Hla-restricted neoantigen polypeptides based on malignant ascites tumor cells and uses thereof

Abstract

The present application relates to the technical field of tumor immunotherapy, and particularly relates to HLA-restricted neoantigen polypeptides based on malignant ascites tumor cells and application thereof. The present application provides cholangiocarcinoma neoantigen polypeptides, and the amino acid sequence of the neoantigen polypeptides is shown as SEQ ID NO. 1, 2 or 3. The present application obtains tumor neoantigen polypeptides from cholangiocarcinoma patient malignant ascites through separation and screening, the neoantigen polypeptides have high affinity with HLA molecules, can stimulate tumor-specific T cells, and further generate immune response targeting tumor cells, and can be used as a target for tumor clinical treatment or diagnosis, and has good application prospect in tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, and in particular to HLA-restricted neoantigen peptides based on malignant ascites tumor cells and their applications. Background Technology

[0002] Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignant tumor of the intrahepatic bile duct epithelium, accounting for 8.2% to 15.0% of primary malignant liver tumors. Its epidemiological characteristics show that its incidence has been increasing globally 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-occupyinglesions: A single-center experience with literature review. World J Gastroenterol. 2011, 17(19): 2372-2378.).Although the pathological type is mainly adenocarcinoma, it has high molecular heterogeneity and extremely 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 nature of early symptoms and the lack of specific biomarkers, approximately 70% to 80% of patients are already in the middle or late stages at initial diagnosis, with vascular invasion or distant metastasis, thus losing the opportunity for radical surgery and 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), authoritative guidelines from the National Comprehensive Cancer Network (NCCN), the European Association for the Study of the Liver (EASL), and the Chinese Society of Clinical Oncology (CSCO) all currently recommend gemcitabine plus cisplatin (GC) combined with durvalumab as a first-line treatment regimen (European Association for the Study of the Liver. EASL-ILCAC Clinical Practice Guidelines on the management of intrahepatic cholangiocarcinoma[J]. J Hepatol, 2023, 79(1): 181-208; Chinese Society of Clinical Oncology Guideline Working Committee. Chinese Society of Clinical Oncology (CSCO) Guidelines for the Diagnosis and Treatment of Malignant Tumors of the Biliary Tract 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 advanced ICC patients in recent years, clinical research results show that the objective response rate (ORR) and median overall survival (mOS) of this combination regimen have not yet reached the expected goals, and the overall efficacy remains unsatisfactory. Therefore, it is necessary to find new therapeutic targets.

[0003] Neoantigens are antigens produced in tumor cells due to factors such as DNA mutations and viral infections. They are not present in normal cells and can be presented to the surface of T cells by binding to human leukocyte antigen (HLA). They are then recognized by T cell receptors (TCR) and elicit a specific immune response (Terai M, Sato T. Individualized neoantigen cancer vaccine therapy. Lancet. 2024, 403(10427):590-591.). Studies on the immunological mechanisms of immune checkpoint blockade (ICB) therapy have found that the lack of clinical response to ICB therapy in cancer patients is closely related to the absence of pre-existing neoantigen-specific T cell responses (Yossef R, Krishna S, Sindiri S, Lowery FJ, Copeland AR, Gartner JJ, et al. Phenotypicsignatures 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 cancer patients are the target of ICB therapy and a core driver for achieving clinical benefits from immunotherapy (Puig-Saus C, Sennino B, Peng S, Wang CL, Pan Z, Yuen B, et al. Neoantigen-targeted CD8 T cells). + T cell responses with PD-1 blockade therapy. Nature. 2023, 615(7953):697-704. Therefore, neoantigen-based immunotherapy will be a new breakthrough in the prevention and treatment of cholangiocarcinoma.

[0004] Currently, neoantigens are mainly obtained from surgical tissue samples, but about 70% to 80% of patients are already in the middle or late stages at the time of initial diagnosis, missing the best time for surgical resection. Therefore, it is impossible to obtain the neoantigen profile of patients who are not treated surgically (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.). Summary of the Invention

[0005] This invention provides HLA-restricted neoantigen peptides based on malignant ascites tumor cells and their applications.

