An antigenic peptide for anti-tumor immune modulation, its screening and application

Abstract

This invention provides an antigenic polypeptide for anti-tumor immune regulation, a screening method thereof, and its application. The antigenic polypeptide is derived from BCG, and its amino acid sequence is shown in SEQ ID NO. 1-8. The screening method combines immunomass spectrometry detection with antigenicity prediction. The antigenic polypeptide can activate CD8. + T cells regulate the tumor immune microenvironment and induce intratumoral CD8. + The increase in T and DC2.4 cells, coupled with the enhanced induction effect of the peptide delivered via iodized oil, provides a strong basis for the screening method of this invention. This invention provides technical support for the development of novel BCG antitumor agents, enriches the application of BCG in antitumor therapy, and offers insights for the development of tumor immunotherapy materials.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and more particularly to an antigenic polypeptide for anti-tumor immune regulation, its screening and application. Background Technology

[0002] Malignant tumors constitute one of the leading causes of death worldwide. Following surgery, chemotherapy, radiotherapy, and targeted therapy, immunotherapy, as an emerging treatment method for cancer, has been widely used clinically and holds promise for providing lasting therapeutic effects. The core objective of tumor immunotherapy is to activate and strengthen the body's immune system, enhancing its ability to recognize, attack, and eliminate tumors, while simultaneously inducing the body to form long-term immune memory to eliminate tumor cells and reduce the likelihood 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 become a bridge for the interaction between cytotoxic CD8+ T cells and tumor cells. Therefore, understanding these peptides is key to developing therapeutic cancer vaccines and eliciting specific anti-tumor adaptive immune responses. By inducing the display of tumor cell surface antigen epitopes, T cell immune responses can be elicited more effectively.

[0004] Currently, tumor immunotherapy strategies include immune checkpoint blockade, adoptive cell therapy, tumor vaccines, and non-specific immune stimulation. These treatments are generally costly and their efficacy is not always satisfactory. Developing novel immunotherapies using existing drugs is another approach, with BCG (Bacillus Calmette-Guérin) attracting attention due to its low cost and easy accessibility. As a non-specific immune stimulant, BCG's local immune-enhancing effect helps attract immune-active cells to kill tumor cells. Intravesical instillation of BCG is considered the gold standard adjuvant therapy for non-muscle-invasive bladder cancer.

[0005] However, some problems exist in the application of BCG. Due to its complex composition and limitations imposed by the expression of fibronectin on the cell surface, the application of BCG is currently relatively limited, mainly confined to the treatment of bladder cancer. Therefore, there is an urgent need to screen out highly effective single antigens from BCG for anti-tumor immunomodulation and to expand its application scope. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an antigenic polypeptide for anti-tumor immune regulation, a screening method thereon, and its application.

[0007] This invention is implemented as follows:

[0008] An antigenic polypeptide for antitumor immune modulation, wherein the amino acid sequence of the antigenic polypeptide is one or more as shown in SEQ ID NO. 1-8.

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

[0010] A method for screening the antigenic polypeptide, comprising the following steps:

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

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

[0013] S3, the MHC-I binding peptide was separated and screened by mass spectrometry to identify antigenic peptides that can be co-presented by MHC-I in DC2.4 cells and HepG2 cells;

[0014] S4, using bioinformatics analysis to theoretically predict the antigenicity of antigenic peptides that can be co-presented by MHC-I in DC2.4 cells and HepG2 cells;

[0015] S5. Based on the overlapping region predicted by mass spectrometry identification and antigenicity theory, the antigenic polypeptide for antitumor immune regulation is identified.

[0016] In some embodiments, the filtering method further includes:

[0017] S6. After pulsating DC2.4 cells with the antigenic peptide for anti-tumor immune regulation, co-culture them with CD8+ T cells, and verify the antigenicity of the antigenic peptide for anti-tumor immune regulation based on the activation level of CD8+ T cells.

[0018] In some embodiments, the 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] The application of the aforementioned antigenic polypeptide in the preparation of antitumor immunomodulatory agents.

[0021] An antitumor immunomodulatory agent comprising the aforementioned antigenic polypeptide.

[0022] In some embodiments, the antitumor immunomodulatory agent further includes a drug delivery carrier and a pharmaceutically acceptable emulsifier.

[0023] In some embodiments, the emulsifier is iodized oil.

