Hla-a*11:01-restricted neoantigen peptides based on malignant pleural effusion tumor cells and applications thereof

Abstract

The present application relates to the technical field of tumor immunotherapy, and particularly relates to HLA-A*11:01 restricted neoantigen peptides based on malignant pleural effusion tumor cells and application thereof. The present application obtains tumor neoantigen polypeptides by separating and screening from malignant pleural effusion of lung cancer patients, the amino acid sequence of the neoantigen polypeptides is shown in any one of SEQ ID NO. 1-9; the tumor neoantigen polypeptides have high affinity with HLA molecules, can stimulate tumor-specific T cells, and can be used as a target for tumor clinical treatment or diagnosis. The present application further provides a method for separating and screening tumor neoantigen polypeptides, which solves the problem that the neoantigen of non-surgical treatment patients cannot be obtained, and provides an effective method for development of tumor neoantigen polypeptides.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, and in particular to HLA-A*11:01 restricted neoantigen peptides based on malignant pleural effusion tumor cells and their applications. Background Technology

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

[0003] Tumor neoantigens are antigens produced in tumor cells due to factors such as DNA mutations and viral infections. These antigens are completely absent in normal cells and can be bound to the human major histocompatibility complex (HLA), presented to T cells, recognized by the TCR, and thus elicit an immune response. Currently, several research teams are using tumor neoantigens to design personalized tumor vaccines, demonstrating good safety and excellent efficacy in phase I clinical trials for patients with melanoma, refractory glioma, liver cancer, and pancreatic cancer (Terai M, Sato T. Individualized neoantigen cancer vaccine therapy. Lancet. 2024, 403(10427):590-591.). Therefore, the development of tumor neoantigen peptides is of great significance for tumor immunotherapy. Summary of the Invention

[0004] This invention provides an HLA-A*11:01-restricted neoantigen peptide based on malignant pleural effusion tumor cells and its application.

[0005] This invention uses malignant pleural effusion from lung cancer patients as a sample, sorts out tumor cells, performs high-throughput sequencing analysis on the isolated tumor cells to identify mutations, uses computer algorithms to predict candidate neoantigens, and analyzes the affinity between the predicted candidate neoantigen peptides and HLA molecules, as well as the frequency of neoantigen-specific T cells, through tetramer staining experiments, to finally obtain tumor neoantigen peptides.

[0006] Specifically, the present invention provides the following technical solutions.

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

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

[0009] Conserved variant sequences that do not affect the function of the aforementioned neoantigen peptides are also within the scope of protection of this invention. For example, one or more amino acids in the amino acid sequence of the aforementioned neoantigen peptides may be conservatively replaced, or one or more amino acids that do not affect the function may be added to the N-terminus or C-terminus of the neoantigen peptides (e.g., adding linker peptides, protein tag sequences, 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] The aforementioned tumor neoantigen peptides have a high affinity for the HLA-A*11:01 molecule and can induce the production of tumor-specific T cells, thus serving as targets for tumor diagnosis, prevention, and treatment.

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

[0014] Preferably, the polypeptide may be a polypeptide that can produce the neoantigen polypeptide by adding one or more amino acid residues to the N-terminus and / or C-terminus of the neoantigen polypeptide;

[0015] Alternatively, the polypeptide 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 activity of each polypeptide.

[0016] Alternatively, the polypeptide can be obtained by fusing the neoantigen polypeptide with an antibody, a carrier, a ligand, albumin, or an Fc fragment.

[0017] Thirdly, the present invention provides a nucleic acid molecule that encodes the tumor neoantigen polypeptide or the polypeptide.

[0018] 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.

[0019] In this invention, the nucleic acid molecule includes DNA or RNA. The RNA includes mRNA.

[0020] Fourthly, the present invention provides biomaterials, said biomaterials comprising any one of the following:

[0021] (1) An expression cassette containing the nucleic acid molecule;

[0022] (2) A vector containing the nucleic acid molecule or the expression cassette described in (1);

[0023] (3) A cell containing the nucleic acid molecule, the expression cassette in (1) or the vector in (2).

[0024] 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.

[0025] In (2) above, the vector includes plasmid vectors, viral vectors, transposons, artificial chromosomes, etc.

[0026] 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.

[0027] Fifthly, the present invention provides any one of the following applications of the tumor 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 lung cancer.

[0037] In a sixth aspect, the present invention provides antigen-presenting cells or tumor-specific T cells, wherein the antigen-presenting cells or tumor-specific T cells are induced by the tumor neoantigen polypeptide and specifically target the tumor neoantigen polypeptide.