[0006] This invention uses malignant ascites from patients with cholangiocarcinoma as a sample. Tumor cells are sorted from the malignant ascites, and mutation status is analyzed by combining high-throughput sequencing. Computer algorithms are used to predict candidate neoantigens. For the predicted candidate neoantigens, the affinity between the candidate neoantigen peptides and HLA molecules and the frequency of neoantigen-specific T cells are analyzed by tetramer staining experiments. Finally, three neoantigen peptides presented by HLA-A*02:01 molecules, namely YLQDVETGTQL (SEQ ID NO.1), ITDDLHFYL (SEQ ID NO.2), and YCLSPHLQYI (SEQ ID NO.3), are obtained.

[0007] Specifically, the present invention provides the following technical solutions.

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

[0009] It should be noted that conserved variant sequences that do not affect the function of the above-mentioned cholangiocarcinoma neoantigen polypeptide generated by mutation are also within the scope of protection of this invention. For example, one or more amino acids in the amino acid sequence of the above-mentioned neoantigen polypeptide can be conservedly replaced, or one or more amino acids that do not affect the function can be added to the N-terminus or C-terminus of the neoantigen polypeptide (e.g., adding a linker peptide, protein tag sequence, etc.).

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

[0011] Furthermore, derivative polypeptides obtained by modifying the amino acid sequence of the aforementioned neoantigen polypeptides one or more times are also within the scope of protection of this invention. These modifications include phosphorylation, PEGylation, amidation, glycosylation, biotinylation, and coupling or fusion with antibodies, carriers, ligands, albumin, Fc fragments, etc.

[0012] Secondly, the present invention provides a polypeptide, wherein the polypeptide is the cholangiocarcinoma neoantigen polypeptide.

[0013] The aforementioned polypeptide may be a polypeptide obtained by adding one or more amino acid residues to the N-terminus and / or C-terminus of the neoantigen polypeptide, which can be cleaved to produce the neoantigen polypeptide.

[0014] The aforementioned polypeptides can be obtained by fusing the tumor neoantigen polypeptide with other functional polypeptides, such as with other known tumor neoantigen polypeptides. Based on conventional polypeptide fusion techniques, those skilled in the art can obtain fusion polypeptides that retain the functional activities of each polypeptide.

[0015] The aforementioned polypeptide can be obtained by fusing the neoantigen polypeptide with an antibody, a carrier, a ligand, albumin, or an Fc fragment.

[0016] Thirdly, the present invention provides a nucleic acid molecule encoding the cholangiocarcinoma neoantigen polypeptide or the polypeptide.

[0017] Based on the amino acid sequence and codon rules of the tumor neoantigen polypeptide provided above, those 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 a single amino acid sequence is not unique, and all nucleic acid molecules capable of encoding the aforementioned tumor neoantigen polypeptide are within the scope of protection of this invention.

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

[0019] Fourthly, the present invention provides a biomaterial, said biomaterial being any of the following:

[0020] (1) An expression cassette containing the nucleic acid molecule;

[0021] (2) A vector containing the nucleic acid molecule or the expression cassette described in (1);

[0022] (3) A cell containing the nucleic acid molecule, the expression cassette in (1) or the vector in (2).

[0023] In (1) above, the expression cassette includes the nucleic acid molecule and transcriptional or translational regulatory elements operatively connected thereto, including but not limited to promoters, terminators, etc.

[0024] In (2) above, the vector includes plasmid vectors, viral vectors, transposons, artificial chromosomes, etc.

[0025] In (3) above, the cells include microbial cells or animal cells. The microbial cells include Escherichia coli, yeast, etc. The animal cells do not have the ability to reproduce into individual animals, including animal cell lines used for polypeptide expression (e.g., CHO cells, HEK293, etc.) or immune cells, etc.

[0026] The neoantigen peptides provided by this invention have a high affinity for HLA molecules and can induce the production of tumor-specific T cells, which can serve as targets for tumor diagnosis, prevention and treatment.

[0027] Fifthly, the present invention provides any one of the following applications of the cholangiocarcinoma neoantigen polypeptide, the polypeptide, the nucleic acid molecule, or the biological material:

[0028] (1) Preparation of antigen-presenting cells;

[0029] (2) Preparation of tumor-specific T cells;

[0030] (3) Preparation of TCR-T or CAR-T cells;

[0031] (4) Preparation of tumor diagnostic reagents;

[0032] (5) Prepare drugs for the prevention or treatment of tumors.

