Tumor neoantigen polypeptide and separation and screening method thereof

Abstract

The invention relates to the technical field of tumor immunotherapy, in particular to tumor neoantigen polypeptide and a separation and screening method thereof. The amino acid sequence of the tumor neoantigen polypeptide provided by the invention is shown as SEQ ID NO.1, 2, 3 or 4. The tumor neoantigen polypeptide has high affinity with HLA molecules, can induce generation of tumor specific T cells, further generates immune response of targeting tumor cells, can be used as a target spot for clinical treatment or diagnosis of tumors, and has a good application prospect in tumor immunotherapy. The method for separating and screening the tumor neoantigen polypeptide provided by the invention solves the problem that the neoantigen of a non-surgical treatment patient cannot be obtained by the existing method, and provides an effective method for developing the tumor neoantigen polypeptide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor immunotherapy, and particularly relates to a tumor neoantigen polypeptide and a separation and screening method thereof. BACKGROUND

[0002] Tumor neoantigens are antigens that do not exist in normal cells but are produced due to DNA mutations, viral infections, and other factors in tumor cells, and can be presented to the surface of T cells by human leukocyte antigen (HLA) and recognized by T cell receptors (TCR) after specific immune responses are triggered (Terai M, Sato T. Individualised neoantigen cancer vaccine therapy. Lancet. 2024, 403(10427):590-591.). Although immune checkpoint blockade (ICB) therapy represented by PD-1 / PD-L1 monoclonal antibodies has shown a lasting and strong clinical effect in liver cancer, it still faces problems such as low response rate and recurrence in some patients (Yang Y, Chen DB, Zhao BG, Ren LY, Huang R, Feng B, et al. The predictive value of PD-L1 expression in patients with advanced hepatocellular carcinoma treated with PD-1 / PD-L1 inhibitors: A systematic review and meta-analysis. Cancer Med. 2023, 12(8):9282-9292.). Studies on the immunological mechanism of ICB therapy found that the clinical non-response of tumor patients to ICB therapy is closely related to the lack of pre-exisiting neoantigen-specific T cell response status in patients (Yossef R, Krishna S, Sindiri S, Lowery FJ, Copeland AR, Gartner JJ, et al. Phenotypic signatures of circulating neoantigen-reactive CD8 T cells in patients with metastatic cancers. Cancer Cell. 2023, 41(12):2154-2165.).This suggests that pre-existing neoantigen-specific T cells in tumor patients are the target of ICB therapy and the core driving force for achieving clinical benefits of immunotherapy (Puig-Saus C, Sennino B, Peng S, Wang CL, Pan Z, Yuen B, et al. Neoantigen-targeted CD8. + T cell responses with PD-1 blockade therapy. Nature. 2023, 615(7953):697-704.)。Therefore, neoantigen-based immunotherapy will be a new breakthrough point in the field of liver cancer prevention and treatment.

[0003] Currently, neoantigen-based immunotherapy has shown good prospects in basic research and clinical trials of liver cancer, but there are still many challenges in its comprehensive application to the clinic. Currently, neoantigens are mainly obtained from surgical tissue samples, but most liver cancer patients are in the middle and advanced stages when they are discovered, missing the best opportunity for surgical resection (Yang S, Deng Y, Zheng Y, Zhang J, He D, Dai Z, et al. Burden, trends, and predictions of liver cancer in China, Japan, and South Korea: analysis based on the Global Burden of Disease Study 2021. Hepatol Int. 2025, doi:10.1007 / s12072-024-10763-6; Yang X, Yang C, Zhang S, Geng H, Zhu AX, Bernards R, et al. Precision treatment in advanced hepatocellular carcinoma. Cancer Cell. 2024, 42(2):180-197.), resulting in the inability to obtain neoantigen profiles for non-surgical treatment patients, limiting the development of neoantigens. SUMMARY

[0004] The present application provides tumor neoantigen polypeptides and methods for isolating and screening the same.

[0005] Specifically, the present application provides the technical solutions described below.

[0006] In a first aspect, the present application provides a tumor neoantigen polypeptide, wherein the amino acid sequence of the tumor neoantigen polypeptide is shown as SEQ ID NO. 1, 2, 3 or 4.