[0024] The positive effects of this invention are mainly reflected in the following aspects:

[0025] (1) This invention utilizes various advanced research methods, including immunopeptidomics, 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 after screening can effectively bind to MHC-I molecules, showing their potential in regulating tumor cell antigen epitopes. These peptides can activate CD8+ T cells, thereby altering the tumor microenvironment and exerting their anti-tumor effects. Therefore, these screened peptides can be further developed and applied as single anti-tumor components in BCG, thus significantly expanding the application scope of BCG in the field of anti-tumor therapy.

[0026] (2) The antigenic peptides involved in this invention can also be delivered by binding with iodized oil. This method provides a new approach and possibility for developing novel anti-tumor immunotherapy materials. In this way, the antigenic peptides can be delivered into the body more effectively, enhancing their role in the anti-tumor immune response and thus improving therapeutic efficacy. This innovative 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. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 The mass spectrometry identification results of the BCG peptide for tumor immunomodulation provided in Example 1 of this invention are shown. In this figure, A represents the Venn diagram analysis results of MHC-I-binding proteins in DC2.4 and HepG2 cells, and B represents the Venn diagram analysis results of HLA-I-binding peptides in DC2.4 and HepG2 cells.

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

[0030] Figure 3 The mass spectrometry identification results of the synthesized BCG peptide provided in Example 3 of this invention.

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

[0032] Figure 5 This is a graph evaluating the immunomodulatory effect of the iodized oil-based BCG peptide provided in Example 5 of the present invention on subcutaneous hepatocellular carcinoma tumors. In this graph, A represents the CD8+ infiltrating tumor tissue in each treatment group. + A) is a representative flow cytometry result of T cells; B) is a quantitative statistical analysis of CD8+ T cells infiltrating tumor tissue in each treatment group; C) is a representative flow cytometry result of dendritic cells (CD11c+) infiltrating tumor tissue in each treatment group; D) is a quantitative statistical analysis of dendritic cells (CD11c+) infiltrating tumor tissue in each treatment group. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Embodiments of this invention relate to specific antigenic peptides for regulating anti-tumor immune responses. The amino acid sequences of these antigenic peptides correspond to any one or more of SEQ ID NO.1 to SEQ ID NO.8. These peptides are obtained from BCG vaccine through a series of advanced techniques, including immunopeptidomics, high-performance liquid chromatography (HPLC), bioinformatics analysis, and in vitro functional screening. These screening methods ensure that the obtained peptides can effectively bind to MHC-I molecules, thereby playing a key role in the regulation of antigenic epitopes in tumor cells. Furthermore, these peptides can 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 some embodiments, particular interest is focused on antigenic peptides whose amino acid sequences match those of SEQ ID NO.1. The specific amino acid sequence of SEQ ID NO.1 is: GLMTSVLMTADGKTVEAEAAHGTVTR. Peptides with this specific sequence exhibit excellent performance in activating CD8+ T cells and can effectively promote the body's immune response against tumor cells, thus playing an important role in anti-tumor therapy. Through further research and clinical application, these peptides hold promise as an effective means of treating various tumors.

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

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

[0039] S2, Next, DC2.4 cells and HepG2 cells were lysed, and the total protein was collected. Then, MHC-I antibody was added to this total protein, and peptides bound to MHC-I molecules were co-precipitated by immunoprecipitation.

[0040] S3, the MHC-I binding peptides obtained in the above steps are separated, and these peptides are identified using mass spectrometry. Mass spectrometry analysis is used to screen for antigenic peptides that can be co-presented by MHC-I from both DC2.4 cells and HepG2 cells.

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

[0042] S5. Based on the overlapping regions identified by mass spectrometry and bioinformatics analysis, antigenic peptides for antitumor immune modulation were determined. These peptides are considered to have the potential to activate the immune system and modulate the tumor microenvironment.

[0043] In some embodiments, the filtering method further includes the following steps:

[0044] In step S6, the selected antigenic peptides for anti-tumor immunomodulation were pulsed onto DC2.4 cells and then co-cultured with CD8+ T cells. The antigenicity of these peptides was verified by detecting the activation level of CD8+ T cells. This verification step helps to confirm whether the peptides can effectively activate immune cells, thereby exerting an anti-tumor effect.

