Immunopeptidomics-based non-classical neoantigen vaccine for liver cancer and immunotherapy application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a liver cancer non-classical neoantigen vaccine based on immunopeptidomics and immunotherapy application of the liver cancer non-classical neoantigen vaccine. MHC-I immunoprecipitation combined mass spectrometry is carried out on a liver cancer sample, a neoantigen with high immunogenicity is identified and screened out, the neoantigen and an adjuvant Poly (I: C) jointly construct the liver cancer vaccine, the effectiveness of the vaccine is verified in a mouse subcutaneous liver cancer model, tumor growth can be inhibited, and the tumor immune microenvironment can be changed. A brand-new strategy is provided for immunotherapy of the liver cancer, development of a more accurate and effective personalized treatment scheme is facilitated, and forward development of the field of immunotherapy of the liver cancer is promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to tumor immunotherapy technology, and in particular to a non-classical neoantigen vaccine for liver cancer based on immunopeptidomics and its immunotherapy application. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the malignant tumors with high morbidity and mortality worldwide. Although existing treatment methods, such as surgery, radiotherapy, and targeted therapy, have improved the prognosis of patients to a certain extent, the 5-year survival rate of patients with advanced hepatocellular carcinoma (HCC) is still low. In recent years, immunotherapy based on tumor neoantigens has become a research hotspot in the field of cancer treatment. Its goal is to activate the body's immune system to achieve specific recognition and killing of tumor cells. However, there are still many challenges in the field of neoantigen identification. For example, traditional gene transcriptome sequencing technology has high false positive and false negative rates, and the immunogenicity of identified neoantigens is limited. The application of mass spectrometry technology in immunopeptidomics provides a powerful means for directly analyzing antigenic peptides presented by MHC molecules on the cell surface, which can more accurately identify the actual presented antigens. However, in the field of liver cancer, there are relatively few related studies, and there is a lack of comprehensive and effective verification of the identified immune peptides. Therefore, a highly specific and immunogenic liver cancer vaccine needs further research and development.

[0003] Non-classical tumor neoantigens are neoantigen epitopes that are specifically expressed by tumor cells and caused by changes in amino acid sequences due to non-point mutations. They are presented to the cell surface through MHC molecules. The generated neoantigen epitopes can be recognized by the immune system, thereby triggering the body's immune response. However, there are many sources of non-classical tumor neoantigens, due to gene recombination, chromosomal translocation, protein post-translational modification, etc. Therefore, there are many challenges in predicting and identifying the generation of neoantigens. First, the accurate proteolysis mechanism of the mature antigen peptide-MHC complex is unknown and complex, and many proteases and peptidases are involved; second, the peptides bound by MHC molecules are very consistent in length and sequence conservation and have relatively low abundance, which increases the difficulty of detection; third, the screening of neoantigens is very challenging, and the algorithm is the core of it. Which mutations will be presented by MHC, which can be recognized by the immune system, and cause a strong enough immune response, the prediction algorithm and process have no unified standard and the analysis process is very complicated; fourth, the traditional NGS method is based on DNA sequencing to predict neoantigens, and the predicted neoantigens may not be expressed and presented on the cell surface, or the expression level is low. Therefore, methods for identifying non-classical tumor neoantigens require further research.

[0004] Nowadays, mass spectrometry (MS)-based proteomics is an advanced large-scale peptide sequencing analysis technology and has become a powerful tool for directly analyzing the immunopeptidome presented by MHC molecules. Mass spectrometry analysis methods can directly identify the neoantigen peptides truly presented on the surface of tumor cells. The principle of its identification is to elute the antigen peptides on the surface of tumor cells from HLA molecules and perform mass spectrometry sequencing on them. Current mass spectrometry technologies make it possible to identify thousands of MHC-presented peptide sequences from cell lines and patient materials. One advantage of mass spectrometry analysis for non-classical neoantigen screening is that it greatly narrows the candidate range of neoantigens and is an unbiased method, which can greatly improve the screening accuracy of neoantigens.

