Fusion polypeptides and uses thereof

Abstract

The application provides a fusion polypeptide and application thereof, takes MG7-Ag single epitope as a target antigen (MG1) of a digestive tract tumor, and is composed of a T helper epitope peptide and three MG1 in series which are connected by a covalent amide bond at an N terminal, wherein, an adapter lysine residue is removed at a splicing position of adjacent MG1 segments of the MG3. The application covalently couples the T helper epitope peptide at the N terminal of the MG3, and relies on a Th epitope to mediate an MHC II class pathway to present, strongly activates a helper T cell, greatly improves an anti-MG1 specific IgG production capacity, and solves a natural immunogenicity deficiency defect of the MG1 from a molecular structure level.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a fusion polypeptide and its applications. Background Technology

[0002] The gastric cancer-associated antigen MG7-Ag is specifically and abnormally highly expressed in various digestive tract tumors, including gastric and colorectal cancer, while it is almost not expressed in normal digestive tract tissues. This makes it an ideal broad-spectrum target for the development of peptide vaccines targeting pan-digestive tract tumors. Developing peptide vaccines based on the MG7-Ag antigen epitope can induce specific humoral and cellular immunity, achieving active immunotherapy for tumors, and is an important research direction for precision immunotherapy of gastric cancer. Current research mostly uses the MG7-Ag single epitope peptide MG1 as an immunogen to prepare vaccines. However, the immunogenicity of natural MG1 peptide is extremely weak, and its single administration is insufficient to stimulate the body to produce high-titer specific antibodies and effective T-cell responses, thus limiting its clinical translation and application.

[0003] In existing technologies, to enhance the immunogenicity or structural stability of multi-epitope peptides, epitope tandem design often employs a strategy of adding one or more flexible amino acid linkers (such as Gly, Ser, Lys, etc.) to ensure independent folding of each epitope and reduce steric hindrance. For example, GnRHm1 peptides are chemically coupled to tetanus toxin T epitopes via glycine-glycine (GG) spacers to construct the GnRHm1-GG-TT fusion peptide (pEHWSYPLRPG-GG-QYIKANSKFIGITEL); or two glycine residues are used as linkers to connect MUC1 glycopeptide (GVTSAPDTRPAPG) to a dipeptide (GK) derivative conjugated with a TLR7 agonist. However, introducing exogenous linker sequences may introduce unexpected new epitopes or increase the risk of immune shift. How to maintain the independent antigenicity of each epitope and synergistically activate humoral and cellular immunity without introducing exogenous linkers remains a pressing technical challenge in this field. Summary of the Invention

[0004] In view of this, the present invention provides a fusion peptide and its application, which improves the overall immunogenicity of the peptide at the molecular structure level.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a fusion polypeptide, which is composed of a T-auxiliary epitope peptide and a MG3 polypeptide consisting of three tandem MG1 peptides linked by an N-terminal covalent amide bond, wherein a linker lysine residue is removed at the splice site of the MG3 adjacent MG1 fragments.

[0006] It should be noted that the MG3 polypeptide composed of three tandem MG1s is specifically formed by the tandem connection of three MG7-Ag antigen single epitope MG1 amino acid sequences.

[0007] Preferably, the T helper epitope peptide includes the diphtheria toxin-derived T helper epitope DT. 331-345 Its amino acid sequence is shown in SEQ ID NO: 1, specifically QSIALSSLMVAQAIP;

[0008] Preferably, the unit price position of each MG1 is as shown in SEQ ID NO: 2, specifically KPHVHTK.

[0009] Preferably, the amino acid sequence of the MG3 polypeptide is as shown in SEQ ID NO: 3, specifically KPHVHTKPHVHTKPHVHTK.

[0010] Secondly, the present invention provides a method for preparing the aforementioned fusion polypeptide, comprising the following steps: S1. MG1 amino acids are sequentially inserted into the resin from the C-terminus to the N-terminus, and three MG1 sequences are continuously connected in series to obtain the MG3 backbone. The lysine residues are omitted at the splicing points of adjacent MG1 to control the generation of non-specific epitopes. The resin is Rink-Amide-AM resin. S2. The full sequence of T-helper epitope peptides is continuously coupled to the N-terminus of the MG3 backbone; S3 was subjected to lysis, ether precipitation, and reversed-phase HPLC purification to obtain the fusion polypeptide DT-MG3.