[0045] This invention employs a combination of methods, including immunopeptidomics, high-performance liquid chromatography (HPLC), bioinformatics analysis, in vitro functional screening, and in vivo functional validation, to conduct in-depth screening of BCG peptides that may regulate tumor antigen epitopes. Through this series of screening processes, this invention successfully discovered a peptide capable of activating CD8+ T cells and altering the tumor microenvironment. This peptide can induce interactions between CD8+ T cells and dendritic cells, thereby contributing to the development of new BCG tumor therapeutic materials and further expanding the application scope 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 and 12 hours. This time range is to ensure that the incubation process can be carried out sufficiently, so that 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 either an H-2 antibody or an HLA-I antibody. These two antibodies target different major histocompatibility complex (MHC) class I molecules. H-2 antibodies are typically used in experimental animal models such as mice, while HLA-I antibodies are used in human samples. Choosing the appropriate antibody is crucial to ensuring the specificity and accuracy of the experiment.

[0048] The application of the aforementioned antigenic polypeptide in the preparation of antitumor immunomodulatory agents. This antigenic polypeptide exhibits significant immunoactivating activity, effectively activating CD8+ T cells and altering the tumor microenvironment, thereby inducing the activation and proliferation of CD8+ T cells and dendritic cells. The application of this polypeptide in antitumor immunomodulatory agents provides new ideas and methods for tumor treatment.

[0049] An antitumor immunomodulatory agent includes the aforementioned antigenic peptide. Through the immunoactivating effect of this peptide, it can effectively enhance the body's antitumor immune response. This agent can be used to treat various cancers, including but not limited to lung cancer, breast cancer, and colorectal cancer. This antitumor immunomodulatory agent also exhibits good safety and tolerability, with few side effects, making it easier for patients to accept and adhere to treatment. In the future, further research on this antigenic peptide and its mechanism of action in antitumor immunomodulation is expected to lead to the development of more highly effective and safe antitumor immunomodulatory agents, bringing more hope and choices to cancer patients.

[0050] In some specific embodiments, the antitumor immunomodulatory agent not only comprises its main active ingredient, but also further includes a drug delivery carrier and a pharmaceutically acceptable emulsifier. This combination helps 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 role of the emulsifier is to uniformly disperse the drug components in the aqueous medium, forming a stable emulsion system, thereby improving the solubility and absorption rate of the drug. The choice of emulsifier is crucial for the stability and safety of the formulation. In certain specific embodiments, iodized oil is used as the emulsifier. Iodized oil, as a commonly used pharmaceutically acceptable emulsifier, has good biocompatibility and safety, effectively aids in the distribution and absorption of drugs in the body, and also provides a certain imaging effect, facilitating monitoring and evaluation by physicians in clinical applications.

[0052] Example 1: Mass spectrometry identification of MHC-I-bound BCG peptides

[0053] (1) Generation and purification of MHC-I polypeptide complex

[0054] DC2.4 and HepG2 cells were co-cultured with BCG (Bacillus Calmette-Guérin) purchased from Ruichu Biotechnology for 10 hours (MOI=7). The supernatant containing BCG was then removed, and the cells were washed twice with PBS. Tumor cell clusters were then collected and resuspended in lysis buffer (containing 0.25% sodium deoxycholate, 0.2mM iodoacetamide, 1mM EDTA, a 1:2000 protease inhibitor cocktail, 1mM PMSF, and 1% octyl-β-D-glucopyranose). The samples were then incubated at 4°C for 1 hour by rotation. Total protein was then collected by centrifugation at 48000g at 4°C for 60 minutes. The protein was then magnetically separated using Protein A / G beads pre-conjugated with HLA-I antibody (Sigma, W6 / 32). The HLA-I antigen-peptide complex was eluted with low-pH elution buffer and neutralized with antibody neutralization buffer. Finally, quantitative proteomics techniques were used to study the MHC-I peptides. The Protein A / G magnetic beads, low pH elution buffer, and antibody neutralization buffer used were all from Beaver Biotech's Protein A / G antibody purification kit.

