Tumor antigens for gliomas and uses thereof

Abstract

The application discloses a tumor antigen for glioma and application thereof, and belongs to the field of biomedical technology. Based on a novel curative effect marker URGCP, HEATR1 and HCMV screening and verification, effective immunogenic peptide segments are obtained, a trivalent vaccine is prepared, and the trivalent vaccine is verified to enhance the antigen presentation ability of DCs in vitro. After overexpression of HCMV and exploration of a suitable radiation dose, in-vivo experiments are used to verify that the trivalent mixed vaccine promotes DC homing, lymph node activation, tumor-specific T cell generation, establishment of humoral immunity and memory immunity. Finally, a preventive model and a therapeutic model are used to verify that the universal DC vaccine can inhibit tumor progression and has good safety.

Description

Technical Field

[0001] This invention relates to tumor antigens for gliomas and their applications, belonging to the field of biomedical technology. Background Technology

[0002] Gliomas, as the most malignant and deadliest primary tumors of the central nervous system, pose a significant threat to human health. Traditional treatments primarily rely on surgery and radiotherapy / chemotherapy to improve patient survival. Currently, even with comprehensive treatment, the survival time for gliomas is only 15-17 months, highlighting the urgent need for new treatment methods! In recent years, immunotherapy has emerged as an effective treatment option following surgery, radiotherapy, and chemotherapy. Because glioma patients experience local and systemic immunosuppression, immunotherapy (especially targeting glioma-associated antigens) combined with other methods can have a strong complementary effect, providing specific anti-tumor efficacy for restoring and rebuilding the patient's damaged immune system, thereby further preventing tumor recurrence and metastasis. Furthermore, it significantly differs from surgery and radiotherapy / chemotherapy, which are unlikely to be repeated frequently, as repeated surgery and radiotherapy / chemotherapy are often intolerable to the body. Immunotherapy, with fewer side effects, can be repeated to enhance the body's anti-tumor capabilities.

[0003] Immunotherapy includes vaccines, T-cell infusion, and immune checkpoint inhibitors. Vaccine therapy includes dendritic cell vaccines, peptide vaccines, and mRNA vaccines. Dendritic cells (DCs), as the most potent antigen-presenting cells known in the human body, efficiently take up and present tumor antigens, activating naive T cells and playing an indispensable role in both innate and adaptive immunity. Therefore, DCs have been developed as a novel anti-tumor tool: DC vaccines. DC vaccines work by contacting circulating T cells in lymph nodes and presenting tumor-specific antigens to naive T cells. These naive T cells are then activated, proliferate, and recruited to the tumor region to specifically kill glioma cells. Currently, a near-phase III clinical trial of a DC vaccine targeting glioma has been successful internationally. Although this study used historical control group data and was not randomized controlled, it suggests that vaccine therapy is very safe, has very few side effects, and can be used multiple times! However, because its antigen uses whole tumor cell lysate (containing various non-tumor components) and the quantity is limited (excluding pathological diagnosis, there are few specimens, and once they are used up, they are gone), it cannot actually be used multiple times. In addition to safety, more effective vaccine use also needs to take into account the requirements of multiple uses and initiating a sufficient and specific anti-glioma effect.

[0004] Therefore, one of the most important factors for vaccine therapy is finding effective, specific, and safe glioma antigens. Tumor antigens are specific molecules expressed on the surface or inside tumor cells, and are key targets for the immune system to recognize and attack tumor cells. They can be divided into tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). Their important roles include: 1. In vivo antigen-presenting cells (such as dendritic cells) process tumor antigens into peptides and present them to T cells via MHC molecules, triggering a specific T cell immune response. mRNA vaccine systems can also be used. 2. Based on effective tumor antigens, new antibody drugs can be developed, including immune checkpoint inhibitors; T-cell receptor (TCR) therapy: modifying T cells to target specific antigens (such as NY-ESO-1); adoptive cell therapy, such as CAR-T cell therapy: directly killing tumor cells by targeting tumor antigens (such as CD19, BCMA). 3. Based on the level of tumor antigen expression, a predictive system for evaluating the efficacy of immune checkpoint inhibitors such as PD-1 can also be established.

[0005] Currently, research on tumor antigens for gliomas has made some progress, mainly focusing on TAAs, TSAs, and neoantigens. This includes targeted vaccines against EGFRvIII, survivin, IDH, etc., multi-target combination vaccines, and personalized vaccines. EGFRvIII: Epidermal growth factor receptor type III mutant, present in approximately 30% of GBMs, but not expressed in normal tissues. However, a phase III clinical trial of the Rindopepimut vaccine targeting the EGFRvIII site in combination with TMZ for the treatment of EGFRvIII-positive nGBMs failed: it did not prolong patient survival compared to TMZ monotherapy, suggesting that using EGFR alone as a tumor antigen target for gliomas is not a good choice. Antigen IL13Rα2: Interleukin-13 receptor α2 chain, highly expressed in GBMs, and lowly expressed in normal brain tissue. EphA2: Tyrosine kinase receptor, overexpressed in GBMs, involved in tumor invasion. HER2 / neu, Survivin, and other tumor-specific neoantigens have been reported by other teams and used in clinical practice abroad, but there is no evidence-based data showing that they can prolong patient survival, especially among Chinese patients, where no relevant reports have been found. Furthermore, the customization process for these neoantigens based on personalized tumor sequencing is cumbersome, and the vaccine production process is lengthy, which is not conducive to the rapid establishment of anti-tumor immunity in patients after surgery (tumors continue to grow after surgery, and there is currently a lack of effective time; it is a race against time! At the same time, the economic cost is enormous, as vaccine preparation requires a large amount of human and material resources. Our team, with funding from the National Natural Science Foundation of China, discovered that gliomas are generally cold tumors with few mutations and very few neoantigens! Therefore, developing other more effective universal antigens is crucial. Once the antigens are established, if these antigens are expressed in the patient's tissues, vaccines can be rapidly produced using these pre-defined antigens to protect postoperative patients in a timely manner (we also found that the higher the tumor burden, the more effective the treatment; timely postoperative vaccine therapy is essential for high-efficiency protection). Additionally, if the patient's intraoperative specimens were not cryopreserved in time, the discovery and preparation of pre-defined antigens can also enable timely vaccine treatment. Of course, gliomas are highly heterogeneous tumors; treatment with a single target has limited and short-lived effects, with rapid recurrence and drug resistance. Therefore, a combined approach using multiple antigens is necessary. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a tumor antigen for glioma and its application, with the aim of developing a universal peptide or DC vaccine to solve the technical problems of long vaccine preparation cycles, huge economic costs, and loss of vaccination opportunities for surgical patients.

[0007] The first technical solution provided by this invention is a tumor-specific antigen for glioma, containing URGCP protein fragment, HEATR1 protein fragment and / or CMV protein fragment;

[0008] The URGCP protein fragment contains at least one peptide with an amino acid sequence as shown in SEQ ID NO.1 to SEQ ID NO.15;

[0009] The HEATR1 protein fragment contains at least one peptide with an amino acid sequence such as SEQ ID NO.16 to SEQ ID NO.30;

[0010] The CMV protein fragment contains at least one peptide with an amino acid sequence such as SEQ ID NO.31 or SEQ ID NO.38.

[0011] The second technical solution provided by the present invention is a polypeptide composition containing the specific antigen described in the first technical solution.

[0012] In some embodiments, the polypeptide composition further contains lysate of irradiated A2B5-positive glioma cells (tumor stem cells, the root cause of tumor recurrence).

[0013] In some embodiments, the polypeptide composition includes, but is not limited to, a DC vaccine or an mRNA vaccine, wherein the DC vaccine comprises dendritic cells loaded with the specific antigen described in the first technical solution.

[0014] In some embodiments, the dendritic cells are obtained by isolation or differentiation from monocytes.

[0015] In some embodiments, the monocytes include at least one of bone marrow-derived monocytes, spleen-derived monocytes, peripheral blood-derived monocytes, and umbilical cord blood-derived monocytes.

[0016] The third technical solution provided by this invention is a trivalent vaccine, which contains a trivalent specific antigen, wherein the trivalent specific antigen is composed of an URGCP protein fragment, a HEATR1 protein fragment, and a CMV protein fragment.

[0017] The URGCP protein fragment contains at least one peptide with an amino acid sequence as shown in SEQ ID NO.1 to SEQ ID NO.15;

[0018] The HEATR1 protein fragment contains at least one peptide with an amino acid sequence such as SEQ ID NO.16 to SEQ ID NO.30;

[0019] The CMV protein fragment contains at least one peptide with an amino acid sequence such as SEQ ID NO.31 or SEQ ID NO.38.

[0020] In some embodiments, the trivalent DC vaccine comprises dendritic cells loaded with trivalent specific antigens.

[0021] In some embodiments, the dendritic cells are obtained by isolation or differentiation from monocytes.

[0022] In some embodiments, the monocytes include at least one of bone marrow-derived monocytes, spleen-derived monocytes, peripheral blood-derived monocytes, and umbilical cord blood-derived monocytes.

[0023] In some embodiments, the URGCP protein fragment contains amino acid sequences such as AVFILTDNISK, GISSPSLSEK, and SLSEKQYFL; the HEATR1 protein fragment contains amino acid sequences such as LLPESIPFL and SVQTKAVNK; and the CMV protein fragment contains amino acid sequences such as NLVPMVATV.