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

[0034] In (2) and (3) above, the application includes: using the neoantigen peptide to stimulate the expansion of the patient's specific T cells or constructing TCR-T cells or CAR-T cells that can recognize the neoantigen peptide. The above-mentioned tumor-specific T cells, TCR-T or CAR-T cells can be used for adoptive immunotherapy.

[0035] In (5) above, the drug includes a vaccine. The vaccine includes DNA vaccines, mRNA vaccines, peptide vaccines, dendritic cell vaccines (DC vaccines), etc.

[0036] In this invention, the tumor is preferably cholangiocarcinoma, and more preferably intrahepatic cholangiocarcinoma.

[0037] In a sixth aspect, the present invention provides antigen-presenting cells induced by the cholangiocarcinoma neoantigen polypeptide and specifically targeting the cholangiocarcinoma neoantigen polypeptide.

[0038] In a seventh aspect, the present invention provides tumor-specific T cells, which are induced by the cholangiocarcinoma neoantigen polypeptide and specifically target the cholangiocarcinoma neoantigen polypeptide.

[0039] Preferably, the tumor-specific T cells include cytotoxic T cells, TCR-T cells, or CAR-T cells.

[0040] Eighthly, the present invention provides a method for preparing tumor-specific T cells, the method comprising: isolating peripheral blood mononuclear cells, co-culturing the cholangiocarcinoma neoantigen polypeptide with the peripheral blood mononuclear cells, and activating and amplifying T cells that specifically target the cholangiocarcinoma neoantigen polypeptide.

[0041] In a ninth aspect, the present invention provides a pharmaceutical composition comprising the cholangiocarcinoma neoantigen polypeptide, the polypeptide, the nucleic acid molecule, or the biological material.

[0042] Preferably, the active ingredient of the pharmaceutical composition includes the above-mentioned tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule, or the biological material.

[0043] Furthermore, the pharmaceutical composition may also contain excipients permitted in the pharmaceutical field.

[0044] In a tenth aspect, the present invention provides a vaccine comprising the cholangiocarcinoma neoantigen polypeptide, the polypeptide, the nucleic acid molecule, or the biological material.

[0045] Preferably, the vaccine includes DNA vaccines, mRNA vaccines, peptide vaccines, dendritic cell vaccines (DC vaccines), etc. Taking an mRNA vaccine as an example, the mRNA vaccine may contain mRNA encoding the tumor neoantigen peptide. Taking a DC vaccine as an example, the DC vaccine can be prepared by loading a peptide with DCs.

[0046] The above-mentioned pharmaceutical composition or vaccine can be used to directly immunotherapize patients with the tumor neoantigen peptide.

[0047] In one aspect, the present invention provides a diagnostic reagent comprising the above-described cholangiocarcinoma neoantigen polypeptide, the polypeptide, the nucleic acid molecule, or the biological material.

[0048] In a twelfth aspect, the present invention provides a method for isolating and screening tumor neoantigen peptides, the method comprising: using malignant ascites from a cancer patient as a sample, and sorting tumor cells from the sample by flow cytometry;

[0049] The tumor cells were subjected to DNA and RNA sequencing, and tumor neoantigen peptides were screened based on the sequencing data.

[0050] The tumor cells are 7-AAD. - Hoechest 33342 + CD45 - EpCAM + Cell subpopulations.

[0051] Malignant ascites is an important component of liquid biopsy samples from cancer patients, and it is frequently present in patients with malignant tumors (such as those with intracranial carcinoma). This invention discovers that tumor cells in malignant ascites can serve as a source for the isolation of tumor neoantigen peptides, specifically 7-AAD from malignant ascites. - Hoechest 33342 + CD45 - EpCAM + Cell subpopulations were identified, and high-throughput sequencing and mutation analysis were performed on these subpopulations to screen for tumor neoantigen peptides. This method, based on liquid biopsy technology, can provide neoantigens for patients with inoperable abdominal tumors, offering novel neoantigen sequences for neoantigen-based vaccine development, ultimately for the treatment of cancer patients.

[0052] In the above method, the tumor cells are 7-AAD. - Hoechest 33342 + CD45 - EpCAM + A subpopulation of cells with fewer than 2000 cells.

