HLA restrictive neoantigen polypeptide based on malignant ascites tumor cells and application of HLA restrictive neoantigen polypeptide

Abstract

The invention relates to the technical field of tumor immunotherapy, in particular to an HLA restrictive neoantigen polypeptide based on malignant ascites tumor cells and application of the HLA restrictive neoantigen polypeptide. The invention provides cholangiocarcinoma neoantigen polypeptide. The amino acid sequence of the neoantigen polypeptide is shown as SEQ ID NO.1, 2 or 3. The tumor neoantigen polypeptide is separated and screened from malignant ascites of a biliary duct cancer patient, has high affinity with HLA molecules, can stimulate generation of tumor specific T cells, further generates immune response of targeted tumor cells, can be used as a target spot for clinical treatment or diagnosis of tumors, and has good application prospects. Good application prospects are realized in tumor immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor immunotherapy, and particularly relates to an HLA-restricted neoantigen polypeptide based on malignant ascites tumor cells and application thereof. BACKGROUND

[0002] Intrahepatic cholangiocarcinoma (ICC) is a highly invasive intrahepatic biliary epithelial malignancy, accounting for 8.2% to 15.0% of primary liver malignancies. Its epidemiological characteristics show a global upward trend in the incidence in recent years (Rumgay H, Ferlay J, de Martel C, Georges D, Ibrahim AS, Zheng R, et al. Global, regional and national burden of primary liver cancer by subtype. Eur J Cancer. 2022, 161: 108-118; Cong WM, Dong H, Tan L, Sun XX, Wu MC. Surgicopathological classification of hepatic space-occupying lesions: A single-center experience with literature review. World J Gastroenterol. 2011, 17(19): 2372-2378.).Although adenocarcinoma is the main pathological type, it has high molecular heterogeneity and poor clinical prognosis, with a 5-year overall survival rate of less than 10% (An L, Zheng R, Zhang S, Chen R, Wang S, Sun K, et al. Hepatocellular carcinoma and intrahepatic cholangiocarcinoma incidence between 2006 and 2015 in China: Estimates based on data from 188 population-based cancer registries. Hepatobiliary Surg Nutr. 2023, 12(1): 45-55; Moris D, Palta M, Kim C, Allen PJ, Morse MA, Lidsky ME. Advances in the treatment of intrahepatic cholangiocarcinoma: An overview of the current and future therapeutic landscape for clinicians. CA Cancer J Clin. 2023, 73(2):198-222.). Due to the insidious early symptoms and lack of specific biomarkers, about 70%~80% of patients are in the middle and advanced stages at the initial diagnosis, combined with vascular invasion or distant metastasis, losing the opportunity for radical surgery, resulting in a median survival of less than 12 months (Moris D, Palta M, Kim C, Allen PJ, Morse MA, Lidsky ME. Advances in the treatment of intrahepatic cholangiocarcinoma: An overview of the current and future therapeutic landscape for clinicians. CA Cancer J Clin. 2023, 73(2):198-222.).For unresectable intrahepatic cholangiocarcinoma (ICC), the National Comprehensive Cancer Network (NCCN), European Association for the Study of the Liver (EASL), and Chinese Society of Clinical Oncology (CSCO) guidelines recommend gemcitabine plus cisplatin (GC) regimen combined with durvalumab as the first-line treatment regimen (European Association for the Study of the Liver. EASL-ILC A Clinical Practice Guidelines on the management of intrahepatic cholangiocarcinoma[J]. J Hepatol, 2023, 79(1): 181-208; Chinese Society of Clinical Oncology Guidelines Working Committee. Chinese Society of Clinical Oncology (CSCO) Biliary Tract Malignant Tumor Diagnosis and Treatment Guidelines 2023. Beijing: People's Medical Publishing House, 2023; National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology. Biliary Tract Cancers (Version 5.2024) [EB / OL].). However, although the application of immune checkpoint inhibitors has brought new breakthroughs in the treatment of patients with advanced ICC in recent years, the results of clinical studies show that the objective response rate (ORR) and median overall survival (mOS) of this combined regimen have not yet reached the expected target, and the overall efficacy is still not ideal. Therefore, new treatment targets need to be found.

