HLA-A*11: 01 restrictive neoantigen peptide based on malignant pleural effusion tumor cells and application of HLA-A*11: 01 restrictive neoantigen peptide

Abstract

The invention relates to the technical field of tumor immunotherapy, in particular to an HLA-A * 11: 01 restrictive neoantigen peptide based on malignant pleural effusion tumor cells and application of the HLA-A * 11: 01 restrictive neoantigen peptide. The tumor neoantigen polypeptide is separated and screened from malignant pleural effusion of a lung cancer patient, and the amino acid sequence of the neoantigen polypeptide is shown as any one of SEQ ID NO.1-9. The tumor neoantigen polypeptide has high affinity with HLA molecules, can stimulate generation of tumor specific T cells, and can be used as a target spot for clinical treatment or diagnosis of tumors. The invention also provides a method for separating and screening the tumor neoantigen polypeptide, the method solves the problem that the neoantigen of a non-surgical treatment patient cannot be obtained, and an effective method is provided for the development of the tumor neoantigen polypeptide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor immunotherapy, and particularly relates to HLA-A*11:01 restricted neoantigen peptides based on malignant pleural effusion tumor cells and application thereof. BACKGROUND

[0002] Malignant pleural effusion (MPE) is a common complication in patients with advanced malignancies, which can seriously affect the respiratory and circulatory functions of patients. Malignant pleural effusion is more common in patients with lung cancer. About 10%~15% of patients with non-small cell lung cancer (NSCLC) have malignant pleural effusion at initial diagnosis, and 50% of NSCLC patients will eventually develop pleural metastasis and cause malignant pleural effusion (Yang Y, Du J, Wang YS, et al. Prognostic impact of pleural effusion in patients with malignancy: a systematic review and meta-analysis. Clin Transl Sci. 2022, 15(6):1340-1354; Siegel RL, Miller KD, Fuchs HE, et al. Cancer statistics, 2021. CA Cancer J Clin. 2021, 71(1):7-33; Feller-Kopman DJ, Reddy CB, DeCamp MM, et al. Management of malignant pleural effusions. An official ATS / STS / STR clinical practice guideline. Am J Respir Crit Care Med. 2018, 198(7): 839-849.). Notably, about 11.16% of patients with small cell lung cancer (SCLC) have malignant pleural effusion at initial diagnosis (Shojaee S, Singh I, Solsky I, et al. Malignant pleural effusion at presentation in patients with small-cell lung cancer[J]. Respiration. 2019, 98(3):198-202.).Malignant pleural effusion usually indicates that the tumor has metastasized to the pleura or progressed to the late stage, and the median survival time of patients is less than 12 months (Ryu JS, Ryu HJ, Lee SN, et al. Prognostic impact of minimal pleural effusion in non-small-cell lung cancer. J Clin Oncol. 2014, 32(9): 960-967; Expert consensus on diagnosis and treatment of lung cancer with malignant pleural effusion. Chinese Journal of Oncology. 2024, 46(1): 40-47.).

[0003] Tumor neoantigens are antigens that are completely absent in normal cells due to DNA mutations, viral infections, and other factors in tumor cells, and can be bound by human major histocompatibility complex (HLA) and presented to T cells for TCR recognition, thereby causing an immune response. Currently, multiple research teams have designed personalized tumor vaccines using tumor neoantigens, which have shown good safety and excellent efficacy in phase I clinical studies of melanoma, refractory glioma, liver cancer, and pancreatic cancer patients (Terai M, Sato T. Individualised neoantigen cancer vaccine therapy. Lancet. 2024, 403(10427): 590-591.). Therefore, developing tumor neoantigen polypeptides is of great significance for the immunotherapy of tumors. SUMMARY

[0004] The present application provides HLA-A*11:01 restricted neoantigen peptides based on malignant pleural effusion tumor cells and applications thereof.

[0005] The present application takes malignant pleural effusion of lung cancer patients as samples, sorts tumor cells from them, performs high-throughput sequencing on the isolated tumor cells to analyze the mutations carried by them, uses computer algorithms to predict candidate neoantigens, and analyzes the affinity between candidate neoantigen polypeptides and HLA molecules and the frequency of neoantigen-specific T cells through tetramer (Tetramer) staining experiments and other methods for the predicted candidate neoantigens, and finally obtains tumor neoantigen polypeptides.

[0006] Specifically, the present application provides the technical solutions described below.

[0007] In a first aspect, the present application provides tumor neoantigen polypeptides, and the amino acid sequence of the neoantigen polypeptide is shown in any one of SEQ ID NO. 1-9.