[0007] The present application takes malignant ascites of a liver cancer patient as a sample, sorts tumor cells from the malignant ascites, analyzes mutation conditions by high-throughput sequencing, predicts candidate neoantigens by computer algorithm, analyzes the affinity between the candidate neoantigen polypeptide and HLA molecules and the frequency of neoantigen-specific T cells by tetramer staining experiment, and finally screens four neoantigen polypeptides IYLENYATSMW (SEQ ID NO. 1), VYQEKLEGDF (SEQ ID NO. 2), IWGISVAWHW (SEQ ID NO. 3) and QFMASTLFI (SEQ ID NO. 4) presented by HLA A*24:02 molecules.

[0008] Conservative variant sequences of the above tumor neoantigen polypeptides which do not affect the functions thereof are also within the protection scope of the present application, for example, one or more amino acids in the amino acid sequence of the above tumor neoantigen polypeptides are conservatively substituted, or one or more amino acids which do not affect the functions thereof are added to the N-terminus or C-terminus of the tumor neoantigen polypeptides (for example, a linker peptide, a protein tag sequence, etc. is added).

[0009] The linker peptide can be a flexible linker peptide rich in GS. The protein tag sequence includes but is not limited to a His tag, a GST tag, an MBP tag, etc.

[0010] In addition, a derivative polypeptide obtained by one or more modifications on the basis of the amino acid sequence of the above tumor neoantigen polypeptide is also within the protection scope of the present application, and these modifications include coupling or fusion with an antibody, a carrier, a ligand, albumin, an Fc fragment, phosphorylation modification, PEGylation modification, amidation modification, glycosylation modification, biotinylation modification, etc.

[0011] In a second aspect, the present application provides a polypeptide, wherein the polypeptide comprises the above-mentioned tumor neoantigen polypeptide.

[0012] The above-mentioned polypeptide can be obtained by fusion of the tumor neoantigen polypeptide with other functional polypeptides, for example, with other known tumor neoantigen polypeptides. Based on conventional fusion polypeptide technology, a person skilled in the art can obtain a fusion polypeptide which retains the functional activity of each polypeptide.

[0013] The above-mentioned polypeptide can also be obtained by fusion of the tumor neoantigen polypeptide with an antibody, a carrier, a ligand, albumin, an Fc fragment.

[0014] The polypeptide can also be a polypeptide with one or more amino acid residues added to the N-terminus and / or C-terminus of the tumor neoantigen polypeptide, which can be enzymatically cleaved to produce the tumor neoantigen.

[0015] In a third aspect, the present application provides a nucleic acid molecule encoding the tumor neoantigen polypeptide described above or the polypeptide.

[0016] Based on the amino acid sequence of the tumor neoantigen polypeptide provided above and the codon rules, a person skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the tumor neoantigen polypeptide. Due to the degeneracy of codons, the nucleotide sequence encoding an amino acid sequence is not unique, and all nucleic acid molecules capable of encoding the tumor neoantigen polypeptide described above are within the scope of the present application.

[0017] In the present application, the nucleic acid molecule includes DNA or RNA. Among them, the RNA includes mRNA.

[0018] In a fourth aspect, the present application provides a biological material, which is 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).

[0019] In (1) above, 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.

[0020] In (2) above, the vector includes plasmid vectors, viral vectors, transposons, artificial chromosomes, etc.

[0021] In (3) above, 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.) such as immune cells, etc.

[0022] 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 described above: (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) a medicament for preventing or treating a tumor.

[0023] The tumor neoantigen polypeptide provided by the present application has high affinity with HLA molecules, can induce the generation of tumor-specific T cells, and can be used as a target for tumor diagnosis, prevention and treatment.

[0024] In the present application, the tumor is preferably a liver tumor, and more preferably hepatocellular carcinoma.

[0025] In the above (2), the tumor-specific T cells include cytotoxic T cells and the like.

[0026] In the above (2) and (3), the application includes stimulating the patient's own specific T cells to expand or constructing TCR-T cells or CAR-T cells that can recognize the tumor neoantigen polypeptide using the tumor neoantigen polypeptide. The tumor-specific T cells, TCR-T or CAR-T cells can be used for adoptive immunotherapy.

[0027] In the above (5), the medicament includes a vaccine. The vaccine includes a DNA vaccine, an mRNA vaccine, a polypeptide vaccine, a dendritic cell vaccine (DC vaccine) and the like.

[0028] In a sixth aspect, the present application provides an antigen presenting cell or a tumor-specific T cell, which is induced by the tumor neoantigen polypeptide described above and specifically targets the tumor neoantigen polypeptide.