[0003] Tumor neoantigens are antigens that do not exist in normal cells 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 receptor (TCR) after specific immune response is triggered (Terai M, Sato T. Individualised neoantigen cancer vaccine therapy. Lancet. 2024, 403(10427): 590-591.). Studies on the immunological mechanism of immune checkpoint blockade (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 the pre-exisiting neoantigen-specific T cells in tumor patients are the targets of ICB therapy and the core driving force to achieve clinical benefit 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 cholangiocarcinoma prevention and treatment.

[0004] Currently, neoantigens are mainly obtained from surgical tissue samples, but about 70-80% of patients are in the middle and late stages at the time of initial diagnosis, missing the best opportunity for surgical resection, so it is impossible to obtain the neoantigen profile of patients who cannot undergo surgery (Moris D, Palta M, Kim C, Allen PJ, Morse MA, Lidsky ME. Advances in the treatment of intra⁃hepatic cholangiocarcinoma: An overview of the current and future therapeutic landscape for clinicians. CA Cancer J Clin. 2023, 73(2): 198-222.). SUMMARY

[0005] The present application provides HLA-restricted neoantigen polypeptides based on malignant ascites tumor cells and applications thereof.

[0006] The present application takes malignant ascites of cholangiocarcinoma patients as samples, sorts tumor cells from the malignant ascites, and analyzes mutations by high-throughput sequencing. Computer algorithms are used to predict candidate neoantigens. For the candidate neoantigens obtained by prediction, the affinity between the candidate neoantigen polypeptides and HLA molecules and the frequency of neoantigen-specific T cells are analyzed by tetramer (Tetramer) staining experiments, and finally three neoantigen polypeptides YLQDVETGTQL (SEQ ID NO. 1), ITDDLHFYL (SEQ ID NO. 2) and YCLSPHLQYI (SEQ ID NO. 3) presented by HLA-A*02:01 molecules are screened.

[0007] Specifically, the present application provides the technical solutions described below.

[0008] In a first aspect, the present application provides cholangiocarcinoma neoantigen polypeptides, the amino acid sequence of the neoantigen polypeptide is shown in SEQ ID NO. 1, 2 or 3.

[0009] It should be noted that conservative variant sequences that do not affect the function of the above-mentioned cholangiocarcinoma neoantigen polypeptides are also within the scope of protection of the present application, for example, one or more amino acids in the amino acid sequence of the above-mentioned neoantigen polypeptide are conservatively substituted, or one or more amino acids that do not affect its function are added to the N-terminus or C-terminus of the neoantigen polypeptide (e.g., adding 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 connecting peptide can be a flexible connecting peptide rich in GS.

[0011] In addition, a derivative polypeptide obtained by performing one or more modifications on the amino acid sequence of the above new antigen polypeptide is also within the protection scope of the present application, and the modifications include phosphorylation modification, PEGylation modification, amidation modification, glycosylation modification, biotinylation modification, and coupling or fusion with an antibody, a carrier, a ligand, albumin, an Fc fragment, etc.

[0012] In a second aspect, the present application provides a polypeptide, which is the cholangiocarcinoma new antigen polypeptide.

[0013] The above polypeptide can be a polypeptide obtained by adding one or more amino acid residues at the N-terminus and / or C-terminus of the new antigen polypeptide, which can be enzymatically cleaved to produce the new antigen polypeptide.

[0014] The above polypeptide can be obtained by fusing the tumor new antigen polypeptide with other functional polypeptides, such as other known tumor new antigen polypeptides. Based on conventional fusion polypeptide technology, a person skilled in the art can obtain a fusion polypeptide that retains the functional activity of each polypeptide.

[0015] The above polypeptide can be obtained by fusing the new antigen polypeptide with an antibody, a carrier, a ligand, albumin, an Fc fragment.