[0008] The amino acid sequences of the tumor neoantigen polypeptides are as follows: VAGSTRIPK (SEQ ID NO. 1), LVAGSTRIPK (SEQ ID NO. 2), SSLFSQAVK (SEQ ID NO. 3), NSSLFSQAVK (SEQ ID NO. 4), LSFVPATTK (SEQ ID NO. 5), CLSFVPATTK (SEQ ID NO. 6), SSSRVFSSR (SEQ ID NO. 7), VALLQLGLK (SEQ ID NO. 8), and YSNRTRLAK (SEQ ID NO. 9).

[0009] Conservative variant sequences of the tumor neoantigen polypeptides that do not affect their functions and are generated by mutations are also within the protection scope of the present application, for example, one or more amino acids in the amino acid sequences of the above-mentioned neoantigen polypeptides are replaced conservatively, or one or more amino acids that do not affect their functions are added to the N-terminus or C-terminus of the neoantigen polypeptides (for example, a linker peptide, a protein tag sequence, etc.).

[0010] The protein tag sequence includes but is not limited to His tag, GST tag, MBP tag, etc. The linker peptide can be a flexible linker peptide rich in GS.

[0011] In addition, the derived polypeptides obtained by one or more modifications on the basis of the amino acid sequences of the above-mentioned neoantigen polypeptides are also within the protection scope of the present application, and these modifications include phosphorylation modification, PEGylation modification, amidation modification, glycosylation modification, biotinylation modification, and coupling or fusion with antibodies, carriers, ligands, albumin, Fc fragments, etc.

[0012] The tumor neoantigen polypeptides have high affinity with HLA-A*11:01 molecules and can induce the production of tumor-specific T cells, and can be used as targets for tumor diagnosis, prevention and treatment.

[0013] In a second aspect, the present application provides a polypeptide comprising the tumor neoantigen polypeptide.

[0014] Preferably, the polypeptide can be a polypeptide obtained by adding one or more amino acid residues to the N-terminus and / or C-terminus of the neoantigen polypeptide, which can be enzymatically cleaved to produce the neoantigen polypeptide. Alternatively, the polypeptide is obtained by fusing the tumor neoantigen polypeptide with other functional polypeptides, for example, with other known tumor neoantigen polypeptides. Based on conventional fusion polypeptide technology, those skilled in the art can obtain fusion polypeptides that retain the functional activities of each polypeptide.

[0015] Alternatively, the polypeptide is obtained by fusing the neoantigen polypeptide with an antibody, a carrier, a ligand, an albumin, an Fc fragment.

[0016] In a third aspect, the present application provides a nucleic acid molecule encoding the tumor neoantigen polypeptide or the polypeptide.

[0017] Based on the amino acid sequences of the tumor neoantigen polypeptides provided above and the codon rules, one skilled in the art can obtain the nucleotide sequences of the nucleic acid molecules encoding the tumor neoantigen polypeptides. Due to the degeneracy of codons, the nucleotide sequences encoding an amino acid sequence are not unique, and all nucleic acid molecules capable of encoding the tumor neoantigen polypeptides described above are within the scope of the present application.

[0018] In the present application, the nucleic acid molecule includes DNA or RNA. Among them, the RNA includes mRNA.

[0019] In a fourth aspect, the present application provides a biological material, which includes any one of the following: (1) an expression cassette comprising the nucleic acid molecule; (2) a vector comprising the nucleic acid molecule or the expression cassette in (1); (3) a cell comprising the nucleic acid molecule, the expression cassette in (1), or the vector in (2).

[0020] In the above (1), the expression cassette comprises the nucleic acid molecule and transcriptional or translational regulatory elements operably linked thereto, including but not limited to promoters, terminators, etc.

[0021] In the above (2), the vector includes plasmid vectors, viral vectors, transposons, artificial chromosomes, etc.

[0022] In the above (3), the cell includes microbial cells or animal cells. Among them, the microbial cells include Escherichia coli, yeast, etc. The animal cells do not have the ability to reproduce into animal individuals, including animal cell lines for polypeptide expression (such as CHO cells, HEK293, etc.) or immune cells, etc.

[0023] In a fifth aspect, the present application provides any one of the following applications of the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule, or the biological material: (1) preparing antigen presenting cells; (2) preparing tumor-specific T cells; (3) preparing TCR-T or CAR-T cells; (4) preparing tumor diagnosis reagents; (5) preparing drugs for preventing or treating tumors.

[0024] In the above (2), the tumor-specific T cells include cytotoxic T cells and the like.

[0025] In the above (2) and (3), the application includes stimulating specific T cells of a patient with the neoantigen polypeptide or constructing TCR-T cells or CAR-T cells that can recognize the neoantigen polypeptide. The tumor-specific T cells, TCR-T cells or CAR-T cells can be used for adoptive immunotherapy.

[0026] In the above (5), the drug includes a vaccine. The vaccine includes a DNA vaccine, an mRNA vaccine, a polypeptide vaccine, a dendritic cell vaccine (DC vaccine) and the like.

