. The major histocompatibility complex class I complex is also denoted as MHC class I complex. Human leukocyte antigen is also known as HLA antigen and histocompatibility antigen, and is a group of human genes that are very important for the function of the immune system. HLA - antigenic peptides can serve as mediators between the corresponding MHC complexes presented on the cell surface and the T - cell receptor. TCR, namely T - cell receptor, has diversity and is an important part for recognizing antigens. CD3 is a unique molecular marker of T cells and is expressed on the surface of all T cells. CD8 is a leukocyte differentiation antigen, which is used to assist TCR in recognizing antigens and participates in the transduction of T - cell activation signals. CD45RO is a marker of memory T cells. FoxP3 is a marker molecule of regulatory T cells (Treg).

[0057] In all the following experimental results, the activity analysis was repeated more than 3 times, and the results were expressed as mean ± standard deviation. Statistical analysis was performed using a t-test (Student’s t-test).

[0058] It should be noted that whether it is the electrophoresis band of protein or DNA, an obvious electrophoresis band means that the expression level of the protein / DNA in this group is higher than that of the protein / DNA in the group corresponding to the non-obvious electrophoresis band.

[0059] In this application, unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial channels. The specific examples described in these implementation cases are only used to explain the present invention, rather than to limit the present invention. In order to better understand the present invention rather than limit the scope of the present invention, all numbers representing amounts, times, percentages, and other numerical values used in the present invention should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to different desired effects.

[0060] Example 1

[0061] This example provides a process for predicting and screening tumor neoantigens using a tumor neoantigen prediction platform.

[0062] A total of 38 ovarian cancer tumor tissue samples and corresponding adjacent tissues were collected clinically. Genomic DNA was extracted and whole-exome sequencing and transcriptome sequencing were performed. First, through paired analysis of the whole exome and adjacent tissues (normal tissues), specific mutations in the tumor tissues (such as Insertion / Deletion, INDEL, etc.) were obtained, and the peptide segments of these specific mutations constituted the first-stage tumor neoantigen database. Secondly, the gene expression level (Fragments Per Kilobase of transcript per Million mapped reads, FPKM) was calculated based on the transcriptome sequencing data of the tumor samples, and compared with the reference genome. Neoantigens with FPKM < 10 in the first-stage tumor neoantigen database were filtered to form the second-stage tumor neoantigen database. Finally, HLA genotyping was performed on each patient, and the HLA typing of each patient and the second-stage tumor neoantigen database were used for antigen affinity prediction. The lower the IC50 value, the higher the affinity and the stronger the immune response may be. Tumor neoantigens with IC50 values < 500 nM were selected to form the final tumor neoantigen database. In this example, a total of 6 tumor neoantigen polypeptides were screened as candidates. Specifically, the amino acid sequences of the tumor neoantigen peptide epitopes include: SLPPLPFWV (SEQ ID NO 6); RVAQDTPHL (SEQ ID NO 5); SSAKRPLWLK (SEQ ID NO 4); ILSVMNVHDF (SEQ ID NO 3); MPVSTTLVA (SEQ ID NO 2); MVRWAAASK (SEQ ID NO 1) (see Table 1).

[0063] Table 1 Information of candidate tumor neoantigen peptides

[0064]

[0065] Table 1 shows that the IC50 values of the neoantigen peptides with amino acid sequences SLPPLPFWV, SSAKRPLWLK, and MVRWAAASK are < 50 nM, proving that these neoantigen peptides have high binding affinity with HLA molecules and strong ability to activate T cells, especially improving the recognition and killing functions of T cells.

[0066] For the new antigen peptide with the amino acid sequence MVRWAAASK, which is determined based on the c.5470_5477del8 mutation of BRCA1, a gene closely related to hereditary breast and ovarian cancers, this antigen peptide may have particular value in the prevention and treatment of these cancers. The high affinity of this antigen peptide for HLA-A*03:01 (IC50 value of 29.71) indicates that this antigen peptide is easily recognized by T cells during immune surveillance, contributing to the early detection and elimination of potential tumor cells by the immune system. This antigen peptide can be effectively presented to the immune system, whether in the case where cancer has already occurred or in healthy individuals with a genetic risk. Specifically, the antigen peptide is first captured and processed by antigen-presenting cells (APCs), such as dendritic cells (DCs), macrophages, and B cells; the processed antigen peptide binds to major histocompatibility complex molecules, especially HLA-A*03:01, and then is presented on the cell surface; when the T cell receptor recognizes the antigen peptide-MHC molecule complex, the T cell is activated, and this recognition is specific and depends on the high match between the antigen peptide and the HLA molecule. The activated T cells begin to proliferate and differentiate into effector T cells and memory T cells. Effector T cells can be further divided into cytotoxic T cells (CTLs) and helper T cells (such as Th1 and Th2). Cytotoxic T cells (CTLs) can recognize the same antigen peptide-MHC complex on the surface of tumor cells and release cytotoxic molecules, such as perforin and granzyme, leading to the lysis and death of tumor cells; helper T cells enhance the immune response by releasing cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). These cytokines can promote the inflammatory response and activate other immune cells, such as natural killer (NK) cells and macrophages. Memory T cells persist in the body for a long time and can respond quickly when encountering the same antigen again, providing long-term immune protection. This antigen peptide can, in the form of a prophylactic vaccine, stimulate the immune system's vigilance against potential tumor cells, thus providing protection before tumor formation. Since this antigen peptide is designed against specific mutations, it can direct the immune system to specifically recognize and kill tumor cells expressing these mutations, reducing damage to normal cells.