[0053] In the above methods, the DNA sequencing is whole exome sequencing, and the RNA sequencing is transcriptome sequencing;

[0054] The screening of tumor neoantigen peptides 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 loss of HLA heterozygosity; predicting mutant proteins from somatic mutations; and identifying potential mutant peptides in the tumor cells.

[0055] Furthermore, based on transcriptome sequencing data, the potential mutant peptides are analyzed to determine the number of mutations in the potential mutant peptides at the RNA level.

[0056] Assess the affinity between mutant peptides and patient HLA, and screen for potential tumor neoantigen peptides after excluding duplication and loss of HLA heterozygosity.

[0057] The above-mentioned identification of mutations carried by the tumor cells based on whole-exome sequencing data includes: identifying (preferably using GATK Mutect2) mutations carried by tumor cells based on WES data of peripheral blood mononuclear cells (PBMCs) from patients, with mutations TLOD ≥ 10 being preserved;

[0058] The OptiType algorithm is preferred for obtaining the patient's HLA type.

[0059] The LOHHLA algorithm is preferred for predicting potential HLA heterozygosity loss in patients.

[0060] The above-mentioned prediction of mutant proteins from somatic mutations and identification of potential mutant peptides in tumor cells includes: using the VEP algorithm to predict and disassemble mutant proteins from somatic mutations and identify potential mutant peptides in tumor cells.

[0061] The above analysis of the potential mutant peptides based on transcriptome sequencing data, and the determination of the number of mutations in the potential mutant peptides at the RNA level, preferably uses the GATK ASEReadCounter algorithm.

[0062] In the above method, the tumor is preferably cholangiocarcinoma, and more preferably intrahepatic cholangiocarcinoma.

[0063] The beneficial effects of the present invention include at least the following: The present invention isolates and screens tumor neoantigen polypeptides from malignant ascites of patients with cholangiocarcinoma. These neoantigen polypeptides have a high affinity for HLA molecules and can stimulate the production of tumor-specific T cells, thereby generating an immune response targeting tumor cells. They can be used as targets for clinical treatment or diagnosis of tumors and have good application prospects in tumor immunotherapy.

[0064] The method for isolating and screening tumor neoantigen peptides provided by this invention solves the problem that existing methods cannot obtain neoantigens from patients undergoing non-surgical treatment, and provides an effective method for the development of tumor neoantigen peptides. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0066] Figure 1 and Figure 2 EpCAM was present in the malignant ascites of a patient with cholangiocarcinoma in Example 1 of this invention. + Tumor cells; among them, Figure 1 To detect chromosomal abnormalities in tumor cells in malignant ascites using the SE-i·FISH method; Figure 2 Flow cytometry plots of tumor cells and statistical analysis of the P7 subset in malignant ascites samples; *** P <0.001.

[0067] Figure 3 , Figure 4 , Figure 5 and Figure 6 This refers to the neoantigens of tumor cells in malignant ascites samples from patients with cholangiocarcinoma screened using specific Tetramer in Example 3 of the present invention. Figure 3 To detect the affinity between candidate neoantigen peptides and HLA molecules using the QuickSwitch™ Quant Tetramer assay kit; Figure 4 and Figure 5 To detect the frequency of neoantigen-specific T cells in malignant ascites of P15 cholangiocarcinoma patients using the QuickSwitch™ Quant Tetramer assay kit; Figure 6 To detect the frequency of neoantigen-specific T cells in peripheral blood and malignant ascites of P15 cholangiocarcinoma patients using the QuickSwitch™ Quant Tetramer assay kit; APAF1-M represents the neoantigen peptide YCLSPHLQYI, SND1-M-1 represents the neoantigen peptide YLQDVETGTQL, and SND1-M-2 represents the neoantigen peptide ITDDLHFYL;* P <0.05,** P <0.01, *** P <0.001, **** P <0.0001.