[0029] 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 a tumor-specific T cell, which comprises: isolating peripheral blood mononuclear cells, co-culturing the tumor neoantigen polypeptide with the peripheral blood mononuclear cells, and activating and expanding T cells that specifically target the tumor neoantigen polypeptide.

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

[0032] In addition to containing the active ingredients such as the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material described above, the pharmaceutical composition can also contain excipients allowed in the pharmaceutical field.

[0033] In a ninth aspect, the present application provides a vaccine comprising the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material described above.

[0034] The vaccine includes a DNA vaccine, an mRNA vaccine, a polypeptide vaccine, a dendritic cell vaccine (DC vaccine), etc. Taking the mRNA vaccine as an example, the mRNA vaccine can include mRNA coding the tumor neoantigen polypeptide. Taking the DC vaccine as an example, the DC vaccine can be prepared by loading the polypeptide into DC.

[0035] The vaccine can be used for immunotherapy of a patient having the tumor neoantigen polypeptide.

[0036] In a tenth aspect, the present application provides a diagnostic reagent including the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material.

[0037] In an eleventh aspect, the present application provides a method for isolating and screening a tumor neoantigen polypeptide, including: taking malignant ascites of a tumor patient as a sample, and sorting tumor cells from the sample by flow cytometry; sequencing the tumor cells by DNA sequencing and RNA sequencing, and screening a tumor neoantigen polypeptide based on the sequencing data; The tumor cells are 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation.

[0038] The malignant ascites is an important component of a liquid biopsy sample of a tumor patient. The present application finds that the tumor cells in the malignant ascites can be used as an isolation source of a tumor neoantigen polypeptide. By isolating 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation from the malignant ascites, and performing high-throughput sequencing and mutation analysis on the cell subpopulation, a tumor neoantigen polypeptide is screened. The above method based on liquid biopsy technology can provide a neoantigen for a patient with an abdominal tumor that cannot be operated on, provide a new neoantigen sequence for vaccine research based on a neoantigen, and ultimately be used for preventing and treating recurrence and metastasis of a tumor patient.

[0039] In the above method, the tumor cells belong to 7-AAD - Hoechest 33342 + CD45 - EpCAM + a cell subpopulation with less than 2000 cells.

[0040] 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 heterozygous loss of the patient; predicting mutant proteins from somatic mutations, and determining potential mutant polypeptides in the tumor cells; And, based on transcriptome sequencing data, analyzing the potential mutant polypeptides to determine the number of mutations of the potential mutant polypeptides at the RNA level; Evaluating the affinity between the mutant polypeptide and the HLA of the patient, and screening the potential tumor neoantigen polypeptide by excluding repetitions and HLA heterozygous loss.

[0041] In the above method, the tumor is preferably a liver tumor, and more preferably hepatocellular carcinoma.

[0042] The beneficial effects of the present application at least include: the tumor neoantigen polypeptide provided by the present application has high affinity with HLA molecules, can induce the production of tumor-specific T cells, and further produce an immune response targeting tumor cells, which can be used as a target for clinical treatment or diagnosis of tumors, and has good application prospect in tumor immunotherapy.

[0043] The method for isolating and screening the tumor neoantigen polypeptide provided by the present application solves the problem that the existing method cannot obtain neoantigens of patients who cannot be treated by surgery, and provides an effective method for the development of tumor neoantigen polypeptides. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. 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.

[0045] Figure 1 And Figure 2 In Example 1 of the present application, EpCAM + of tumor cells in malignant ascites of a liver cancer patient; wherein, Figure 1 Chromosome abnormalities of tumor cells in malignant ascites were detected by SE-i·FISH method; Figure 2 Flow cytometry analysis chart of tumor cells in ascites samples and flow cytometry statistical chart of P7 subpopulation; P<0.05.

[0046] Figure 3 , Figure 4 And Figure 5 In Example 3 of the present application, the neoantigens of tumor cells in the malignant ascites samples of a liver cancer patient were screened by specific Tetramer; whereinFigure 3 A represents the affinity between candidate neoantigen peptides and HLA molecules detected using the QuickSwitch™ Quant Tetramer Assay Kit; Figure 3 B and Figure 4 The frequencies of T cells specific for neoantigens in the malignant ascites of patients with liver cancer at P10 and P14 were detected using the QuickSwitch™ Quant Tetramer Assay Kit, respectively; Figure 5 The frequencies of T cells specific for neoantigens in peripheral blood and malignant ascites of P10 patients were detected using the QuickSwitch™ Quant Tetramer Assay Kit; TCP1-M represents IYLENYATSMW, ABCD4-M represents VYQEKLEGDF, ACKR2-M represents IWGISVAWHW, and CLDND1-M represents QFMASTLFI; Negative represents control; *P<0.05, **P<0.01, ***P<0.001.