[0016] In a third aspect, the present application provides a nucleic acid molecule encoding the cholangiocarcinoma new antigen polypeptide or the polypeptide.

[0017] Based on the above-provided amino acid sequence of the tumor new antigen polypeptide and the codon rules, a person skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the tumor new antigen 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 above tumor new antigen polypeptide are within the protection scope of the present application.

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

[0019] 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).

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

[0021] In the above (2), the vector comprises a plasmid vector, a viral vector, a transposon, an artificial chromosome, and the like.

[0022] In the above (3), the cell comprises a microbial cell or an animal cell. The microbial cell comprises Escherichia coli, yeast, and the like. The animal cell does not have the ability to reproduce into an animal individual, and comprises an animal cell line (such as CHO cells, HEK293, and the like) for polypeptide expression or an immune cell, and the like.

[0023] The new-born antigen 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.

[0024] In a fifth aspect, the application provides the following applications of the cholangiocarcinoma new-born antigen 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.

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

[0026] In the above (2) and (3), the applications comprise stimulating the specific T cells of a patient to expand or constructing TCR-T cells or CAR-T cells that can recognize the new-born antigen polypeptide by using the new-born antigen polypeptide. The tumor-specific T cells, TCR-T cells, or CAR-T cells can be used for adoptive immunotherapy.

[0027] In the above (5), the drugs comprise vaccines. The vaccines comprise DNA vaccines, mRNA vaccines, polypeptide vaccines, dendritic cell vaccines (DC vaccines), and the like.

[0028] In the application, the tumor is preferably cholangiocarcinoma, and more preferably intrahepatic cholangiocarcinoma.

[0029] In a sixth aspect, the application provides antigen presenting cells, which are induced by the cholangiocarcinoma new-born antigen polypeptide and specifically target the cholangiocarcinoma new-born antigen polypeptide.

[0030] In a seventh aspect, the present application provides tumor-specific T cells induced by the cholangiocarcinoma neoantigen polypeptide and specifically targeting the cholangiocarcinoma neoantigen polypeptide.

[0031] Preferably, the tumor-specific T cells include cytotoxic T cells, TCR-T cells or CAR-T cells.

[0032] In an eighth aspect, the present application provides a method for preparing tumor-specific T cells, which comprises isolating peripheral blood mononuclear cells, co-culturing the cholangiocarcinoma neoantigen polypeptide with the peripheral blood mononuclear cells, and activating and expanding T cells specifically targeting the cholangiocarcinoma neoantigen polypeptide.

[0033] In a ninth aspect, the present application provides a pharmaceutical composition comprising the cholangiocarcinoma neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material.

[0034] Preferably, the active ingredients of the pharmaceutical composition include the above tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material.

[0035] Further, the pharmaceutical composition can further comprise excipients allowed in the pharmaceutical field.

[0036] In a tenth aspect, the present application provides a vaccine comprising the cholangiocarcinoma neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material.

[0037] 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 comprise mRNA encoding the tumor neoantigen polypeptide. Taking the DC vaccine as an example, the DC vaccine can be prepared by loading polypeptides into DCs.

[0038] The above pharmaceutical composition or vaccine can be directly used for immunotherapy of patients with the tumor neoantigen polypeptide.

[0039] In an eleventh aspect, the present application provides a diagnostic reagent comprising the above cholangiocarcinoma neoantigen polypeptide, the polypeptide, the nucleic acid molecule or the biological material.

[0040] In a twelfth aspect, the present application provides a method for isolating and screening tumor neoantigen polypeptides, which comprises using 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 tumor neoantigen polypeptides based on the sequencing data; wherein the tumor cells are 7-AAD- Hoechest 33342 + CD45 - EpCAM + cell subpopulation.

[0041] Malignant ascites is an important component of liquid biopsy samples of tumor patients, and malignant ascites often accompanies malignant tumor patients (e.g. ICC patients). The present application finds that tumor cells in malignant ascites can be used as a source of isolation of tumor neoantigen polypeptides. By isolating 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation, and performing high-throughput sequencing and mutation analysis on the cell subpopulation, and screening tumor neoantigen polypeptides. The above method is based on liquid biopsy technology, which can provide neoantigens for patients with abdominal tumors who 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.

