Antigen polypeptide for anti-tumor immunoregulation as well as screening and application of antigen polypeptide

Abstract

The invention provides an antigen polypeptide for anti-tumor immunoregulation and a screening method and application thereof. The antigen polypeptide is derived from BCG (Bacillus Calmette Guerin) vaccine, and the amino acid sequence is shown as SEQ ID NO.1-8. According to the screening method, immunomass spectrometry detection is combined with antigenicity prediction, and the antigen polypeptide can activate CD8 + T cells and regulate the tumor immune microenvironment. CD8 + T and DC2.4 cells in a tumor are induced to be increased, and the induction effect of delivering the polypeptide by utilizing iodophol is stronger. The screening method provides technical support for development of a novel BCG anti-tumor preparation, the provided antigen polypeptide enriches the application of BCG in tumor resistance, and a thought is provided for development of tumor immunotherapy materials.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to an antigen polypeptide for anti-tumor immune regulation and the screening and application thereof. Background Art

[0002] Malignant tumors are one of the main causes of death in the world today. After surgery, chemotherapy, radiotherapy and targeted therapy, immunotherapy, as an emerging tumor treatment method, has been widely used in the clinic and is expected to bring lasting therapeutic effects. The core goal of tumor immunotherapy is to activate and strengthen the body's immune system, improve its ability to identify, attack and eliminate tumors, and induce the body to form long-term immune memory to eliminate tumor cells and reduce the possibility of recurrence.

[0003] Antigens on the surface of tumor cells are crucial to the immune system because their immunogenicity is determined by key epitope peptides that bind to major histocompatibility complex (MHC) molecules. Once these epitope peptides are recognized as targets by the immune system, they serve as a bridge for cytotoxic CD8+ T cells to interact with tumor cells. Therefore, understanding these peptides is key to developing therapeutic cancer vaccines and stimulating specific anti-tumor adaptive immune responses. By inducing the display of antigen epitopes on the surface of tumor cells, T cell immune responses can be more effectively stimulated.

[0004] Currently, strategies for tumor immunotherapy include immune checkpoint blockade, adoptive cell therapy, tumor vaccines, and nonspecific immune stimulation. These treatments are usually costly and have unsatisfactory efficacy. Using existing drugs to develop new immunotherapeutic drugs is another approach, among which BCG has attracted attention due to its low cost and easy access. As a nonspecific immunostimulator, the local immune-enhancing effect of BCG helps attract immune-active cells to kill tumor cells. Intravesical instillation of BCG is considered the gold standard adjuvant treatment for non-muscle invasive bladder cancer.

[0005] However, there are also some problems in the application of BCG. Due to its complex composition and the restriction of cell surface fibronectin expression, the application of BCG is currently limited, mainly to the treatment of bladder cancer. Therefore, it is urgent to screen out highly effective single antigens from BCG for anti-tumor immune regulation and expand its scope of application. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an antigen polypeptide for anti-tumor immune regulation and a screening method and application thereof.

[0007] The present invention is achieved in that:

[0008] An antigen polypeptide for anti-tumor immune regulation, wherein the amino acid sequence of the antigen polypeptide is one or more of those shown in SEQ ID NO.1-8.

[0009] In some embodiments, the amino acid sequence of the antigenic polypeptide is as shown in SEQ ID NO.1.

[0010] A method for screening antigenic polypeptides comprises the following steps:

[0011] S1, BCG was co-incubated with DC2.4 cells and tumor cells HepG2;

[0012] S2, DC2.4 cells and HepG2 cells were lysed, total proteins were collected, MHC-I antibodies were added to the total proteins, and MHC-I binding peptides were co-precipitated;

[0013] S3, separating the MHC-I binding peptides and identifying them by mass spectrometry to screen out the antigenic peptides that can be co-presented by MHC-I of DC2.4 cells and HepG2 cells;

[0014] S4, theoretical prediction of antigenicity of antigenic peptides that can be co-presented by DC2.4 cells and HepG2 cells MHC-Ⅰ was performed through bioinformatics analysis;

[0015] S5, determining the antigenic polypeptide for anti-tumor immune regulation according to the overlapping region identified by mass spectrometry and predicted by antigenicity theory.