[0005] Through the research and identification of non-classical neoantigens and the simultaneous use of the existing adjuvant Poly(I:C), it is beneficial to deepen the development of highly specific and highly immunogenic liver cancer neoantigen vaccines and their curative effects in the treatment of hepatocellular carcinoma, providing more ideas for the clinical treatment of hepatocellular carcinoma. Summary of the Invention

[0006] The present invention aims to provide a non-classical liver cancer neoantigen vaccine based on immunopeptidomics and its immunotherapeutic application. Based on the mass spectrometry platform, the present invention efficiently and accurately screens out immunogenic non-classical tumor neoantigens, constructs a mature and optimized polypeptide vaccine system, and explores its curative effect in the treatment of hepatocellular carcinoma, providing more ideas for the clinical treatment of hepatocellular carcinoma.

[0007] To achieve the above object, the technical solutions adopted by the present invention are as follows: The first aspect of the present invention provides a group of non-classical neoantigen polypeptides, which are composed of the following 1)-7) neoantigen polypeptides: 1) Neoantigen polypeptide PNC-L3: The amino acid sequence is KAAIVCYDFTKLHLTDS; 2) Neoantigen polypeptide PNC-L5: The amino acid sequence is DACASGPCFCATLLDVS; 3) Neoantigen polypeptide PNC-L7: The amino acid sequence is PGGVSLLNLCETLLHDC; 4) Neoantigen polypeptide PNC-L9: The amino acid sequence is YCMWRNYEYGCRLDGQT; 5) Neoantigen polypeptide PNC-L10: The amino acid sequence is FRRDASMLNYSYLLTSQ; 6) Neoantigen polypeptide PNC-L11: The amino acid sequence is FRRDASMLNSYYLKCLE; 7) Neoantigen polypeptide PNC-L13: The amino acid sequence is TAIASMLSYFAALMTTM.

[0008] The second aspect of the present invention provides the use of the above-mentioned set of non-classical neoantigen polypeptides in the preparation of tumor immunotherapy drugs; Further, the tumor is liver cancer; Further, the drug is a vaccine.

[0009] The third aspect of the present invention provides a tumor immunotherapy drug, which comprises the above-mentioned non-classical neoantigen polypeptides; Further, the tumor is liver cancer; Further, the drug is a vaccine.

[0010] The fourth aspect of the present invention provides a preparation method of the above-mentioned tumor immunotherapy drug, which mixes the neoantigen polypeptide PNC-L3, the neoantigen polypeptide PNC-L5, the neoantigen polypeptide PNC-L7, the neoantigen polypeptide PNC-L9, the neoantigen polypeptide PNC-L10, the neoantigen polypeptide PNC-L11, the neoantigen polypeptide PNC-L13 with PBS and Poly(I:C) to obtain the tumor immunotherapy drug; Further, in the tumor immunotherapy drug, the mass ratio of the neoantigen polypeptide PNC-L3, the neoantigen polypeptide PNC-L5, the neoantigen polypeptide PNC-L7, the neoantigen polypeptide PNC-L9, the neoantigen polypeptide PNC-L10, the neoantigen polypeptide PNC-L11, the neoantigen polypeptide PNC-L13 is 1:1:1:1:1:1:1; Furthermore, 2.5 μl of the neoantigen polypeptide PNC-L3 at 40 μg / μl, 2.5 μl of the neoantigen polypeptide PNC-L5 at 40 μg / μl, 2.5 μl of the neoantigen polypeptide PNC-L7 at 40 μg / μl, 2.5 μl of the neoantigen polypeptide PNC-L9 at 40 μg / μl, 2.5 μl of the neoantigen polypeptide PNC-L10 at 40 μg / μl, 2.5 μl of the neoantigen polypeptide PNC-L11 at 40 μg / μl, 2.5 μl of the neoantigen polypeptide PNC-L13 at 40 μg / μl are mixed with 132.5 μl of 0.01M PBS at pH 7.4 and 50 μl of Poly(I:C) at 1 μg / μl to obtain the tumor immunotherapy drug.