[0042] In the above method, the tumor cells are 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation, and performing high-throughput sequencing and mutation analysis on the cell subpopulation, and screening tumor neoantigen polypeptides. The above method is based on liquid biopsy technology, which can provide neoantigens for patients with abdominal tumors who 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.

[0043] In the above method, 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 HLA type of the patient; predicting the potential HLA heterozygous loss of the patient; predicting mutant proteins from somatic mutations to determine 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 potential mutant polypeptides at the RNA level; evaluating the affinity between the mutant polypeptides and the HLA of the patient, and screening potential tumor neoantigen polypeptides after excluding duplicates and HLA heterozygous loss.

[0044] The identification of mutations carried by the tumor cells based on whole exome sequencing data includes: identifying (preferably using GATK Mutect2) mutations carried by the tumor cells based on WES data of peripheral blood mononuclear cells (PBMC) of the patient, and mutations with TLOD ≥ 10 are retained. The HLA typing of the patient is preferably performed using the OptiType algorithm.

[0045] The prediction of the potential HLA loss of heterozygosity of the patient is preferably performed using the LOHHLA algorithm.

[0046] The prediction of the mutant protein from the somatic mutation and the determination of the potential mutant polypeptide in the tumor cell comprises: predicting and disassembling the mutant protein from the somatic mutation using the VEP algorithm to determine the potential mutant polypeptide in the tumor cell.

[0047] The analysis of the potential mutant polypeptide based on the transcriptome sequencing data and the determination of the number of mutations of the potential mutant polypeptide at the RNA level are preferably performed using the GATK ASEReadCounter algorithm.

[0048] In the method, the tumor is preferably cholangiocarcinoma, and more preferably intrahepatic cholangiocarcinoma.

[0049] The present application has at least the following beneficial effects: the tumor neoantigen polypeptide obtained by the present application from the malignant ascites of the cholangiocarcinoma patient is screened, the neoantigen polypeptide has high affinity with the HLA molecule, 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.

[0050] The method for isolating and screening the tumor neoantigen polypeptide provided by the present application solves the problem that the existing method cannot obtain the neoantigen of the patient who is not treated by surgery, and provides an effective method for the development of tumor neoantigen polypeptide. BRIEF DESCRIPTION OF DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the present application or the 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.

[0052] Figure 1 And Figure 2 The tumor cells in the malignant ascites of the cholangiocarcinoma patient in Example 1 of the present application express EpCAM + ; wherein, Figure 1 The SE-i·FISH method is used to detect the chromosomal abnormalities of the tumor cells in the malignant ascites. Figure 2 The flow cytometry analysis chart of the tumor cells in the malignant ascites sample and the flow cytometry analysis chart of the P7 subpopulation P <0.001.

[0053] Figure 3 、 Figure 4 、 Figure 5 and Figure 6 are the neoantigens of tumor cells in malignant ascites samples of cholangiocarcinoma patients screened by specific Tetramer in Example 3 of the present application; Figure 3 is the detection of the affinity between the candidate neoantigen peptide and the HLA molecule by using the QuickSwitch™ Quant Tetramer Detection Kit; Figure 4 and Figure 5 are the detection of the frequency of specific T cells against the neoantigens in the malignant ascites of P15 cholangiocarcinoma patients by using the QuickSwitch™ Quant Tetramer Detection Kit; Figure 6 is the detection of the frequency of specific T cells against the neoantigens in the peripheral blood and malignant ascites of P15 cholangiocarcinoma patients by using the QuickSwitch™ Quant Tetramer Detection Kit; APAF1-M represents the neoantigen polypeptide YCLSPHLQYI, SND1-M-1 represents the neoantigen polypeptide YLQDVETGTQL, and SND1-M-2 represents the neoantigen polypeptide ITDDLHFYL; P <0.05, P <0.01, P <0.001, P <0.0001.