[0027] In the present application, the tumor is preferably lung cancer.

[0028] In a sixth aspect, the present application provides antigen presenting cells or tumor-specific T cells induced by the tumor neoantigen polypeptide and specifically targeting the tumor neoantigen polypeptide.

[0029] Preferably, the tumor-specific T cells include cytotoxic T cells, TCR-T cells or CAR-T cells.

[0030] In a seventh aspect, the present application provides a preparation method of tumor-specific T cells, which includes isolating peripheral blood mononuclear cells, co-culturing the tumor neoantigen polypeptide with the peripheral blood mononuclear cells, and activating and amplifying T cells specifically targeting the tumor neoantigen polypeptide.

[0031] In an eighth aspect, the present application provides a pharmaceutical composition containing the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material.

[0032] Preferably, the active ingredients of the pharmaceutical composition include the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material.

[0033] Further, the pharmaceutical composition can further contain excipients allowed in the pharmaceutical field.

[0034] In a ninth aspect, the present application provides a vaccine containing the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material.

[0035] Preferably, the vaccine includes a DNA vaccine, an mRNA vaccine, a polypeptide vaccine, a dendritic cell vaccine (DC vaccine) and the like. Taking the mRNA vaccine as an example, the mRNA vaccine can contain mRNA encoding the tumor neoantigen polypeptide. Taking the DC vaccine as an example, the DC vaccine can be prepared by loading polypeptides on DCs.

[0036] The pharmaceutical composition or vaccine can be used for immunotherapy of a patient having the tumor neoantigen polypeptide.

[0037] In a tenth aspect, the present application provides a diagnostic reagent comprising the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material as described above.

[0038] In an eleventh aspect, the present application provides a method for isolating and screening a tumor neoantigen polypeptide, comprising: using flow cytometry to sort tumor cells from a malignant pleural effusion sample of a tumor patient; sequencing the DNA and RNA of the tumor cells, and screening a tumor neoantigen polypeptide based on the sequencing data; wherein the tumor cells are 7-AAD + CD45- EpCAM + cell subpopulation.

[0039] Malignant pleural effusion is an important component of liquid biopsy samples of tumor patients. The present application finds that tumor cells in malignant pleural effusion can be used as a source for isolating tumor neoantigen polypeptides. By isolating 7-AAD - Hoechest 33342 + CD45- EpCAM + cell subpopulation from malignant pleural effusion, and performing high-throughput sequencing and mutation analysis on the cell subpopulation, and then screening a tumor neoantigen polypeptide. The above method based on liquid biopsy technology can provide neoantigens for patients with chest tumors that cannot be operated on, provide new neoantigen sequences for the development of neoantigen-based vaccines, and ultimately be used for the treatment of tumor patients.

[0040] In some embodiments of the present application, by obtaining the neoantigen profile of a patient with advanced lung cancer from malignant pleural effusion, tumor neoantigens with application potential are screened, and a new treatment scheme is provided for patients with advanced lung cancer.

[0041] In the above method, the tumor cells are 7-AAD - Hoechest 33342 + CD45 - EpCAM + a population of less than 2000 cells in the cell subpopulation.

[0042] In the above method, the DNA sequencing is whole-exome sequencing, and the RNA sequencing is transcriptome sequencing. The screening of the tumor neoantigen polypeptide based on the sequencing data comprises: identifying mutations carried by the tumor cells based on whole-exome sequencing data; obtaining HLA types of the patient; predicting potential HLA loss of heterozygosity of the patient; predicting mutant proteins from somatic mutations, and determining potential mutant polypeptides in the tumor cells; and analyzing the potential mutant polypeptides based on transcriptome sequencing data to determine the number of mutations of the potential mutant polypeptides at the RNA level. The affinity between the mutant polypeptide and the HLA of the patient is evaluated, and the potential tumor neoantigen polypeptide is screened by excluding repetitions and HLA loss of heterozygosity.

[0043] The above-mentioned identification of mutations carried by the tumor cells based on whole-exome sequencing data comprises: identifying (preferably using GATK Mutect2) mutations carried by the tumor cells based on WES data of peripheral blood mononuclear cells (PBMCs) of the patient, and mutations with TLOD ≥ 10 are retained. The above-mentioned obtaining of HLA types of the patient preferably uses the OptiType algorithm.

[0044] The above-mentioned prediction of potential HLA loss of heterozygosity of the patient preferably uses the LOHHLA algorithm.

[0045] The above-mentioned prediction of mutant proteins from somatic mutations, and determination of potential mutant polypeptides in the tumor cells comprises: predicting and disassembling mutant proteins from somatic mutations using the VEP algorithm, and determining potential mutant polypeptides in the tumor cells.

[0046] The above-mentioned analysis of the potential mutant polypeptides based on transcriptome sequencing data to determine the number of mutations of the potential mutant polypeptides at the RNA level is preferably performed using the GATK ASEReadCounter algorithm.