[0011] The test method adopted by the present invention is as follows: 1. Sample acquisition and pretreatment: A subcutaneous liver cancer model was established using C57BL / 6 mice, and MHC-I molecular complexes were enriched by grinding liver cancer tissues. After the mouse liver cancer tissues were snap-frozen in liquid nitrogen, they were ground into powder using a tissue grinder, lysate buffer was added, and after incubation on ice for 60 min, sonication was performed for 5 min. After high-speed centrifugation, the supernatant was collected for subsequent enrichment of the immunopeptidome. The enrichment antibody was immobilized on an agarose enrichment material by chemical cross-linking. At the same time, experimental samples were taken for SDS-PAGE experiments to examine the antibody cross-linking efficiency. The MHC-peptides complex was purified by immunoprecipitation, and the immunopeptides were fractionated by the purification column method to obtain immunopeptide segments, and the collected fractions were analyzed by LC-MS for mass spectrometry. 18 The purified column method was used for fractional separation to obtain immunopeptide segments, and the collected fractions were analyzed by LC-MS for mass spectrometry.

[0012] 2. Mass spectrometry data acquisition: The above high-resolution mass spectrometer was used for data acquisition. The data was acquired in the DDA mode, with an acquisition duration of 90 min, a scan range of the mass-to-charge ratio of 400 - 650 m / z, a resolution of 120,000 for MS1, a resolution of 30,000 for MS2, a fragmentation mode of HCD, a fragmentation energy of 35, and a total cycle time of 2 sec, generating raw mass spectrometry detection data (.raw).

[0013] 3. Database search: The mass spectrometry MS / MS spectrum data was obtained using the DDA acquisition mode. All raw data files were analyzed using the DeepImmu TM neoantigen analysis platform, and peptide identification results were obtained by combining analysis methods such as de novo sequencing, database search, and homology search. Peptides with a length of 7 - 16 amino acids were screened out from them.

[0014] 4. Quality assessment and feature analysis: Quality assessment was performed on all the above data, including the cross-linking efficiency of the antibody and magnetic beads during immunoprecipitation; monitoring the mass error of mass spectrometry detection; comparing the experimental retention time with the predicted retention time and calculating the correlation coefficient between the two; statistically analyzing the identified immunopeptides for feature analysis, comparing with known public databases and relevant reported literature to verify their consistency; using in vitro mouse experiments to verify the immunogenicity of candidate neoantigens.

[0015] 5. Vaccine construction: The selected highly immunogenic short peptides (8 - 10 amino acids) may have immune tolerance. By chemical synthesis, auxiliary sequences of 3 - 5 amino acids were added to their N-terminal and C-terminal respectively to extend them into long peptides (17 - 25 amino acids). The selected non-classical long peptides were combined and mixed with PBS and Poly(I:C) to construct a vaccine against hepatocellular carcinoma.

[0016] 6. Animal model establishment and vaccination: A subcutaneous liver cancer model was constructed using C57BL / 6 mice, and the above-constructed vaccine was used for immunotherapy. During this period, the body weight and tumor burden of the mice were observed and recorded, etc.

[0017] 7. Vaccine safety detection: HE staining and biochemical index detection were used to observe the morphological changes of tumor tissues and simultaneously evaluate the effects of the vaccine on normal organs.

[0018] 8. Evaluation of treatment effect: The treatment effect was mainly reflected in the tumor growth curve, enzyme-linked immunospot assay (Elispot), detection of changes in immune cell phenotypes by flow cytometry, and exploration of the infiltration of immune cells in tumors by immunohistochemistry.

[0019] After strict screening, the present invention has developed a brand-new non-classical tumor neoantigen vaccine. This vaccine can be applied to the field of immunotherapy research for hepatocellular carcinoma, providing a solid basis for the precision medicine of HCC and promising to bring significant benefits to a large number of patients. Its significance is mainly reflected as follows: 1) The present invention has successfully developed neoantigens by mass spectrometry and then constructed neoantigen polypeptide vaccines, providing a brand-new treatment plan for the treatment of hepatocellular carcinoma.

[0020] 2) The mass spectrometry identification of neoantigens and verification strategies provided by the present invention offer a brand-new idea for the development of polypeptide vaccines for different cancer types, helping to develop more targeted vaccines and promoting the implementation of new treatment strategies. Description of the Drawings

[0021] Figure 1 : SDS-PAGE analysis of the cross-linking efficiency of MHC I-specific antibodies. 1: Protein Marker; 2: Solution before antibody cross-linking; 3: Flow-through solution after cross-linking; 4: Solution for washing beads; 5: Beads after cross-linking.