[0024] The fourth technical solution provided by the present invention is a trivalent mixed vaccine, which includes a trivalent specific antigen and irradiated A2B5 positive glioma cell lysate. The trivalent specific antigen is composed of URGCP protein fragment, HEATR1 protein fragment and CMV protein fragment. The URGCP protein fragment contains at least one peptide with an amino acid sequence as shown in SEQ ID NO.1 to SEQ ID NO.15.

[0025] The HEATR1 protein fragment contains at least one peptide with an amino acid sequence such as SEQ ID NO.16 to SEQ ID NO.30;

[0026] The CMV protein fragment contains at least one peptide with an amino acid sequence such as SEQ ID NO.31 or SEQ ID NO.38.

[0027] In some embodiments, the amount of lysis buffer used for the irradiated A2B5 positive glioma cells is 10 μg (1 μg / μl = 100 μl).

[0028] In some embodiments, the glioma cells include GL261 cells, primary glioma cells, and primary glioblastoma cells.

[0029] In some embodiments, the irradiated A2B5-positive glioma cells

[0030] The lysis buffer was prepared by treating A2B5 positive glioma cells with a total irradiation dose of 2–8 Gary.

[0031] The fifth technical solution provided by the present invention is a drug containing the specific antigen described in the first technical solution, or the composition described in the second technical solution, or the trivalent vaccine described in the third technical solution, or the trivalent mixed vaccine described in the fourth technical solution.

[0032] In some embodiments, the pharmaceutical product also includes a pharmaceutically acceptable carrier.

[0033] Furthermore, the carrier may be a microcapsule, microsphere, nanoparticle, or liposome.

[0034] In some embodiments, the dosage form of the drug may be a medically conventionally selectable dosage form, including but not limited to injection, lyophilized powder for injection, suspension, implant, embolization, capsule, tablet, pill or oral liquid.

[0035] The sixth technical solution provided by the present invention is the use of the specific antigen described in the first technical solution, or the composition described in the second technical solution, or the trivalent vaccine described in the third technical solution, or the trivalent mixed vaccine described in the fourth technical solution in the preparation of drugs for the prevention, relief and / or treatment of glioma.

[0036] In some ways, the application includes at least one of the following functions:

[0037] (1) Promotes DC homing of individuals;

[0038] (2) Promotes the activation of lymph nodes in individuals;

[0039] (3) Promotes the generation of tumor-specific T cells in individuals;

[0040] (4) Promote the establishment of humoral immunity and memory immunity in individuals;

[0041] (5) Inhibit the progression of individual tumors.

[0042] The technical effects of this invention are as follows:

[0043] This invention utilizes novel therapeutic markers URGCP, HEATR1, and HCMV to screen and validate effective immunogenic peptides, preparing a trivalent mixed vaccine. During the antigen preparation process, selecting an appropriate irradiation dose to inactivate tumor cells, ensuring their loss of tumorigenic potential while maximizing immunogenic cell death, is crucial for improving the efficacy of the trivalent mixed vaccine. Through a series of experiments, it was found that A2B5+GL261 cells receiving a total irradiation dose of 6Gray or higher exhibited significantly reduced proliferation and completely lost their tumorigenic ability. Simultaneously, 6Gray was found to maintain the integrity of the A2B5+GL261 cell membrane, and flow cytometry analysis revealed increased expression of the H2Kb MHCI-like molecule on its surface. Higher doses of irradiation directly led to the disintegration of A2B5+GL261 cells. In vitro validation demonstrated that the trivalent mixed vaccine enhanced the antigen presentation ability of dendritic cells (DCs). After overexpression of HCMV, in vivo experiments verified that the trivalent mixed vaccine promoted DC homing, lymph node activation, tumor-specific T cell generation, humoral immunity, and the establishment of memory immunity. Ultimately, the results were validated using both preventative and therapeutic models, demonstrating that the universal trivalent vaccine can inhibit tumor progression and exhibits good safety. Attached Figure Description

[0044] Figure 1 The diagram illustrates the principle and schematic of the overlapping peptide library and NetMHCpan; A: Overlapping peptide library construction strategy (each peptide contains 9 amino acids, with 1 amino acid shifted each time); B: Working principle of NetMHCpan.

[0045] Figure 2 Immunohistochemical staining of HCMV pp65 in tumor tissues of the DC vaccine group; A: HCMV pp65 expression in each sample of the DC vaccine responder group (left 40×; right 80×); B: HCMV pp65 expression in each sample of the DC vaccine non-responder group (left 40×; right 80×).

[0046] Figure 3 To screen and validate immunogenic peptides for tetramer replacement experiments; A: Flow cytometry detection of FITC signals in control groups 1, 2, and 3 of the tetramer replacement experiment; B: Flow cytometry detection of FITC signals after incubation of HCMV#1, URGCP#1, and HEATR1#1 peptides with HLA-A11:01 subtype; C: Heatmap showing the replacement efficiency of different peptides with HLA molecules (X-axis represents different peptides of HCMV pp65, URGCP, and HEATR1, Y-axis represents HLA-A molecule subtype, darker color represents higher replacement efficiency, black box represents replacement efficiency greater than 98%).

[0047] Figure 4To screen immunogenic peptides using Elispot and flow cytometry; A: Flow cytometry detection of CD3, CD4, and CD8 expression in cells sorted with CD3e magnetic beads; B: Elispot detection of IFN-γ secretion from T cells after 24 h of co-incubation with DMSO, a specified peptide, or CD3 / CD28 stimulating antibodies; C: Statistical analysis of IFN-γ spots in each group; D: Flow cytometry detection of CD137 (top) and CD107a (bottom) expression on the surface of T cells after 24 h of co-incubation with DMSO, a specified peptide, or CD3 / CD28 stimulating antibodies; E: Statistical analysis of CD137 (left) and CD107a (right) expression levels on the surface of T cells in each group. Significant differences between groups are indicated as: ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0048] Figure 5 To construct and validate a GL261 cell line overexpressing HCMV.

[0049] Figure 6 A2B5 + Optimization of optimal irradiation dose for GL261 glioma stem cells; A: Flow cytometry detection of A2B5 marker expression in GL261 cells after A2B5 magnetic bead sorting; B: CCK-8 assay for A2B5 marker expression in GL261 cells after treatment with different irradiation doses (0, 2, 4, 6, 8, 10 Gray). + Changes in the proliferation activity of GL261 cells (days 1, 2, 3, and 4); C: Flow cytometry analysis of A2B5 cells after 6Gray treatment. + H2Kb expression level in GL261 cells; D: In vivo imaging of small animals to detect A2B5 cells after different irradiation doses. + Subcutaneous tumorigenicity of GL261 cells (days 10, 17, and 24); E: Statistical analysis of fluorescence signal intensity in subcutaneously tumorigenic mice to evaluate the effect of different irradiation doses on A2B5 cells. + The effect of GL261 cell tumorigenicity.

[0050] Figure 7 Flow cytometry analysis of DC vaccine maturity and monocyte markers: A: Flow cytometry detection of Ly6C expression in 5 DC vaccine groups; B: Flow cytometry detection of Maturation markers (MHC II(I / ab), CD86, CD80, CD40) in 5 DC vaccine groups; F: Statistical analysis of CD40 expression level in 5 DC vaccine groups. Significant differences between groups are indicated as: ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0051] Figure 8Comparison of maturation markers and antigen presentation capabilities of four groups of DC cells; A: Heatmap showing the expression levels of maturation markers (CD40, CD80, CD86) and MHC genes in different groups of DCs; B: Heatmap showing the GSVA enrichment analysis scores of designated pathways in each group of DCs.

[0052] Figure 9 To trace the accumulation of DID dye-prestained DC cells in draining lymph nodes.

[0053] Figure 10 The expression profiles of chemokines and chemokine receptors in DC cells from different groups.

[0054] Figure 11 Changes in draining lymph node volume after different vaccinations; A: Three days after vaccination, the draining lymph node volume of mice in the combined peptide library group and the mixed antigen group was significantly larger than that in the PBS group and the irradiated antigen group; B: Statistical analysis of lymph node volume in mice three days after vaccination with the four vaccine groups. Significance markers for intergroup differences: ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0055] Figure 12 Immunofluorescence analysis of GL-7 in draining lymph nodes; A: GL-7 immunofluorescence staining showing the expression of GL-7 in draining lymph nodes of different DC vaccine groups; B: Statistical analysis of the proportion of GL-7 positive areas to compare the effects of different vaccine groups on lymph node activation. Significance between groups is indicated by: ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0056] Figure 13 DC vaccine induces IFN-γ secretion from tumor-specific T cells. Significant differences between groups are indicated as follows: ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0057] Figure 14 To dynamically monitor the tumor-killing effect of tumor-specific T cells using RTCA.

[0058] Figure 15 The table shows the changes in serum total IgG, IgG1, and IgG2a concentrations in mice. A: Changes in serum total IgG concentration after multiple vaccinations with the four DC vaccines; B: Changes in serum IgG1 concentration after multiple vaccinations with the four DC vaccines; C: Changes in serum IgG2a concentration after multiple vaccinations with the four DC vaccines. Significance between groups is indicated by: ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0059] Figure 16 Changes in the proportion of CD8+ effector memory T cells in mice; A: Spleen CD8+ + Effector memory T cell gating logic; B: Flow cytometry detection of vaccine-induced spleen CD8 + The proportion of effector memory T cells; C: Statistical analysis of spleen CD8 cells induced by different vaccine groups. + The proportion of effector memory T cells. Significance between groups is indicated by: ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0060] Figure 17 To evaluate the prophylactic DC vaccination model and its anti-tumor effect; A: Schematic diagram of the prophylactic DC vaccination process and the establishment of intracranial tumors; B: In vivo fluorescence imaging to track changes in tumor fluorescence signals in mice with different DC vaccines and assess tumor growth; C: HE staining analysis of intracranial tumor volume in mice with different DC vaccines to verify the inhibitory effect of DC vaccines on tumor growth; D: Mouse survival curve analysis to compare the impact of different DC vaccination strategies on mouse survival.