[0016] In some embodiments, the screening method further comprises:

[0017] S6, after pulsing DC2.4 cells with the antigen polypeptide for anti-tumor immunomodulation, co-culture with CD8+T cells, and verify the antigenicity of the antigen polypeptide for anti-tumor immunomodulation according to the degree of activation of CD8+T cells.

[0018] In some embodiments, the co-incubation time in step S1 is 8-12 hours.

[0019] In some embodiments, the MHC-I antibody is an H-2 antibody or an HLA-I antibody.

[0020] A use of the antigen polypeptide in the preparation of an anti-tumor immunomodulatory preparation.

[0021] An anti-tumor immunomodulatory preparation comprises the antigen polypeptide.

[0022] In some embodiments, the anti-tumor immunomodulatory preparation further comprises a drug delivery carrier and a pharmaceutically acceptable emulsifier.

[0023] In some embodiments, the emulsifier is lipiodol.

[0024] The positive effects brought by the present invention are mainly reflected in the following aspects:

[0025] (1) The present invention uses a variety of advanced research methods such as immunopeptidomics technology, high performance liquid chromatography (HPLC), bioinformatics analysis, and in vitro functional screening to conduct in-depth screening and research on peptides in BCG that may have the function of regulating tumor antigen epitopes. The antigenic peptides obtained through screening can effectively bind to MHC-I class molecules, showing their potential in regulating tumor cell antigen epitopes. These peptides can activate CD8+T cells and then change the tumor microenvironment, thereby exerting their anti-tumor effects. Therefore, these screened polypeptides can be further developed and applied as a single anti-tumor component in BCG, thereby significantly expanding the scope of application of BCG in the field of anti-tumor treatment.

[0026] (2) The antigen polypeptides involved in the present invention can also be delivered by combining with iodized oil, which provides new ideas and possibilities for developing novel anti-tumor immunotherapy materials. In this way, the antigen polypeptides can be more effectively delivered into the body, enhancing their role in anti-tumor immune response, thereby improving the therapeutic effect. This innovation in delivery strategy not only provides a new direction for the anti-tumor application of BCG, but also provides a useful reference for the development of other immunotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The mass spectrometry identification results of the tumor immunomodulatory BCG polypeptide provided in Example 1 of the present invention. A is the Venn diagram analysis result of the protein bound to MHC-Ⅰ of DC2.4 and HepG2 cells, and B is the Venn diagram analysis result of the polypeptide bound to HLA-Ⅰ of DC2.4 and HepG2 cells.

[0029] Figure 2 This is the MHC-Ⅰ affinity prediction diagram provided in Example 2 of the present invention. A is a scatter plot of peptides predicted by IDEB to bind to HLA-A*02:01, IC 50 <5000; B is the first 100 ICs 50 Scatter plot of the ranking of peptides with p<50.

[0030] Figure 3 This is the mass spectrometry identification result of the synthetic BCG polypeptide provided in Example 3 of the present invention.

[0031] Figure 4 CD8 provided in Example 4 of the present invention + Analysis results of T cell intracellular factor IFN-γ staining.

[0032] Figure 5 This is a graph evaluating the immunomodulatory effect of lipiodol BCG polypeptide on subcutaneous liver cancer tumors provided in Example 5 of the present invention. A is the CD8 + A is a representative flow cytometry result graph of T cells; B is a quantitative statistical analysis graph of CD8+T cells infiltrating tumor tissues in each treatment group; C is a representative flow cytometry result graph of dendritic cells (CD11c+) infiltrating tumor tissues in each treatment group; D is a quantitative statistical analysis graph of dendritic cells (CD11c+) infiltrating tumor tissues in each treatment group. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] Embodiments of the present invention relate to a specific antigenic polypeptide for regulating anti-tumor immune response. The amino acid sequences of these antigenic polypeptides correspond to any one or more of SEQ ID NO.1 to SEQ ID NO.8. These polypeptides are screened from BCG through a series of advanced technical means, including immunopeptidomics technology, high performance liquid chromatography (HPLC), bioinformatics analysis, and in vitro functional screening. These screening methods ensure that the obtained polypeptides can effectively bind to MHC-I class molecules, thereby playing a key role in the regulation of antigenic epitopes of tumor cells. In addition, these polypeptides can also activate CD8+T cells, thereby enhancing the body's immune response to tumor cells and exerting a significant anti-tumor effect.