[0022] Figure 2 : Mass error distribution map and retention time error distribution map of the samples.

[0023] Figure 3 : Motif clustering results of neoantigen polypeptide sequences.

[0024] Figure 4 : Enzyme-linked immunospot image results of neoantigen immunogenicity and analysis of the difference in the number of spots.

[0025] Figure 5 : Tumor growth curve of the neoantigen vaccine and the size of tumor tissues in each group.

[0026] Figure 6 : HE staining and biochemical detection to verify the safety of the vaccine.

[0027] Figure 7 Flow cytometry and immunohistochemistry were used to explore the immune response mechanism stimulated by the vaccine. Detailed implementation manners

[0028] To make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific implementation manners, but the present invention is not limited thereto.

[0029] Example 1: Identification of neoantigen immune polypeptides 1. Sample information Sample source: Mouse hepatoma cell line Hepa1-6 was purchased from ATCC (Manassas, VA, USA); male C57BL / 6 mice, 6-8 weeks old, weighing 20-25 g each, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (License No.: SCXK(Hu)2022-0004) and raised under specific pathogen-free conditions.

[0030] 2. Immune purification of MHC class I peptides (1) A subcutaneous tumor model of liver cancer was established using C57BL / 6 mice, and then the liver cancer tissue was mechanically ground under liquid nitrogen conditions. Subsequently, lysis buffer was added and incubated on ice for 60 minutes. Then, ultrasonic treatment was performed for 5 minutes. After high-speed centrifugation, the supernatant and cell lysate were collected.

[0031] (2) BCA assay was used to quantify the protein concentration to determine the mass concentration of protein extraction. Then, the supernatant was taken for enriching MHC-peptide complexes with MHC-H-Kb / Db (MHC-I) antibody and incubated overnight at 4°C in the dark. At the same time, experimental samples were taken for SDS-PAGE experiments to test the antibody cross-linking efficiency.

[0032] (3) MHC-binding peptides were separated and washed through a C 18 column and dried by high-speed vacuum. For in-depth identification, we took the above flow-through samples, divided them into two parts, and enriched them with H-2-Kb antibody (Bioxcell Cat BE0172) and H-2-Db antibody (Thermo Fisher Cat MA5-17992) respectively.

[0033] (4) Antibody cross-linking efficiency ( Figure 1 ): The results showed that the vast majority of the antibodies were successfully cross-linked to the Beads, which was suitable for subsequent enrichment experiments.

[0034] 3. LC-MS analysis of MHC class I peptides (1) Dissolve the above-mentioned immune polypeptide in 10 μL of LC-MS solvent A (0.1% formic acid in water), and add it to a nano-scale liquid chromatography (nEasy-nLC 1200, Thermo, USA), which is connected to an Orbitrap Eclipse triple quadrupole mass spectrometer (ThermoFisher Science, Waltham, MA, USA).

[0035] (2) Separate the immune peptide onto a C 18 analytical column with a 90-minute gradient at a constant flow rate of 300 nL / min (0 - 78 minutes), 2 - 20% solvent B (0.1% formic acid in acetonitrile), 79 - 85 minutes; 20 - 100% solvent B, and hold at 100% solvent B for 5 minutes. MS is operated in the data-dependent acquisition (DDA) mode with a mass range of 400 - 650 m / z.

[0036] (3) The resolution of MS1 is set to 120,000, and the precursors selected for MS2 analysis are fragmented by higher energy collision dissociation (HCD) using a normalized collision energy of 34%.

[0037] 4. Proteomics database search and immunopeptidome analysis (1) The acquired mass spectrometry data is analyzed using PEAKS online v1.8 and against the Uniprot mouse database (17,162 entries).

[0038] (2) For immunopeptidome identification, the PSM error range is required to be within 10 ppm; spectra with a peptide FDR ≤ 0.1% are set as reliable; oxidation of methionine (+15.9949 Da) is set as a variable modification; the enzyme is set to none; the Deep novo score is set to ≥ 80.