[0011] Thirdly, the present invention provides an anti-tumor vaccine raw material for targeting MG7-Ag gastric cancer, comprising: the fusion polypeptide as described above, or the fusion polypeptide prepared by the preparation method described above.

[0012] It should be noted that the T helper epitope peptide of the present invention can be co-encapsulated with the NKT cell agonist αGalCer / GCS-11 / GCS-12 to prepare a liposomal antitumor vaccine. After multiple immunizations via the intraperitoneal route, the vaccine can efficiently induce specific IgG secretion, upregulate IFN-γ cytokine levels, and activate CD4+. + / CD8 + T cells kill tumor cells through the CDC pathway, significantly inhibiting tumor proliferation in vivo and prolonging the survival time of tumor-bearing animals.

[0013] Preferably, it also includes liposomes containing a Th1-type GCS-11 adjuvant.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In this invention, T helper epitope peptides are covalently coupled at the N-terminus of MG3, and the MHC class II pathway is mediated by Th epitope to present the peptides, which strongly activates helper T cells and greatly enhances the ability to generate anti-MG1 specific IgG. This solves the problem of insufficient natural immunogenicity of MG1 at the molecular structure level.

[0015] (2) Unlike existing epitope tandem techniques that rely on exogenous amino acid linkers (such as Gly, Ser, Lys, etc.) to maintain epitope conformation, in the fusion peptides of this invention, the T helper epitope peptide is directly linked to the N-terminus of the MG3 backbone, and the MG1 triplet fragments are directly linked to each other via peptide bonds, without introducing any exogenous linker residues. This greatly reduces the risk of unexpected new epitopes or immune interference that may be introduced by exogenous sequences. Experiments have shown that this direct fusion method not only does not affect the independent antigenicity of each MG1 epitope, but also synergizes with the T helper epitope and Th1 adjuvant to significantly improve the specific IgG titer and CTL killing activity, demonstrating the technical superiority of this simplified linking strategy in maintaining the integrity of functional epitopes and enhancing immunogenicity.

[0016] (3) The present invention integrates Fmoc solid-phase synthesis, and the MG3 backbone and T auxiliary epitope peptide are continuously synthesized in the same resin without the need for segmented preparation and in vitro chemical coupling, which shortens the production process; the product is purified by HPLC with a purity of ≥95%, with small batch-to-batch differences, which is convenient for large-scale industrial preparation.

[0017] (4) In this invention, a T-helper epitope peptide is covalently coupled to the N-terminus of MG3. After coupling, the amphiphilicity of the peptide is enhanced, and it can autonomously assemble into a liposome structure. This liposome formulation can effectively resist the degradation by proteases in vivo, significantly delay the drug clearance rate in vivo, and prolong the retention time in vivo. Relying on the core advantages of liposomes, such as strong targeting, good biocompatibility, and sustained release, it can greatly improve the efficiency of the immune response to antigens in the body and enhance the overall immune effect. Attached Figure Description