[0068] Figure 7 and Figure 8 In Example 4 of this invention, CD39, which is reactive to neoantigens, was found in fluid biopsy samples such as peripheral blood and malignant ascites from patients with cholangiocarcinoma.+ T cells; among them, Figure 7 To use FCM to detect and statistically analyze the expression level of CD39 on T cells in peripheral blood and malignant ascites of patients with cholangiocarcinoma; Figure 8 To analyze the gene mutation status of SND1 and APAF1 in different cancer types in the TCGA database; where ACC represents adrenocortical carcinoma, BLCA represents urothelial carcinoma of the bladder, BRCA represents breast cancer, BRCA-Basal represents Basal-like breast cancer, BRCA-Her2 represents Her2-like breast cancer, BRCA-LumA represents LumA-like breast cancer, CESC represents cervical squamous cell carcinoma and adenocarcinoma, CHOL represents cholangiocarcinoma, COAD represents colon cancer, ESCA represents esophageal cancer, GBM represents glioblastoma, HNSC represents head and neck squamous cell carcinoma, HNSC-HPV- represents head and neck squamous cell carcinoma originating from HPV-negative, HNSC-HPV+ represents head and neck squamous cell carcinoma originating from HPV-positive, KIRC represents clear cell renal carcinoma, KIRP represents papillary renal 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 PAAD represents ovarian cancer, PAAD represents pancreatic cancer, READ represents rectal adenocarcinoma, SARC represents sarcoma, SKCM represents cutaneous melanoma, STAD represents gastric cancer, THCA represents thyroid cancer, THYM represents thymic carcinoma, UCEC represents endometrial cancer, and UCS represents uterine sarcoma. P <0.01.

[0069] Figure 9 The detection of IFN-γ secreted by neoantigen-stimulated T cells after co-incubation with tumor cells in Example 5 of this invention (A) and the detection of cytotoxicity of neoantigen-stimulated T cells killing tumor cells (B); *** P <0.001; where APAF1-M represents the neoantigen polypeptide YCLSPHLQYI, SND1-M-1 represents the neoantigen polypeptide YLQDVETGTQL, and SND1-M-2 represents the neoantigen polypeptide ITDDLHFYL. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0071] The malignant ascites samples from patients with cholangiocarcinoma used in the following examples were collected and used with the approval of the Ethics Committee of Peking University People's Hospital, and the patients or their families were informed and signed informed consent forms.

[0072] Example 1: Obtaining tumor cells from malignant ascites in patients with cholangiocarcinoma using flow cytometry

[0073] The prediction of neoantigens primarily relies on validation at the tumor cell DNA mutation and RNA levels. In the tumor tissue of cholangiocarcinoma patients, tumor cells typically constitute a high proportion, allowing for direct acquisition of neoantigens through DNA and RNA extraction. However, cholangiocarcinoma is often diagnosed at an intermediate or advanced stage, frequently missing the opportunity for surgical resection, making it difficult to obtain a neoantigen profile through tissue biopsy in non-surgical patients. Malignant ascites, a common complication in advanced cholangiocarcinoma patients, suggests the potential presence of tumor cells, providing a potential source for neoantigen profile acquisition. However, white blood cells often constitute over 90% of malignant ascites from cholangiocarcinoma, making it difficult to effectively obtain tumor-specific gene mutation profiles through next-generation sequencing by directly extracting DNA and RNA from all cells in the ascites, thus hindering the generation of a neoantigen profile. Therefore, flow cytometry (FCM) is necessary to sort viable tumor cells without damaging their DNA and RNA integrity.

[0074] Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein responsible for mediating calcium-independent adhesion in epithelial cells. 2+It is a homologous cell adhesion. It is specifically expressed in epithelial tissues and tumors of epithelial origin, and is therefore considered a potential marker of circulating tumor cells (CTCs). Under normal circumstances, cells expressing EpCAM are virtually non-existent in body fluids, so its detection in ascites usually indicates the presence of metastatic 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 a tumor derived from epithelial cells, highly expresses EpCAM in its tissues (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, combined with other specific markers, to effectively isolate tumor cells from ascites in patients with cholangiocarcinoma. Furthermore, studies have shown that various 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.). This invention utilizes differential phase enrichment-multiplex tumor marker immunofluorescence staining-chromosome fluorescence in situ hybridization (SE-i·FISH) to detect liquid biopsy samples, and found that chromosomally abnormal tumor cells were present in the malignant ascites of patients with advanced cholangiocarcinoma. In addition to expressing CEP8, these tumors also expressed tumor markers such as EpCAM, CEA, CA199, and CA125. Figure 1However, tumor markers such as CEA, CA199, and CA125 are mainly expressed intracellularly, which is not conducive to the sorting of viable tumor cells from malignant ascites. Therefore, this invention utilizes FCM technology to obtain viable (7-AAD) tumor cells from malignant ascites of patients with cholangiocarcinoma. - ), containing a cell nucleus (Hoechest 33342) + ) and eliminate white blood cells (CD45) - EpCAM tumor cells + This is used to obtain the DNA and RNA of tumor cells, and then further obtains the neoantigen profile through next-generation sequencing. 7-AAD in liquid biopsy samples is analyzed using FCM. - Hoechest 33342 + CD45 - EpCAM + The number of cell subsets (hereinafter referred to as P7 subsets) was analyzed, and it was found that the number of P7 subsets in the ascites of patients with cholangiocarcinoma was significantly higher than that in the ascites of patients with cirrhosis. P <0.05)( Figure 2 Therefore, the P7 subset can be obtained through FCM, and thus tumor cells can be obtained.