[0038] Preferably, the tumor-specific T cells include cytotoxic T cells, TCR-T cells, or CAR-T cells.

[0039] In a seventh aspect, the present invention provides a method for preparing tumor-specific T cells, the method comprising: separating 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.

[0040] Eighthly, the present invention provides a pharmaceutical composition comprising the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule, or the biological material.

[0041] 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.

[0042] Furthermore, the pharmaceutical composition may also contain excipients permitted in the pharmaceutical field.

[0043] In a ninth aspect, the present invention provides a vaccine comprising the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule, or the biological material.

[0044] 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.

[0045] The above-mentioned pharmaceutical composition or vaccine can be used to directly immunotherapize patients with the tumor neoantigen peptide.

[0046] In a tenth aspect, the present invention provides a diagnostic reagent comprising the tumor neoantigen polypeptide, the polypeptide, the nucleic acid molecule, or the biological material described above.

[0047] Eleventhly, the present invention provides a method for isolating and screening tumor neoantigen peptides, the method comprising: using malignant pleural effusion of a cancer patient as a sample, and sorting tumor cells from the sample by flow cytometry;

[0048] The tumor cells were subjected to DNA and RNA sequencing, and tumor neoantigen peptides were screened based on the sequencing data.

[0049] The tumor cells were 7-AAD-Hoechest 33342. + CD45-EpCAM + Cell subpopulations.

[0050] Malignant pleural effusion is an important component of liquid biopsy samples from cancer patients. This invention discovers that tumor cells in malignant pleural effusion can serve as a source for isolating tumor neoantigen peptides, through the isolation of 7-AAD from malignant pleural effusion. - 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 thoracic tumors, offer novel neoantigen sequences for neoantigen-based vaccine development, and ultimately be used to treat cancer patients.

[0051] In some specific embodiments of the present invention, by obtaining the neoantigen spectrum of patients with advanced lung cancer from malignant pleural effusion, tumor neoantigens with application potential are screened to provide new treatment options for patients with advanced lung cancer.

[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 lung cancer.

[0063] The beneficial effects of this invention include at least the following: Tumor neoantigen peptides were isolated and screened from malignant pleural effusions of lung cancer patients. These tumor neoantigen peptides have a high affinity for HLA molecules, can stimulate the production of tumor-specific T cells, and thus generate an immune response targeting tumor cells. They can serve as targets for clinical treatment or diagnosis of tumors and have promising applications in tumor immunotherapy. This invention also provides a method for the isolation and screening of tumor neoantigen peptides, which solves the problem of not being able to obtain neoantigens from patients undergoing non-surgical treatment, providing an effective method for the development of tumor neoantigen peptides. Attached Figure Description

[0064] 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.

[0065] Figure 1 and Figure 2 EpCAM was present in the malignant pleural effusion of a lung cancer patient in Example 1 of this invention. + Tumor cells; among them, Figure 1 To detect chromosomal abnormalities in tumor cells in malignant pleural effusion using the SE-i·FISH method; Figure 2 Flow cytometry plots of tumor cells in malignant pleural effusion samples and flow cytometry statistics of the P7 subset; ***P<0.001.

[0066] Figure 3 , Figure 4 , Figure 5 and Figure 6 In Example 3 of this invention, neoantigens of tumor cells in malignant pleural effusion samples from lung cancer patients are screened using specific Tetramer; wherein, Figure 3 To detect the affinity between candidate neoantigen peptides derived from P19 and P23 patients 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 pleural effusions of P19 and P23 lung cancer patients using the QuickSwitch™ Quant Tetramer assay kit; Figure 6To detect the frequency of neoantigen-specific T cells in peripheral blood and malignant pleural effusion of P19 (top image) and P23 (bottom image) lung cancer patients using the QuickSwitch™ QuantTetramer assay kit; where HSPA8-M-1 represents AGSTRIPK, HSPA8-M-2 represents LVAGSTRIPK, CCDC89-M-1 represents SSLFSQAVK, CCDC89-M-2 represents NSSLFSQAVK, FAM173B-M-1 represents LSFVPATTK, FAM173B-M-2 represents CLSFVPATTK, VKORC1-M represents SSSSRVFSSR, PIGO-M-2 represents VALLQLGLK, and HSP90B1-M represents YSNRTRLAK; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0067] Figure 7 In Example 4 of this invention, FCM was used to detect and statistically analyze the expression level of CD39 on T cells in peripheral blood and malignant pleural effusion of lung cancer patients; **P<0.01.