[0018] Figure 1 Figure 1 shows the solid-phase synthesis route and structural confirmation diagram of the pure polypeptide antigen in Example 1 of this invention; wherein, Figure A is the synthesis route of the gastric cancer-specific polypeptide antigen MG3, Figure B is the synthesis route of the fusion polypeptide antigen DT-MG3, Figure C is the high performance liquid chromatography (HPLC) chromatogram of the fusion polypeptide antigen DT-MG3, and Figure D is the mass spectrum of the fusion polypeptide antigen DT-MG3. Figure 2 Figure 4 shows a comparison of the levels of cytokine polarization in mice immunized with different liposomal vaccines according to Example 4 of this invention. Figure A shows the expression level of IFN-γ in the peripheral serum of mice 24 hours after immunization, Figure B shows the expression level of IL-4 in the peripheral serum of mice 2 hours after immunization, Figure C shows the activation fold ratio of the cytokine secretion level of each group relative to the classic αGalCer group, and Figure D shows the concentration ratio of IFN-γ to IL-4 in the peripheral serum. Figure 3Figure 4 shows the humoral immune response and complement-dependent cytotoxicity evaluation of mice induced by different vaccine formulations in Example 4 of this invention. Figure A shows the antibody titer of anti-MG1 specific total IgG produced in mouse peripheral serum on day 42 post-immunization; Figures B to E show the subtype distribution of anti-MG1 specific IgG antibodies in mouse serum on day 42 post-immunization, namely IgG1, IgG3, IgG2a, and IgG2b, respectively; Figure F shows the survival rate of EAC target cells under complement-dependent cytotoxicity as determined by the MTT assay; Figure G is a flow cytometry histogram showing the comparison of the specific binding ability of different groups of immune serum to EAC target cells overexpressing MG1 (the percentage represents the binding rate of antigen-positive cells). Figure 4 This refers to the cellular immune response and cytotoxic T lymphocyte (CTL) activity induced by different vaccine formulations in Example 4 of this invention; Figure A shows the spleen CD4 count after in vitro restimulation with MG3 peptide on day 42. + and CD8 + Representative flow cytometry atlases of IFN-γ and TNF-α expression in T cells; Figures B and C show IFN-γ... + TNF-α + Double positive CD4 + and CD8 + Quantitative analysis of the proportion of T cells; Figure D shows the specific killing rate of spleen cells (effect cells) against EAC target cells when the ratio of effector cells to target cells (E:T) is 10:1, as determined by the LDH release assay. Figure 5 Figure 5 shows the in vivo antitumor efficacy and survival rate curves of the anti-gastric cancer liposome vaccine as a single agent or in combination with the small molecule PD-L1 inhibitor BMS-202 in a mouse EAC gastric cancer model. Among them, Figure A shows the time axis of tumor inoculation and immunization administration, Figure B shows the morphological photographs of solid tumors resected in each treatment group at the experimental endpoint, Figure C shows the average tumor volume growth curve during the 20-day observation period, Figure D shows the comparison of the average tumor weight resected in each group at the experimental endpoint, and Figure E shows the Kaplan-Meier survival rate curves of each group of mice during the 40-day observation period. Figure 6 Figure A shows the systemic biosafety assessment of mice in each treatment group in Example 5 of this invention; Figure A shows H&E stained histopathological microscopic sections of each tissue and organ (heart, liver, spleen, lung, kidney, brain); Figure B shows the quantitative measurement results of liver function biochemical index (ALT) and kidney function biochemical index (UREA, CREA) in peripheral serum of mice on day 42 after immunization. Figure 7 This is a component composition diagram of the liposome vaccine provided in Example 5 of the present invention; Figure 8This is a schematic diagram illustrating the synergistic enhancement of the immune response by combining a gastric cancer vaccine based on a fusion peptide with a PD-L1 inhibitor (BMS-202) as provided in Example 5 of the present invention. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0020] Example 1: Synthesis of fusion peptide antigen DT-MG3 Synthesized via solid-phase peptide synthesis (SPPS) on Rink amide AM resin (0.65 mmol / g loading) using a Fmoc protection strategy (9-fluorenylmethoxycarbonyl protection strategy). Conventionally Fmoc-protected amino acids were coupled to the resin, and 0.2 mmol / L LPyBOP and 0.6 mmol / L DIEA (N,N-diisopropylethylamine) were administered in DMF (N,N-dimethylformamide) for 1 h. All coupling reactions were monitored using a standard Kaiser assay. Subsequently, the N-terminal Fmoc protecting group was removed by treatment with a solution of 20% piperidine in DMF. The MG3 three-epitope sequence (KPHVHTKPHVHTKPHVHTK) and DT... 331-345 The sequence QSIALSSLMVAQAIP is coupled sequentially, DT 331-345 The peptide was attached to the N-terminus of MG3. After coupling, the peptide was treated with a mixture of 95% TFA (trifluoroacetic acid), 2.5% TIS (triisopropylsilane), and 2.5% water for 2 hours to cleave the peptide from the resin and remove side-chain protecting groups. The residue was precipitated with diethyl ether, dissolved in an aqueous acetonitrile solution, and purified by reversed-phase HPLC (high performance liquid chromatography) to obtain the fusion peptide antigen DT-MG3 (yield 35%, HPLC purity ≥95%). The structure was confirmed by HR-ESI-TOF-MS. Figure 1 As shown, the synthetic route for the pure polypeptide antigen of this invention is extremely simple, and the reaction conditions are mild and controllable. Experiments confirmed that the crude product yield of DT-MG3 consistently reached 35%, and the purity after RP-HPLC purification was ≥95%. This structural simplification not only significantly reduces the difficulty of producing and preparing fully synthetic vaccines and the cost of industrial scale-up, but also greatly improves batch-to-batch consistency and drug quality controllability, overcoming the shortcomings of traditional glycopeptide vaccines with complex structures and high drug development barriers.