[0047] In the above-mentioned method, the tumor is preferably lung cancer.

[0048] The present application has at least the following beneficial effects: the present application separates and screens tumor neoantigen polypeptides from malignant pleural effusion of lung cancer patients, the tumor neoantigen polypeptides have high affinity with HLA molecules, can stimulate the production of tumor-specific T cells, and further produce an immune response targeting tumor cells, and can be used as a target for clinical treatment or diagnosis of tumors, and has good application prospect in tumor immunotherapy. The present application also provides a method for isolating and screening tumor neoantigen polypeptides, which solves the problem of being unable to obtain neoantigens of non-surgical treatment patients, and provides an effective method for the development of tumor neoantigen polypeptides. BRIEF DESCRIPTION OF DRAWINGS

[0049] In order to more clearly illustrate the technical solutions of the present application or the prior art, the drawings required to be used in the following examples or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0050] Figure 1 and Figure 2 Tumor cells in malignant pleural effusion of lung cancer patients in Example 1 of the present application exist EpCAM + ; wherein, Figure 1 Chromosome abnormalities of tumor cells in malignant pleural effusion are detected by SE-i·FISH method; Figure 2 Flow cytometry diagram of tumor cells in malignant pleural effusion sample and flow cytometry statistical diagram of P7 subpopulation;***P<0.001.

[0051] Figure 3 , Figure 4 , Figure 5 and Figure 6 New antigens of tumor cells in malignant pleural effusion sample of lung cancer patients are screened by specific Tetramer in Example 3 of the present application; wherein, Figure 3 Affinity between candidate neoantigen polypeptides derived from P19 and P23 patients and HLA molecules is detected by QuickSwitch™ Quant Tetramer detection kit; Figure 4 and Figure 5 Specific T cell frequency against neoantigens in malignant pleural effusion of P19 and P23 lung cancer patients is detected by QuickSwitch™ Quant Tetramer detection kit; Figure 6To detect the specific T cell frequency against neoantigens in peripheral blood and malignant pleural effusion of P19 (upper picture) and P23 (lower picture) lung cancer patients by using QuickSwitch™ QuantTetramer detection kit; wherein, HSPA8-M-1 represents AGSTRIPK, HSPA8-M-2 represents LVAGSTRIPK, CCDC89-M-1 represents SSLFSQAVK, CCDC89-M-2 represents NSSLFSQAVK, FAM173B-M-1 represents LSFVPATTK, FAM173B-M-2 represents CLSFVPATTK, VKORC1-M represents SSSRVFSSR, PIGO-M-2 represents VALLQLGLK and HSP90B1-M represents YSNRTRLAK; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0052] Figure 7 To detect the expression level of CD39 on T cells in peripheral blood and malignant pleural effusion of lung cancer patients by using FCM in embodiment 4 of the present application; **P<0.01.

[0053] Figure 8 、 Figure 9 and Figure 10 To analyze the gene mutation of HSPA8, CCDC89, FAM173B, VKORC1, PIGO and HSP90B1 in different cancers in TCGA database in embodiment 4 of the present application; wherein, ACC represents adrenocortical carcinoma, BLCA represents bladder urothelial carcinoma, BRCA represents breast cancer, BRCA-Basal represents basal-like breast cancer, BRCA-Her2 represents Her2-like breast cancer, BRCA-LumA represents LumA-like breast cancer, CESC represents cervical squamous cell carcinoma and adenocarcinoma, CHOL represents cholangiocarcinoma, COAD represents colon cancer, ESCA represents esophageal cancer, GBM represents glioblastoma, HNSC represents head and neck squamous cell carcinoma, HNSC-HPV- represents head and neck squamous cell carcinoma derived from HPV-negative, HNSC-HPV+ represents head and neck squamous cell carcinoma derived from HPV-positive, KICH represents kidney chromophobe carcinoma, KIRC represents kidney clear cell carcinoma, KIRP represents kidney papillary cell carcinoma, LGG represents brain low-grade glioma, LIHC represents hepatocellular carcinoma, LUAD represents lung adenocarcinoma, LUSC represents lung squamous carcinoma, OV represents ovarian cancer, PAAD represents pancreatic cancer, READ represents rectal adenocarcinoma, SKCM represents skin melanoma, STAD represents gastric cancer, TGCT represents testicular cancer, THCA represents thyroid cancer, UCEC represents endometrial cancer, UCS represents uterine sarcoma, UVM represents ocular melanoma.

[0054] Figure 11 IFN-γ secretion (A) and cytotoxicity (B) of neoantigen-stimulated T cells after co-incubation with tumor cells; *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0055] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below with reference to the drawings in the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0056] The malignant pleural effusion of lung cancer patients used in the following examples was collected and used under the approval of the Ethics Committee of Peking University People's Hospital, and the patients or their families were informed, and both parties signed the informed consent form.