[0068] Figure 8 , Figure 9 and Figure 10 In Example 4 of this invention, the gene mutation status of HSPA8, CCDC89, FAM173B, VKORC1, PIGO, and HSP90B1 in different cancer types was analyzed in the TCGA database. Wherein, 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 individuals, and HNSC-HPV+ represents... The following are cancer cell carcinomas: KICH (for head and neck squamous cell carcinoma originating from HPV-positive cells), KIRC (for kidney chromophobe carcinoma), KIRP (for kidney papillary cell carcinoma), LGG (for low-grade glioma of the brain), LIHC (for hepatocellular carcinoma), LUAD (for lung adenocarcinoma), LUSC (for lung squamous cell carcinoma), OV (for ovarian cancer), PAAD (for pancreatic cancer), READ (for rectal adenocarcinoma), SKCM (for cutaneous melanoma), STAD (for gastric cancer), TGCT (for testicular cancer), THCA (for thyroid cancer), UCEC (for endometrial cancer), UCS (for uterine sarcoma), and UVM (for ocular melanoma).

[0069] Figure 11 The results are as follows: (A) Detection of IFN-γ secreted by neoantigen-stimulated T cells after co-incubation with tumor cells in Example 5 of this invention, and (B) Detection of cytotoxicity of neoantigen-stimulated T cells killing tumor cells; *P<0.05, **P<0.01, ***P<0.001. 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 pleural effusions from lung cancer patients 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 pleural effusion of lung cancer patients using flow cytometry

[0073] Neoantigen prediction relies heavily on a comprehensive analysis of tumor cell DNA mutations and RNA expression levels. In lung cancer patients, the high proportion of tumor cells in tumor tissue allows for direct extraction of DNA and RNA for neoantigen prediction. However, patients with advanced lung cancer often lose the opportunity for surgery, making it difficult to obtain a neoantigen profile through traditional tissue biopsies. Malignant pleural effusion, a common complication of advanced lung cancer, provides an alternative source of neoantigen profiles due to its tumor cells. Notably, white blood cells often account for over 90% of malignant pleural effusions from lung cancer. Direct extraction of total cellular DNA and RNA for next-generation sequencing results in a low proportion of tumor cells, hindering accurate acquisition of tumor-specific mutation profiles and impacting the reliability of neoantigen prediction. Therefore, establishing a technique capable of specifically enriching live tumor cells is crucial.

[0074] Epithelial cell adhesion molecule (EpCAM) is a tumor marker with important clinical value. As a transmembrane glycoprotein, EpCAM mediates calcium channel blockade. 2+EpCAM-positive cells exhibit non-isomorphic cell adhesion and are specifically expressed in epithelial tissues and tumors of their origin. In normal body fluids, EpCAM-positive cells are almost non-existent; therefore, their detection in malignant pleural effusions has clear diagnostic value for tumors (Zhang X, Wang X, Wen Y, Chen S, Zhou C, Wu F. Single-cell transcriptomics reveal metastatic CLDN4). + Cancer cells underlying the occurrence of malignant pleural effusion in patients with advanced non-small-cell lung cancer. Clin Transl Med. 2024, 14(4):e1649.). Multiple studies have confirmed that EpCAM is widely expressed in lung cancer tissues (Rehulkova A, Chudacek J, Prokopova A, Vidlarova M, Stranska J, Drabek J, et al. Clinical and prognostic significance of detecting CEA , EGFR , LunX , c-met and EpCAMmRNA-positive cells in the peripheral blood, tumor-draining blood and bone marrow of non-small cell lung cancer patients. TranslLung Cancer Res. 2023, 12(5):1034-1050.), which provides a theoretical basis for its use as a lung cancer cell sorting marker. In addition, chromosomal abnormalities are another important feature of tumor cells. For example, abnormalities of chromosome 8 are common in a variety of solid tumors (Cheng H, Wang S, Luan W, Ye X, Dou S, Tang Z, et al. Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH inovarian cancer. Chin J Cancer Res. 2021, 33(2):256-270.). This invention uses differential phase enrichment-multiplex tumor marker immunofluorescence staining-chromosome fluorescence in situ hybridization (SE-i·FISH) to detect tumor cells that simultaneously express markers such as EpCAM, CEA, CA199 and CA125 and are accompanied by chromosomal abnormalities in the malignant pleural effusion of patients with advanced lung cancer. Figure 1 Considering that markers such as CEA, CA199, and CA125 are mainly located intracellularly, which is not conducive to live cell sorting, EpCAM is more technically feasible as a surface marker.