[0021] Example 2: Synthesis of GCS-11 GCS-11 was synthesized according to a previously reported method (Wen et al., J Med Chem 2024): using αGalCer as the parent nucleus, sulfonamide groups were introduced at specific positions on the acyl chain. The product was purified by column chromatography, with an HPLC purity ≥98%.

[0022] Example 3: Preparation of DT-MG3 / GCS-11 liposomal vaccine 23 nmol of distearate phosphatidylcholine (DSPC), 18.4 nmol of cholesterol, 4.6 nmol of polypeptide antigen, and 2.3 nmol of NKT cell agonist were dissolved in a dichloromethane-methanol mixture (1:1, v / v) and placed in a round-bottom flask. The organic solvent was removed by rotary evaporation under reduced pressure at 37°C, forming a uniform lipid film on the inner wall of the flask. Sterile HEPES buffer (pH 7.4) was added, and the film was hydrated at 37°C for 30 min to allow it to fully swell, yielding a multilayer vesicle suspension. After sonication for 15 min, the suspension was repeatedly extruded through a 400 nm polycarbonate membrane using an Avanti micro-extruder 10 times to obtain uniformly sized monolayer liposomes.

[0023] Example 4: In vivo immunogenicity evaluation BALB / c female mice (6-8 weeks old) were randomly assigned to groups (n=5) and intraperitoneally injected with liposomal vaccines of DT-MG3 / GCS-11, DT-MG3 / αGalCer, DT-MG3 / GCS-12, DT-MG3 (without adjuvant), and MG3 / GCS-11 (without DT epitope), respectively. Primitive vaccination was administered on day 1, booster vaccinations were given on days 15 and 29, and blood was collected from the orbital sinus and spleen cells were harvested on day 42. MG1-specific total IgG titers and subclass distribution were determined by ELISA; CTL cytotoxicity was detected by LDH assay; and multifunctional CD4 was detected by ICS assay. + and CD8 + T cell ratio.

[0024] Technical results: 24 hours after immunization, the DT-MG3 / GCS-11 group had the highest IFN-γ secretion, which was twice the concentration of the DT-MG3 / αGalCer group and the DT-MG3 / GCS-12 group. Figure 2 A); Compared with the DT-MG3 / GCS-11 group, the IFN-γ secretion level in the MG3 / GCS-11 control group was significantly decreased. Two hours post-immunization, the IL-4 expression level in the DT-MG3 / GCS-12 group reached its peak, being 3.5 times and 2.8 times higher than that in the DT-MG3 / αGalCer group and the DT-MG3 / GCS-11 group, respectively. Figure 2 B). Changes in fold changes in cytokine expression ( Figure 2 C) and the IFN-γ / IL-4 concentration ratio ( Figure 2D) Analysis: The DT-MG3 / GCS-11 group had the highest IFN-γ / IL-4 ratio, while the DT-MG3 / GCS-12 group had a significantly lower ratio. The high IFN-γ / IL-4 ratio induced by DT-MG3 / GCS-11 is beneficial for constructing an IFN-γ-dominated Th1 immune microenvironment, thereby promoting subsequent CTL proliferation and activation. The above results confirm that the vaccine co-assembled with the iNKT cell agonist GCS-11 and diphtheria toxin (DT) epitopes can efficiently induce the secretion of Th1 cytokines.

[0025] The DT-MG3 / GCS-11 vaccine can induce high titers of MG1-specific IgG antibodies, with an average antibody titer of approximately 112,780. Figure 3 A), which were 5.6 times that of the unadjuvanted DT-MG3 group and 110 times that of the MG3 / GCS-11 control group lacking the Th epitope, respectively. These results confirm that diphtheria toxin originates from the T-cell epitope (DT). 331-345 It has an immune synergistic effect with the NKT agonist GCS-11 and can significantly enhance the IgG response; the IgG level induced by this vaccine is about 1.5 times higher than that of the DT-MG3 / αGalCer and DT-MG3 / GCS-12 groups.