[0047] Figure 6 The expression level of CD39 on T cells in the peripheral blood and malignant ascites of liver cancer patients was detected and statistically analyzed using FCM in Example 4 of the present invention; **P<0.01.

[0048] Figure 7 and Figure 8To analyze the gene mutations of TCP1, ABCD4, ACKR2 and CLDND1 in different cancers in the TCGA database, ACC represents adrenocortical carcinoma, CESC represents cervical squamous carcinoma and adenocarcinoma, 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, COAD represents colon cancer, DLBC represents diffuse large B-cell lymphoma, 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, KIRC represents kidney clear cell carcinoma, KIRP represents kidney papillary cell carcinoma, LAML represents acute myeloid leukemia, 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, SARC represents sarcoma, SKCM represents skin melanoma, STAD represents gastric cancer, THCA represents thyroid cancer, THYM represents thymus cancer, UCEC represents endometrial cancer, UCS represents uterine sarcoma; **P<0.01.

[0049] Figure 9 To detect the IFN-γ secreted by the T cells stimulated by the neoantigens in Example 5 of the present application after co-incubation with tumor cells (A) and the cytotoxicity of the T cells stimulated by the neoantigens to kill tumor cells (B); **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0050] 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 embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0051] The malignant ascites of liver cancer patients used in the following examples were 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.

[0052] Example 1: Obtaining tumor cells in malignant ascites of liver cancer patients by flow cytometry Since the prediction of neoantigens is mainly obtained by mutations in tumor cell DNA and further verified by mutations in RNA, in hepatocellular carcinoma (HCC, hereinafter referred to as liver cancer) patients, tumor cells account for more than 90% of tumor tissues, and neoantigens can be obtained directly by extracting DNA and RNA from tumor tissues. Most liver cancer patients are in the middle and advanced stages when they are discovered, missing the best opportunity for surgical resection, resulting in the inability to obtain the neoantigen profile of non-surgical treatment patients. However, some patients with advanced tumors are accompanied by malignant ascites, which suggests that tumor cells in malignant ascites have the potential to become a source of neoantigen profiles. However, since more than 90% of the malignant ascites of most HCC patients are white blood cells, not tumor cells, if the DNA and RNA of all cells in the ascites are directly extracted, it is difficult to obtain the gene mutation profile by next-generation sequencing, and it is also impossible to further obtain the neoantigen profile. Therefore, it is necessary to obtain live tumor cells by sorting without damaging the structure (RNA and DNA) of tumor cells in malignant ascites. Epithelial cell adhesion molecule positive (EpCAM+) HCC cells are considered a subpopulation with tumor-initiating cell (TIC) or cancer stem cell (CSC) characteristics, which is closely related to poor prognosis in patients. Studies have found that EpCAM + CTC) count can be used as a new predictor of the prognosis of radical resection of hepatocellular carcinoma, that is, compared with hepatocellular carcinoma radical resection patients with EpCAM + CTC value less than 2, hepatocellular carcinoma radical resection patients received hepatocellular carcinoma radical resection EpCAM + CTC value greater than 2, 7.5 mL of peripheral blood from hepatocellular carcinoma radical resection patients received hepatocellular carcinoma radical resection EpCAM +CTC number is greater than or equal to 2, the risk of HCC recurrence is higher (Sun YF, Xu Y, Yang XR, Guo W, Zhang X, Qiu SJ, et al. Circulating stem cell-like epithelial cell adhesion molecule-positive tumor cells indicate poor prognosis of hepatocellular carcinoma after curative resection. Hepatology. 2013, 57(4): 1458-68.). In addition, studies have shown that many solid tumors are often accompanied by chromosome 8 abnormalities (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 liquid biopsy samples, and it is found that malignant ascites of patients with advanced liver cancer all contain tumor cells with chromosome abnormalities, these tumor cells not only express CEP8, but also express EpCAM, in addition, part of the tumor cells also express liver cancer specific marker AFP (Sun YF, Xu Y, Yang XR, Guo W, Zhang X, Qiu SJ, et al. Circulating stem cell-like epithelial cell adhesion molecule-positive tumor cells indicate poor prognosis of hepatocellular carcinoma after curative resection. Hepatology. 2013, 57(4): 1458-68.). Figure 1 ). Therefore, the present application uses FCM technology to obtain live (7-AAD - ), tumor cells containing nuclei (Hoechest 33342 + ) and excluding white blood cells (CD45 - ) (EpCAM + ) in malignant ascites, for obtaining DNA and RNA, and further obtaining neoantigen spectrum by second-generation sequencing. By FCM analysis of the number of 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation (hereinafter referred to as P7 subpopulation) in liquid biopsy samples, it is found that the number of P7 subpopulation in malignant ascites of liver cancer patients is significantly higher than that in ascites of cirrhosis patients (P<0.05) P <0.05) Figure 2). Therefore, P7 subpopulation can be obtained by FCM method, and then tumor cells can be obtained.