[0055] (2) Peptide sample pretreatment

[0056] The total eluent was reduced with 10 mM dithiothreitol (DTT) at 37°C for 30 min, and then alkylated with 20 mM iodoacetamide (IAA) at room temperature in the dark for 30 min. The resulting reduced and alkylated MHC-peptide samples were quality controlled using short SDS-PAGE gels. Based on the sample molecular weight and expression abundance, the gel was digested with acetyltrypsin (12.5 ng / μl) (the preparation method of acetyltrypsin is described in patent CN201510083691.0) at 37°C for 12-14 hours. Then, C... 18 StageTip was used for desalting and sample purification, followed by evaporation and storage at -80℃ 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, 500 ng of MHC-I binding peptide was dissolved in buffer A [0.1% formic acid (FA), 99.9% ddH2O] and the peptide was detected using an LC-MS / MS platform. This platform included an Orbitrap Fusion Lumos mass spectrometer and an EASY-nLC 1200system liquid chromatography system manufactured by Thermo Fisher Scientific. The sample was eluted using a 78-minute nonlinear gradient at a flow rate of 600 nL / min. The detailed gradient was as follows: 6-12% buffer B elution for 8 min, 12-30% buffer B elution for 50 min, 30-40% buffer B elution for 12 min, 40-95% buffer B elution for 1 min, and 95% buffer B elution for 7 min [buffer B, 0.1% FA, 99.9% acetonitrile (ACN)]. The MS1 detection parameters are as follows: quality scan range of 300–1400 m / z, resolution of 1.2 × 10⁻⁶ m / z. 5 Automatic gain control (AGC) is set to 5×10. 5 The maximum implantation time (MIT) was set to 50 ms. The MS2 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%, the 20 strongest ions were selected for fragmentation, the AGC target for each MS2 scan was 5000, the MIT was 35 ms, and the dynamic exclusion time was 20 s.

[0059] In the experiment of BCG-infected HepG2 cells, 1 μg of extracted MHC-I binding peptide was dissolved in the same buffer A as above. The peptide was detected using an LC-MS / MS platform comprising an Orbitrap Exploris 480 mass spectrometer and an EASY-nLC 1200system liquid chromatography system (Thermo Fisher Scientific). The sample was eluted using a nonlinear gradient for 75 minutes at a flow rate of 300 nL / min. The detailed gradient was as follows: 7–12% buffer B for 6 min, 12–30% buffer B for 51 min, 30–45% buffer B for 10 min, 45–95% buffer B for 1 min, and a hold in 95% buffer B for 7 min. The composition of eluent B was the same as above. DDA acquisition mode was used, employing FAIMS dual-compensation voltage switching mode with CV parameters set to -45V and -65V. The MS1 mass scan range was 350–1500 m / z, and the resolution was 6 × 10⁻⁶ m / s. 4 AGC is set to automatic mode, and MIT is set to 50ms. The filter strength threshold is set to a minimum of 20,000. MS2 uses High Energy Collision Dissociation (HCD) with a normalized collision energy (NCE) of 27% and a resolution of 1.5 × 10⁻⁶. 4The MIT is 22ms, the split window is 1.6m / z, and the loop time is set to 1.3s.

[0060] (4) Proteomics Database Search: Using pFind (v3.1.5) software, the Mycobacterium bovis BCG / Pasteur 1173P2 protein data of UniProt (UP000001472, containing 3891 proteins) was used as the database for searching. The specific search parameters were set as follows: 1) The protein digestion 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 cysteine ​​alkylation modification 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 precursor ion mass was 20 ppm, and the mass tolerance of daughter ion mass 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: The quality of the BCG peptide MS2 spectrum was assessed at different levels, including b / y ion coverage and noise peaks. Spectra with high b / y ion coverage and fewer and lower noise peaks were selected, and Venn diagrams were constructed, as shown below. Figure 1 As shown.

[0062] according to Figure 1 A showed that 47 BCG protein peptides could be presented by MHC-I molecules in DC2.4 cells, while 147 peptides could be presented by MHC-I molecules in HepG2 tumor cells. Furthermore, 11 BCG protein peptides could be co-presented by MHC-I molecules from both DC2.4 and HepG2 cells. Figure 1 B revealed that 61 BCG peptides could be presented by MHC-I molecules in DC2.4 cells, while 171 peptides could be presented by MHC-I molecules in HepG2 tumor cells. Notably, nine BCG peptides could be co-presented by HepG2 tumor cells and DC2.4 cells. These nine BCG peptides originated from seven of the eleven BCG proteins (of which 1A could be co-presented by MHC-I molecules in both DC2.4 and HepG2 cells), 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: Immunomodulatory prediction of BCG peptides for tumor immune regulation

[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 in identified DC 2.4 and HepG2 cells was used as a potential immune protein to further predict the affinity of the immune peptides that may be present among the seven proteins identified by mass spectrometry in Example 1.