[0057] Example 1 Obtaining tumor cells in malignant pleural effusion of lung cancer patients by flow cytometry The prediction of neoantigens mainly depends on the comprehensive analysis of DNA mutations and RNA expression levels of tumor cells. In the tumor tissues of lung cancer patients, since the proportion of tumor cells is usually high, DNA and RNA can be extracted directly from tissue samples for neoantigen prediction. However, lung cancer patients in the later stage often lose the opportunity for surgery, making it difficult to obtain the neoantigen profile through traditional tissue biopsy. Malignant pleural effusion (i.e., malignant pleural effusion) as a common complication of advanced lung cancer provides an alternative source of samples for obtaining the neoantigen profile. It is worth noting that the proportion of white blood cells in lung cancer malignant pleural effusion often exceeds 90%, and if total cell DNA and RNA are directly extracted for next-generation sequencing, it will be difficult to accurately obtain the tumor-specific mutation profile due to the low proportion of tumor cells, thereby affecting the reliability of neoantigen prediction. Therefore, it is crucial to establish a technical method that can specifically enrich viable tumor cells.

[0058] Epithelial cell adhesion molecule (EpCAM) is a tumor marker with important clinical value. As a transmembrane glycoprotein, EpCAM mediates Ca 2+EpCAM is an independent homotypic cell-cell adhesion protein expressed specifically in epithelial tissues and their derived tumors. EpCAM-positive cells are almost absent in normal body fluids, so its detection in malignant pleural effusions has a clear value in tumor diagnosis (Zhang X, Wang X, Wen Y, Chen S, Zhou C, Wu F. Single-cell transcriptomics reveal metastatic CLDN4 + Cancer cells underlying therecurrence of malignant pleural effusion in patients with advanced non-small-cell lung cancer. Clin Transl Med. 2024, 14(4):e1649.). Several studies have confirmed that lung cancer tissues generally highly express EpCAM (Rehulkova A, Chudacek J, Prokopova A, Vidlarova M, StranskaJ, Drabek J, et al. Clinical and prognostic significance of detecting CEA , EGFR , LunX , c-met and EpCAMmRNA-positive cells in the peripheral blood, tumor-draining blood and bone marrow of non-small cell lung cancer patients. TranslLung Cancer Res. 2023, 12(5):1034-1050.), which provides a theoretical basis for its use as a lung cancer cell sorting marker. In addition, chromosomal abnormalities are another important feature of tumor cells, such as chromosome 8 abnormalities, which are common in various solid tumors (Cheng H, Wang S, Luan W, Ye X, Dou S, Tang Z, et al. Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH in ovarian cancer. Chin J Cancer Res. 2021, 33(2):256-270.). The present application uses differential enrichment-multiple tumor marker immunofluorescence staining-chromosome fluorescence in situ hybridization (SE-i·FISH) to detect that in advanced lung cancer patients, there are tumor cells in malignant pleural effusion that express EpCAM, CEA, CA199 and CA125 markers and are accompanied by chromosomal abnormalities (P7 subpopulation) Figure 1 ). Considering that CEA, CA199 and CA125 markers are mainly located intracellularly, which is not conducive to live cell sorting, therefore, selecting EpCAM as a surface marker is more technically feasible.

[0059] The present application establishes an optimized flow cytometry (FCM) sorting strategy: through multi-parameter gating, specific sorting of tumor cells with activity (7-AAD - ), containing intact nuclei (Hoechest 33342 + ), excluding leukocytes (CD45 - ) and expressing EpCAM (EpCAM + ), i.e. P7 subpopulation. This method realizes efficient enrichment of rare tumor cells under the premise of maintaining cell integrity. Verification experiments show that the number of P7 subpopulation in the malignant pleural effusion of lung cancer patients is significantly higher than that in the pleural effusion of non-tumor patients (P7 P <0.001) (P7 Figure 2 ), confirming the specificity of the method. The DNA and RNA quality of the P7 subpopulation cells sorted by BD FACSAria II flow cytometer can meet the requirements of second-generation sequencing, providing a reliable technical guarantee for the accurate prediction of neoantigen profile in the subsequent.

[0060] Example 2 Determination of potential neoantigens of tumor cells in malignant pleural effusion of lung cancer patients based on computer algorithm First, 7-AAD - Hoechest 33342 + CD45 - EpCAM + Cell subpopulation (P7 subpopulation), P7 population less than 2000 cells were extracted and amplified RNA and DNA by Smart-seq 2 technology and Discover-sc single cell, respectively. Subsequently, transcriptome sequencing (RNA-seq) and whole-exome sequencing (WES) were performed on RNA and DNA, respectively, using illumina novaseq 6000 platform.

