Tumor neoantigen polypeptides and methods of isolation and screening thereof

Abstract

The present application relates to the technical field of tumor immunotherapy, and particularly relates to a tumor neoantigen polypeptide and a method for isolating and screening the same. The amino acid sequence of the tumor neoantigen polypeptide is shown in SEQ ID NO. 1, 2, 3 or 4. The tumor neoantigen polypeptide has high affinity with HLA molecules, can induce tumor-specific T cells, and further produce an immune response targeting tumor cells. The tumor neoantigen polypeptide can be used as a target for tumor clinical treatment or diagnosis, and has good application prospect in tumor immunotherapy. 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 are not treated by surgery, and provides an effective method for developing tumor neoantigen polypeptides.

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 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:

[0019] (1) an expression cassette comprising the nucleic acid molecule;

[0020] (2) a vector comprising the nucleic acid molecule or the expression cassette in (1);

[0021] (3) a cell comprising the nucleic acid molecule, the expression cassette in (1), or the vector in (2).

[0022] 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.

[0023] In the above (2), the vector includes plasmid vectors, viral vectors, transposons, artificial chromosomes, etc.

[0024] 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 (such as CHO cells, HEK293, etc.) for polypeptide expression, for example, or immune cells, etc.

[0025] 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:

[0026] (1) preparing antigen presenting cells;

[0027] (2) preparing tumor-specific T cells;

[0028] (3) preparing TCR-T or CAR-T cells;

[0029] (4) preparing tumor diagnostic reagents;

[0030] (5) preparing drugs for preventing or treating tumors.

[0031] The tumor neoantigen polypeptide provided by the 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.

[0032] In the application, the tumor is preferably a liver tumor, and more preferably hepatocellular carcinoma.

[0033] In the above (2), the tumor-specific T cells include cytotoxic T cells and the like.

[0034] 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.

[0035] 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.

[0036] In a sixth aspect, the 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.

[0037] The tumor-specific T cell comprises a cytotoxic T cell, a TCR-T cell or a CAR-T cell.

[0038] In a seventh aspect, the application provides a method for preparing 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.

[0039] In an eighth aspect, the application provides a pharmaceutical composition comprising the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material described above.

[0040] 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.

[0041] 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.

[0042] 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 comprise mRNA encoding the tumor neoantigen polypeptide. Taking the DC vaccine as an example, the DC vaccine can be prepared by loading the polypeptide into DC.

[0043] The vaccine described above can be used for immunotherapy of patients with the tumor neoantigen polypeptide.

[0044] 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 described above.

[0045] In an eleventh aspect, the present application provides a method for isolating and screening a tumor neoantigen polypeptide, which comprises: using malignant ascites of a tumor patient as a sample, and sorting tumor cells from the sample by flow cytometry;

[0046] sequencing the tumor cells by DNA sequencing and RNA sequencing, and screening a tumor neoantigen polypeptide based on the sequencing data;

[0047] The tumor cells are 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation.

[0048] Malignant ascites is an important component of liquid biopsy samples of tumor patients. The present application finds that tumor cells in malignant ascites can be used as a source for isolating tumor neoantigen polypeptides. By isolating 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation from malignant ascites, and performing high-throughput sequencing and mutation analysis on the cell subpopulation, a tumor neoantigen polypeptide can be screened. The above method based on liquid biopsy technology can provide neoantigens for patients with abdominal tumors that cannot be operated on, provide new neoantigen sequences for vaccine research based on neoantigens, and ultimately be used for preventing and treating recurrence and metastasis of tumor patients.

[0049] In the above method, the tumor cells belong to 7-AAD - Hoechest 33342 + CD45- EpCAM + a population of less than 2000 cells in a cell subpopulation.

[0050] In the above method, the DNA sequencing is whole-exome sequencing, and the RNA sequencing is transcriptome sequencing.