[0068] In IEBDB, the prediction of MHC-I binding peptides was performed using the Consensus model (v2.24). For the human MHC-I allele, HLA-A*02:01 was selected. For the 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 binding capacity of the immune peptides was classified using the Ann method. 50 <ANN IC 50 <5000 and IC 50 <50 represents peptides with low and high affinity, respectively. IDEB predicted IC50 for binding to HLA-A*02:01. 50 A scatter plot of peptides <5000 is shown below. Figure 2 As shown in A; IC 50 A scatter plot of the top 100 peptides out of <50 is shown below. Figure 2 As shown in B. In NetMHCpan-4.1, the human MHC-I allele was selected as the target of the above IEDB analysis, and the mouse MHC alleles H-2-Dq, H-2-Kq, and H-2-Lq were also selected. MHC-I affinity peptides in the proteins to which the peptides identified in Example 1 belonged were predicted. Peptides with binding ability were retained in the analysis, and their sequence listing is shown in Table 2:

[0069] Table 2. MHC-I peptide affinity predictions for the proteins containing the 7 mass spectrometry-identified peptides.

[0070]

[0071]

[0072] Example 3: BCG peptide synthesis and mass spectrometry verification

[0073] The predicted MHC-I affinity peptide sequences from Examples 1 and 2 were compared for position. Seven proteins with overlapping regions identified by mass spectrometry and predicted by software were selected as target peptides. The final synthesized peptides contained the eight peptides listed in Table 3 (the actual synthesized peptides were a combination of the peptides identified by mass spectrometry in Example 1 and predicted by software in Example 2). The BCG peptides were synthesized and identified by mass spectrometry by Nanjing Genscript Biotech Co., Ltd. Solubility tests were performed by Genscript Biotech; SEQ ID NO. 1–SEQ ID NO. 7 used water as solvent, and SEQ ID NO. 8 used DMSO as solvent. The dissolved peptides… Store in a refrigerator. Its mass spectrometry identification spectrum is as follows: Figure 3 As shown, its sequence and solubility are shown in Table 3:

[0074] Table 3 Synthetic peptides

[0075]

[0076] Example 4: Analysis results of intracellular IFN-γ production in CD8+ T cells

[0077] Use CD8 + T-cell sorting kit (Beaver, 70902) for sorting mouse spleen CD8 cells + After T cell inoculation, cells were cultured in 5% CO2 (37℃) with RPMI 1640, 10% fetal bovine serum, 100 IU / mL penicillin, 100 mg / mL streptomycin, 50 μM β-mercaptoethanol (Maclin), 1 M HEPES (basalmedia), and 100 mM sodium pyruvate (basalmedia), respectively. Monodisperse cells from mouse femoral and tibial bone marrow were cultured in RPMI 1640 medium containing 10% heat-inactivated FBS and 20 ng / ml GM-CSF (MCE, HY-P7361). After 7 days, bone marrow-derived dendritic cells (4 × 10⁶ cells / mL) were obtained by pulsed administration of BCG peptide (80 μg / ml). 4 / well). After 24 hours, aspirate the culture medium containing the BCG peptide synthesized in Example 1 and mix it with spleen CD8. + T cells (2×10) 5 / well) were co-cultured in 96-well round-bottom plates for 24 h. To assess the responsiveness of T cells to BCG peptide, CD8 cells were stimulated with 5 μg / ml brefeldin A (MKBio, MZ2103). +T cells were collected for 4 hours, then resuspended in FACS buffer, and Fc receptors were blocked with anti-mouse CD16 / 32 antibody (Biolegend, 101301). Cells were then stained with FITC anti-mouse CD8a antibody (Biolegend, 100706) at 4°C for 20 minutes, washed with FACS buffer, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized for 30 minutes. Cells were then stained with PE anti-mouse IFN-γ antibody (Biolegend, 505807) on ice for 30 minutes. Cells were then washed with FACS buffer, collected using a Cytoflex LX flow cytometer, and CD8a analysis was performed. + T cell activation. Results as follows: Figure 4 CD8+ induced by pulsed DC2.4 cells compared to solvent control + Compared to T activity, the synthetic BCG peptide pulsed into DC2.4 cells significantly induced CD8+ activity. + T cell activity, exhibiting antigenicity. Among them, SEQ ID NO.1 (peptide 1) induces CD8... + T cells exhibit the strongest activity.