[0054] Figure 7 and Figure 8 are the CD39 + T cells in the peripheral blood and malignant ascites of cholangiocarcinoma patients in Example 4 of the present application, which have a response to the neoantigens; wherein, Figure 7 is the detection of the expression level of CD39 on T cells in the peripheral blood and malignant ascites of cholangiocarcinoma patients by using FCM and statistics; Figure 8To analyze the gene mutation status of SND1 and APAF1 in different cancer types in the TCGA database; 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 bile duct carcinoma, 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, KIRC represents renal clear cell carcinoma, KIRP represents renal papillary cell carcinoma, LGG represents low-grade glioma of the brain, LIHC represents hepatocellular carcinoma, LUAD represents lung adenocarcinoma, LUSC represents lung squamous cell carcinoma, MESO represents mesothelioma, OV represents ovarian cancer, and EGFR represents glioma. stands for ovarian cancer, PAAD stands for pancreatic cancer, READ stands for rectal adenocarcinoma, SARC stands for sarcoma, SKCM stands for cutaneous melanoma, STAD stands for gastric cancer, THCA stands for thyroid cancer, THYM stands for thymic carcinoma, UCEC stands for endometrial cancer, and UCS stands for uterine sarcoma;** P <0.01.

[0055] Figure 9 Detection of IFN-γ secretion by neoantigen-stimulated T cells after co-incubation with tumor cells (A) and cytotoxicity of neoantigen-stimulated T cells against tumor cells (B) in Example 5 of the present invention; *** P <0.001; APAF1-M represents the neoantigen peptide YCLSPHLQYI, SND1-M-1 represents the neoantigen peptide YLQDVETGTQL, and SND1-M-2 represents the neoantigen peptide ITDDLHFYL. DETAILED DESCRIPTION

[0056] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0057] The malignant ascites from patients with cholangiocarcinoma used in the following examples was collected and used with the approval of the Ethics Committee of Peking University People's Hospital. The patients or their families were informed and both parties signed an informed consent.

[0058] Example 1 Obtaining tumor cells in malignant ascites of cholangiocarcinoma patients by flow cytometry The prediction of neoantigens mainly relies on the verification of DNA mutation and RNA of tumor cells. In the tumor tissues of cholangiocarcinoma patients, the proportion of tumor cells is usually high, and neoantigens can be directly obtained by extracting DNA and RNA. However, cholangiocarcinoma patients are often diagnosed at an advanced stage, and often miss the opportunity for surgical resection, resulting in non-surgical patients who are difficult to obtain neoantigen profiles through tissue biopsy. Malignant ascites is a common complication in patients with advanced cholangiocarcinoma, suggesting that it may contain tumor cells, providing a potential source for obtaining neoantigen profiles. However, the proportion of white blood cells in cholangiocarcinoma malignant ascites is often more than 90%, and it is difficult to effectively obtain tumor-specific gene mutation profiles through second-generation sequencing by directly extracting DNA and RNA from all cells in the ascites, and thus it is impossible to generate neoantigen profiles. Therefore, flow cytometry (FCM) technology is needed to sort live tumor cells without damaging the integrity of tumor cell DNA and RNA.