[0051] 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 the HLA type of the patient; predicting potential HLA heterozygous loss of the patient; predicting mutant proteins from somatic mutations to determine potential mutant polypeptides in the tumor cells;

[0052] and, based on the transcriptome sequencing data, analyzing the potential mutant polypeptides to determine the number of mutations of the potential mutant polypeptides at the RNA level;

[0053] The affinity between the mutant polypeptide and the HLA of the patient is evaluated, and the potential tumor neoantigen polypeptide is screened after excluding repetitions and HLA heterozygous loss.

[0054] In the above method, the tumor is preferably a liver tumor, and more preferably hepatocellular carcinoma.

[0055] 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, and can be used as a target for clinical treatment or diagnosis of tumors, and has good application prospects in tumor immunotherapy.

[0056] 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 are not treated by surgery, and provides an effective method for the development of tumor neoantigen polypeptides. BRIEF DESCRIPTION OF DRAWINGS

[0057] 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 those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0058] Figure 1 and Figure 2 EpCAM + tumor cells in malignant ascites of a liver cancer patient in Example 1 of the present application; wherein, Figure 1 chromosome abnormalities of tumor cells in malignant ascites are detected by SE-i·FISH method.Figure 2 Flow cytometry analysis of tumor cells in ascites samples and flow cytometry analysis statistics of P7 subgroups; *P<0.05.

[0059] Figure 3 , Figure 4 and Figure 5 are the neoantigens of tumor cells in malignant ascites samples of liver cancer patients screened by specific Tetramer in Example 3 of the present application; wherein Figure 3 A of is the affinity between the candidate neoantigen peptide and the HLA molecule detected by using the QuickSwitch™ Quant Tetramer detection kit; Figure 3 B and Figure 4 respectively are the specific T cell frequencies against the neoantigens in the malignant ascites of P10 and P14 liver cancer patients detected by using the QuickSwitch™ Quant Tetramer detection kit; Figure 5 is the specific T cell frequency against the neoantigens in the peripheral blood and malignant ascites of the P10 patient detected by using the QuickSwitch™ Quant Tetramer detection kit; TCP1-M represents IYLENYATSMW, ABCD4-M represents VYQEKLEGDF, ACKR2-M represents IWGISVAWHW, CLDND1-M represents QFMASTLFI; Negative represents the control; *P<0.05, **P<0.01, ***P<0.001.

[0060] Figure 6 is the expression level of CD39 on T cells in the peripheral blood and malignant ascites of liver cancer patients detected by FCM in Example 4 of the present application and statistics are performed; **P<0.01.

[0061] 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 carcinoma, UCEC represents endometrial carcinoma, UCS represents uterine sarcoma; **P<0.01.

[0062] 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

[0063] 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.

[0064] 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.

[0065] Example 1: Obtaining tumor cells in malignant ascites of liver cancer patients by flow cytometry

[0066] 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). Thus, P7 subpopulation can be obtained by the method of FCM, and then tumor cells can be obtained.

[0067] 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 a BD FACSAria II flow cytometer to sort 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation.

[0068] Example 2 Determination of potential neoantigens of tumor cells in malignant ascites of liver cancer patients based on computer algorithm

[0069] First, using a BD FACSAria II flow cytometer, 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation (i.e., P7 subpopulation) is sorted from the malignant ascites of liver cancer patients, and the P7 population of less than 2000 cells is extracted and amplified by RNA and DNA by Smart-seq 2 technology and Discover-sc single cell, respectively. Subsequently, using an illumina novaseq 6000 platform, RNA and DNA are subjected to transcriptome sequencing (RNA-sequencing, RNA-seq) and whole-exome sequencing (WES), respectively.