[0075] Based on the above experimental results, a method for separating tumor cells from malignant ascites by flow cytometry (FCM) was constructed, including: separating the P7 cell tumor subpopulation from malignant ascites of patients with cholangiocarcinoma using a BD FACSAria II flow cytometer.

[0076] Example 2: Identifying Potential Neoantigens in Tumor Cells from Malignant Ascites of Cholangiocarcinoma Patients Based on Computer Algorithms

[0077] First, 7-AAD was isolated from malignant ascites in patients with cholangiocarcinoma using a BD FACSAria II flow cytometer. - Hoechest 33342 + CD45 - EpCAM + Cell subpopulations (P7 subpopulation): RNA and DNA were extracted and amplified from P7 populations with fewer than 2000 cells using Smart-seq 2 technology and Discover-sc Single Cell. Subsequently, RNA and DNA were sequenced using the Illumina novaseq 6000 platform for transcriptome sequencing (RNA-sequencing, RNA-seq) and whole-exome sequencing (WES), respectively.

[0078] WES data of the aforementioned P7 subpopulation in malignant ascites were compared with WES data of peripheral blood mononuclear cells (PBMCs) from patients. GATK Mutect2 was used to identify mutations in the P7 subpopulation of patient ascites, with mutations TLOD ≥ 10 preserved. The OptiType algorithm was used to obtain the patients' HLA type. The LOHHLA algorithm was used to predict potential HLA loss of heterozygosity in patients. The VEP algorithm was used to predict and disassemble mutant proteins from somatic mutations, identifying potential mutant peptides in the P7 subpopulation of ascites from cholangiocarcinoma patients. Further, combined with RNA-seq data of the P7 subpopulation of ascites from cholangiocarcinoma patients, the GATK ASEReadCounter algorithm was used to analyze potential mutant peptides from WES, determining the mutation count (MutCount) of potential mutant peptides at the RNA level.

[0079] Finally, the affinity between the mutant peptide and patient HLA was calculated using the NetMHCpan-4.1 algorithm. After excluding duplication and loss of HLA heterozygosity, potential neoantigens were predicted using thresholds of MutAff ≤ 200 nM or RefAff / MutAff ≥ 10.

[0080] Using the methods described above, potential neoantigens were identified in the malignant ascites of patients with P12, P13, and P15 cholangiocarcinoma (Table 1). These included three neoantigen peptides presented by HLA-A*02:01 molecules: YLQDVETGTQL, YCLSPHLQYI, and ITDDLHFYL; two neoantigen peptides presented by HLA-A*31:01 molecules: LTHRRHHCR and ALTHRRHHCR; and one neoantigen peptide presented by HLA-A*03:04 molecules: SSVSSCGAM. These neoantigen peptides could be used to prepare tumor vaccines for the treatment of cholangiocarcinoma.

[0081] Table 1. Linear sequences and characteristic parameters of six neoantigens predicting cholangiocarcinoma and their paired WT antigens.

[0082]

[0083] Example 3: Identification of neoantigens of high-frequency HLA-presented tumor cells in malignant ascites of patients with cholangiocarcinoma using the Tetramer assay.