[0053] Based on the above research results, the embodiment constructs a set of methods for sorting tumor cells from malignant ascites by flow cytometry (FCM), which includes: using BD FACSAria II flow cytometer to sort 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation.

[0054] Example 2 Determination of potential neoantigens of tumor cells in malignant ascites of liver cancer patients based on computer algorithm First, using BD FACSAria II flow cytometer to sort 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation (i.e. P7 subpopulation), the P7 population of less than 2000 cells was extracted and amplified RNA and DNA by Smart-seq 2 technology and Discover-sc single cell, respectively. Subsequently, using illumina novaseq 6000 platform, respectively, transcriptome sequencing (RNA-sequencing, RNA-seq) and whole-exome sequencing (WES) were performed on RNA and DNA.

[0055] The WES data of the above malignant ascites P7 population was compared with the WES data of the patient's peripheral blood mononuclear cells (PBMC), and the patient's ascites P7 subpopulation mutations were identified using GATK Mutect2, and mutations with TLOD ≥ 10 were 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 patient's malignant ascites P7 subpopulation was determined. Further combined with the RNA-seq data of the malignant ascites P7 subpopulation, the potential mutant polypeptide derived from WES was analyzed using GATK ASEReadCounter algorithm, and the mutation number (Mutation Count, MutCount) of the potential mutant polypeptide was determined at the RNA level.

[0056] Finally, the affinity between the mutant polypeptides and the patient's HLA was calculated using the NetMHCpan-4.1 algorithm, and potential neoantigens were predicted using the MutAff < 200 nM or RefAff / MutAff > 10 threshold, after excluding duplicates and HLA heterozygosity loss.

[0057] The above method was used to find potential neoantigens in the tumor cells of malignant ascites of liver cancer patients P10 and P14 (Table 1), which included four neoantigen peptides IYLENYATSMW (TCP1), VYQEKLEGDF (ABCD4), IWGISVAWHW (ACKR2), and QFMASTLFI (CLDND1) presented by HLA A*24:02 molecules, neoantigen peptides MALPFATPS and NPRAGGPPA presented by HLA-B*54:01 molecules, neoantigen peptide FMASTLFIWAA presented by HLA-A*02:06 molecules, neoantigen peptide APLQFMASTL presented by HLA-B*07:02 molecules, and neoantigen peptide AESNMNDLI presented by HLA-B*40:01 molecules. These neoantigen peptides have the potential to be used to make tumor vaccines to prevent the recurrence and metastasis of liver cancer.