[0067] For the neoantigen peptide with the amino acid sequence SLPPLPFWV, the high affinity of this antigen peptide for HLA-A*02:01 (IC50 value of 12.05 nM) means that it is particularly targeted at individuals with this HLA genotype, that is, this antigen peptide has a higher immune activation potential in the population with this genotype. Due to the low IC50 value of this antigen peptide, it is easily recognized by T cells during the immune surveillance process, which helps immune cells detect and eliminate tumor cells at an early stage. TP53 is a tumor suppressor gene, and its mutations are common in various cancers, including breast cancer, ovarian cancer, lung cancer, colorectal cancer, etc. Therefore, this antigen peptide may have broad application prospects in a variety of tumor types. Since the p53 protein encoded by the TP53 gene is involved in various biological processes such as cell cycle control, DNA repair, and apoptosis, the immune activation process involving this antigen peptide may promote the apoptosis of tumor cells, reduce the survival rate of tumor cells, and thus inhibit tumor growth. TP53 can also inhibit tumor angiogenesis by regulating the expression of angiogenesis inhibitors, and the immune activation process involving this antigen peptide may help reduce the blood supply to tumors, thereby limiting tumor growth and metastasis. After immune activation, the immune system can form a memory of TP53 mutant cells, which helps to rapidly generate an immune response when encountering the same or similar tumor cells in the future, providing long-term immune protection.

[0068] For the neoantigen peptide with the amino acid sequence SSAKRPLWLK, it can be effectively presented to the immune system, especially CD8 +T cells. This enhances the ability of T cells to recognize mutant PIK3CA tumor cells, thus helping the immune system to more accurately locate and attack tumors. The high affinity of this antigenic peptide for HLA-A*11:01 (IC50 value of 15.14 nM) gives it significant immune activation ability in individuals with this HLA genotype. The relatively low IC50 value indicates that this antigenic peptide has high potential in immune activation and may play an important role in immunotherapy. PIK3CA is a key regulator of the PI3K / AKT / mTOR signaling pathway, which plays a crucial role in cell growth, proliferation, survival, and metabolism, and its mutations are common in various cancers. This antigenic peptide may enhance the immune system's attack on tumors by affecting this key signaling pathway. PIK3CA gene mutations often lead to abnormal activation of PI3K, which in turn activates downstream AKT and mTOR. By activating the immune system, this antigenic peptide may indirectly inhibit the activity of PI3K, blocking or weakening its signal transduction. This antigenic peptide may also inhibit the abnormal progression of the cell cycle by affecting the activity of AKT. This antigenic peptide can also indirectly affect the activity of AKT by activating immune cells such as T cells to release cytokines, thereby promoting apoptosis and reducing the survival of tumor cells. mTOR is a key regulator of cell metabolism, affecting protein synthesis, lipid metabolism, and glucose uptake. This antigenic peptide may change the metabolic state of tumor cells by affecting the activity of mTOR, thereby inhibiting their energy supply and biosynthesis.

[0069] In summary, MVRWAAASK, SLPPLPFWV, and SSAKRPLWLK have high affinity for HLA molecules and can target specific HLA types (highly matched with specific HLA molecules), ensuring that they can be effectively presented to the immune system in individuals with the corresponding HLA types. These characteristics make the selected neoantigenic peptides have significant potential in cancer immunotherapy, thus enabling the provision of individualized treatment plans for patients and enhancing the immune system's ability to attack tumors.

[0070] Example 2

[0071] This example provides the results of immune infiltration assessment based on immunohistochemical staining.

[0072] In this example, the tumor microenvironment immune infiltration of the above 38 ovarian cancer patients was evaluated respectively. The specific methods included: completing immunohistochemical staining of four molecular markers, CD3 / CD8 / CD45RO / FOXP3, and the results are as Figure 1 shown (only listing the staining results of some patients); completing the analysis of the immune infiltration score Immune Score of tumor patients, and the results are as Figure 2 shown; completing the analysis of the infiltration level of 1O types of cells in tumor tissues.Figure 2 Pt01 - Pt38 in it respectively represent 38 ovarian cancer patients, Figure 1 and the immunohistochemical staining results of patients Pt09 - Pt12 are shown in it.