[0059] Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein responsible for mediating Ca 2+homotypic cell adhesion. It is specifically expressed in epithelial tissues and their epithelial-derived tumors, and is therefore considered a potential marker of circulating tumor cells (CTCs). Normally, cells expressing EpCAM are essentially absent in body fluids, so their detection in ascites usually suggests the presence of metastasis from tumor tissue (Patriarca C, Macchi RM, Marschner AK, Mellstedt H. Mellstedt Epithelial cell adhesion molecule expression (CD326) in cancer: a short review. Cancer Treat. Rev. 2012, 38(1):68-75.). Cholangiocarcinoma, as an epithelial-derived tumor, highly expresses EpCAM in its tissue (Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, et al. Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. J Hepatol. 2017, 67(2):282-292.). Therefore, EpCAM can be used as a marker to effectively isolate tumor cells in ascites of cholangiocarcinoma patients in combination with other specific markers. 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.), and the present application uses the method of differential phase enrichment-multiple tumor marker immunofluorescence staining-chromosome fluorescence in situ hybridization (SE-i·FISH) to detect liquid biopsy samples, and found that malignant ascites of patients with advanced cholangiocarcinoma all contain tumor cells with chromosome abnormalities, and these tumors express not only CEP8, but also EpCAM, CEA, CA199 and CA125, etc. tumor markers (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.), which is consistent with the results of previous studies (Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, et al. Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. J Hepatol. 2017, 67(2):282-292.). Figure 1), but tumor markers such as CEA, CA199 and CA125 are mainly expressed in cells, which is not conducive to the separation of live tumor cells from malignant ascites as markers. Therefore, the present application uses FCM technology to obtain live (7-AAD - ), containing nuclei (Hoechest 33342 + ) and tumor cells excluding leukocytes (CD45 - ) (EpCAM + ) in the malignant ascites of cholangiocarcinoma patients, for obtaining DNA and RNA of tumor cells, and further obtaining neoantigen spectrum by second-generation sequencing. By FCM analysis 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 the malignant ascites of cholangiocarcinoma patients is significantly higher than that in the ascites of cirrhosis patients (P7 P <0.05) (P7 Figure 2 ). Therefore, P7 subpopulation can be obtained by FCM method, and then tumor cells can be obtained.

[0060] Based on the above experimental results, a set of methods for sorting tumor cells from malignant ascites by flow cytometry (FCM) is constructed, including: using BD FACSAria II flow cytometer to sort P7 cell tumor subpopulation from malignant ascites of cholangiocarcinoma patients.

[0061] Example 2 Determination of potential neoantigens of tumor cells in malignant ascites of cholangiocarcinoma patients based on computer algorithm First, using BD FACSAria II flow cytometer to sort 7-AAD - Hoechest 33342 + CD45 - EpCAM + cell subpopulation (P7 subpopulation) from malignant ascites of cholangiocarcinoma patients, and using Smart-seq 2 technology and Discover-sc Single cell to extract and amplify RNA and DNA of P7 population below 2000 cells, respectively. Subsequently, using illumina novaseq 6000 platform to perform transcriptome sequencing (RNA-sequencing, RNA-seq) and whole-exome sequencing (whole-exome sequencing, WES) on RNA and DNA, respectively.

[0062] The WES data of the above-mentioned malignant ascites P7 population is compared with the WES data of the peripheral blood mononuclear cells (PBMC) of the patient, GATK Mutect2 is used to identify the mutations of the patient ascites P7 subpopulation, and the mutations with TLOD ≥ 10 are retained; the HLA type of the patient is obtained using the OptiType algorithm; the potential HLA heterozygous deletion of the patient is predicted using the LOHHLA algorithm; the mutant protein is predicted and disassembled from the somatic mutation using the VEP algorithm, and the potential mutant polypeptide in the ascites P7 subpopulation of the cholangiocarcinoma patient is determined. Further combined with the RNA-seq data of the ascites P7 subpopulation of the cholangiocarcinoma patient, the potential mutant polypeptide derived from WES is analyzed using the GATK ASEReadCounter algorithm, and the mutation number (Mutation Count, MutCount) of the potential mutant polypeptide at the RNA level is determined.

[0063] Finally, the affinity between the mutant polypeptide and the patient HLA is calculated using the NetMHCpan-4.1 algorithm, and the potential neoantigen is predicted using the MutAff ≤ 200 nM or RefAff / MutAff ≥ 10 threshold value after excluding duplicates and HLA heterozygous deletion.