[0061] Figure 18 This is a schematic diagram of a therapeutic model vaccination.

[0062] Figure 19 To evaluate the antitumor effects of different DC vaccination strategies: A: In vivo fluorescence imaging was used to track changes in tumor fluorescence signals in different groups of mice to assess tumor growth; B: HE staining was used to analyze intracranial tumor volume in mice with different DC vaccines to verify the inhibitory effect of different DC vaccination strategies on tumor growth; C: Mouse survival curve analysis was used to compare the impact of different DC vaccination strategies on mouse survival.

[0063] Figure 20 Analysis of immune cell infiltration in tumor tissues of different DC vaccine groups in a preventive model (10×).

[0064] Figure 21 Analysis of T cell exhaustion status in tumor tissues from different DC vaccine groups in a prophylactic model; A: Immunofluorescence staining analysis of CD4+ in tumor tissues from different DC vaccine groups. + The co-expression of T cells (green) and CTLA-4 (orange) was used to assess CD4. + T cell exhaustion state (left: 10×, right: 80×); B: Immunofluorescence staining analysis of CD8 in tumor tissues of different DC vaccine groups. + Co-expression of T cells (green) and PD-1 (orange) to assess CD8 +T cell exhaustion state; (Left side: 10×, Right side: 80×).

[0065] Figure 22 HE staining of mouse body weight and major organs during DC vaccination; A: Dynamic monitoring of mouse body weight during vaccination in different groups; B: HE staining (10×) of heart, liver, spleen, lung and kidney tissues of DC vaccine therapeutic model mice. Detailed Implementation

[0066] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0067] Test method:

[0068] 1. The glioma tumor tissue used in this patent originated from a phase-one clinical trial of the DC vaccine. This clinical trial has been approved by the Ethics Committee of Huashan Hospital Affiliated to Fudan University (Ethics No.: KY2021-547) and registered in the Chinese Clinical Trial Registry (ChiCTR2100048592) and the US Clinical Trial Registry (NCT04888611). A total of 21 patients with recurrent glioblastoma were enrolled in this clinical trial, including 11 patients in the PD-1 inhibitor monotherapy group and 10 patients in the DC vaccine combined with PD-1 inhibitor group. All enrolled patients signed written informed consent forms, clearly understanding the research objectives, potential risks, and rights protection measures. This patent also involves animal experiments, which have been approved by the Ethics Committee of Fudan University (Animal Ethics Approval No.: 202309001S). All animal experiments and cultures strictly followed the ARRIVE principle (the principle of transparency and reproducibility in animal research reporting). The C57 / B6 mice used in the animal experiments were purchased from Nanjing Jicui Pharmaceutical Co., Ltd., and all had animal quality certificates. All animal breeding and management are carried out at the Animal Center of Fudan University School of Medicine. The Animal Center complies with relevant animal welfare regulations and provides appropriate husbandry environments, including controlled temperature and humidity, a 12-hour light-dark cycle, and sufficient water and food, to ensure the good welfare and health of the animals.

[0069] 2. Immunohistochemical staining of glioma tumor tissue

[0070] 1) Sample collection and fixation: After collection, the tumor samples were rinsed with sterile PBS to remove blood, and the electrocoagulated portion was cut off with sterile scissors. After cutting the tumor tissue to an appropriate size, the samples were placed in test tubes and submerged in sufficient 4% paraformaldehyde.

[0071] 2) Paraffin embedding and sectioning: The paraffin embedding and sectioning of the tumor tissue were performed by Savill Company.

[0072] 3) Dewaxing of paraffin sections: Place the paraffin sections in xylene I for 30 min, then in xylene II for 15 min. After slightly drying the sections, place them in anhydrous ethanol I for 5 min, then in anhydrous ethanol II for 3 min, then in 90% ethanol for 3 min, then in 80% ethanol for 3 min, then in 75% ethanol for 3 min, then in 50% ethanol for 3 min, and finally wash with distilled water for 3 min. Repeat 3 times.

[0073] 4) Blocking endogenous peroxidase activity: After washing the sections, drain them on absorbent paper and use an oil-based pen to circle the periphery of the tissue. Titrate the sections with 0.3% H2O2 solution and incubate at room temperature for 20 minutes. Afterward, rinse three times with distilled water.

[0074] 5) Antigen retrieval: Place the dewaxed slides into a container containing sodium citrate antigen retrieval solution. Microwave on high until boiling (ensuring both the antigen retrieval solution and the water in the pot reach approximately 95°C), then immerse in preheated hot water for 20 minutes. Allow to cool naturally afterward, then rinse three times with distilled water.

[0075] 6) Blocking solution: Take an appropriate amount of blocking solution and titrate it to cover the section, then block for 30 minutes (to block the non-specific binding of tissue and antibody and reduce background staining).

[0076] 7) Primary antibody incubation: Discard the blocking solution, add an appropriate amount of antibody diluent to dilute the primary antibody to the specified ratio, and incubate overnight at 4°C in a humidified chamber. The next day, remove the chamber and allow it to warm to room temperature for 1-2 hours. Rinse thoroughly three times with TBST for 5 minutes each time.

[0077] 8) Secondary antibody incubation: Dilute the secondary antibody to the specified ratio with an appropriate amount of TBST, titrate 200 μl of secondary antibody to cover the slide, place it in a humidified chamber, and incubate at room temperature for 30 min. After incubation, rinse thoroughly with TBST 3 times, 5 min each time.

[0078] 9) Developing: Add developing solution (one drop of white and two drops of red) to 2.5 ml TBST, titrate an appropriate amount of developing solution to cover the section, develop for about 2 minutes, and then stop developing in water.

[0079] 10) Cell nucleus staining: Counterstain cell nuclei with hematoxylin and eosin for 30 seconds, then recover the staining solution.

[0080] 11) Washing: Wash with running water for 15 minutes.

[0081] 12) Dehydration: Place the sections in 50% ethanol for 1 second, 75% ethanol for 1 second, 80% ethanol for 1 second, 90% ethanol for 1 second, anhydrous ethanol I for 1 second, and anhydrous ethanol II for 1 second in sequence. Place the sections in a fume hood to dry.

[0082] 13) Mounting: Drop an appropriate amount of neutral resin to the center of the slide, and gently mount the slide with a coverslip to avoid air bubbles.

[0083] 3. Immunohistochemical scoring method

[0084] Immunohistochemistry was performed using Aipathwell software, an AI-based digital pathology image analysis software. Employing AI deep learning principles, the algorithm is trained on massive datasets and integrated into an automated image analysis system. The specific process is as follows: 1) Tracing: Automatically locates the tissue and delineates the test area along the tissue. Manual positioning is possible based on specific requirements. 2) Color Selection: Automatically determines positives and classifies them according to HSI (Hue, Saturation, Intensity): weak positive (pale yellow, 1 point); moderate positive (brownish yellow, 2 points); strong positive (brownish brown, 3 points). Manual adjustments are possible based on specific circumstances. 3) Calculation: The software automatically locates cell nuclei and expands the cytoplasmic range; calculates the number and area of ​​weak, moderate, and strong positive cells; calculates the integrated optical density (IOD); and measures tissue area and other parameters. 4) Analysis: The test area is calculated step-by-step under high magnification. After completion, the software automatically calculates each item based on the original baseline data and algorithm formulas to obtain the analysis results.

[0085] Analysis of parameters related to the number of positive cells includes: 1) Positive cell ratio = Number of positive cells / Total number of cells. This reflects the quantity of positive cells of the same type. 2) H-score: An abbreviation for Histochemistry score, it is a histological scoring method for immunohistochemistry. It converts the percentage of positive cells and their staining intensity within each slide into corresponding numerical values, achieving a comprehensive semi-quantitative analysis of both the depth and quantity of positive staining in tissue immunostaining. H-Score (∑(pi×i)=(percentage of weak intensity cells×1)+(percentage of moderate intensity cells×2)+(percentage of strong intensity cells×3), where i represents the positive cell grade: negative (no staining), 0 points; weakly positive (pale yellow), 1 point; moderately positive (brownish-yellow), 2 points; strongly positive (brownish-brown), 3 points. pi represents the percentage of positive cells in the corresponding grade). The H-score ranges from 0 to 300; a higher value indicates a stronger overall positive intensity in terms of both depth and quantity. It has the widest applicability.

[0086] 4. Tetramer displacement experiment

[0087] Using QuickSwitch TM Tetramer replacement kit, all steps were performed according to the manufacturer's instructions.

[0088] Part 1: Using peptide exchange to generate new specific tetramers.

[0089] 1) Dissolve each lyophilized peptide to be tested in DMSO solution to prepare a 10 mM solution (approximately 10 mg / ml for a peptide containing 9 amino acids). This peptide solution can be further diluted with water to the desired concentration. For high-affinity peptides, a 1 mM stock solution is a reasonable starting concentration. For low-affinity peptides, higher concentrations may be required, but this may lead to tetramer aggregation.