[0035] Specifically, the amino acid sequence of SEQ ID NO.1 is: GLMTSVLMTADGKTVEAEAAHGTVTR. The amino acid sequence of SEQ ID NO.2 is: DAGQIAGLNVLRIVNEPTAAALAYGLDK. The amino acid sequence of SEQ ID NO.3 is: KAWKYLMALFSSKIDEHADPKVQIQQAIEEAQRTHQALTQQAAQVIGNQR. The amino acid sequence of SEQ ID NO.4 is: TGEVLSVPVGDGFLGRVVNPLGQPIDGR. The amino acid sequence of SEQ ID NO.5 is: RVTGPVVDVEFPRGSIPELFNAL. The amino acid sequence of SEQ ID NO.6 is: FLRELISNASDALDKLRIEALRNKDLEV. The amino acid sequence of SEQ ID NO.7 is: NNNSYGLQQPPRLG. The amino acid sequence of SEQ ID NO.8 is: EAYPGDVFYLHSRLLERCAKLSDDLGGGSL.

[0036] In certain embodiments, particular attention is paid to those antigenic polypeptides whose amino acid sequences match SEQ ID NO.1. The amino acid sequence of SEQ ID NO.1 is specifically: GLMTSVLMTADGKTVEAEAAHGTVTR. The polypeptides of this specific sequence perform well in activating CD8+T cells and can effectively promote the body's immune response to tumor cells, thereby playing an important role in anti-tumor therapy. Through further research and clinical application, these polypeptides are expected to become an effective means of treating a variety of tumors.

[0037] A method for screening antigenic peptides for anti-tumor immune regulation, the specific steps are as follows:

[0038] S1, first, BCG was co-incubated with DC2.4 cells and tumor cells HepG2, respectively. This process is to simulate the interaction between BCG and immune cells and tumor cells in vivo.

[0039] S2, then, DC2.4 cells and HepG2 cells were lysed to collect the total proteins, and then MHC-I antibodies were added to the total proteins to co-precipitate the peptides bound to the MHC-I molecules by immunoprecipitation.

[0040] S3, separating the MHC-I binding peptides obtained in the above step, and identifying these peptides using mass spectrometry. Through mass spectrometry analysis, those antigenic peptides that can be co-presented by MHC-I of DC2.4 cells and HepG2 cells are screened.

[0041] S4, bioinformatics analysis of the screened antigenic peptides to theoretically predict their antigenicity. This analysis process includes a comprehensive evaluation of the sequence, structure and binding ability of the peptide to MHC-I molecules.

[0042] S5, based on the overlapping regions of mass spectrometry identification results and bioinformatics analysis, identify those antigenic peptides for anti-tumor immune regulation. These peptides are considered to have the potential to activate the immune system and regulate the tumor microenvironment.

[0043] In some embodiments, the screening method further comprises the following steps:

[0044] S6, after pulsing DC2.4 cells with the selected antigenic peptides for anti-tumor immune regulation, they are co-cultured with CD8+T cells. The antigenicity of these antigenic peptides is verified by detecting the degree of activation of CD8+T cells. This verification step helps to confirm whether the peptides can effectively activate immune cells and thus exert anti-tumor effects.

[0045] The present invention combines immunopeptidomics, high performance liquid chromatography (HPLC), bioinformatics analysis, in vitro functional screening and in vivo functional verification to conduct in-depth screening of peptides in BCG that may have tumor antigen epitopes. Through this series of screening processes, the present invention successfully discovered a peptide that can activate CD8+T cells and change the tumor microenvironment. This polypeptide can induce the interaction between CD8+T cells and dendritic cells, thereby contributing to the development of new BCG tumor treatment materials and further expanding the scope of application of BCG in the field of tumor treatment.