[0064] The above method is used to find potential neoantigens in the malignant ascites of cholangiocarcinoma patients P12, P13 and P15 (Table 1), which include three neoantigen peptides YLQDVETGTQL, YCLSPHLQYI and ITDDLHFYL presented by HLA A*02:01 molecules, neoantigen peptide LTHRRHHCR and ALTHRRHHCR presented by HLA-A*31:01 molecules, and neoantigen peptide SSVSSCGAM presented by HLA-A*03:04 molecules. These neoantigen peptides can be used to prepare tumor vaccines for treating cholangiocarcinoma.

[0065] Table 1 Linear sequence and characteristic parameters of 6 predicted cholangiocarcinoma neoantigens and their paired WT antigens Example 3 Determination of neoantigens of high-frequency HLA-presented tumor cells in malignant ascites of cholangiocarcinoma patients by Tetramer detection method To screen the neoantigens of cholangiocarcinoma patients, the Tetramer staining technique was used for preliminary verification. For the detection of the affinity of neoantigen peptides to HLA molecules, the commercially available MBL QuickSwitch™ Quant Tetramer detection kit is widely used for the detection of antigen peptides of human high-frequency HLA subtypes (such as HLA-A 02:01, HLA-A 11:01 and HLA-A 24:02） antigen peptide detection 。 The QuickSwitch™ Quant Tetramer HLA-A02:01 Kit-APC kit was used to evaluate the affinity of the candidate neoantigen peptides derived from SND1 and APAF1 in Table 1 to HLA-A*02:01 molecules.

[0066] The experiment was performed according to the kit instructions. First, the candidate peptide solution at a concentration of 2 mM and the 1 mM reference peptide solution were warmed to room temperature. 50 μL of Tetramer was dispensed into an EP tube, 1 μL of candidate peptide or reference peptide was added to each tube, respectively, and 1 μL of polypeptide displacement factor was added, mixed and incubated at room temperature for 5 hours in the dark, and then stored at 4°C for standby. Multiple controls were set up, 20 μL of magnetic beads were added to each well of a round-bottom 96-well plate, and the following groups were treated: (1) Well 1 (Control #1) and Well 3 (Control #3): add 5 μL Tetramer; (2) Well 2 (Control #2): add 5 μL 1x Assay Buffer; (3) The remaining wells: add 5 μL peptide-displaced Tetramer. After incubation at 550 rpm with aluminum foil in the dark for 45 minutes, 150 μL of 1x Assay Buffer was added to each well, and the magnetic stand was placed for 5 minutes before the supernatant was discarded, and vortexed for 2 seconds. Except for Well 1, which added 25 μL of 1x Assay Buffer, the remaining wells added 25 μL of freshly prepared 1x working concentration of Exiting Peptide Antibody, and were shaken at 550 rpm in the dark for 55 minutes before repeating the washing step. Finally, each well was resuspended with 200 μL of 1x Assay Buffer, and 200 μL of buffer and 5 μL of magnetic beads were added to Well 4 as a magnetic bead control group. The samples were analyzed by flow cytometry (FCM), and the displacement efficiency of the candidate neoantigen peptides, i.e., their affinity to HLA-A*02:01, was calculated. The results showed that the peptide displacement efficiencies of the candidate neoantigen peptides YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1), and ITDDLHFYL (SND1) to HLA-A*02:01 were 98.26%, 99.42%, and 98.24%, respectively, which were significantly higher than the 75% threshold set by the instructions Figure 3). This indicates that the above four candidate neoantigen peptides have high affinity with HLA-A*02:01 molecules, providing reliable candidate targets for further immunotherapy research.