[0061] The WES data of P7 population in the above malignant pleural effusion were compared with the WES data of peripheral blood mononuclear cells (PBMC) of the patient, and the patient's P7 subpopulation mutation in the pleural effusion was identified using GATK Mutect2, and the mutation with TLOD ≥ 10 was retained; the patient's HLA type was obtained using OptiType algorithm; the patient's potential HLA heterozygous deletion was predicted using LOHHLA algorithm; the mutant protein was predicted and disassembled from somatic mutations using VEP algorithm, and the potential mutant polypeptide in the P7 subpopulation of the patient's malignant pleural effusion was determined. Further combined with the RNA-seq data of the P7 subpopulation of the malignant pleural effusion, the potential mutant polypeptide derived from WES was analyzed using GATK ASEReadCounter algorithm, and the mutation number (MutationCount, MutCount) of the potential mutant polypeptide was determined at the RNA level.

[0062] Finally, the affinity between the mutant polypeptide and the patient's HLA was calculated using NetMHCpan-4.1 algorithm, and the potential neoantigen was predicted using MutAff ≤ 200 nM or RefAff / MutAff ≥ 10 threshold value after excluding duplicates and HLA heterozygous deletion.

[0063] Using the above method, potential neoantigens were found to exist in the tumor cells of malignant pleural effusion of lung cancer patients at P19, P23 (Table 1), including 12 predicted neoantigen peptides, including neoantigen peptides VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2), and YSNRTRLAK (HSP90B1-M) presented by HLA A*A11:01 molecules. These neoantigen peptides have the potential to be used to make tumor vaccine to treat lung cancer.

[0064] Table 1. Linear sequences and characteristic parameters of 12 predicted neoantigens and their paired WT antigens Example 3 Determination of neoantigens of high-frequency HLA-presenting tumor cells in malignant pleural effusion of lung cancer patients by Tetramer detection method To screen the neoantigens of lung cancer patients, the Tetramer staining technique was used for preliminary verification. For the detection of the affinity of neoantigen peptides and HLA molecules, the commercially available MBL QuickSwitch™ Quant Tetramer detection kit is widely used for the detection of antigen peptides of high-frequency HLA subtypes (such as HLA-A 02:01, HLA-A 11:01 and HLA-A 24:02） 。 Using QuickSwitch™ Quant Tetramer HLA-A *11:01 Kit-PE kit, the affinity of candidate neoantigen peptides derived from VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2), YVALLQLGLK (PIGO-M-1), YSNRTRLAK (HSP90B1-M), SETHSFLHPR (SKIL-M), and SRVFSSRWGR (RP11-M) and HLA-A*11:01 molecules was evaluated.

[0065] ​The experiment was performed according to the kit instructions. First, the candidate peptide solution with a concentration of 2 mM and the reference peptide solution with a concentration of 1 mM were warmed to room temperature. 50 μL of Tetramer was aliquoted into EP tubes, and 1 μL of candidate peptide or reference peptide and 1 μL of polypeptide displacement factor were added to each tube, respectively. After mixing, the mixture was incubated at room temperature for 5 hours in the dark, and then stored at 4°C for standby. Multiple control groups were set up. In a round-bottom 96-well plate, 20 μL of magnetic beads were added to each well, and the following groups were treated: (1) Well 1 (Control #1) and Well 3 (Control #3): 5 μL of Tetramer was added; (2) Well 2 (Control #2): 5 μL of 1x Assay Buffer was added; (3) The remaining wells: 5 μL of Tetramer after peptide displacement was added. After the sample was incubated at 550 rpm for 45 minutes in an aluminum foil in the dark, 150 μL of 1x Assay Buffer was added to each well. After 5 minutes of standing in a magnetic stand, the supernatant was discarded, and the mixture was vortexed for 2 seconds. Except that 25 μL of 1x Assay Buffer was added to Well 1, 25 μL of freshly prepared 1x working concentration of Exiting Peptide Antibody was added to the remaining wells. After 550 rpm shaking in the dark for 55 minutes, the washing step was repeated. Finally, 200 μL of 1x Assay Buffer was used to resuspend the magnetic beads in each well, and 200 μL of buffer and 5 μL of magnetic beads were added to Well 4 as a magnetic bead control group. The sample was analyzed by flow cytometry (FCM), and the displacement efficiency of the candidate neoantigen peptide, i.e., its affinity for HLA-A*11:01, was calculated.The results show that the affinity of the candidate neoantigen peptide VAGSTRIPK (HSPA8-M-1) to HLA-A*11:01 is 95.24%, the affinity of LVAGSTRIPK to HLA-A*11:01 (HSPA8-M-2) is 93.03%, the affinity of SSLFSQAVK (CCDC89-M-1) to HLA-A*11:01 is 95.93%, the affinity of NSSLFSQAVK (CCDC89-M-2) to HLA-A*11:01 is 89.01%, the affinity of LSFVPATTK (FAM173B-M-1) to HLA-A*11:01 is 89.35%, the affinity of CLSFVPATTK (FAM173B-M-2) to HLA-A*11:01 is 80.06%, the affinity of SSSRVFSSR (VKORC1-M) to HLA-A*11:01 is 89.89%, the affinity of VALLQLGLK (PIGO-M-2) to HLA-A*11:01 is 87.98%, and the affinity of YSNRTRLAK (HSP90B1-M) to HLA-A*11:01 is 95.74%, all of which are significantly higher than the threshold of 75% set in the specification; and the affinity of YVALLQLGLK (PIGO-M-1) to HLA-A*11:01 is 63.62%, the affinity of SETHSFLHPR (SKIL-M) to HLA-A*11:01 is 47.85%, and the affinity of SSSRVFSSRWGR (RP11-M) to HLA-A*11:01 is 57.57%, which are poor affinities of the three polypeptides to HLA*A11:01. Figure 3 ). This indicates that VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2), and YSNRTRLAK (HSP90B1-M) have high affinities to HLA-A*11:01 molecules, providing reliable candidate targets for further immunotherapy research.