[0084] To screen for neoantigens in patients with cholangiocarcinoma, Tetramer staining was used for preliminary validation. For detecting the affinity of neoantigen peptides for HLA molecules, the commercially available MBL QuickSwitch™ Quant Tetramer assay kit is widely used for detecting high-frequency human HLA subtypes (such as HLA-A). 02:01, HLA-A 11:01 and HLA-A 24:02） antigen peptide detection 。 The affinity of candidate neoantigen peptides derived from SND1 and APAF1 in Table 1 to the HLA-A*02:01 molecule was evaluated using the QuickSwitch™ Quant Tetramer HLA-A02:01 Kit-APC.

[0085] The experiment was conducted according to the kit instructions. First, the 2 mM candidate peptide solution and the 1 mM reference peptide solution were warmed to room temperature. 50 μL of Tetramer was dispensed into EP tubes, and 1 μL of candidate peptide or reference peptide and 1 μL of peptide replacement factor were added to each tube. After mixing, the tubes were incubated at room temperature in the dark for 5 hours, and then stored at 4°C for later use. Multiple control groups were set up in the experiment. 20 μL of magnetic beads were added to each well of a 96-well round-bottom plate and treated as follows: (1) Well 1 (Control #1) and Well 3 (Control #3): 5 μL of Tetramer was added; (2) Well 2 (Control #2): 5 μL of 1×Assay Buffer was added; (3) The remaining wells: 5 μL of peptide-replaced Tetramer was added. After shaking the samples at 550 rpm and incubating them in the dark with aluminum foil for 45 minutes, 150 μL of 1×Assay Buffer was added to each well. After standing on a magnetic rack for 5 minutes, the supernatant was discarded, and the samples were vortexed for 2 seconds. Except for well 1, which contained 25 μL of 1×Assay Buffer, all other wells contained 25 μL of freshly prepared 1× working concentration Exiting Peptide Antibody. The mixture was shaken at 550 rpm in the dark for 55 minutes, and the washing step was repeated. Finally, each well was resuspended with 200 μL of 1×Assay Buffer, and well 4 contained 200 μL of buffer and 5 μL of magnetic beads as a control group. Samples were analyzed by flow cytometry (FCM) to calculate the displacement efficiency of candidate neoantigen peptides, i.e., their affinity for HLA-A*02:01. The results showed that the peptide displacement efficiencies of candidate neoantigen peptides YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1), and ITDDLHFYL (SND1) with HLA-A*02:01 were 98.26%, 99.42%, and 98.24%, respectively, all significantly higher than the 75% threshold set in the manufacturer's instructions. Figure 3This indicates that the four candidate neoantigen peptides mentioned above have a high affinity for the HLA-A*02:01 molecule, providing reliable candidate targets for further immunotherapy research.

[0086] To further validate the specific T-cell response to candidate neoantigens in cholangiocarcinoma, specific Tetramer staining was used to detect the specific T-cell frequencies targeting candidate neoantigen peptides YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1), and ITDDLHFYL (SND1) in malignant ascites and peripheral blood from P15 patients. The experimental procedure was as follows: cells from ascites or peripheral blood were adjusted to 1×102 6 ~1×10 7 / mL concentration, take 100 μL of cell suspension, add 10 μL 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 greater than 75% to each tube, incubate at room temperature for 30 minutes, and then add CD8 antibody (incubate at 4℃ for 20 minutes). Wash cells with PBS (centrifuge at 400 g for 5 minutes), discard the supernatant, resuspend cells in 500 μL PBS, add 20 μL Cell Viability Solution, store at 4℃ in the dark, and complete FCM analysis within 24 hours. The results showed that specific T cells that could recognize the antigenic peptides YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1), and ITDDLHFYL (SND1) could be detected in the malignant ascites of P15 cholangiocarcinoma patients, with a T cell frequency of more than 20% ( Figure 4 and Figure 5 This suggests that these antigens may be neoantigen peptides for cholangiocarcinoma. In P15 patients, no specific T cells were detected in peripheral blood without stimulation by the candidate neoantigen peptides, but after stimulation with a final concentration of 2 μM neoantigen peptides for 13 days, specific T cells recognizing the neoantigen peptides YLQDVETGTQL (SND1) and YCLSPHLQYI (AFAP1) could be detected. Figure 6 ).