[0046] In some specific embodiments, the incubation time involved in step S1 is set to be between 8 hours and 12 hours. This time range is to ensure that the incubation process can be fully carried out, so that the subsequent experimental steps can be carried out smoothly and reliable results can be obtained.

[0047] In some specific embodiments, the MHC-I antibody used can be an H-2 antibody or an HLA-I antibody. These two antibodies target different major histocompatibility complex (MHC) class I molecules, respectively. H-2 antibodies are usually used in experimental animal models such as mice, while HLA-I antibodies are used in human samples. The purpose of selecting the appropriate antibody is to ensure the specificity and accuracy of the experiment.

[0048] An application of the above antigenic polypeptide in the preparation of an anti-tumor immunomodulatory preparation. The antigenic polypeptide has a significant immune activation effect, can effectively activate CD8+T cells, and can change the tumor microenvironment, thereby inducing the activation and proliferation of CD8+T cells and dendritic cells. The application of the polypeptide in an anti-tumor immunomodulatory preparation provides a new idea and method for tumor treatment.

[0049] An anti-tumor immunomodulatory preparation, comprising the above-mentioned antigenic polypeptide. Through the immune activation effect of this polypeptide, the body's anti-tumor immune response can be effectively enhanced. This preparation can be used to treat a variety of tumors, including but not limited to lung cancer, breast cancer, colorectal cancer, etc. This anti-tumor immunomodulatory preparation also has good safety and tolerability, with fewer side effects, and patients are more likely to accept and adhere to treatment. In the future, with further research on this antigenic polypeptide and its mechanism of action in anti-tumor immunomodulation, it is expected that more efficient and safe anti-tumor immunomodulatory preparations will be developed, bringing more hope and choices to tumor patients.

[0050] In certain specific embodiments, the anti-tumor immunomodulatory preparation not only contains its main active ingredient, but also further includes a drug delivery carrier and a pharmaceutically acceptable emulsifier. Such a combination helps to improve the stability and bioavailability of the drug, ensuring that the drug can more effectively reach the target site and exert its therapeutic effect.

[0051] In these embodiments, the effect of emulsifier is to disperse the drug ingredients in aqueous medium, to form a stable emulsion system, thereby to improve the solubility and absorption rate of medicine. The selection of emulsifier is most important for the stability and safety of preparation. In some specific embodiments, selected emulsifier is iodized oil. As a kind of pharmaceutically acceptable emulsifier commonly used, iodized oil has good biocompatibility and safety, can effectively help medicine to distribute and absorb in vivo, can also provide certain imaging effect simultaneously, is convenient for the doctor to monitor and evaluate in clinical application.

[0052] Example 1 Mass Spectrometric Identification of MHC-Ⅰ-Bound BCG Peptides

[0053] (1) Production and purification of MHC-I peptide complexes

[0054] DC2.4 and HepG2 cells were co-cultured with BCG purchased from Ruichu Bio for 10 hours (MOI = 7), and then the supernatant containing BCG was removed and washed twice with PBS. Then, the tumor cell pellet was collected and resuspended in lysis buffer (the buffer contained 0.25% sodium deoxycholate, 0.2mM iodoacetamide, 1mM EDTA, 1:2000 protease inhibitor cocktail, 1mM PMSF, and 1% octyl-β-D-pyranose). The sample was then rotated and incubated at 4°C for 1 hour. Then, the total protein was collected by centrifugation at 48000g for 60 minutes at 4°C. After that, magnetic separation was performed by Protein A / G magnetic beads pre-bound to HLA-Ⅰ antibody (Sigma, W6 / 32). The HLA-Ⅰ-antigen polypeptide complex was eluted using a low pH eluent, and the antibody neutralization buffer was added. Finally, the MHC-Ⅰ peptide segment was studied by quantitative proteomics technology. The Protein A / G magnetic beads, low pH elution buffer, and antibody neutralization buffer used were all from the Protein A / G antibody purification kit from Beaver Biotech.