[0066] To further validate specific T cell responses to candidate neoantigens in lung cancer, specific tetramer staining was used to detect the frequency of T cells specific for candidate neoantigen peptides, including VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2), and YSNRTRLAK (HSP90B1-M), in the malignant pleural effusion and peripheral blood of patients with P19 and P23 lung cancer. The experimental procedure was as follows: cells from pleural effusion or peripheral blood were adjusted to 1×10 6 ~1×10 7 / mL concentration, take 100 μL of cell suspension, add 10 μL ClearBack (incubate at room temperature in the dark for 5 minutes to block nonspecific binding). Subsequently, add 10 μL of Tetramer with a peptide replacement efficiency greater than 75% to each tube, incubate at room temperature for 30 minutes, and then add CD8 antibody (incubate at 4°C for 20 minutes). Wash the cells with PBS (centrifuge at 400g for 5 minutes), discard the supernatant and resuspend the cells with 500 μL PBS, add 20 μL Cell Viability Solution, store at 4°C in the dark, and complete FCM analysis within 24 hours. The results showed that specific T cells that can recognize the above 9 antigen peptides were detected in the malignant pleural effusions of P19 and P23 lung cancer patients ( Figure 4 and Figure 5 ), but CD8 + In P19 and P23 lung cancer patients, no specific CD8 T cells were detected in the peripheral blood without stimulation with candidate neoantigen peptides. + However, after 13 days of stimulation with a final concentration of 2 μM neoantigen peptides, specific T cells CD8 T cells that could recognize the above 9 antigen peptides could be detected. + T cell frequencies were higher than 5% ( Figure 6 ). This suggests that these 9 antigenic peptides are neoantigenic peptides with immunogenicity.

[0067] Example 4 CD39 responsive to neoantigens is present in the peripheral blood and malignant pleural effusion of lung cancer patients + T cells Multiple reports (e.g., Liu T, Tan J, Wu M, Fan W, Wei J, Zhu B, et al. High-affinity neoantigens correlate with better prognosis and trigger potent antihepatocellular carcinoma (HCC) activity by activating CD39+ CD8+ T cells. Gut. 2021, 70(10): 1965-1977.) and the applicant's previous studies (Chen P, Chen DB, Bu DC, et al. Dominant neoantigen verification in hepatocellular carcinoma by a single-plasmid system coexpressing patient HLA and antigen. J Immunother Cancer. 2023, 11(4):e006334.) have found that CD39 + T cells can have better ability to recognize neoantigens, and dominant neoantigens can activate CD39 + T cells to exert anti-tumor effect. Further studies have found that CD8 + CD39 + T cells in the peripheral blood and malignant pleural effusion of lung cancer patients have a response to neoantigens Figure 7 Compared with the peripheral blood of lung cancer patients, the expression proportion of CD8 + CD39 + T cells in the malignant pleural effusion is significantly increased Figure 7 .

[0068] In addition, analysis of the TCGA database found that HSPA8, CCDC89, FAM173B, VKORC1, PIGO and HSP90B1 (HSPA8, CCDC89, FAM173B, VKORC1, PIGO and HSP90B1) Figure 8 , Figure 9 and Figure 10 have gene mutations not only in lung cancer but also in other cancer types, which suggests that these neoantigen peptides discovered by the present application can be used as tumor vaccines for treating tumors in the future.