[0026] Antibody subtype analysis showed that compared with the DT-MG3 / GCS-11 group, the expression of IgG1, IgG2a, IgG2b, and IgG3 in the DT-MG3 / GCS-11 group was significantly upregulated; compared with DT-MG3 / αGalCer and DT-MG3 / GCS-12, IgG1 and IgG3 were slightly increased in this group, while the levels of IgG2a and IgG2b were not significantly different. Figure 3 B~E).

[0027] The MTT assay was used to investigate the vaccine serum-mediated target cell lysis ability based on complement-dependent cytotoxicity (CDC). Figure 3 F): Serum from mice immunized with DT-MG3 / GCS-11 significantly reduced the survival rate of EAC tumor cells; the cell survival rate in the DT-MG3 group was 42% higher than that in the DT-MG3 group, and the cell survival rates in the DT-MG3 / αGalCer and DT-MG3 / GCS-12 groups were also higher than those in the DT-MG3 / GCS-11 group after serum treatment. The underlying mechanism is that the Fc fragments of IgG2a and IgG3 can efficiently bind complement C1q, and the increased antibody titers of these two antibodies are an important reason for the enhanced killing effect of CDC cells, corroborating the fact that the DT epitope and the Th1-biased GCS-11 can synergistically enhance complement-dependent tumor cell killing. Flow cytometry results showed ( Figure 3 (G), the binding ability of the antiserum in the DT-MG3 / GCS-11 group to EAC cells was significantly better than that in the other groups, and the quantitative results of MUC1 specific antibody showed a consistent trend.

[0028] Intracellular cytokine flow cytometry results ( Figure 4 A~C): PBS blank group and DT-MG3 group IFN-γ + TNF-α + The proportion of double-positive T cells was less than 0.75%; significant differences in T cell responses were observed after combining different iNKT adjuvants, with the DT-MG3 / GCS-11 group showing higher CD4 counts. + CD8 + The proportions of double-positive T cells reached 1.63% and 1.45%, respectively, which were significantly better than those of αGalCer and the Th0-biased GCS-12 adjuvant group, confirming that Th1-type iNKT agonists are key to enhancing antigen-specific cellular immunity.

[0029] LDH release assay to detect specific in vitro tumor-killing activity ( Figure 4 D): Under the effector-target ratio of 10:1, the baseline lysis rate of the PBS group was only 6.0%; the specific lysis rate of mouse spleen cells against EAC cells in the DT-MG3 / GCS-11 group reached 17.8%, and the anti-tumor killing effect was comprehensively better than the other experimental groups.

[0030] Example 5: In vivo therapeutic effects of peptide vaccines and their synergistic anti-tumor effects with PD-L1 inhibitors Female BALB / c mice aged 4–6 weeks were subcutaneously inoculated with Ehrlich ascites carcinoma (EAC) cells. On day 8, they were randomly assigned to one of the following groups (n=5): PBS control group, DT-MG3 group, DT-MG3 / αGalCer group, DT-MG3 / GCS-12 group, DT-MG3 / GCS-11 group, and DT-MG3 / GCS-11+BMS-202 combination group. The vaccine was administered intraperitoneally or peritumorally (on days 8, 12, and 16). The combination group received a simultaneous intraperitoneal injection of BMS-200 (200 μg / injection). Figure 5 A). Measure the tumor's major and minor axes every 48 hours to plot growth curves and Kaplan-Meier survival curves.

[0031] Technical efficacy: The DT-MG3 / GCS-11 vaccine formulation can effectively inhibit tumor growth, with an average tumor volume of approximately 500 mmHg as the experimental endpoint. 3 The tumor-suppressing effect was significantly better than that of the αGalCer adjuvant group and the GCS-12 adjuvant group. Figure 5 (B) To further overcome the potential immune escape of tumors, this study combined the vaccine with the immune checkpoint inhibitor BMS-202. Results showed that tumor growth was significantly inhibited in the DT-MG3 / GCS-11 combined with BMS-202 group, with the endpoint mean tumor volume below 400 mm. 3 Significantly lower than all control groups ( Figure 5 C).