[0073] When the body gets sick, macrophages will infiltrate into the diseased tissue, participating in the clearance of pathogens and tissue repair. The degree of infiltration can be used as an indicator to evaluate the severity and mixed prognosis of the disease. Through immunohistochemical staining evaluation, it is found that: 1) The overall immune infiltration of ovarian cancer patients is not high; 2) There are differences in the immune levels of different ovarian cancer patients.

[0074] Example 3

[0075] This example provides an in vitro cell experiment to evaluate the effectiveness of tumor neoantigen polypeptide sequences.

[0076] The specific steps are as follows:

[0077] 1) Collection of peripheral blood mononuclear cells (PBMC)

[0078] Take 5 mL of the patient's peripheral blood and add an equal volume of PBS buffer. Take another 50 mL centrifuge tube, add 6 mL of Ficoll (lymphocyte separation solution), add the diluted blood sample, centrifuge at 1500 rpm at room temperature for 30 min, remove the upper plasma layer, collect the middle buffy coat layer (PBMC), and store it in liquid nitrogen.

[0079] 2) Incubation of T cells

[0080] Take out the cryopreserved PBMC cells from liquid nitrogen and quickly thaw and recover the cells in a 37 °C water bath; centrifuge at 1600 rpm at room temperature for 5 min to remove the supernatant, resuspend the cells with a culture medium containing 5% fetal bovine serum, mix well and stain with trypan blue, and count the number of live cells; after counting, adjust the cell concentration to 2×10 5 / mL, and culture in a 24 - well plate; add the 6 synthetic peptide segments in Example 1 and the cell growth factors IL - 2 / IL - 7 (working concentration 50 ng / mL), and culture in a 37 °C incubator; after ten days, collect the cells and wash the cells with 3 volumes of PBS buffer (centrifuge at 1600 rpm for 5 min).

[0081] 3) Detection and counting by enzyme - linked immunospot assay (ELISPOT)

[0082] First, add 100 μL of the coating antibody to each well of the ELISPOT plate and incubate overnight at 4 - 8 °C. Secondly, add 200 μL of 1640 culture medium and incubate at room temperature for at least 30 min. Add the peptide segments in Example 1 (working concentration: 5 μg / mL) respectively, and set up positive and negative control groups (water) for each. Incubate in a 37 °C incubator (5% CO2) for 12 - 48 h. Finally, perform a color reaction. Add the detection antibody and streptavidin to the wells. After washing away the excess antibody, add the colorimetric substrate, wash three times with sterile water, air-dry the plate, and detect and count the number of spots under a dissecting microscope.

[0083] 4) Result analysis

[0084] Through ELISPOT plate spot counting, the activation effect of the 6 selected novel tumor antigen polypeptides on patients' T cells was systematically analyzed, as Figure 3 shown. The increase in spot counting directly reflects the activation degree of T cells. Figure 3 The activation effects of the synthetic peptide segment numbered 6 and the synthetic peptide segment numbered 2 on patients' T cells are respectively shown. Among them, lectin (Phytohemagglutinin, PHA) is the positive control, and the working concentration of PHA is 5 μg / mL. Medium is the negative control group, which is an equal volume of sterile water. The spot counting results in this experiment are shown in Figure 4 . After calculation, the spot-forming units (SFU) / million cells of the polypeptide numbered 6 is 297.5, which is greater than the positive threshold of 50 (Porter M et al., J Invest Dermatol. 2022). The number of spots caused by the peptide segment numbered 6 is significantly more than that of the control group and the peptide segment numbered 2, indicating that this polypeptide can significantly activate T lymphocytes and release the cytokine IFN-γ, that is, the polypeptide produced by the BRCA1 gene carrying the c.5470_5477del8 mutation can stimulate the body to produce an immune response, has strong immunogenicity, and increases the killing ability of T cells against cancer cells with the c.5470_5477del8 mutation in the BRCA1 gene. In contrast, the spot-forming units (SFU) / million cells of the peptide segment numbered 2 is 7.5, which is less than the positive threshold, indicating that it cannot stimulate T lymphocyte activation and release the cytokine IFN-γ, and has no immunogenicity or weak immunogenicity. The polypeptide numbered 6 is a polypeptide derived from the c.5470_5477del8 mutation of the BRCA1 gene. The strong T cell response caused by the peptide segment numbered 6 not only contributes to the current tumor clearance but also may promote the formation of immune memory, providing long-term protection against possible future tumor recurrence. The strong immunogenicity of the peptide segment numbered 6 makes it a strong candidate for developing cancer vaccines, especially for patients with the corresponding HLA types.