[0069] Example 5 Peripheral blood T cells stimulated by neoantigen peptides have killing function on tumor cells derived from malignant pleural effusion of lung cancer patients 1. Activation and expansion of neoantigen-specific T cells First, mononuclear cells from P19 and P23 lung cancer patients peripheral blood were extracted. Then, 9 neoantigen peptides from P19 and P23 lung cancer patients were respectively co-incubated with mononuclear cells from each patient's peripheral blood to activate and expand patient neoantigen-specific T cells, in the following way: culture in 12-well plates, 1x10 6 cells per well, 1 mL of X-VIVO 15 + 5% human AB serum + 1% penicillin / streptomycin + 100 IU / mL IL-2 + 4 μM polypeptide per well, half-volume replacement every 2-3 days, and culture for 13 days to collect T cells.

[0070] 2. Tumor cell organoid culture and expansion Tumor cells from P19 and P23 lung cancer patients malignant pleural effusion were isolated and expanded by organoid culture. The specific method is as follows: tumor cells in malignant pleural effusion were mixed with culture medium (containing insulin growth factor-2 at a concentration of 8 ng / mL) to make the concentration of tumor cells 2x10 4 / mL, then 5% Matrigel was added and mixed on ice to obtain the culture medium. The above culture medium was inoculated into a 24-well low-adsorption plate and cultured in a 37°C incubator for 30 min, then 200 μL of culture medium was added to each well. During the culture process, 150 μL of culture medium was added to each well every three days. When the diameter of the organoid reached 200-500 μm, the original culture medium was removed, TrypLE Express was added to each well of the 24-well plate, and after 1 min of enzyme digestion, culture medium containing 2% FBS was added to terminate digestion and collect the enzyme solution. The enzyme solution was centrifuged in a centrifuge (300 g, 7 min) to collect the tumor cell precipitate.

[0071] 3. Evaluation of T cell killing function 1x10 6 effector cells (T cells) stimulated by neoantigens and 1x10 5The researchers co-incubated the cells with target cells (tumor cells) for 24 hours, while the control group used T cells that were not stimulated with neoantigens and tumor cells to co-incubate. The IFN-γ secretion level was detected by ELISA. The results showed that the IFN-γ secretion of T cells stimulated with neoantigens VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2) and YSNRTRLAK (HSP90B1-M) was significantly higher than that of the control group ( Figure 11 A). The level of lactate dehydrogenase (LDH) release was further detected by ELISA to evaluate the killing effect of T cells on tumor cells. The results showed that the amount of LDH released by T cells stimulated by the above 9 neoantigen peptides was significantly higher than that in the control group ( Figure 11 This indicates that peripheral blood T cells stimulated by neoantigen peptides can kill tumor cells derived from malignant pleural effusion of lung cancer patients.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A tumor neoantigen polypeptide, characterized in that: The amino acid sequence of the neoantigen polypeptide is shown in any one of SEQ ID NOs. 1 to 9.

2. A polypeptide, characterized in that The polypeptide comprises the tumor neoantigen polypeptide according to claim 1.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the tumor neoantigen polypeptide according to claim 1 or the polypeptide according to claim 2.

4. Biomaterial, characterized in that The biological material includes any one of the following: (1) an expression cassette comprising the nucleic acid molecule of claim 3; (2) A vector comprising the nucleic acid molecule of claim 3 or the expression cassette of (1); (3) A cell comprising the nucleic acid molecule according to claim 3, the expression cassette according to (1), or the vector according to (2).

5. Any of the following uses of the tumor neoantigen polypeptide of claim 1, the polypeptide of claim 2, the nucleic acid molecule of claim 3, or the biomaterial of claim 4: (1) Preparation of antigen-presenting cells; (2) Preparation of tumor-specific T cells; (3) Preparation of TCR-T or CAR-T cells; (4) Preparation of tumor diagnostic reagents; (5) Preparation of drugs for preventing or treating tumors.

6. The use according to claim 5, characterized in that The tumor is lung cancer.

7. Antigen presenting cells or tumor-specific T cells, characterized in that The antigen-presenting cells or tumor-specific T cells are induced by the tumor neoantigen polypeptide according to claim 1 and specifically target the tumor neoantigen polypeptide according to claim 1.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the tumor neoantigen polypeptide according to claim 1, the polypeptide according to claim 2, the nucleic acid molecule according to claim 3, or the biomaterial according to claim 4.

9. A vaccine, characterized in that The vaccine comprises the tumor neoantigen polypeptide of claim 1, the polypeptide of claim 2, the nucleic acid molecule of claim 3 or the biological material of claim 4.

10. A method for separating and screening tumor neoantigen polypeptides, characterized in that: The method comprises: using malignant pleural effusion of a tumor patient as a sample, and sorting tumor cells from the sample by flow cytometry; performing DNA sequencing and RNA sequencing on the tumor cells, and screening tumor neoantigen polypeptides based on the sequencing data; Wherein, the tumor cells are 7-AAD - Hoechest 33342 + CD45 - EpCAM + Cell subsets.

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