[0039] (3) Peptide sequence information is used for immunogenicity prediction using DeepImmu software, which can rank peptide immunogenicity based on a deep learning model of TCR - neoantigen - MHC complex binding.

[0040] (4) Quality control detection ( Figure 2 ): The identified PSM error range is within 10 ppm, and there is a good correlation between the experimentally detected retention time and the predicted retention time, indicating the accuracy of the identification results.

[0041] 5. MHC motif and MHC binding affinity prediction (1) Perform Gibbs enrichment analysis on the above-mentioned peptides using the GibbsCluster 2.0 tool on the DTU Health Tech - Bioinformatics Tools website. Cluster with 1 - 5 clusters, maximum deletion length of 1, maximum insertion length of 4, and heuristic sequence weighting. Known MHC motifs are obtained from the MotifViewer section of NetMHCpan - 4.1.

[0042] (2) Use NetMHCpan - 4.1 software for MHC binding affinity prediction. Set the species to Mouse and select alleles H - 2 - Db and H - 2 - Kb. Polypeptides with a binding percentage rank ≤ 0.5% are classified as strong binders, those with a rank between 0.5% and 2% are classified as weak binders, and polypeptides with a rank exceeding 2% are considered "non - binders".

[0043] (3) Motif analysis results ( Figure 3 ):According to the Motif clustering results obtained using Gibbs sampling, visualize through NetMHCpan / Motifs Viewer. The results show that the identified peptides cover the expected binding motifs of MHC I alleles. In addition, the MHC - I binding motifs of non - classical immunopeptides closely match those of classical immunopeptides, further supporting the reliability of non - classical polypeptide identification.

[0044] (4) Thirteen non - classical candidate neoantigens were selected for further verification based on an affinity ranking in the top 2% and a length of 8 - 10 amino acids (Table 1).

[0045] Table 1 Example 2: Verification of neoantigen immunopeptides 1. Verify the immunogenicity of candidate neoantigens by enzyme - linked immunospot assay (1) Solid - phase synthesize each neoantigen, then take 20 μg and 50 μg respectively and mix with Poly(I:C), and immunize female C57BL / 6 mice subcutaneously at the flanks on day 0, day 4, and day 8 respectively.

[0046] (2) Sacrifice the mice on day 14, take out the spleen, and use a syringe to completely blow the cells in the spleen into a 6 - cm dish. Use lymphocyte separation medium Ficoll (GE) for gradient centrifugation to obtain the middle cloudy layer of T cells. Then use erythrocyte lysate to stand for 3 min, and place the supernatant in RPMI1640 complete medium containing 10% FBS, 20 ng / μl mGM - CSF, and rmil - 4 for culture.

[0047] (3) Take out the lower limb bones of the mice, use a syringe to completely blow the bone marrow into a 6-cm dish and filter it under a 40-μm cell strainer. Then collect the supernatant and let it stand for 3 min with red blood cell lysate (Gibco). Place the supernatant in the medium and culture the DC cells until they mature.

[0048] (4) DC polypeptide loading: Use the Mabtech ELISpot Mouse IFN-y ELISpot kit. Wash the ELISpot plate 4 times with PBS, 200 μl each time. Then incubate it with 1640 medium containing 10% serum, 200 μl / well, at 37 °C for 30 - 40 min.

[0049] (5) Adjust the concentration of im DC to 2×10 4 / well with 1640 medium containing 10% FBS and 20 ng / μl GM-CSF and IL-4. Take 100 μl / well of the cell suspension and place it in a 200-μl PCR tube for loading and plating with the polypeptide.

[0050] (6) Load the polypeptide onto the DC: Mix each polypeptide (dissolved in 2 mg / 100 μl DMSO), take 10 μl and dilute it in 50 μl of 1640 medium containing 20 ng / μl GM-CSF and IL-4 and 10% FBS. Take 10 μl of the diluted solution and place it in a PCR tube containing 100 μl of the cell suspension to make the final working amount 4 μg / well.