[0085] Figure 3and Figure 4 The experimental results of Figure 4 show that the polypeptide MVRWAAASK (synthetic peptide segment No. 6) targeting the BRCA1-c.5470_5477del8 somatic mutation can significantly activate T lymphocytes in the body of ovarian cancer patients and release the cytokine IFN-γ, and this polypeptide has obvious immunogenicity. The results of this example indicate that the neoantigen polypeptide targeting the c.5470_5477del8 mutation site of the BRCA1 gene can be used in standardized and individualized tumor immunotherapy in the future.

[0086] Example 4

[0087] This example provides the exploration process of the in vitro killing of tumor cells by neoantigen-specific CTL.

[0088] The synthetic peptide segment 2 and synthetic peptide segment 6 in Example 1 were respectively presented by dendritic cells and co-cultured with cytotoxic T lymphocytes (CTL cells), and the antigen peptide 2- and antigen peptide 6-specific CTL cells obtained were used as effector cells. Using the human ovarian cancer cell line A2780 as the target cell, the effector-to-target ratios were set at 1:1 and 4:1 respectively. The lactate dehydrogenase (LDH) method was used to detect the killing level of CTL on tumor cells, and the killing efficiency of CTL cells was calculated. Figure 5 The results showed that when the effector-to-target ratio was 1:1, the tumor killing rate of neoantigen peptide 6 was 5.5%, and the tumor killing rate of neoantigen peptide 2 was 0.2%; when the effector-to-target ratio was 4:1, the tumor killing rate of neoantigen peptide 6 was 18.8%, and the tumor killing rate of neoantigen peptide 2 was 3.7%. When the ratio of effector cells to target cells was 1:1 or 4:1, the tumor killing rate of peptide segment No. 6 was significantly higher than that of peptide segment No. 2, which indicates that under the same conditions, peptide segment No. 6 can more effectively activate effector cells, that is, peptide segment No. 6 has a higher tumor cell killing rate, reflecting its wider effector cell coverage and stronger activation intensity in immune activation. The experimental results show that the neoantigen peptide 6 (MVRWAAASK) can promote the effective killing of tumor cells by CTL cells under different effector-to-target ratio conditions. When the ratio of effector cells to target cells increased to 4:1, the tumor killing rate of peptide segment No. 6 further increased, while the increase amplitude of peptide segment No. 2 was smaller, which shows that peptide segment No. 6 can more effectively play its killing role when the number of effector cells increases. The high tumor killing rate of peptide segment No. 6 may be related to its specific immune response. This specific response can more accurately identify and attack tumor cells, reducing damage to normal cells. The results indicate that the DC-CTL cell immunotherapy targeting MVRWAAASK has potential efficacy for the treatment of ovarian cancer, and this antigen peptide provides a basis for further laboratory research and clinical development, helping to explore new cancer treatment strategies.

[0089] It should be noted that the above specific embodiments are exemplary. Those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also fall within the scope of the disclosure of the present invention and within the protection scope of the present invention. Those skilled in the art should understand that the description and drawings of the present invention are illustrative and do not constitute a limitation on the claims. The protection scope of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts. For example, "preferably" and "according to a preferred embodiment" indicate that the corresponding paragraphs disclose an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept. Throughout the text, the features guided by "preferably" are only optional and should not be understood as being necessarily provided. Therefore, the applicant reserves the right to waive or delete relevant preferred features at any time.

Claims

1. An immunogenic composition capable of eliciting a T cell response, characterized in that, It comprises: An antigenic peptide, polynucleotide and / or population of antigen-presenting cells; And a pharmaceutically acceptable buffer, carrier or excipient, and / or having an adjuvant; The amino acid sequence of the antigenic peptide is as shown in SEQ ID NO.1, The polynucleotide comprises a base sequence encoding an antigenic peptide with an amino acid sequence of SEQ ID NO.1, The population of antigen-presenting cells is obtained by pulsing with an antigenic peptide having an amino acid sequence of SEQ ID NO.1 or transfecting with a polynucleotide comprising a base sequence encoding an antigenic peptide having an amino acid sequence of SEQ ID NO.

1.

2. Use of an antigenic peptide, polynucleotide or population of antigen-presenting cells in the preparation of a medicament for preventing or treating ovarian cancer, characterized in that, The amino acid sequence of the antigenic peptide is as shown in SEQ ID NO.1, The polynucleotide comprises a base sequence encoding an antigenic peptide with an amino acid sequence of SEQ ID NO.1, The population of antigen-presenting cells is obtained by pulsing with an antigenic peptide having an amino acid sequence of SEQ ID NO.1 or transfecting with a polynucleotide comprising a base sequence encoding an antigenic peptide having an amino acid sequence of SEQ ID NO.1.

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