[0055] (2) Peptide sample pretreatment

[0056] The total eluate was reduced with 10 mM dithiothreitol (DTT) at 37°C for 30 minutes, and then alkylated with 20 mM iodoacetamide (IAA) at room temperature in the dark for 30 minutes. The quality of the reduced and alkylated MHC-peptide samples was controlled using a short SDS-PAGE gel. The gel was cut according to the molecular weight and expression abundance of the sample, and the enzyme was digested with Ac-trypsin (12.5 ng / μl) (for the preparation method of Ac-trypsin, see patent CN201510083691.0) at 37°C for 12-14 hours. Then C 18 StageTip was used for desalting, sample purification, evaporation and storage at -80°C for LC-MS / MS analysis.

[0057] (3) LC-MS / MS analysis of MHC-I binding peptides

[0058] In the experiment of BCG infection of DC 2.4 cells, buffer A [0.1% formic acid (FA), 99.9% ddHO] was used. 2O] After dissolving 500 ng of MHC-I binding peptide, the peptides were detected using the LC-MS / MS platform. The platform includes the Orbitrap Fusion Lumos mass spectrometer and the EASY-nLC 1200system liquid chromatography system produced by Thermo Fisher Scientific, USA. The sample was eluted using a 78-minute nonlinear gradient with a flow rate of 600 nL / min. The detailed gradient is as follows: 6-12% buffer B for 8 minutes, 12-30% buffer B for 50 minutes, 30-40% buffer B for 12 minutes, 40-95% buffer B for 1 minute, and 95% buffer B for 7 minutes [buffer B, 0.1% FA, 99.9% acetonitrile (ACN)]. MS 1 The detection parameters were as follows: mass scanning range was 300-1400 m / z, and the resolution was 1.2×10 5 The automatic gain control (AGC) is set to 5×10 5 , the maximum injection time (MIT) is set to 50ms. MS 2 The detection parameters were as follows: data dependent acquisition (DDA) mode was used, high energy collision dissociation (HCD) was used, normalized collision energy (NCE) was 35%, and the 20 most intense ions were selected for fragmentation. 2 The scan's AGC target was 5000, MIT was 35ms, and dynamic exclusion time was 20s.

[0059] In the experiment of BCG-infected HepG2 cells, 1 μg of extracted MHC-I binding peptides were dissolved in the same buffer A as above, and the peptides were detected using an LC-MS / MS platform, which included an Orbitrap Exploris 480 mass spectrometer and an EASY-nLC 1200system liquid chromatography system produced by Thermo Fisher Scientific, USA. The sample was eluted through a 75-minute nonlinear gradient with a flow rate of 300 nL / min. The detailed gradient is as follows: 7-12% buffer B for 6 minutes, 12-30% buffer B for 51 minutes, 30-45% buffer B for 10 minutes, 45-95% buffer B for 1 minute, and 95% buffer B for 7 minutes. The composition of eluent B is the same as above. Use the DDA acquisition mode, and use the FAIMS dual compensation voltage switching mode for acquisition. The parameters are set to -45V and -65V CV, MS 1 The mass scan range was 350-1500 m / z with a resolution of 6 × 10 4 The AGC was set to automatic mode and the MIT was set to 50ms. The filter strength threshold was set to a minimum of 20,000. MS 2 The high energy collision dissociation (HCD) mode was used with a normalized collision energy (NCE) of 27% and a resolution of 1.5 × 104 , MIT was 22 ms, separation window was 1.6 m / z, and cycle time was set to 1.3 s.