[0051] (7) DC-T co-culture: Add T cells after 24 h, adjust the cell density to (1×10^5 / well) with 1640 medium containing 10% FBS, and add 100 μl / well (i.e., 1×10 5 cells / well) to the corresponding wells in the ELISpot plate for co-culture with the DC cells. Develop the color after 24 - 36 h.

[0052] (8) ELISpot assay: Aspirate the cells in the wells, wash the wells with PBS 3 - 5 times, 200 μl / well; Take the R4 - 6A2 - biotin antibody in DMEM medium with 0.5% FBS, at a concentration of 1 μg / ml, add 100 μl / well into the wells, and incubate at room temperature for 2 h; Aspirate the liquid, wash with PBS 3 - 5 times, 200 μl / well; Take ALP (1:1000) in the medium, add 100 μl / well into the wells, and incubate at room temperature in the dark for 1 h; Aspirate the liquid, wash with PBS 3 - 5 times, 200 μl / well; Add 100 μl / well of ready - to - use chromogenic solution, observe the color development, and terminate the color development with a large amount of deionized water (ddH2O has been filtered) at an appropriate time (10 - 20 min).

[0053] (9) Image processing and data analysis: Use an enzyme - linked immunospot image analyzer to observe the IFN - γ secretion of T cells under different peptide restimulation conditions to evaluate the specificity of the T - cell response to single - peptide restimulation.

[0054] (10) Differential analysis of neoantigen peptides ( Figure 4 ): Select the top seven with significant differences (P < 0.05) and immunogenicity as candidate neoantigens for our vaccine construction.

[0055] 2. Elongation strategy of candidate neoantigen immunopeptides For non - classical neoantigens, the origin and extension of peptides are more challenging. After analyzing the sequences of the top 7 non - classical neoantigens with pre - immunogenicity, we found that 2 of them might be caused by single - amino - acid insertion or deletion because they differed from the known gene sequences by only one amino - acid insertion or deletion. For such peptides, we also referred to the known genomic sequences for peptide extension. In addition, the sequences of 5 peptides showed obvious splicing characteristics between two proteins, with the splicing sequence accounting for more than 40% (4 / 10) of the total sequence. This enabled us to perform splicing extension based on the amino - acid sequences of these two proteins. According to this strategy, the final neoantigen peptide sequences used for vaccine development are as follows: Table 2 Example 3: Antitumor efficacy of neoantigen polypeptide vaccine 1. In - vivo antitumor treatment and efficacy evaluation (1) Inject 3×10 6 Hepa1 - 6 cells into the posterior thigh of C57BL / 6 mice. One week later, when the tumor volume reaches about 120 m 3When there was no significant difference in the body weight of the mice, the model was successfully established. Subsequently, the mice were divided into 3 groups for subsequent vaccination, with 5 mice in each group, including the non-classical long peptide (PNC_L) group, the PBS group, and the adjuvant Poly(I:C) group.

[0056] PNC_L group: Mix 2.5 μl of 40 μg / μl of the neoantigen polypeptide PNC-L3 solution, 2.5 μl of 40 μg / μl of the neoantigen polypeptide PNC-L5 solution, 2.5 μl of 40 μg / μl of the neoantigen polypeptide PNC-L7 solution, 2.5 μl of 40 μg / μl of the neoantigen polypeptide PNC-L9 solution, 2.5 μl of 40 μg / μl of the neoantigen polypeptide PNC-L10 solution, 2.5 μl of 40 μg / μl of the neoantigen polypeptide PNC-L11 solution, 2.5 μl of 40 μg / μl of the neoantigen polypeptide PNC-L13 solution with 132.5 μl of 0.01 M PBS at pH 7.4 and 50 μl of 1 μg / μl of the Poly(I:C) solution to obtain a tumor immunotherapy drug for vaccination. Among them, each neoantigen polypeptide was prepared by solid-phase synthesis method. The neoantigen polypeptide solution was prepared with a 50 wt% DMSO aqueous solution as the solvent, and the Poly(I:C) solution was prepared with 0.01 M PBS at pH 7.4 as the solvent.

[0057] PBS group: 132.5 μl of 0.01 M PBS at pH 7.4 for vaccination.