[0070] The WES data of the above-mentioned malignant ascites P7 population was compared with the WES data of the peripheral blood mononuclear cells (PBMC) of the patient, GATK Mutect2 was used to identify the mutations of the patient's ascites P7 subpopulation, and the mutations with TLOD ≥ 10 were retained; the HLA type of the patient was obtained using the OptiType algorithm; the potential HLA heterozygous deletion of the patient was predicted using the LOHHLA algorithm; the mutant protein was predicted and disassembled from the somatic mutation using the VEP algorithm, and the potential mutant polypeptide in the malignant ascites P7 subpopulation of the patient 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 the GATK ASEReadCounter algorithm, and the mutation number (Mutation Count, MutCount) of the potential mutant polypeptide at the RNA level was determined.

[0071] Finally, the affinity between the mutant polypeptide and the patient's HLA was calculated using the NetMHCpan-4.1 algorithm, and the potential neoantigens were predicted using the MutAff ≤ 200 nM or RefAff / MutAff ≥ 10 threshold value after excluding duplicates and HLA heterozygous deletion.

[0072] The potential neoantigens were found in the tumor cells of the malignant ascites of liver cancer patients P10 and P14 using the above method (Table 1), including 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 make tumor vaccines to prevent and treat the recurrence and metastasis of liver cancer.

[0073] Table 1. Linear sequences and characteristic parameters of 9 predicted neoantigens in liver cancer malignant ascites and their paired WT antigens

[0074]

[0075] Example 3 Determination of neoantigens of high frequency HLA-presenting tumor cells in malignant ascites of hepatocarcinoma patients by Tetramer detection method

[0076] 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.

[0077] 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 cells / mL, 50 μL cell suspension was added to 10 μL Clear Back (room temperature, 5 min, block non-specific binding). Then 10 μL peptide-replaced Tetramer (replacement rate >75%) was added to each tube, incubated at room temperature for 30 min, and then CD8 antibody was added (4°C, incubate for 20 min); after PBS washing (400xg, 5 min), the supernatant was discarded, 500 μL PBS was used to resuspend the cells, 20 μL Cell Viability Solution was added, and the mixture was stored at 4°C in the dark and FCM analysis was completed 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) could be detected after 13 days of stimulation with a final concentration of 2 μM of neoantigen polypeptide. Figure 5 This also suggested that the presence of long-term malignant ascites might lead to the exhaustion or apoptosis of specific T cells against neoantigens.

[0078] Example 4 CD39 T cells with reaction to neoantigens exist in peripheral blood and malignant ascites of hepatocellular carcinoma patients +

[0079] ​​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 T cells in the peripheral blood of patients with liver cancer. + CD39 + T cells in malignant ascites were significantly increased (CD39 Figure 6 ).

[0080] In addition, analysis of the TCGA database found that TCP1, ABCD4, ACKR2 and CLDND1 all 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.

[0081] Example 5 Peripheral blood stimulated by neoantigen peptides has killing function on tumor cells derived from malignant ascites of patients with hepatocellular carcinoma

[0082] 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.

[0083] 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.

[0084] 1x10 6 effector cells (T cells) and 1x10 5 target cells (tumor cells) were co-incubated for 12 h, while the control group used T cells and tumor cells that were not stimulated by neoantigens 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) neoantigens (Figure 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.

[0085] 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 in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the tumor neoantigen polypeptide of claim 1.

3. A biomaterial, characterized in that, The biomaterial is any one of the following: (1) An expression cassette comprising the nucleic acid molecule of claim 2; (2) A carrier comprising the nucleic acid molecule of claim 2 or the expression cassette of (1); (3) A cell comprising the nucleic acid molecule of claim 2, the expression cassette of (1) or the vector of (2).

4. The use of the tumor neoantigen polypeptide of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of hepatocellular carcinoma-specific T cells.

5. A hepatocellular carcinoma-specific T cell, characterized in that, The hepatocellular carcinoma-specific T cells are induced by the tumor neoantigen polypeptide of claim 1 and specifically target the tumor neoantigen polypeptide of claim 1.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the tumor neoantigen polypeptide of claim 1, the nucleic acid molecule of claim 2, or the biological material of claim 3.

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

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