[0090] 2) Add 50μl of QuickSwitch TM Transfer the tetramer into the wells of a microcentrifuge tube or a 96-well microtiter plate with a round or conical bottom. Add 1 μl of peptide and gently mix by pipetting. Add 1 μl of peptide exchange factor from the vial with the green cap and gently mix by pipetting. Incubate at room temperature in the dark for at least 4 hours.

[0091] Part 2: Quantitative peptide exchange assay using flow cytometry sandwich immunoassay.

[0092] 5) Prepare 1× assay buffer as follows: For 1-5 peptide exchanges, mix 750 μl of 10× concentrated assay buffer with 6.75 ml of distilled water to prepare 7.5 ml of 1× buffer. For 6-10 peptide exchanges, double the volume.

[0093] 6) Immediately before use, vibrate the tetramer trapping beads for 60 seconds, then sonicate them for 30 seconds in a water bath ultrasonic processor. If an ultrasonic processor is unavailable, vibrate for an additional 30 seconds.

[0094] 7) Mix the three control wells and each peptide exchange tetramer to be tested in a round-bottom or conical-bottom 96-well microplate for 60 seconds, and add 20 μl of magnetic trap beads to each well.

[0095] 8) Add 5 μl of 1× assay buffer to well #2. Add 5 μl of QuickSwitch buffer to wells #1 and #3. TM Tetramer. In well #4, add 5 μl of solution taken from the first peptide exchange microtube. For each additional peptide exchange, repeat this process, adding the solution to the adjacent well. After gently mixing, shake the plate at 550 rpm for 45 min and protect it from light, for example, by covering it with aluminum foil.

[0096] 9) Add 150 μl of 1× assay buffer to each well (add the buffer first, then place the plate on the 96-well magnet). Attach the microplate firmly to the magnet, protect from light, and let it stand for 5 minutes. Gently shake off the supernatant and tap the plate lightly. Then gently tap the plate on a paper towel to completely remove the supernatant. Attach the microplate firmly to the magnet, along with the plate and magnetic holder, and shake for 2 seconds to disperse the beads.

[0097] 10) Dilute the 25-fold existing peptide antibody to 1-fold as follows: Determine the number of samples (n) to be stained with the antibody, including control wells #2 and #3. Add 1 (+1) to account for pipetting error. In a microtube, add (n+1) × 24 μl of assay buffer, then add (n+1) × 1 μl of the existing peptide antibody. Mix by pipetting. Add 25 μl of 1× existing peptide antibody to all wells (except well #1). Add 25 μl of 1× assay buffer to well #1. Gently mix, then vortex at 550 rpm for 45 min and incubate in the dark.

[0098] 11) Rinse with 150 μl / well of 1× assay buffer. Place the microplate firmly against the magnet and let it stand in the dark for 5 minutes. Gently shake off the supernatant and tap the plate lightly. Then gently tap the plate on a paper towel to completely remove the supernatant. Place the microplate firmly against the magnet, along with the plate and magnetic holder, and shake for 2 seconds to disperse the beads.

[0099] 12) Resuspend the beads in 200 μl of 1× assay buffer, mix gently, and acquire the data on a flow cytometer, preferably within 3 hours. Collect at least 300-500 events per sample to obtain reliable data.

[0100] Part Three: Flow Cytometry Setup and Data Analysis

[0101] 13) Take 5 μl of magnetic capture beads from the red-capped vial and add them to a flow cytometer tube containing 200 μl of 1× assay buffer as a “beads only” control.

[0102] 14) Adjust the voltage, gain, and threshold of FSC and SSC to keep bead events within range. Set thresholds for single beads based on FSC and SSC parameters to exclude dimers and aggregates.

[0103] 15) Set the voltage and gain for FITC and the second fluorescent dye (PE, APC, or BV421) (compensate for the beads group and Control#1 group) so that the average fluorescence intensity (MFI) of "beads only" is at the first logarithmic scale. Record the MFI of the FITC channel (MFI_FITC).

[0104] 16) Run Comparison #1 (QuickSwitch with Bead Capture) TM(Tetramer), adjust the compensation so that the MFI_FITC of control #1 beads is equal to the MFI_FITC of the "beads only" control. The values ​​shown are for demonstration purposes only, and the actual values ​​will vary depending on the experiment and flow cytometry conditions.

[0105] 17) Run control #2, beads that did not capture any tetramers and therefore had no expelled peptides. Low MFI_FITC corresponds to 0% expelled peptides or 100% peptide exchange (no tetramers and antibody added). Record MFI_FITC.

[0106] 18) Running comparison #3, QuickSwitch was captured. TM For tetrameric beads, the MFI_FITC corresponds to 100% expelled peptide or 0% peptide exchange (only tetramer and antibody group added), and the MFI_FITC is recorded.

[0107] 19) Run the samples from well #4 and subsequent peptide exchange samples, and record MFI_FITC. The tetramers of peptide exchange will show different amounts of expelled peptides, which are inversely proportional to the amount of peptide newly loaded onto the MHC molecule. Therefore, the measured MFI_FITC will fall between the MFI values ​​obtained from the beads of control #2 and control #3.

[0108] 20) Replacement efficiency calculation: The peptide-HLA molecule replacement efficiency is calculated according to the following formula.

[0109] 5. Sorting mouse spleen T cells with magnetic beads

[0110] 1) Spleen isolation: Six- to eight-week-old C57BL / 6 mice were anesthetized and euthanized by cervical dislocation. After soaking in 75% alcohol for 5 minutes, the mice were removed and placed on a sterile operating table with their left ventral side facing upwards. A small incision was made in the middle of the left ventral side of the mouse, the skin was torn open, and the abdominal wall was exposed, revealing a long, red, strip-shaped spleen. The peritoneum was lifted from the lower side of the spleen, cut open, and turned upwards to expose the spleen. The spleen was lifted with forceps, and the connective tissue beneath the spleen was separated using ophthalmic scissors. The spleen was removed and immersed in RPMI-1640 medium.

[0111] 2) Grind spleen tissue: After chopping and grinding the spleen, transfer the ground tissue through a 40μm filter to a 50ml centrifuge tube, centrifuge at 300g for 5min, and discard the supernatant.

[0112] 3) Lysis of red blood cells: Add 5 ml of red blood cell lysis buffer, mix well by pipetting, and incubate at room temperature for 5 min; add 20 ml of PBS, centrifuge at 300 g for 5 min, and discard the supernatant.

[0113] 4) Cell filtration: Add 20 ml PBS, resuspend the cells, filter the cell suspension through a 40 μm filter, centrifuge at 300 g for 5 min, and discard the supernatant. 5) Antibody incubation: Resuspend the cells in 10 ml PBS and count them. Centrifuge at 300 g for 5 min at 4 °C, discard the supernatant, and resuspend each × 10^7 cells / cell in 90 μl of MACS buffer. Add 10 μl of mouse CD3e-Biotin or other antibody to each × 10^7 cells, mix thoroughly by pipetting, and incubate at 4 °C in the dark for 15-20 min.

[0114] 6) Washing: Add 5 times the volume of MACS buffer to wash, centrifuge at 300g for 5 min, and discard the supernatant; add 80μl of MACS buffer to resuspend each 1×107 cells.

[0115] 7) Magnetic bead capture: Add 20 μl of Anti-Biotin MicroBeads to each 1×10^7 cells, mix thoroughly by pipetting, and incubate at 4°C in the dark for 10 min.

[0116] 8) Wash again: Add 1-2 ml of MACS buffer to wash, centrifuge at 300 g for 10 min, and discard the supernatant.

[0117] 9) Magnetic field sorting: Resuspend the cells in an appropriate amount of MACS buffer until slightly transparent, up to 1.25 × 10^8 cells / 500 μl. Add 3 ml of MACS buffer to activate the sorting column; pass the resuspended cell suspension through the column in batches.

[0118] 10) Cell washing: After all the cell suspension has dripped down, add 3-5 ml of MACS buffer to wash the column again; add 3-5 ml of MACS buffer and use a pusher to collect the cells.

[0119] 11) Cell culture: In this study, mouse spleen T lymphocytes were cultured in RPMI 1640 containing 10% fetal bovine serum, 1% penicillin / streptomycin, and 20 ng / ml mouse IL-2, and cultured at 37°C in a 5% CO2 incubator.

[0120] 6. HCMV virus overexpression

[0121] 1) Cell preparation: Digest and count GL261 cells the day before infection, and add 1×10^5 cells / well to each well of a 24-well plate to ensure that the cell confluence is between 40% and 60% when the virus is infected the next day.

[0122] 2) Viral infection (1 / 2 volume method): Begin viral infection when cell confluence reaches 40%-60%. Before infection, remove the virus and thaw it on ice. Aspirate the original culture medium from the cells. Mix 200 μl of fresh culture medium, 50 μl of control virus or overexpression virus, and 0.5 μl of Polybrene and add the mixture to the wells. Four hours after lentiviral infection, bring the DMEM medium to a final volume of 500 μl.

[0123] 3) Cell medium change and passage: Change the medium approximately 24 hours after infection, removing all culture medium and adding 500 μl of fresh culture medium. When the cells reach 90-100% confluence, digest and passage them sequentially into 6-well plates and 10 cm cell culture dishes.

[0124] 4) Cell screening: 48 h post-infection, cells carrying green fluorescent protein (GFP) began to express GFP, reaching peak expression 72 h post-infection. After the cells stabilized, they were screened by adding fresh culture medium containing 1 μg / ml puromycin.