[0058] Adjuvant Poly(I:C) group: 50 μl of 1 μg / μl of the Poly(I:C) solution for vaccination. The Poly(I:C) solution was prepared with 0.01 M PBS at pH 7.4 as the solvent.

[0059] (2) Subcutaneously inject the vaccine on days 0, 4, and 8 respectively. Monitor tumor growth and body weight with a caliper every 2 days for 20 days.

[0060] (3) Continuously monitor the tumor burden and draw a growth curve. Sacrifice the mice after 20 days of vaccination and take pictures to obtain the actual tumor images ( Figure 5 ), and the results showed that the PNC_L group had obvious anti-tumor efficacy, further proving that the neoantigens in Table 2 had anti-tumor effects.

[0061] 2. Biosafety detection (1) Two days before sacrificing the mice, tail vein blood was collected and sent to the testing laboratory (Clinical Laboratory of Mengchao Hepatobiliary Hospital, Fujian Medical University) for serum biochemical tests, including total bilirubin (TBil), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (urea), serum creatinine (SCr), glucose (Glu), and triglyceride (TG), to evaluate the safety of the vaccine.

[0062] (2) After 20 days of treatment, the mice were euthanized, and single-cell suspensions were prepared from the spleen, lymph nodes, and tumor tissues for flow cytometry analysis. The heart, liver, spleen, lungs, kidneys, and tumors were fixed in formalin for H&E staining to further verify whether there were any effects on normal organs after treatment.

[0063] (3) Biosafety ( Figure 6 ): Biochemical test and H&E results showed no significant differences between the PNC_L group and normal healthy C57BL / 6 mice, demonstrating that this neoantigen is suitable for designing into a vaccine and used for the treatment of liver cancer.

[0064] 3. Neoantigens can promote the increase of immune cells in the body (1) Preparation of single-cell suspension: After 20 days of treatment, the mice were euthanized, the tumor tissues of the mice were chopped with scissors, and digested with digestive enzymes (1 mg / mL type II collagenase and 2 mg / mL DNase I, Sigma) for 1 hour. The digested tissues were filtered through a 40 μm cell strainer and then centrifuged at 800 g for 20 minutes. The pellet was resuspended and separated by Ficoll gradient centrifugation to obtain a single-cell suspension. Lymph node tissues were mechanically disrupted with a syringe plunger through a 40 μm filter and then centrifuged at 800 g for 5 minutes to obtain a single-cell suspension of lymphocytes.

[0065] (2) Flow cytometry analysis: The isolated cell suspension was centrifuged at 300 g for 3 minutes, washed twice with PBS, and blocked with 0.5% BSA for 30 minutes. Then the cells were stained with fluorescently labeled antibodies and incubated on ice for 1 hour in the dark. The antibodies used included: anti-CD3-APC (cat: 17-0032-82), anti-CD44-PE (cat: 12-0081-82), anti-CD44-PE-Cyanine7 (cat: 25-0441-82), anti-CD62L-PerCP / Cy5.5 (cat: 45-0621-82), anti-4-1BB-APC (cat: 17-1371-82), anti-CD80-PE (cat: 12-0801-82), anti-CD86-PE (cat: 25-0862-82), and anti-CD11c-APC (cat: 17-0801-82). The antibody mixture was incubated in 100 μL according to the manufacturer's instructions. The stained samples were analyzed using a BD Accuri 6C Plus flow cytometer (BD Bioscience), and the data were processed using FlowJo software (v.10).

[0066] (3) Immunohistochemistry: The tumors of each mouse were fixed with formalin and embedded in paraffin. The slides were baked at 60 °C for 2 hours, and then subjected to gradient dewaxing and EDTA antigen retrieval; endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and the sections were blocked with 5% bovine serum albumin; then incubated with the secondary antibody at room temperature for 30 minutes; the slides were developed with DAB chromogenic solution for 3 minutes, counterstained with hematoxylin for 30 seconds, blued for 30 seconds, dehydrated with alcohol, air-dried, and fixed and sealed with neutral resin; image processing was performed under a scanning microscope, and finally data quantification analysis was performed using Image J.