[0060] (4) Proteomics database search: pFind (v3.1.5) software was used to search the Mycobacterium bovis BCG / Pasteur 1173P2 protein data of UniProt (UP000001472, containing 3891 proteins). The specific search parameters were set as follows: 1) The enzyme protein was set to Trypsin, with a maximum of 2 missed cleavage sites allowed; 2) The peptide length was ≥ 7 amino acids; 3) The fixed modification was alkylation modification of cysteine ​​Carbamidomethyl [C]; 4) The variable modification was methionine oxidation modification Oxidation [M] and protein acetylation modification Acetyl [Protein N-term]; 5) The mass tolerance of the precursor ion was 20 ppm, and the mass tolerance of the daughter ion was 0.5 Da; 6) The false discovery rate (FDR) of peptide, protein and secondary spectrum identification was ≤ 5%.

[0061] (5) Selection of BCG protein: BCG peptide MS analysis based on different levels of b / y ion coverage, noise peaks, etc. 2 The quality of the spectra was tested. Among them, spectra with higher b / y ion coverage, fewer and lower noise peaks were screened out to make Venn diagrams, such as Figure 1 shown.

[0062] according to Figure 1 A shows that 47 peptides of BCG protein can be presented by MHC-Ⅰ molecules of DC2.4 cells, while 147 peptides can be presented by MHC-Ⅰ molecules of HepG2 tumor cells. In addition, 11 peptides of BCG protein can be presented by MHC-Ⅰ molecules of DC2.4 cells and HepG2 cells. Figure 1 B revealed that 61 peptides of BCG peptides can be presented by MHC-Ⅰ molecules of DC2.4 cells, while 171 peptides can be presented by MHC-Ⅰ molecules of HepG2 tumor cells. It is worth noting that 9 peptides of BCG peptides can be co-presented by HepG2 tumor cells and DC2.4 cells. These 9 peptides of BCG peptides are derived from 7 BCG proteins among the 11 BCG proteins (which can be co-presented by MHC-Ⅰ molecules of DC2.4 cells and HepG2 cells in 1A), as shown in Table 1:

[0063] Table 1 BCG peptides co-presented by HepG2 tumor cells and DC2.4 cells

[0064]

[0065]

[0066] Example 2 Prediction of the Immunity of Tumor Immunomodulatory BCG Peptides

[0067] Using the Immune Epitope Database (IEDB, http: / / www.iedb.org / ) and the NetMHCpan-4.1 online website (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ), the BCG protein co-bound to MHC-I of DC 2.4 and HepG2 cells was identified as a potential immune protein, and the affinity of the immune peptides that may exist in the 7 proteins identified by mass spectrometry in Example 1 was further predicted.

[0068] In IEDB, the prediction of MHC-I binding peptides was performed using the Consensus model (v2.24). For human MHC-I alleles, HLA-A*02:01 was selected. For mouse MHC-I alleles, H-2-Db, H-2-Dd, H-2-Kb, H-2-Kd, H-2-Kk, and H-2-Ld were selected. The Ann method was used to classify the binding ability of immune peptides. 50 <ANN IC 50 <5000 and IC 50 <50 represents peptides with lower and higher affinity, respectively. IC values ​​predicted by IDEB for binding to HLA-A*02:01 50 The scatter plot of peptides <5000 is as follows Figure 2 As shown in A; IC 50 The scatter plot of the top 100 peptides in <50 is as follows Figure 2 B. In NetMHCpan-4.1, human MHC-I alleles were selected as the objects of the above IEDB analysis, and mouse MHC alleles H-2-Dq, H-2-Kq and H-2-Lq were also selected. The MHC-I affinity peptides in the proteins to which the peptides identified in Example 1 belonged were predicted, and the peptides with binding ability were retained in the analysis, and their sequence list is shown in Table 2:

[0069] Table 2 Prediction of MHC-Ⅰ peptide affinity for the proteins containing the 7 peptides identified by mass spectrometry

[0070]

[0071]