[0125] 5) Sample collection and cryopreservation: After cell expansion, cryopreserve cells at a density of 1×10^6 / ml. If necessary, take a portion of the cells for relevant experiments.

[0126] Raw materials used in the examples:

[0127] 1. The peptides were synthesized by Nanjing Genscript Biotech Co., Ltd., and the purity was required to be >95%;

[0128] 2. The tetramer replacement kit was purchased from MBL Corporation;

[0129] 3. C57 / B6 mice (6-8 weeks old, weighing approximately 18-22g) were purchased from Jicui Pharmaceutical Co., Ltd.

[0130] 4. The Elispot test kit was purchased from Mabtech.

[0131] 5. The ELISA test kit was purchased from Thermo Fisher Scientific;

[0132] 6. The RTCA chip was purchased from Agilent Technologies.

[0133] 7. Flow cytometry antibodies were purchased from BioLegend.

[0134] 8. The GL261 cell line was purchased from Fuheng Biotechnology Co., Ltd.

[0135] Example 1: Screening for Immunogenic Peptides

[0136] Because MHC class I molecules have specific binding pockets deep within their binding grooves, they can only accept shorter peptide chains with lengths between 9 and 11 amino acids. Most MHC class I peptide ligands contain 9 amino acids, therefore prediction tends to focus on peptides of this length. Using the DC vaccine efficacy biomarkers URGCP and HEATR1 as targets (disclosed in publication number CN118818055A), an overlapping peptide library technique was used to systematically screen for their immunogenic epitopes. The specific steps are as follows:

[0137] URGCP and HEATR1 proteins were analyzed using the overlapping peptide library method. Nine-mer overlapping peptide libraries (1 amino acid step) were designed for the full-length sequences of both proteins, generating 1030 peptides (URGCP) and 2146 peptides (HEATR1), respectively. Figure 1 A). This method covers all potential T-cell epitopes, effectively addressing HLA polymorphisms and reducing treatment resistance due to antigen loss.

[0138] Next, NetMHCpan v4.1 was used to predict the affinity of peptides for HLA molecules, and peptide adaptability was optimized by combining this with high-frequency HLA typing in the population (HLA-A11:01: 20.893%; HLA-A02:01: 12.036%). Figure 1 B). Neoantigens identified by NetMHCpanv4.1 software were classified according to their RNAK values. The RNAK value is a correction for affinity scoring (the ratio of predicted peptide affinity to the affinity of a set of random natural peptides). A smaller RNAK value indicates a smaller deviation and a stronger binding affinity between the peptide and MHC class I molecules. In this embodiment, RNAK < 0.5 was used as the screening threshold. If RNAK < 0.5, the short peptide was considered a strong-binding antigenic peptide of MHC class I molecules. Ultimately, 37 high-affinity epitopes for URGCP (e.g., STVGVPGTGK, RNAK = 0.03399) and 80 epitopes for HEATR1 (e.g., RVFSLLQKK, RNAK = 0.0059) were obtained. The 10 protein fragments with the best affinity for each were selected as candidate targets for the two universal DC vaccines, URGCP and HEATR1, respectively, as shown in Table 1.

[0139] Table 1. Affinity of the top 10 URGCP and HEATR1 peptides to HLA molecules

[0140]

[0141]

[0142] Example 2: The Association Between Human Cytomegalovirus and Immunotherapy

[0143] After identifying URGCP and HEATR1 as candidate targets for two universally applicable DC vaccines in Example 1, further exploration of other suitable antigen targets was conducted. Several studies have already confirmed that HCMV is an effective target for glioma treatment. Therefore, immunohistochemical staining was used to quantitatively detect the expression of HCMV pp65 in tumor samples from clinical trial patients. The results showed that all tumor samples in the vaccine group (glioma tumor tissue) highly expressed the HCMV pp65 fragment, confirming its theoretical basis as a targeted antigen. Figure 2 (AB). Therefore, it is inferred that HCMV pp65 is one of the ideal antigens for developing a universal DC vaccine. Based on this, the following eight peptide fragments were selected as candidate targets for vaccines containing HCMV pp65 antigen (Table 2):

[0144] Table 2 HCMV peptides

[0145]

[0146]

[0147] Example 3: Performance Verification of Candidate Peptides

[0148] (1) Tetramer substitution assay identifies peptides with high affinity for HLA-I molecules.

[0149] Due to the availability of mouse spleen T cells and the convenience of subsequent in vivo mouse experiments, the high-affinity antigenic peptides screened in Examples 1 and 2 were subjected to human-mouse homology analysis. The human-mouse homologous high-affinity peptides were then synthesized in vitro, requiring a peptide purity greater than 95%. Next, two HLA-A subtypes (HLA-A11:01 and HLA-1 02:01) with a distribution greater than 5% in the Chinese population were selected for subsequent tetramer replacement experiments.

[0150] The experimental results showed that in Control 1, the magnetic beads captured tetramers (APC positive) but did not undergo peptide substitution (FITC negative), indicating that there was no spontaneous dissociation or non-specific binding in the experimental system; in Control 2, the magnetic beads did not capture tetramers (APC negative) and did not undergo peptide substitution (FITC negative), which was used to exclude interference from free tetramers; in Control 3, the magnetic beads captured tetramers (APC positive) and underwent peptide substitution (FITC positive), confirming the sensitivity and specificity of the detection system. Figure 3 A).

[0151] Using the formula for calculating replacement efficiency

[0152]

[0153] The substitution efficiencies of peptides with matching HLA-A subtypes were obtained. Specifically, the substitution efficiency of HCMV peptide 1 with HLA-A 02:01 was 96.1%, URGCP#1 with HLA-A 02:01 was 97.9%, and HEATR1 peptide #1 with HLA-A 02:01 was 94.8%. Figure 3 B). The positive rate of most peptides was between 90% and 98%, while the positive rate of a small number of peptides was greater than 98% or less than 90%. Among them, peptides with a replacement efficiency greater than 98% included 3 HCMV-HLA-A 02:01 interacting peptides, 1 HCMV-HLA-A 11:01 interacting peptide, 2 URGCP-HLA-A 02:01 interacting peptides, 2 URGCP-HLA-A 11:01 interacting peptides, 1 HEATR1-HLA-A 02:01 interacting peptide, and 1 HEATR1P-HLA-A 11:01 interacting peptide. Figure 3 C). Ultimately, we defined peptides with a replacement efficiency greater than 90% as high-affinity peptides for subsequent analytical validation.

[0154] (2) In vitro experiments to verify the immunogenicity of candidate peptides

[0155] After confirming the high affinity of HCMV pp65, URGCP, and HEATR1 peptides for HLA-A molecules, further investigation was conducted to determine whether these peptides could induce a sufficient immune response, i.e., whether these peptides possess immunogenicity. Human-mouse homologous peptides with a replacement efficiency greater than 90% were selected for further validation using enzyme-linked immunospot assays (Elispot). CD3+ was sorted from mouse spleen cells using magnetic bead sorting. + T cells were analyzed, and their positivity rate was determined by flow cytometry. The results showed that approximately 88.41% of the sorted cells expressed CD3 molecules. Figure 4 A). After activating the Elispot plate according to the instructions, we set up negative control wells (DMSO group), positive control group (CD3 / CD28 antibody stimulation group), and peptide stimulation group. The final results showed that peptides such as URGCP peptide #2, URGCP peptide #9, and HEATR1 peptide #5 stimulated T cells to produce large amounts of IFN-γ secretion. Figure 4 B). Spot statistical analysis also showed that URGCP peptide #2 (average 151 IFN-γ spots, p = 0.0032), URGCP peptide #9 (average 438 IFN-γ spots, p < 0.0001), and HEATR1 peptide #5 (average 613 IFN-γ spots, p = 0.0021) were significantly different from the DMSO control group (average 29 IFN-γ spots). Simultaneously, the CD3 / CD28 positive stimulation group also showed significantly higher expression of IFN-γ (…). Figure 4 C).

[0156] In addition, the expression of T cell activation markers CD137 and CD107a was detected. Flow cytometry results demonstrated that peptides URGCP peptide #2, URGCP peptide #3, URGCP peptide #9, HEATR1 peptide #5, and HEATR1 peptide #10 all significantly stimulated T cell activation. Figure 4 D). Notably, URGCP peptide #2, URGCP peptide #3, URGCP peptide #9, HEATR1 peptide #5, and HEATR1 peptide #10 all stimulated CD137 expression, showing a significant difference compared to the DMSO control group (p<0.05). However, only URGCP peptide #9 and HEATR1 peptide #5 stimulated CD107a secretion. Figure 4 E).

[0157] Example 4: Constructing HCMV overexpressing HCMV OE -GL261 cell line

[0158] DC vaccines induce anti-tumor immunity dependent on activated T cells recognizing corresponding tumor antigen fragments. The mouse GL261 cell line itself lacks the viral genome of HCMV pp65, therefore it cannot be directly used to study the anti-tumor effect induced by the HCMV pp65 peptide. The HCMV pp65 protein sequence was extracted for designing an overexpression virus containing known HCMV immunogenic epitopes and antibody recognition epitopes (pp65: 251-350 / 561). The HCMV pp65 protein fragment sequence was overexpressed in the GL261 cell line, and resistance selection was performed using 1 μg / ml puromycin, ultimately constructing the HCMVOE-GL261 cell line.