[0075] This invention establishes an optimized flow cytometry (FCM) sorting strategy: through multi-parameter gating, it specifically sorts cells containing 7-AAD. - ), containing an intact nucleus (Hoechest 33342) + ), eliminate white blood cells (CD45) - Tumor cells expressing EpCAM (EpCAM) + This method, specifically the P7 subset, achieves highly efficient enrichment of rare tumor cells while maintaining cell integrity. Validation experiments showed that the number of P7 subsets in malignant pleural effusions from lung cancer patients was significantly higher than that in pleural effusions from non-tumor patients. P <0.001)( Figure 2 The specificity of this method was confirmed. The P7 subpopulation cells obtained by BD FACSAria II flow cytometry had DNA and RNA quality that met the requirements of next-generation sequencing, providing a reliable technical guarantee for the accurate prediction of subsequent neoantigen profiles.

[0076] Example 2: Identification of potential neoantigens in tumor cells from malignant pleural effusions of lung cancer patients using computer algorithms.

[0077] First, 7-AAD was isolated from malignant pleural effusions of lung cancer patients 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 P7 population in the malignant pleural effusion was compared with WES data of peripheral blood mononuclear cells (PBMCs) from the patients. GATK Mutect2 was used to identify mutations in the P7 subset of the pleural effusion, with mutations TLOD ≥ 10 preserved. The OptiType algorithm was used to obtain the patients' HLA type. The LOHHLA algorithm was used to predict potential loss of HLA heterozygosity. The VEP algorithm was used to predict and disassemble mutant proteins from somatic mutations, identifying potential mutant peptides in the P7 subset of malignant pleural effusion from lung cancer patients. Further, combined with RNA-seq data of the P7 subset of malignant pleural effusion, the GATK ASEReadCounter algorithm was used to analyze potential mutant peptides from WES, determining the mutation count (MutationCount, 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 tumor cells from malignant pleural effusions of lung cancer patients at levels P19 and P23 (Table 1). These included 12 predicted neoantigen peptides presented by the HLA A*A11:01 molecule, such as VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2), and YSNRTRLAK (HSP90B1-M). These neoantigen peptides have the potential to be used to develop tumor vaccines for the treatment of lung cancer.

[0081] Table 1. Linear sequences and characteristic parameters of 12 predicted neoantigens and their paired WT antigens

[0082]

[0083] Example 3: Identification of high-frequency HLA-presented neoantigens of tumor cells in malignant pleural effusion of lung cancer patients using the Tetramer assay.

[0084] To screen for neoantigens in lung cancer patients, Tetramer staining technology 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 。 use QuickSwitch™ Quant Tetramer HLA-A*11:01 Kit-PE kit, evaluating the affinity of candidate neoantigen peptides derived from VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2), YVALLQLGLK (PIGO-M-1), YSNRTRLAK (HSP90B1-M), SETHSFLHPR (SKIL-M), and SSRVFSSRWGR (RP11-M) to the HLA-A*11:01 molecule.

[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 samples were 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 the candidate neoantigen peptide, i.e., its affinity for HLA-A*11:01.The results showed that the affinity of the candidate neoantigen peptides VAGSTRIPK (HSPA8-M-1) for HLA-A*11:01 was 95.24%, LVAGSTRIPK (HSPA8-M-2) for HLA-A*11:01 was 93.03%, SSLFSQAVK (CCDC89-M-1) for HLA-A*11:01 was 95.93%, NSSLFSQAVK (CCDC89-M-2) for HLA-A*11:01 was 89.01%, and LSFVPATTK (FAM173B-M-1) for HLA-A*11:01 was [not specified in the original text]. The affinity of the following compounds to HLA-A*11:01 was 89.35%; the affinity of CLSSFVPATTK (FAM173B-M-2) to HLA-A*11:01 was 80.06%; the affinity of SSSSRVFSSR (VKORC1-M) to HLA-A*11:01 was 89.89%; the affinity of VALLQLGLK (PIGO-M-2) to HLA-A*11:01 was 87.98%; and the affinity of YSNRTRLAK (HSP90B1-M) to HLA-A*11:01 was 95.74%, all significantly higher than the 75% specified in the instructions. Threshold; while the affinity of YVALLQLGLK (PIGO-M-1) to HLA-A*11:01 was 63.62%, the affinity of SETHSFLHPR (SKIL-M) to HLA-A*11:01 was 47.85%, and the affinity of SSRVFSSRWGR (RP11-M) to HLA-A*11:01 was 57.57%, these three peptides had poor affinity for HLA*A11:01. Figure 3 This indicates that the aforementioned VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2), and YSNRTRLAK (HSP90B1-M) have high affinity for the HLA-A*11:01 molecule, providing reliable candidate targets for further immunotherapy research.