[0072] Example 3 BCG polypeptide synthesis and mass spectrometry verification

[0073] The predicted MHC-I affinity peptide sequences in Example 1 and Example 2 were aligned, and the overlapping regions of mass spectrometry identification and software prediction in 7 proteins were selected as target peptides. Finally, it was determined that the synthesis contained 8 peptide segments as described in Table 3 (the actual synthesized peptide was a combination of the peptide identified by mass spectrometry in Example 1 and the peptide predicted by software in Example 2). The BCG peptide was synthesized and identified by mass spectrometry by Nanjing GenScript, and the solubility test was performed by GenScript. SEQ ID NO.1 to SEQ ID NO.7 used water as solvent, and SEQ ID NO.8 used DMSO as solvent. The dissolved peptide was Store in refrigerator. Its mass spectrometry identification spectrum is as follows Figure 3 Its sequence and solubility are shown in Table 3:

[0074] Table 3 Synthetic peptides

[0075]

[0076] Example 4 Analysis results of the production of intracellular factor IFN-γ by CD8+ T cells

[0077] Use CD8 + T cell isolation kit (Beaver, 70902) was used to isolate mouse spleen CD8 + After T cells were grown, RPMI1640, 10% fetal bovine serum, 100 IU / mL penicillin, 100 mg / mL streptomycin, 50 μM β-mercaptoethanol (Maclin), 1 M HEPES (basal media), and 100 mM sodium pyruvate (basal media) were used to culture the cells in 5% CO. 2 Monodispersed cells from the femoral and tibia bone marrow of mice were cultured in RPMI 1640 medium containing 10% heat-inactivated FBS and 20 ng / ml GM-CSF (MCE, HY-P7361). After 7 days, they were pulsed with BCG peptide (80 μg / ml) to obtain bone marrow-derived dendritic cells (4×10 4 / well). After 24 hours, the culture medium containing the BCG polypeptide synthesized in Example 1 was aspirated and mixed with spleen CD8 + T cells (2×10 5 / well) in a 96-well round-bottom plate for 24 h. To evaluate the responsiveness of T cells to BCG peptides, 5 μg / ml brefeldin A (MKBio, MZ2103) was used to stimulate CD8 +T cells were cultured for 4 hours and then resuspended in FACS buffer. After blocking Fc receptors with anti-mouse CD16 / 32 antibody (Biolegend, 101301), cells were stained with FITC anti-mouse CD8a antibody (Biolegend, 100706) at 4°C for 20 minutes. The cells were then washed with FACS buffer, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized for 30 minutes. They were stained with PE anti-mouse IFN-γ antibody (Biolegend, 505807) on ice for 30 minutes. Then, the cells were washed with FACS buffer, collected and analyzed for CD8 in a Cytoflex LX flow cytometer. + T cell activation. Figure 4 DC2.4 cells pulsed with solvent control elicited CD8 + Compared with the above synthetic BCG peptide-pulsed DC2.4 cells, the CD8 + T cell activity, showing antigenicity. Among them, SEQ ID NO.1 (peptide 1) induced CD8 + T cells are the most active.

[0078] Example 5 Evaluation of the immunomodulatory effect of BCG polypeptide on subcutaneous liver cancer tumors

[0079] Female BALB / c mice aged 5 to 6 weeks were subcutaneously inoculated on the right side with 5×10 6 H22 cells (Wuhan Punosai), when the tumor volume reaches 100mm 3 At about 1:10 pm, the mice were divided into 4 groups: blank group, SEQ ID NO.1 (50 μg / mouse), iodized oil (WOR=1:2, 50 μl / mouse) and iodized oil emulsified SEQ ID NO.1 (50 μg, WOR=1:2, 50 μl / mouse), and intratumoral injection was performed. The preparation method of iodized oil emulsified SEQ ID NO.1 is as follows: Tween-80 with a final volume concentration of 0.2% is added to the iodized oil and mixed in advance, the SEQ ID NO.1 polypeptide is dissolved in physiological saline, the two are respectively drawn with a sterile syringe, and the sterile three-way pipe is manually mixed. After 21 days, the mouse tumors were collected, and the tumor-infiltrated tissues were cut into small pieces with ophthalmic scissors. Tumor tissue was digested with collagenase IV (Biosharp, BS165) and DNase I (Golden Clone, EZ0380) in a 37°C water bath for 30 minutes; tumor tissue was separated by density gradient centrifugation with Percoll separation solution (Biosharp, BS909) and centrifuged at 500g for 30 minutes at room temperature; after centrifugation, the centrifuge tube was divided into 4 layers, from top to bottom: diluent layer, lymphocyte layer, transparent separation layer, and red blood cell layer. Milky white lymphocytes were collected, washed with flow cytometry buffer, and then flow cytometry antibody staining was performed to detect tumor-infiltrating CD4 + CD8+ T lymphocytes and dendritic cells (DCs). The tumor cells obtained above were first incubated with anti-CD16 / 32 antibodies on ice for 15 minutes, and then stained with anti-mouse CD4-FITC antibody (Biolegend, 100406, GK15), anti-mouse CD8-APC antibody (Biolegend, 100711, 53-6.7) and anti-mouse CD11c-PE antibody (Biolegend, 117307, N418) on ice in the dark for 30 minutes. After staining, the cells were washed twice with flow buffer, resuspended in 300 μl and passed through a 300-mesh sieve, detected with a Cytoflex LX flow cytometer (Beckman, Germany), and analyzed with Flowjo software (Version 10.0).