[0087] Example 4: CD39, which is responsive to neoantigens, was found in the peripheral blood and malignant ascites of patients with cholangiocarcinoma. + T cells

[0088] 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 research (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 all found that: CD39 + T cells may have a better ability to recognize neoantigens, and dominant neoantigens can activate CD39. + T cells exert their anti-tumor effects. Further research revealed that CD8+ cells, which respond to neoantigens, are present in the peripheral blood and malignant ascites of patients with cholangiocarcinoma. + CD39 + T cells ( Figure 7 Compared with peripheral blood from patients with cholangiocarcinoma, CD8+ in malignant ascites... + CD39 + The proportion of T cells expressed was significantly increased. Figure 7 ).

[0089] Furthermore, analysis of the TCGA database revealed that SND1 and APAF1 are not only present in cholangiocarcinoma, but also in other types of cancer. Figure 8 This suggests that the neoantigen peptides discovered in this invention could be used as tumor vaccines to treat tumors in the future.

[0090] Example 5: Ascites T cells stimulated by neoantigen peptides have cytotoxic effects against tumor cells derived from malignant ascites in patients with cholangiocarcinoma.

[0091] 1. Activation and expansion of neoantigen-specific T cells

[0092] First, mononuclear cells were extracted from the ascites fluid of P15 patients. Then, YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1), and ITDDLHFYL (SND1) neoantigen peptides derived from P15 patients were co-incubated with the mononuclear cells from the patient's ascites fluid to activate and expand the patient's neoantigen-specific T cells. The specific method is as follows: cells were cultured in 12-well plates, with 1 × 10⁶ cells per well. 6 The culture medium consisted of 1 mL of X-VIVO 15 + 5% human AB serum + 1% penicillin / streptomycin + 100 IU / mL IL-2 + 4 μM polypeptide. Half of the medium was changed every 2-3 days, and T cells were collected after 13 days of culture.

[0093] 2. Organoid culture and expansion of tumor cells

[0094] Tumor cells were isolated from malignant ascites in P15 patients and expanded using organoid culture. The specific method was as follows: tumor cells from the malignant ascites were mixed with a culture medium (containing insulin-like growth factor-2 at a concentration of 8 ng / mL) to achieve a tumor cell concentration of 2 × 10⁻⁶. 4 = / mL, then add 5% matrix gel and mix well on ice to obtain the culture. The culture was seeded into 24-well low-absorption plates and incubated at 37°C for 30 min, then 200 μL of culture medium was added to each well. During the culture, 150 μL of culture medium was added to each well every three days. When the organoid diameter reached 200-500 μm, the original culture medium was aspirated, and TrypLEExpress was added to each well of the 24-well plate. After 1 min of enzymatic digestion, culture medium containing 2% FBS was added to stop the digestion and the enzymatic digest was collected. The enzymatic digest was centrifuged (300 g, 7 min) and the tumor cell pellet was collected.

[0095] 3. Assessment of T cell killing function

[0096] 1×10 stimulatory neoantigens 6 1 effector cells (T cells) and 1×10 5 T cells (tumor cells) were co-incubated with target cells for 24 h, while the control group was co-incubated with unstimulated T cells and tumor cells. IFN-γ secretion levels were detected by ELISA. Results showed that the IFN-γ secretion level of T cells stimulated with the neoantigens YCLSPHLQYI (APAF1), YLQDVETGTQL (SND1), and ITDDLHFYL (SND1) was significantly higher than that of the control group. Figure 9(A). The release level of lactate dehydrogenase (LDH) was further detected by ELISA to assess the killing effect of T cells on tumor cells. The results showed that the LDH release induced by T cells stimulated with the above three neoantigen peptides was significantly higher than that in the control group (A). Figure 9 (B). This indicates that ascites T cells stimulated by neoantigen peptides can kill tumor cells derived from malignant ascites in patients with cholangiocarcinoma.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

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. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the cholangiocarcinoma 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 cholangiocarcinoma neoantigen polypeptide of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of cholangiocarcinoma-specific T cells.

5. The use of the cholangiocarcinoma neoantigen polypeptide of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of TCR-T cells targeting cholangiocarcinoma.

6. The use of the cholangiocarcinoma neoantigen polypeptide of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of a medicament for treating cholangiocarcinoma.

7. Cholangiocarcinoma-specific T cells, characterized in that, The cholangiocarcinoma-specific T cells are obtained by stimulation with the cholangiocarcinoma neoantigen polypeptide of claim 1, and specifically recognize the cholangiocarcinoma neoantigen polypeptide of claim 1.

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

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

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