[0058] Table 1. Linear sequences and characteristic parameters of 9 predicted liver cancer malignant ascites neoantigens and their paired WT antigens Example 3 Determination of high-frequency HLA-presented neoantigens of tumor cells in malignant ascites of liver cancer patients by Tetramer detection method The method of Tetramer staining was used to preliminarily screen the neoantigens of hepatocellular carcinoma (HCC). Due to the detection of the affinity between the neoantigen peptide and HLA on the market, the QuickSwitch™ QuantTetramer detection kit of MBL Company is mainly used for the detection of antigen peptides presented by HLA-A*02:01, HLA-A*11:01 and HLA-A*24:02 with high frequency in humans. Therefore, the QuickSwitch™ QuantTetramer HLA-A*24:02 Kit-PE kit was used to detect the affinity between the candidate neoantigen polypeptides derived from TCP1, ABCD4, ACKR2 and CLDND1 in Table 1 and the HLA-A*24:02 molecule. According to the instructions, first, the candidate polypeptide solution with a concentration of 2 mM and the 1 mM reference peptide solution were warmed to room temperature. 50 μL Tetramer was dispensed into an EP tube, 1 μL of candidate peptide or reference peptide was added to each tube, followed by the addition of 1 μL of polypeptide displacement factor. After gentle mixing, it was incubated at room temperature for 5 hours in the dark, and finally stored at 4°C for standby. During this process, different control groups were set according to the instructions. In a round-bottom 96-well plate, 20 μL magnetic beads were added to each well, and the following groups were processed: 1# hole (Control #1) and 3# hole (Control #3) added with 5 μL Tetramer, 2# hole (Control #2) added with 5 μL 1x Assay Buffer, and the remaining holes added with 5 μL Tetramer after peptide replacement. After aluminum foil was used to avoid light, it was incubated at 550 rpm for 45 minutes, 150 μL of 1x Assay Buffer was added to each well, and the magnetic stand was placed for 5 minutes to discard the supernatant. After vortexing for 2 seconds, it was removed; except for the 1# hole added with 25 μL of 1x Assay Buffer, the remaining holes were added with 25 μL of 1x working concentration of Exiting Peptide Antibody (freshly prepared), and avoided light. After shaking at 550 rpm 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, 200 μL of buffer and 5 μL of magnetic beads were added to the 4# hole as a magnetic bead control group, and FCM analysis was performed to calculate the displacement efficiency of the candidate neoantigen peptide, i.e. the affinity between the candidate neoantigen peptide and HLA. It was found that the peptide displacement efficiency between the candidate neoantigen peptides IYLENYATSMW (TCP1), VYQEKLEGDF (ABCD4), IWGISVAWHW (ACKR2) and QFMASTLFI (CLDND1) and HLA A*24:02 molecules was 96.45%, 96.35%, 97.52% and 97.33% respectively, which was significantly higher than the threshold value of 75% set in the instructions. Figure 3A), which indicated that these four candidate neoantigen peptides had good affinity with HLA-A*24:02 molecules.

[0059] Subsequently, the frequency of specific T cells against IYLENYATSMW (TCP1), VYQEKLEGDF (ABCD4), IWGISVAWHW (ACKR2) and QFMASTLFI (CLDND1) candidate neoantigens in malignant ascites and peripheral blood of P10 and P14 hepatocellular carcinoma patients were detected by specific Tetramer staining method. Figure 3 Figure 4 Firstly, the concentration of ascites or peripheral blood-derived cells was controlled at 1x10 6-7 6 / mL, 50 μL of cell suspension was added to 10 μL of Clear Back (5 min at room temperature in the dark, to block non-specific binding). Then 10 μL of peptide-replaced Tetramer (replacement rate >75%) was added to each tube, and incubated at room temperature for 30 min, followed by the addition of CD8 antibody (4°C, incubation for 20 min); after PBS washing (400xg, 5 min), the supernatant was discarded, 500 μL of PBS was used to resuspend the cells, 20 μL of Cell Viability Solution was added, and the mixture was stored at 4°C in the dark and subjected to FCM analysis within 24 h. It was found that in addition to IYLENYATSMW (TCP1) polypeptide, specific T cells derived from VYQEKLEGDF (ABCD4), IWGISVAWHW (ACKR2) and QFMASTLFI (CLDND1) antigen peptides could be detected in malignant ascites, which suggested that these antigens might be hepatocellular carcinoma neoantigens. In P10 patients, no specific T cells were detected in peripheral blood without candidate neoantigen polypeptide stimulation, but more than 5% of specific T cells against three neoantigen peptides including IYLENYATSMW (TCP1) were detected after 13 days of stimulation with a final concentration of 2 μM of neoantigen polypeptide Figure 5 , which also suggested that the presence of long-term malignant ascites might lead to the exhaustion or apoptosis of specific T cells against neoantigens.

[0060] Example 4 CD39 T cells with reactivity to neoantigens exist in peripheral blood and malignant ascites of hepatocellular carcinoma patients + ​​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 may have better ability to recognize neoantigens, and dominant neoantigens can exert anti-tumor effect by activating CD39 + T cells. Further studies have found that CD8 + CD39 + T cells (CD39 Figure 6 ) in the peripheral blood and malignant ascites of liver cancer patients have a response to neoantigens. Compared with the peripheral blood of liver cancer patients, the expression ratio of CD8 + CD39 + T cells in the malignant ascites is significantly increased (CD39 Figure 6 ).