[0159] Immunofluorescence staining revealed the expression and distribution of URGCP, HEATR1, and HCMV in GL261 cells: URGCP and HEATR1 proteins were preferentially expressed in the cytoplasm, with a small amount distributed in the nucleus, while overexpressed HCMV was mainly distributed in the cytoplasm. Figure 5 ).

[0160] Example 5: Deactivation of A2B5 + Exploration of the appropriate irradiation dose for GL261 stem cell-like tumor antigen

[0161] First, A2B5 was enriched from the GL261 mouse glioma cell line using flow cytometry. + Glioma stem cells, flow cytometry analysis showed A2B5 + The positive rate of cells was as high as 84.56% ( Figure 6A). Since the stemness characteristics of different tumor cell populations affect their sensitivity to radiotherapy, the selection of the total irradiation dose becomes a key factor. To clarify A2B5... + The optimal irradiation dose for GL261 glioma stem cells was determined using whole-cell irradiation, with dose gradients of 0, 2, 4, 6, 8, and 10 Gray. Cellular biological effects were assessed. Following successful irradiation, A2B5 levels were dynamically monitored at 1, 2, 3, and 4 days post-irradiation using the CCK-8 assay. + The proliferative capacity of GL261 cells ( Figure 6 B). The results showed that the cell viability of the 2- and 4-Gray treatment groups was slightly decreased compared to the unirradiated control group (0Gray); the cell viability of the 6-Gray treatment group remained relatively stable during the monitoring period, with no obvious proliferation trend; the 8- and 10-Gray treatment groups showed obvious cell death patterns from day 1, and cell viability was significantly decreased. In summary, 6Gray was the optimal irradiation dose, which could completely inhibit A2B5. + The proliferation capacity of GL261 cells was assessed while preventing complete apoptosis, in order to further study the expression changes of MHC-I molecules and their immunological effects.

[0162] To investigate the effect of radiotherapy on the expression of MHC-I molecules, flow cytometry was used to detect the expression of H2Kb (mouse MHC-I molecule) at the A2B5 molecule. + GL261 cell surface expression level ( Figure 6 (C) The results showed that the H2Kb expression level was lower in the unirradiated group, while the H2Kb expression was significantly upregulated in the 6Gray-treated group. This finding demonstrates that radiotherapy can promote the migration of endogenous MHC-I molecules to the surface of tumor cells, thereby enhancing the immunogenicity of tumor cells and making them more easily recognized by antigen-specific T cells.

[0163] To further verify the effects of irradiation on A2B5 + The in vivo proliferation capacity of GL261 cells was assessed by treating A2B5 cells with different irradiation doses. + GL261 cells were subcutaneously inoculated into the left lower abdomen of C57BL / 6 mice, and tumor growth was monitored by in vivo fluorescence imaging. Figure 6 D). Statistical analysis of fluorescence signals confirmed that 6Gray effectively inhibits A2B5. + The appropriate dose for GL261 cell proliferation and tumorigenesis in vivo ( Figure 6 E).

[0164] Example 7: Performance Verification of the Trivalent DC Vaccine

[0165] (1) Combination antigen library and mixed antigen loading enhance the antigen presentation ability of DC.

[0166] Different antigen groups, including PBS, irradiated A2B5-positive GL261, a combined antigen library (a mixture of five peptides screened in Example 3 and CMV fragments NLVPMVATV), and a mixed group (irradiated A2B5-positive GL261 combined with the combined antigen library), were applied to the antigen loading stage of immature DCs. Mature DC vaccine experiments were divided into four groups: PBS group, irradiated A2B5-positive GL261 group, combined antigen group, and mixed group. These four experimental groups were simplified and named DC@PBS, DC@GL261, DC@Peptide (trivalent vaccine), and DC@Combined (trivalent mixed vaccine), respectively.

[0167] After the DC vaccine matured, flow cytometry was used to detect the monocyte marker Ly6C and maturation markers (MHC II (I / ab), CD40, CD80, CD86, etc.). In each DC vaccine group, the majority of cells (59.03% to 63.60%) expressed the monocyte marker Ly6C, indicating that most DCs were induced from differentiation of bone marrow monocytes, and a minority were differentiated from DC precursor cells already present in the bone marrow cells themselves. Figure 7 A). Furthermore, the DC maturation markers MHC II (I / ab), CD80, and CD86 were all highly expressed in different vaccine groups, demonstrating that different combinations had little impact on DC maturation. Figure 7 BD).

[0168] CD40 plays a crucial role in dendritic cell (DC) antigen presentation, serving as a key molecule for DC activation and immune responses. By binding to its ligand CD40L, CD40 activates dendritic cells and promotes their maturation, a process involving the upregulation of co-stimulatory molecules on the dendritic cell surface (such as CD80 and CD86). In mature dendritic cells, CD40 signaling not only enhances the cells' antigen-presenting capacity, thereby more effectively initiating T-cell immune responses, but also strengthens the secretion of cytokines (such as IL-12), further promoting T-cell activation and differentiation. IL-12 release is particularly important for Th1 cell generation, contributing to enhanced cell-mediated immune responses. Significant differences in CD40 expression exist among different types of vaccines. Figure 7 E). Among them, the CD40 expression level of DC@Combined was significantly higher than that of DC@PBS (p<0.0001), DC@GL261 (p=0.0002) and DC@Peptide (p=0.0013). Figure 7 F). The CD40 expression induced by DC@Peptide was also higher than that induced by DC@GL261. This further demonstrates that both DC@Peptide and DC@Combined can significantly enhance the antigen presentation ability of DC vaccines by increasing CD40 expression.

[0169] To better analyze the function and status of different DC vaccines, bulk transcriptome sequencing was performed on DC cells loaded with different experimental groups. The DC@Combined group showed the highest CD40 expression, followed by the combined peptide library group. The expression levels of maturation markers CD80, CD86, and MHC class II molecules did not differ significantly among the groups. Furthermore, DCs derived from C57 / B6 mouse bone marrow cells did not express the H2-Ea gene (…). Figure 8 A). Further GSVA analysis of gene expression matrices using the GO and Reactome databases revealed that the antigen presentation level of DC@Combined was significantly higher than that of other vaccine groups, including T-cell antigen presentation, MHC class I antigen presentation, and MHC class II antigen presentation. Furthermore, the activation level of the antigen extraction pathway in DC@Peptide was also higher than that in DC@GL261. Figure 8 B). The above findings further demonstrate that DC@Peptide and DC@Combined have the characteristic of significantly enhancing the presentation ability of DC antigens.

[0170] (2) DC@Peptide (trivalent vaccine) and DC@Combined (trivalent mixed vaccine) promote the infiltration of DC vaccine into draining lymph nodes.

[0171] To track the migration pathway of dendritic cells (DCs), four groups of DC vaccines (DC@PBS, DC@GL261, DC@Peptide, and DC@Combined) were labeled with DID near-infrared fluorescent dye and injected subcutaneously into the paw pads of mice (1*10^7 DC cells / 100ul PBS). Subsequently, the distribution of DID dye signals was observed on days 1, 2, and 3 post-inoculation using an in vivo fluorescence imaging system.

[0172] The results showed that DID signal infiltration into the inguinal lymph nodes was significantly detectable on day 3 after DC@Peptide and DC@Combined inoculation. In contrast, no obvious signal was observed in the inguinal lymph nodes of mice inoculated with DC@PBS and DC@GL261 on day 3, suggesting weaker DC homing ability. Furthermore, mice inoculated with DC@Combined showed higher DID signal intensity in the lymph nodes, indicating that this group of DC cells had stronger migration ability and more effectively reached secondary lymphoid tissues. Figure 9 ).

[0173] DC cell homing is regulated by chemokines, adhesion molecules, and cell migration mechanisms. To investigate the mechanisms by which DC@Peptide and DC@Combined promote DC vaccine infiltration into draining lymph nodes, the expression of homing-related chemokines and adhesion molecules in four DC vaccine groups was analyzed. Bulk transcriptome data ( Figure 10The results showed that DC@Peptide and DC@Combined significantly higher expressed chemokines CCR7, LFA-1 (encoded by the Itgal gene), and CD62L (encoded by the Sell gene) than DC@PBS and DC@GL261. CCL19 and CCL21 are secreted by lymphatic endothelial cells and attract CCR7. + DCs migrate to lymphatic vessels, and DCs with high CCR7 expression exhibit stronger lymphatic chemotaxis, thus accelerating homing to draining lymph nodes. LFA-1 (encoded by the Sell gene) mediates DC attachment to ICAM-1-positive lymphatic vessel walls and promotes DC entry into lymphatic vessels through endothelial cells; DCs with high LFA-1 expression can more easily cross the endothelial barrier, thereby enhancing migration ability. Furthermore, CD62L (encoded by the Sell gene) binds to PNAd (peripheral lymph node addressin) in highly endothelial microveins, enabling DCs to smoothly enter the T-cell region of lymph nodes from lymphatic vessels. DC@Peptide and DC@Combined significantly enhance the expression of CCR7, LFA-1, and CD62L in DCs, thereby improving DC adhesion and homing ability. Enhanced DC homing ability can promote more effective migration of DC vaccines to draining lymph nodes, contributing to stronger antigen presentation and T-cell activation, ultimately enhancing the anti-tumor immune response.

[0174] DC@Peptide and DC@Combined can effectively increase the expression levels of DC homing-related molecules, thereby enhancing the ability of DCs to migrate to secondary lymphoid organs.