[0080] like Figure 5 As shown, Figure 5 A and Figure 5 C shows the typical flow cytometry results of CD8+ T cells and dendritic cells in the tumor tissues of mice in each treatment group. Figure 5 B and Figure 5 D shows the corresponding quantitative analysis data. It can be seen that SEQ ID NO.1 (peptide 1) treatment significantly promoted the CD8+T cells in tumor tissue ( Figure 5 A, B) and dendritic cells ( Figure 5 When iodized oil was used as the delivery carrier, the infiltration of CD8+T cells and dendritic cells induced by the above polypeptide SEQ ID NO.1 (polypeptide 1) was more significant, indicating that iodized oil delivery enhanced the induction effect of the polypeptide.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antigen polypeptide for anti-tumor immune regulation, characterized in that: The amino acid sequence of the antigenic polypeptide is one or more of those shown in SEQ ID NO. 1-8.

2. The antigen polypeptide according to claim 1, characterized in that The amino acid sequence of the antigen polypeptide is shown in SEQ ID NO.

1.

3. A method for screening antigen polypeptides according to claim 1 or 2, characterized in that: The following steps are involved: S1, BCG was co-incubated with DC2.4 cells and tumor cells HepG2; S2, DC2.4 cells and HepG2 cells were lysed, total proteins were collected, MHC-I antibodies were added to the total proteins, and MHC-I binding peptides were co-precipitated; S3, separating the MHC-I binding peptides and identifying them by mass spectrometry to screen out the antigenic peptides that can be co-presented by MHC-I of DC2.4 cells and HepG2 cells; S4, theoretical prediction of antigenicity of antigenic peptides that can be co-presented by DC2.4 cells and HepG2 cells MHC-Ⅰ was performed through bioinformatics analysis; S5, determining the antigenic polypeptide for anti-tumor immune regulation according to the overlapping region identified by mass spectrometry and predicted by antigenicity theory.

4. The screening method according to claim 3, characterized in that Also includes: S6, after pulsing DC2.4 cells with the antigen polypeptide for anti-tumor immunomodulation, co-culture with CD8+T cells, and verify the antigenicity of the antigen polypeptide for anti-tumor immunomodulation according to the degree of activation of CD8+T cells.

5. The screening method according to claim 3, characterized in that The total incubation time in step S1 is 8-12 hours.

6. The screening method according to claim 3, characterized in that The MHC-I antibody is an H-2 antibody or an HLA-I antibody.

7. Use of the antigen polypeptide according to claim 1 or 2 in the preparation of an anti-tumor immunomodulatory preparation.

8. An anti-tumor immunomodulatory preparation, characterized in that: Comprising the antigen polypeptide according to claim 1.

9. The anti-tumor immunomodulatory preparation according to claim 8, characterized in that: Also included are drug delivery vehicles and pharmaceutically acceptable emulsifiers.

10. The anti-tumor immunomodulatory preparation according to claim 9, characterized in that: The emulsifier is iodized oil.

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