[0061] In addition, analysis of the TCGA database found that TCP1, ABCD4, ACKR2 and CLDND1 have gene mutations in multiple cancers (CD39 Figure 7 and Figure 8 ), which suggests that these neoantigen polypeptides discovered by the present application can be used as tumor vaccines for treating tumors in the future.

[0062] Example 5 Peripheral blood stimulated by neoantigen peptides has a killing function on tumor cells derived from the malignant ascites of liver cancer patients First, PBMCs from P10 and P14 patients were extracted, respectively. Then, P10 patient-derived IYLENYATSMW (TCP1), VYQEKLEGDF (ABCD4) and IWGISVAWHW (ACKR2) and P14 patient-derived QFMASTLFI (CLDND1) neoantigen peptides were co-incubated with patient-derived PBMCs, respectively, to activate and expand patient neoantigen-specific T cells, as follows: 1x10 6 PBMCs were cultured in 12-well plates, 1 mL of X-VIVO 15 + 5% human AB serum + 1% penicillin / streptomycin + 100 IU / mL IL-2 + 4 mM polypeptide per well, half-volume replacement every 2-3 days, and T cells were collected at 13 days.

[0063] Second, patient's own tumor cells were obtained from patient malignant ascites and expanded by organoid culture for use in subsequent experiments. First, tumor cells in malignant ascites were mixed with culture medium (containing insulin growth factor-2 at a concentration of 8 ng / mL) to obtain a tumor cell concentration of 2x10 4 / mL, and then 5% Matrigel was added and mixed on ice to obtain a culture medium. The culture medium was inoculated into a 24-well low-adsorption plate and incubated in a 37°C incubator for 30 min, and 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 culture in a single culture well 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.

[0064] 1x10 6 effector cells (T cells) stimulated by neoantigens were co-incubated with 1x10 5 target cells (tumor cells) for 12 h, and the control group used T cells not stimulated by neoantigens and tumor cells for co-incubation. ELISA was used to detect IFN-γ secreted by neoantigen-specific T cells. Compared with the control group, more IFN-γ was secreted by T cells co-incubated with tumor cells stimulated by IYLENYATSMW (TCP1), VYQEKLEGDF (ABCD4) and IWGISVAWHW (ACKR2) and QFMASTLFI (CLDND1) neoantigensFigure 9 ). The level of lactate dehydrogenase (LDH) released from tumor cells killed by neoantigen-specific T cells was further detected by ELISA. Compared with the control group, the level of LDH released from tumor cells killed by T cells stimulated by IYLENYATSMW (TCP1), VYQEKLEGDF (ABCD4), IWGISVAWHW (ACKR2) and QFMASTLFI (CLDND1) neoantigens was higher (p < 0.05) Figure 9 ). This indicates that peripheral blood stimulated by neoantigen peptides can produce killing function on tumor cells derived from malignant ascites of hepatocellular carcinoma patients.

[0065] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A tumor neoantigen polypeptide, characterized in that: The amino acid sequence of the tumor neoantigen polypeptide is shown as SEQ ID NO. 1, 2, 3 or 4.

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 is 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. An antigen-presenting cell or tumor-specific T cell, 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.

7. 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.

8. 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.

9. A method for separating and screening tumor neoantigen polypeptides, characterized in that: The method comprises: using malignant ascites 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.

10. The method according to claim 9, characterized in that The tumor cells belong to 7-AAD - Hoechest33342 + CD45 - EpCAM + A population of less than 2000 cells in a cell subpopulation; The DNA sequencing is whole exome sequencing, and the RNA sequencing is transcriptome sequencing; The screening of tumor neoantigen polypeptides based on sequencing data includes: identifying mutations carried by the tumor cells based on whole exome sequencing data; obtaining the patient's HLA type; Predicting the patient's potential HLA loss of heterozygosity; predicting mutant proteins from somatic mutations and determining potential mutant polypeptides in the tumor cells; and, analyzing the potential mutant polypeptide based on transcriptome sequencing data to determine the number of mutations in the potential mutant polypeptide at the RNA level; Evaluate the affinity between the mutant peptide and the patient's HLA, and screen potential tumor neoantigen peptides while excluding duplications and HLA loss of heterozygosity.

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