[0175] (3) DC@Peptide and DC@Combined induce lymph node inflammation and immune activation.

[0176] Four vaccines (DC@PBS, DC@GL261, DC@Peptide, and DC@Combined) were subcutaneously injected into the paw pads of mice, and draining lymph nodes were collected 3 days post-vaccination. Analysis revealed that mice vaccinated with DC@Peptide and DC@Combined exhibited significantly larger draining lymph node volumes, suggesting that this vaccine strategy effectively promotes lymph node immune activation. Statistical analysis showed that the lymph node volumes of both vaccines were significantly larger than those of DC@PBS and DC@GL261, further confirming that DC@Peptide and DC@Combined can more effectively induce lymph node amplification and immune activation. Figure 11 AB).

[0177] Furthermore, considering that lymphocytes within lymph nodes are the core immune cells for antigen presentation and induction of adaptive immune responses by DC vaccines, we further investigated whether the infiltration of four types of DC vaccines into draining lymph nodes could effectively induce activation of the lymphoid region. GL-7 is a key marker of germinal center lymphocyte activation; its high expression represents the entry of B cells into the germinal center response and participates in antibody affinity maturation and adaptive immune enhancement. An increase in GL-7-positive areas reflects the degree of lymphocyte activation induced by the DC vaccine and suggests a stronger immune response. Immunofluorescence staining was used to observe the expression of GL-7 in draining lymph nodes of mice. Ten random fields of view were selected from the immunofluorescence sections of each group of mice, and the proportion of GL-7-positive areas in each field of view was calculated (…). Figure 12 A). The results showed that the proportion of GL-7 positive areas was highest in DC@Peptide and DC@Combined inoculated groups, suggesting that they can effectively promote the formation of germinal centers and lymphocyte activation in lymph nodes. DC@GL261 induced local expression of GL-7 to some extent, but the expression level was still lower than that of DC@Peptide and DC@Combined, indicating that DC homing ability and antigen loading strategy have an important impact on the formation of germinal centers. The DC@PBS group had the lowest GL-7 expression, indicating that the level of lymphocyte activation in lymph nodes was low in the absence of antigen presentation. Figure 12 B).

[0178] (4) DC@Peptide and DC@Combined induce the generation of tumor-specific T cells

[0179] Tumor-specific T cells are key effector cells in the immune activation mediated by dendritic cell (DC) vaccines and are also important indicators for assessing the strength of the vaccine-induced immune response and its anti-tumor efficacy. After DC cells complete antigen presentation in draining lymph nodes, they can activate naive T cells and promote their proliferation and differentiation into effector T cells and memory T cells. Subsequently, some activated T cells enter the peripheral circulation to perform anti-tumor immune functions. The spleen is an important component of peripheral lymphoid organs and plays a crucial role in T cell storage, activation, and secondary immune responses. In particular, memory T cells can be rapidly activated upon re-encounter with corresponding antigens, mediating a stronger anti-tumor immune response and ultimately exerting a specific killing effect.

[0180] To further investigate whether four types of DC vaccines (DC@PBS, DC@GL261, DC@Peptide, and DC@Combined) can effectively induce the generation of tumor-specific T cells and promote their functional activation, mice were randomly assigned to four groups. Starting from day 1, mice were vaccinated every 3 days. After 5 doses, the mice were euthanized, and their spleens were collected. Splenic T cells were sorted for functional testing. Subsequently, the DC vaccine-induced activated splenic T cells from each group were co-incubated with the corresponding antigen group for 24 hours. IFN-γ secreted by the T cells was then detected using Elispot to assess the antigen-specific activation capacity of the T cells.

[0181] The results confirmed that the DC@PBS group (DC vaccine without antigen loading) failed to effectively induce T cell IFN-γ secretion, indicating that the DC vaccine without antigen loading could not induce a specific immune response; DC@GL261 could induce T cell IFN-γ secretion to some extent, but its IFN-γ secretion level was significantly lower than that of DC@Peptide and DC@Combined; both DC@Peptide and DC@Combined significantly enhanced T cell IFN-γ secretion, indicating that they more effectively induced the activation of tumor-specific T cells, thereby enhancing the strength of the anti-tumor immune response. Figure 13 ).

[0182] To further investigate whether splenic T cells reactivated by antigens possess the ability to directly kill tumor cells, real-time label-free cell analysis (RTCA) was used to monitor the cytotoxic effect of T cells on tumor cells in real time. This allowed for quantitative assessment of tumor cell growth status and T cell-mediated cytotoxicity by detecting changes in the electrical impedance of tumor cells during adherence. HCMV was used... OE -GL261 cells were seeded at a density of 50,000 per well. After cell adhesion, splenic T cells activated with antigen were added at a ratio of 2:1, and cell viability was dynamically monitored using RTCA over the following 24 hours. Consistent with the previous results, tumor cells in the DC@PBS group continued to proliferate, and the RTCA impedance curve steadily increased, indicating that this vaccine group failed to effectively induce tumor-specific T cells and could not produce a significant killing effect. Tumor cell proliferation in the DC@GL261 group was slightly inhibited, but the decrease in impedance was small, indicating that the T cells in this group had some killing ability, but the killing efficiency was low. The results of the DC@Peptide group showed a significant decrease in tumor cell impedance, indicating that the T cells in this group could effectively recognize and kill tumor cells. The DC@Combined group had the strongest T cell killing effect, and the tumor cell impedance decreased the fastest and the largest magnitude, suggesting that the T cells in this group had the strongest cytotoxic effect. Figure 14 ).

[0183] The above results indicate that both DC@Peptide and DC@Combined can induce the production of tumor-specific T cells and significantly enhance the tumor-killing ability of T cells, with the mixed antigen-loaded DC vaccine showing the best effect.

[0184] (5) Humoral immunity and the generation of effector memory T cell immunity

[0185] Mice were randomly assigned to four groups (DC@PBS, DC@GL261, DC@Peptide, and DC@Combined). Mice were vaccinated with one dose of DC vaccine every two weeks. Plasma was collected and separated via tail vein microinjection before the initial vaccination and three days after each vaccination. After four doses of DC vaccine, mice were euthanized and their spleens were collected. The plasma from all mice was diluted 100,000 times, and the concentrations and trends of total IgG, IgG1, and IgG2a in serum from different groups and batches of mice were measured using ELISA. It was found that the total IgG concentration in the DC@PBS group remained at a low level and did not change significantly with increasing vaccination frequency. The total IgG concentration in the DC@GL261, DC@Peptide, and DC@Combined groups increased rapidly after the second vaccination and was higher than that in the PBS group. The DC@Combined group showed the fastest increase in total IgG concentration, and its total IgG concentration was significantly higher than all other groups after the second vaccination, suggesting that this group of vaccines could most effectively induce the production of tumor-specific antibodies. Figure 15 A). The overall trend of IgG1 is similar to that of total IgG; its concentration gradually increases with the extension of inoculation time, but the overall increase is smaller than that of total IgG. Figure 15 B). In the DC@Combined group, IgG2a concentration continued to rise rapidly after the second vaccination, peaking after the third vaccination, and then slightly declining, remaining at around 200-300 μg / ml. In the DC@GL261 and DC@Peptide groups, IgG2a concentrations increased only after the third vaccination, indicating that the two vaccines had insufficient ability to induce IgG2a. Figure 15 C).

[0186] The above results demonstrate that DC vaccines can induce tumor-specific IgG production and enhance humoral immune responses, with DC@Combined exhibiting the strongest immunogenicity. DC@Combined can simultaneously promote Th1 and Th2 immune responses, manifested as a rapid and significant increase in IgG1 and IgG2a levels, suggesting that it not only enhances antibody-dependent immunity (IgG1-induced antibody-dependent cell-mediated cytotoxicity) but also promotes cytotoxic T cell activation (IgG2a-related Th1 responses).

[0187] In addition, CD3e magnetic beads were used to label and sort the spleen T cells of the mice that had received four doses of the vaccine, and flow cytometry was used to detect their CD3 content. + CD8 + T cell memory phenotype (CD44) high CD62L low ()( Figure 16 A). The results showed that the DC@Peptide group, DC@Peptide group, and DC@Combined group could all induce a high proportion of effector memory T cell subsets, with the DC@Combined group showing a significantly higher proportion than the other three groups. Figure 16 BC).

[0188] In summary, DC vaccines can induce humoral immunity and effector memory T-cell immunity, and DC vaccines prepared based on a mixed antigen strategy can more effectively enhance long-term immune memory.

[0189] Example 8: Application of trivalent DC vaccine in the treatment of glioma

[0190] (1) The prophylactic model reveals the differences in in vivo antitumor effects mediated by DC vaccines among different groups.

[0191] To evaluate the in vivo antitumor effect of DC vaccines, a prophylactic immunization model of glioma was established and the impact of different vaccine strategies on tumor growth and survival was monitored.