[0032] The average tumor weight in the DT-MG3 / GCS-11 monotherapy group decreased to below 1g; while the average tumor weight in the DT-MG3 / GCS-11 combined with BMS-202 group further decreased to approximately 0.5g. Figure 5 D). Analysis of the survival rate of tumor-bearing mice further validated the therapeutic effect of the vaccine. Figure 5 E). At the end of the 40-day observation period, the survival rate of mice in the DT-MG3 / GCS-11 monotherapy group reached 40%; after being combined with BMS-202, the overall survival rate of mice increased to 80%.

[0033] The above results indicate that the combination of tumor vaccines and small molecule PD-L1 inhibitors can effectively inhibit tumor growth in mice and significantly prolong the survival of tumor-bearing mice. Figure 5 A is a schematic diagram of the tumor inoculation and immunotherapy administration process.

[0034] Example 6: In the polypeptide vaccine combination group, H&E staining of major organs showed no pathological changes, and liver and kidney function biochemical indicators were within the normal range. To further evaluate the safety of the candidate vaccine, histopathological examinations were performed on the heart, liver, spleen, lungs, kidneys, brain, and other major organs of immunized mice.

[0035] Technical results: Microscopic observation revealed no obvious pathological damage to the organs of mice immunized with the vaccine in any group, indicating that the vaccine does not induce severe inflammation or organ damage in mice. Figure 6 A). Furthermore, there were no statistically significant differences in blood biochemical parameters between the vaccine-treated group and the PBS control group ( Figure 6 (B) indicates that the vaccine has no significant toxic side effects. These results confirm that the candidate vaccine has good safety, laying the foundation for subsequent preclinical research and clinical translation.

[0036] Example 7: Synergistic anti-tumor effect mechanism of gastric cancer vaccine combined with PD-L1 inhibitor This study will use the T helper cell epitope DT 331-345 A novel gastric cancer liposomal vaccine was successfully constructed by covalently coupling the MG3 antigen peptide with the fusion peptide and then co-encapsulating the fusion peptide with the Th1-biased iNKT agonist GCS-11 in liposomes. Figure 7 The DT-MG3 / GCS-11 liposomal vaccine not only significantly enhances MG3-specific humoral immune responses and activates complement-dependent cytotoxicity, but also effectively promotes T-cell immune activation. Most importantly, this liposomal vaccine, when used in combination with the PD-L1 inhibitor BMS-202, produces a potent synergistic anti-tumor effect, inhibiting tumor progression and prolonging the survival of tumor-bearing mice. Figure 8 ).

[0037] In conclusion, this study demonstrates the great application potential of liposomal vaccines combined with immune checkpoint blockade therapy, providing a feasible technical strategy for developing precise combined immunotherapy regimens for gastric cancer.

[0038] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0039] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fusion polypeptide, characterized in that, The MG3 polypeptide, composed of a T-auxiliary epitope peptide and a triplet MG1 peptide, is covalently linked at the N-terminus by an amide bond, wherein the linker lysine residue is removed at the splice site between adjacent MG1 fragments of MG3. T-helper epitope peptides include diphtheria toxin-derived T-helper epitope DT. 331-345 Its amino acid sequence is shown in SEQ ID NO: 1; The unit price table for each MG1 is shown in SEQ ID NO: 2; The amino acid sequence of the MG3 polypeptide is shown in SEQ ID NO:

3.

2. The method for preparing the fusion polypeptide according to claim 1, characterized in that, Includes the following steps: S1. MG1 amino acids are sequentially inserted into the resin from the C-terminus to the N-terminus, and three MG1 sequences are continuously connected in series to obtain the MG3 backbone. The lysine residues are omitted at the splicing points of adjacent MG1 to control the generation of non-specific epitopes. The resin is Rink-Amide-AM resin. S2. The full sequence of T-helper epitope peptides is continuously coupled to the N-terminus of the MG3 backbone; S3 was subjected to lysis, ether precipitation, and reversed-phase HPLC purification to obtain the fusion polypeptide Th epitope-MG3.

3. A raw material for an antitumor vaccine targeting MG7-Ag gastric cancer, characterized in that, It includes: the fusion polypeptide as described in claim 1, or the fusion polypeptide prepared by the preparation method described in claim 2.

4. The antitumor vaccine raw material according to claim 3, characterized in that, It also includes liposomes containing Th1-type GCS-11 adjuvant.

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