[0078] Example 5: Evaluation of the immunomodulatory effect of BCG peptides on subcutaneous hepatocellular carcinoma tumors.

[0079] Female BALB / c mice aged 5–6 weeks were subcutaneously injected on the right side with 5 × 10⁵ gram of phytoalexins in the logarithmic growth phase. 6 H22 cells (Wuhan Pronosai), when the tumor volume reaches 100 mm. 3 At approximately 21 days, mice were divided into four groups: a blank control group, a group receiving SEQ ID NO.1 (50 μg / mouse), a group receiving iodized oil (WOR = 1:2, 50 μl / mouse), and a group receiving iodized oil emulsified SEQ ID NO.1 (50 μg, WOR = 1:2, 50 μl / mouse), and all were injected intratumorally. The preparation method for iodized oil emulsified SEQ ID NO.1 was as follows: Iodized oil was mixed with Tween-80 to a final volume concentration of 0.2% beforehand. The SEQ ID NO.1 polypeptide was dissolved in physiological saline. Both were aspirated separately using sterile syringes and manually mixed using sterile three-way stopcocks. Twenty-one days later, mouse tumors were collected, and the tumor-infiltrating tissue was cut into small pieces using ophthalmic scissors. Tumor tissue was digested with collagenase IV (Biosharp, BS165) and DNase I (Kinkrono, EZ0380) in a 37°C water bath for 30 minutes. Tumor tissue was then separated using Percoll separation buffer density gradient centrifugation (Biosharp, BS909) at 500g for 30 minutes at room temperature. After centrifugation, the centrifuge tubes separated into four layers from top to bottom: dilution buffer layer, lymphocyte layer, clear separation buffer layer, and erythrocyte layer. Milky white lymphocytes were collected, washed with flow cytometry buffer, and then subjected to flow cytometry antibody staining to detect CD4+ infiltration in tumors. + CD8+ T lymphocytes and dendritic cells (DCs) were collected. The tumor cells obtained were first incubated with anti-CD16 / 32 antibody on ice for 15 minutes, 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 cytometry buffer, resuspended in 300 μl, and passed through a 300-mesh sieve. Detection was performed using a Cytoflex LX flow cytometer (Beckman, Germany) and analyzed using Flowjo software (Version 10.0).

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

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. An antigenic polypeptide for antitumor immunomodulation, characterized in that, The amino acid sequence of the antigenic polypeptide is shown in SEQ ID NO.

1.

2. A method for screening antigenic polypeptides according to claim 1, characterized in that, Includes the following steps: S1, BCG vaccine was co-incubated with DC2.4 cells and HepG2 tumor cells, respectively; S2, DC2.4 cells and HepG2 cells were lysed, total protein was collected, MHC-I antibody was added to the total protein, and MHC-I binding peptides were co-precipitated; S3, the MHC-I binding peptide was separated and screened by mass spectrometry to identify antigenic peptides that can be co-presented by MHC-I in DC2.4 cells and HepG2 cells; S4, using bioinformatics analysis to theoretically predict the antigenicity of antigenic peptides that can be co-presented by MHC-I in DC2.4 cells and HepG2 cells; S5. Based on the overlapping region predicted by mass spectrometry identification and antigenicity theory, the antigenic polypeptide for antitumor immune regulation is identified.

3. The screening method according to claim 2, characterized in that, Also includes: S6. After pulsating DC2.4 cells with the antigenic peptide for anti-tumor immune regulation, co-culture them with CD8+ T cells, and verify the antigenicity of the antigenic peptide for anti-tumor immune regulation based on the activation level of CD8+ T cells.

4. The screening method according to claim 2, characterized in that, The incubation time in step S1 is 8-12 hours.

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

6. The use of the antigenic polypeptide according to claim 1 in the preparation of an antitumor immunomodulatory agent, characterized in that, The tumor is liver cancer.

7. An antitumor immunomodulatory agent, characterized in that, Includes the antigenic polypeptide as described in claim 1.

8. The antitumor immunomodulatory agent according to claim 7, characterized in that, It also includes drug delivery carriers and pharmaceutically acceptable emulsifiers.

9. The antitumor immunomodulatory agent according to claim 8, characterized in that, The emulsifier is iodized oil.

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