[0192] First, mice were randomly assigned to four groups (DC@PBS, DC@GL261, DC@Peptide, and DC@Combined), and each group received a subcutaneous injection of DC vaccine every 3 days in the nape of the neck for a total of 3 doses. After vaccination, 1×10⁻⁶ DC vaccine as described in Example 4 was implanted intracranially into the mice. 5 HCMV OE -GL26 cells were used, and in vivo fluorescence imaging was employed to track tumor growth. Figure 17 A). In vivo imaging analysis was performed on days 7, 14, and 21 after tumor establishment. The results showed that the DC@PBS group exhibited the strongest fluorescence signal, indicating the fastest tumor growth; the DC@GL261 group showed a lower fluorescence signal, suggesting slower tumor growth; the DC@Peptide group had a slightly lower tumor fluorescence signal than the DC@GL261 group, indicating it was more effective at inhibiting tumor growth than DC@GL261; and the DC@Combined group showed the lowest fluorescence signal, significantly lower than DC@PBS, DC@GL261, and DC@Peptide, indicating the strongest inhibitory effect on tumor growth. Figure 17B). Hematoxylin-eosin (HE) staining also revealed differences in the in vivo antitumor effects among different vaccine groups. The tumors in the DC@Combined group were significantly smaller than those in other groups, further confirming its optimal antitumor effect, while the tumor volume in the DC@Peptide group was slightly smaller than that in the DC@GL261 group. Figure 17 C). Furthermore, mouse survival curves showed that DC@Combined significantly prolonged the overall survival of mice, while the survival of mice in the DC@GL261 and DC@Peptide groups was longer than that in the DC@PBS group, but the difference was not statistically significant (p = 0.0505), suggesting that a single antigen strategy may not be sufficient to maintain long-term anti-tumor immunity. Figure 17 D).

[0193] Prophylactic DC vaccination effectively inhibited glioma growth and prolonged the survival of mice. Among them, DC@Combined showed the best in vivo antitumor effect, suggesting that this strategy can more effectively activate antitumor immune responses and has great application potential.

[0194] (2) Treatment models reveal differences in in vivo antitumor effects caused by different vaccination strategies.

[0195] After validating the effectiveness of preventive vaccines, we will further explore the effects of tumor vaccines on therapeutic models and optimize immunization strategies.

[0196] In the therapeutic model, experimental mice were randomly assigned to six groups: the first group of mice received one dose of DC@PBS on the day of tumor formation, followed by one dose every 3 days. Figure 18 A); The second group of mice were injected with one dose of DC@GL261 on the day of tumor formation, followed by one dose every 3 days. Figure 18 B); The third group of mice were injected with one dose of DC@Peptide on the day of tumor formation, followed by one dose every 3 days. Figure 18 C); The fourth group of mice were injected with one dose of DC@Peptide on the day of tumor formation, followed by one dose of DC@Combined (C) every three days. Figure 18 D); The fifth group of mice were injected with one dose of DC@Combined on the day of tumor formation, followed by one dose every 3 days. Figure 18 E); Group 6 mice were injected with one dose of DC@Combined on day 3 after tumor formation, followed by one dose every 3 days. Figure 18 F).

[0197] Consistent with the preventative model, the therapeutic DC vaccine inhibited tumor growth and prolonged the survival of mice. In group 5, which employed an optimized vaccination strategy (initial vaccination with DC@Peptide followed by a booster vaccination with DC@Combined), the tumor fluorescence signal value and tumor volume were significantly reduced compared to groups 2 (DC@GL261) and 3 (DC@Peptide), and the survival of mice was significantly prolonged. Overall, the efficacy of the optimized vaccination strategy in group 5 was comparable to that in group 4 (DC@Combined). In group 6, the initial vaccination was delayed to day 3 after tumor formation, while the boost time point was delayed to day 6. The tumor fluorescence signal value and tumor volume in this group were significantly greater than those in groups 2 to 5, suggesting that early initial vaccination and induction of adaptive immunity at low tumor burden levels are crucial to improving the clinical benefit of vaccine immunotherapy. Figure 19 AC).

[0198] (3) DC vaccines reverse the immunosuppressive state of the local tumor microenvironment.

[0199] CD3, CD11b, and F4 / 80 were used as markers for T lymphocytes, myeloid cells, and macrophages. Immunohistochemical staining was performed on the brain tissue of mice in four groups after DC vaccination to assess whether the DC vaccine affected the composition of different immune cell populations within the tumor. Results showed that the proportions of myeloid cells (CD11b positive) and macrophages (F4 / 80 positive) did not change significantly among the groups after DC vaccination, suggesting that the DC vaccine had no significant effect on the recruitment of myeloid cells in the tumor microenvironment. Conversely, the proportion of T cells (CD3 positive) changed. Compared with the DC@PBS group, the proportion of T cells in the DC@GL261 and DC@Peptide groups increased slightly, while the proportion of T cell infiltration in the tumor periphery of the DC@Combine group increased significantly, suggesting that this strategy can effectively promote T cell infiltration into the tumor area and may enhance the local anti-tumor immune effect. Figure 20 ).

[0200] To further investigate the effects of DC vaccines on the phenotype and functional status of tumor-infiltrating T cells, immunofluorescence analysis was performed. CTLA-4 was used to label CD4+. + T cell exhaustion state, PD-1 markers CD8 + The study aimed to assess whether different DC vaccines could affect T cell activation and functional exhaustion. Results showed that the proportions of CD4-positive and CD8-positive T cells were increased in the DC@Peptide, DC@GL261, and DC@Combined groups compared to the DC@PBS group. Furthermore, the proportions of CD4-positive and CD8-positive T cells were higher in the DC@Combined group. + CTLA4 +and CD8 + PD1 + The proportion of [a specific cell type] decreased compared to the DC@PBS group, suggesting that it induces a decrease in the expression of T cell exhaustion genes. Figure 21 AB).

[0201] DC@Combined can not only promote T cell infiltration into the tumor microenvironment, but also reduce the functional exhaustion of T cells. It may reverse the immunosuppressive state of the local tumor microenvironment and enhance the efficacy of anti-tumor immune response by enhancing antigen presentation and T cell activation.

[0202] (4) Safety issues of DC vaccines

[0203] During the DC vaccine administration, the body weight of the prophylactic model mice was monitored. The monitoring results showed that the body weight of mice in the control group and each vaccine injection group was within the normal range, and there were no significant differences in any component. Figure 22 A). Furthermore, on day 20, we collected heart, liver, spleen, lung, and kidney tissues from mice in the DC vaccine therapeutic model for HE staining to observe for any organ damage. The results showed that the DC vaccine demonstrated good safety during injection, and no mild inflammatory response was observed in any of the mice's organs. Figure 22 (B) The above observations confirm that the DC vaccine-induced immune response targets tumor cells and does not attack antigens in the mice's own normal tissues. Consistent with previous studies, no serious adverse reactions occurred during the DC vaccine administration period.

[0204] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A polypeptide composition, characterized in that, The composition contains URGCP protein fragments, HEATR1 protein fragments and CMV protein fragments; The URGCP protein fragments are at least one of the peptide segments with the amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 3 and / or SEQ ID NO. 9; The HEATR1 protein fragments are at least one of the peptide segments with the amino acid sequence as shown in SEQ ID NO. 20 and / or SEQ ID NO. 25; The CMV protein fragments are the peptide segment with the amino acid sequence as shown in SEQ ID NO.

31.

2. The polypeptide composition of claim 1, wherein, The polypeptide composition further contains irradiated A2B5-positive glioma cell lysate.

3. A trivalent vaccine, characterized in that, The trivalent vaccine contains trivalent specific antigens, which are composed of URGCP protein fragments, HEATR1 protein fragments and CMV protein fragments, The URGCP protein fragments are at least one of the peptide segments with the amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 3 and / or SEQ ID NO. 9; The HEATR1 protein fragments are at least one of the peptide segments with the amino acid sequence as shown in SEQ ID NO. 20 and / or SEQ ID NO. 25; The CMV protein fragments are the peptide segment with the amino acid sequence as shown in SEQ ID NO.

31.

4. The trivalent vaccine of claim 3, characterized in that, The trivalent vaccine includes dendritic cells loaded with trivalent specific antigens.

5. A trivalent combination vaccine characterized in that, The trivalent mixed vaccine includes trivalent specific antigens and irradiated A2B5-positive glioma cell lysate, which are composed of URGCP protein fragments, HEATR1 protein fragments and CMV protein fragments, The URGCP protein fragments are at least one of the peptide segments with the amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 3 and / or SEQ ID NO. 9; The HEATR1 protein fragments are at least one of the peptide segments with the amino acid sequence as shown in SEQ ID NO. 20 and / or SEQ ID NO. 25; The CMV protein fragments are the peptide segment with the amino acid sequence as shown in SEQ ID NO.

31.

6. The trivalent combination vaccine of claim 2, characterized in that, The preparation method of the irradiated A2B5-positive glioma cell lysate is that A2B5-positive glioma cells are treated with a total irradiation dose of 2-8 Gary.

7. A medicine, characterized in that, The drug of claim 1 or 2, or the trivalent vaccine of claim 3 or 4, or the trivalent mixed vaccine of claim 5 or 6.

8. The pharmaceutical product according to claim 7, characterized in that The drug further includes a pharmaceutically acceptable carrier, or an antibody, mRNA sequence, ligand corresponding to the above antigens; the carrier is a microcapsule, microsphere, exosome, nanoparticle or liposome.

9. The pharmaceutical product according to claim 7, characterized in that The dosage form of the drug can be a commonly selected dosage form in medicine, including but not limited to injection, injection lyophilized powder, suspension, implant, embolization agent, capsule, tablet, aerosol, pill or oral liquid.

10. Use of the polypeptide composition according to claim 1 or 2, or the trivalent vaccine according to claim 3 or 4, or the trivalent cocktail vaccine according to claim 5 or 6 for the preparation of a medicament for the prevention, alleviation and / or treatment of glioma.

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