[0067] To further verify the specificity of the T cell response of the candidate neoantigen of cholangiocarcinoma, the specific Tetramer staining technique was used to detect the frequency of T cells specific to YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1), and ITDDLHFYL (SND1) candidate neoantigen peptides in the malignant ascites and peripheral blood of P15 patients. The experimental operation is as follows: adjust the cells from ascites or peripheral blood to a concentration of 1 x 10 6 ~1x10 7 / mL, take 100 μL of cell suspension, add 10 μL of Clear Back (incubate at room temperature in the dark for 5 minutes to block non-specific binding). Then, add 10 μL of Tetramer with a peptide replacement efficiency of more than 75% per 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 (400 g centrifugation for 5 minutes), resuspend the cells with 500 μL of PBS after discarding the supernatant, add 20 μL of Cell Viability Solution, store at 4°C in the dark, and complete FCM analysis within 24 hours. The results show that in the malignant ascites of P15 cholangiocarcinoma patients, specific T cells that can recognize YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1), and ITDDLHFYL (SND1) antigen peptides can be detected, and the T cell frequency is more than 20% ( Figure 4 and Figure 5 ), which suggests that these antigens may be cholangiocarcinoma neoantigen polypeptides. In the peripheral blood of P15 patients without candidate neoantigen polypeptide stimulation, no specific T cells were detected, but after 13 days of stimulation with a final concentration of 2 μM of neoantigen polypeptide, specific T cells that can recognize YLQDVETGTQL (SND1) and YCLSPHLQYI (AFAP1) neoantigen peptides were detected ( Figure 6 ).

[0068] Example 4 CD39 T cells that react to neoantigens exist in the peripheral blood and malignant ascites of cholangiocarcinoma 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 ascites of patients with cholangiocarcinoma have a response to neoantigens. Figure 7 Compared with the peripheral blood of patients with cholangiocarcinoma, the expression ratio of CD8 + CD39 + T cells in malignant ascites is significantly increased. Figure 7

[0069] In addition, analysis of the TCGA database found that SND1 and APAF1 have gene mutations in cholangiocarcinoma and other cancers (Table 1) 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.

[0070] Example 5: Ascites T cells stimulated by neoantigen peptides have killing function on tumor cells derived from malignant ascites of patients with cholangiocarcinoma 1. Activation and expansion of neoantigen-specific T cells ​First, single nucleated cells from P15 patient ascites were extracted. Then, P15 patient-derived YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1) and ITDDLHFYL (SND1) neoantigen peptides were co-incubated with the single nucleated cells in the patient ascites to activate and expand patient neoantigen-specific T cells, in the following way: 1x10 6 cells per well in a 12-well plate, 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 change every 2-3 days, and T cells were collected at 13 days.

[0071] 2. Tumor cell organoid culture and expansion Tumor cells from P15 patient malignant ascites were isolated and expanded by organoid culture. In the following way: tumor cells from malignant ascites 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 organoids 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 enzymatic digestion, culture medium containing 2% FBS was added to terminate digestion and collect the enzymatic solution. The enzymatic solution was centrifuged in a centrifuge (300 g, 7 min) to collect the tumor cell precipitate.

[0072] 3. T cell killing function evaluation 1x10 6 effector cells (T cells) stimulated by neoantigens and 1x10 5 target cells (tumor cells) were co-incubated for 24 h, while the control group used T cells not stimulated by neoantigens and tumor cells to co-incubate, and the IFN-γ secretion level was detected by ELISA. The results showed that the IFN-γ secretion level of the T cell group stimulated by YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1) and ITDDLHFYL (SND1) neoantigens was significantly higher than that of the control group (p<0.05). Figure 9A). Further, the killing effect of T cells on tumor cells was evaluated by detecting the lactate dehydrogenase (LDH) release level through ELISA. The results showed that the amount of LDH release induced by T cells stimulated by the three kinds of neoantigen peptides was significantly higher than that of the control group (P < 0.05, Fig. 2B). This indicated that the ascites T cells stimulated by the neoantigen peptides could produce killing function on tumor cells derived from malignant ascites of cholangiocarcinoma patients. Figure 9

[0073] 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 cholangiocarcinoma neoantigen polypeptide, characterized in that: The amino acid sequence of the neoantigen polypeptide is shown in SEQ ID NO. 1, 2 or 3.

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

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the cholangiocarcinoma 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 cholangiocarcinoma 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 bile duct 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 cholangiocarcinoma neoantigen polypeptide according to claim 1 and specifically target the cholangiocarcinoma neoantigen polypeptide according to claim 1.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cholangiocarcinoma 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 cholangiocarcinoma 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 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.

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