[0086] To further validate the specific T-cell responses to candidate neoantigens in lung cancer, specific Tetramer staining was used to detect the specific T-cell frequencies of candidate neoantigen peptides VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2), and YSNRTRLAK (HSP90B1-M) in malignant pleural effusions and peripheral blood from P19 and P23 lung cancer patients. The experimental procedure was as follows: cells from pleural effusion or peripheral blood were adjusted to 1×102 6 ~1×10 7 At a concentration of / mL, 100 μL of cell suspension was taken, and 10 μL of ClearBack was added (incubated at room temperature in the dark for 5 minutes to block non-specific binding). Then, 10 μL of Tetramer with a peptide replacement efficiency greater than 75% was added to each tube, and the cells were incubated at room temperature for 30 minutes, followed by the addition of CD8 antibody (incubated at 4°C for 20 minutes). Cells were washed with PBS (centrifuged at 400g for 5 minutes), the supernatant was discarded, and the cells were resuspended in 500 μL of PBS. 20 μL of Cell Viability Solution was added, and the cells were stored at 4°C in the dark. FCM analysis was performed within 24 hours. Results showed that specific T cells recognizing the above nine antigenic peptides could be detected in the malignant pleural effusions of P19 and P23 lung cancer patients. Figure 4 and Figure 5 ), but CD8 + T cell frequencies were all below 5%. In P19 and P23 lung cancer patients, specific CD8+ was undetectable in peripheral blood unstimulated by candidate neoantigen peptides. + T cells, however, after 13 days of stimulation with a final concentration of 2 μM neoantigen peptides, were found to contain specific T cell CD8 that could recognize the aforementioned nine antigenic peptides. + T cell frequencies were all above 5% ( Figure 6 This suggests that these nine antigenic peptides are neoantigen peptides with immunogenicity.

[0087] Example 4: CD39, which is responsive to neoantigens, is found in the peripheral blood and malignant pleural effusion of lung cancer patients. + 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+ CD8+ T cells are more likely to be positive for hepatocellular carcinoma. + 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 pleural effusions of lung cancer patients. + CD39 + T cells ( Figure 7 Compared with peripheral blood from lung cancer patients, CD8+ levels in malignant pleural effusions were higher. + CD39 + The proportion of T cells expressed was significantly increased. Figure 7 ).

[0089] Furthermore, analysis of the TCGA database revealed HSPA8, CCDC89, FAM173B, VKORC1, PIGO, and HSP90B1 ( Figure 8 , Figure 9 and Figure 10 In addition to gene mutations in lung cancer, these new antigenic peptides also exist in other types of cancer, suggesting that these new antigenic peptides discovered in this invention could be used as tumor vaccines for cancer treatment in the future.

[0090] Example 5: Peripheral blood T cells stimulated by neoantigen peptides have cytotoxic effects against tumor cells derived from malignant pleural effusion in lung cancer patients.

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

[0092] First, mononuclear cells were extracted from peripheral blood of patients with P19 and P23 lung cancer. Then, nine neoantigen peptides derived from P19 and P23 lung cancer patients were co-incubated with mononuclear cells from their respective peripheral blood to activate and expand neoantigen-specific T cells. The specific method was 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 pleural effusions of P19 and P23 lung cancer patients and expanded using organoid culture. The specific method was as follows: tumor cells from the malignant pleural effusion 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 5T 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 T cells stimulated with the neoantigens VAGSTRIPK (HSPA8-M-1), LVAGSTRIPK (HSPA8-M-2), SSLFSQAVK (CCDC89-M-1), NSSLFSQAVK (CCDC89-M-2), LSFVPATTK (FAM173B-M-1), CLSFVPATTK (FAM173B-M-2), SSSRVFSSR (VKORC1-M), VALLQLGLK (PIGO-M-2), and YSNRTRLAK (HSP90B1-M) had significantly higher IFN-γ secretion levels than the control group. Figure 11 (A). The release level of lactate dehydrogenase (LDH) was further detected by ELISA to assess the cytotoxic effect of T cells on tumor cells. The results showed that the LDH release induced by T cells stimulated with the above nine neoantigen peptides was significantly higher than that in the control group (A). Figure 11 (B). This indicates that peripheral blood T cells stimulated by neoantigen peptides can kill tumor cells derived from malignant pleural effusion in lung cancer patients.

[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 tumor neoantigen polypeptide, characterized in that, The amino acid sequence of the neoantigen polypeptide is shown in SEQ ID NO.

7.

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 lung cancer-specific T cells.

5. 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 TCR-T for lung cancer.

6. 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 a medicament for treating lung cancer.

7. 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.

8. 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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