[0067] (4) Data analysis ( Figure 7 ): According to the data obtained from the above steps, in flow cytometry analysis, it was observed that the PNC_L group showed a significant increasing trend in CD8T+, DC cell presentation, and tumor T cells, while a large infiltration of immune cells was observed in the PNC_L group in the immunohistochemistry results.

[0068] In summary, the present invention uses mass spectrometry technology to screen a group of neoantigens and applies them to the field of liver cancer treatment. Verified by in vitro Elispot and PRM experiments on mice, this group of neoantigens has good immunogenicity and reliability, indicating that the neoantigens screened by mass spectrometry can be used for the development of anti-tumor vaccines. Through in vivo experiments, namely mouse liver cancer treatment experiments, the results show that the screened neoantigens have significant efficacy in treating tumors. Through flow cytometry and immunohistochemistry experiments, the results show that the neoantigens have obvious advantages in inducing immune responses in vivo. In summary, the neoantigen vaccine can be used for liver cancer treatment and has good efficacy.

[0069] The above are only the preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. A group of non-classical neoantigen polypeptides, characterized in that: Composed of the following neoantigen polypeptides 1)-7): Composition: 1) Neoantigen polypeptide PNC-L3: The amino acid sequence is KAAIVCYDFTKLHLTDS; 2) Neoantigen polypeptide PNC-L5: The amino acid sequence is DACASGPCFCATLLDVS; 3) Neoantigen polypeptide PNC-L7: The amino acid sequence is PGGVSLLNLCETLLHDC; 4) Neoantigen polypeptide PNC-L9: The amino acid sequence is YCMWRNYEYGCRLDGQT; 5) Neoantigen polypeptide PNC-L10: The amino acid sequence is FRRDASMLNYSYLLTSQ; 6) Neoantigen polypeptide PNC-L11: The amino acid sequence is FRRDASMLNSYYLKCLE; 7) Neoantigen polypeptide PNC-L13: The amino acid sequence is TAIASMLSYFAALMTTM.

2. Use of the non-classical neoantigen polypeptide according to claim 1 in the preparation of a tumor immunotherapy drug.

3. The application according to claim 2, characterized in that: The tumor is liver cancer.

4. The application according to claim 2, wherein: The drug is a vaccine.

5. A tumor immunotherapy drug, characterized in that: It contains the non-classical neoantigen polypeptide according to claim 1.

6. The tumor immunotherapy drug according to claim 5, wherein: The tumor is liver cancer.

7. The tumor immunotherapy drug according to claim 5, wherein: The drug is a vaccine.

8. The preparation method of the tumor immunotherapy drug according to any one of claims 5 to 7, characterized in that: Mix the neoantigen polypeptide PNC-L3, neoantigen polypeptide PNC-L5, neoantigen polypeptide PNC-L7, neoantigen polypeptide PNC-L9, neoantigen polypeptide PNC-L10, neoantigen polypeptide PNC-L11, neoantigen polypeptide PNC-L13 with PBS and Poly(I:C) to obtain a tumor immunotherapy drug.

9. The preparation method according to claim 8, characterized in that: The mass ratio of the neoantigen polypeptide PNC-L3, neoantigen polypeptide PNC-L5, neoantigen polypeptide PNC-L7, neoantigen polypeptide PNC-L9, neoantigen polypeptide PNC-L10, neoantigen polypeptide PNC-L11, neoantigen polypeptide PNC-L13 is 1:1:1:1:1:1:

1.

10. The preparation method according to claim 9, characterized in that: Mix 2.5 μl of 40 μg / μl neoantigen polypeptide PNC-L3, 2.5 μl of 40 μg / μl neoantigen polypeptide PNC-L5, 2.5 μl of 40 μg / μl neoantigen polypeptide PNC-L7, 2.5 μl of 40 μg / μl neoantigen polypeptide PNC-L9, 2.5 μl of 40 μg / μl neoantigen polypeptide PNC-L10, 2.5 μl of 40 μg / μl neoantigen polypeptide PNC-L11, 2.5 μl of 40 μg / μl neoantigen polypeptide PNC-L13 with 132.5 μl of 0.01 M PBS at pH 7.4 and 50 μl of 1 μg / μl Poly(I:C) to obtain a tumor immunotherapy drug.

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