A polypeptide vaccine delivery vehicle and methods of making the same

Abstract

The application provides a polypeptide vaccine delivery carrier, which is a phenylalanine-based polyester amide polymer prepared from triethylamine, p-nitrophenol, L-phenylalanine and butanediol. + The polypeptide vaccine has good stability, high antigen presentation effect, and can induce the body to produce effective antitumor CD8 T cell immune response, inhibit tumor growth and metastasis, and improve the effect of immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a polypeptide vaccine delivery vector and its preparation method. Background Technology

[0002] As a promising immunotherapy approach, tumor vaccines promote the presentation of tumor antigens by antigen-presenting cells, inducing the immune system to produce an immune response against these antigens, thereby specifically killing tumor cells. This aims to control tumor progression, eliminate residual tumor cells, and generate lasting anti-tumor immune memory. These advantages give tumor vaccines enormous clinical application potential in preventing tumor development, improving existing immunotherapy regimens, and eradicating latent tumor cells in recovered patients. However, despite numerous reports of research and clinical trials on tumor vaccines, current tumor vaccines still face challenges such as weak immunogenicity of tumor antigens and the inability to induce sufficient antigen-specific CD8+. + The challenges include low T-cell response and overall low vaccine efficacy. Therefore, overcoming these challenges and developing more effective cancer vaccines is a crucial issue.

[0003] Tumor vaccines consist of tumor antigens, vaccine adjuvants, and delivery systems. Based on the form of the antigen, tumor vaccines are classified into DNA vaccines, RNA vaccines, peptide vaccines, cell vaccines, and viral vaccines. Among these, peptide vaccines, because the antigen exists in the form of synthetic peptide chains, offer guaranteed safety, stability, and efficacy, and are currently the widely used formulation in clinical trials of tumor vaccines. Adjuvants are non-antigen components that enhance the immunogenicity of vaccines and typically have an immune-stimulating effect. Although existing vaccine adjuvants, such as aluminum salts, MF59, and the ASO family, often activate innate immune responses, challenges remain in overcoming the immunosuppressive tumor microenvironment and enhancing the immunogenicity of low-immunogenic tumor antigens. Therefore, it is necessary to find new immunostimulatory molecules for use in the design of tumor vaccines.

[0004] Delivery systems have a significant impact on the efficacy of tumor vaccines. Effectively transporting tumor antigens into the body, presenting them to antigen-presenting cells, and inducing a significant anti-tumor immune response is a crucial challenge in designing an effective tumor vaccine. In recent years, tumor nanovaccines have shown great potential to enhance vaccination efficacy and have gradually become a research hotspot in the field of vaccinology. Compared to previous tumor vaccines that have not achieved significant clinical benefits, nanovaccines can efficiently deliver tumor-specific antigens and immune adjuvants to antigen-presenting cells to stimulate cytotoxic T-cell responses, enabling patients to produce effective, specific, and durable anti-tumor responses, making them a promising next-generation immunotherapy. However, despite numerous research reports on tumor nanovaccines, existing nanovaccines still suffer from the limitation of not being able to induce a sufficient number of antigen-specific T cells in vitro, especially CD8 cells. + T cells. Furthermore, preclinical, late-stage, and clinical trials have all presented challenges to existing nanovaccine design schemes, as large-scale production requires the simplest and most scalable operational process possible, while existing nanovaccines are often quite complex to prepare. In addition, the toxic side effects of existing nanovaccines are a critical issue that must be considered when translating and applying them.

[0005] Therefore, existing research on tumor nanovaccines is not yet perfect and needs to be explored more deeply to find out whether there are other new and effective nanovaccines for the treatment of tumors, or to combine them with existing immunotherapies to synergistically enhance the treatment effect. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing a polypeptide nanoparticle vaccine delivery carrier that is stable, efficient, low in toxicity, safe and effective.

[0007] Another object of the present invention is to provide a method for preparing the polypeptide nanovaccine delivery carrier.

[0008] Another object of the present invention is to provide a polypeptide vaccine.

[0009] Another object of the present invention is to provide a method for preparing the polypeptide vaccine.

[0010] Therefore, the present invention provides a polypeptide vaccine delivery carrier, which is a phenylalanine-based polyester amide polymer, wherein the phenylalanine-based polyester amide polymer is prepared from triethylamine, p-nitrophenol, L-phenylalanine and butanediol.

[0011] Preferably, the molar ratio of triethylamine to p-nitrophenol in a single reaction for synthesizing type I monomers is 32:31, and the molar ratio of L-phenylalanine to butanediol in a single reaction for synthesizing type II monomers is 2:1.

[0012] The present invention also provides a method for preparing the polypeptide vaccine delivery vector, comprising the following steps:

[0013] (1) Synthesis of Class I monomers: Triethylamine and p-nitrophenol were mixed in a molar ratio of 32:31, acetone was added, the mixture was placed in an ice bath, and then sebacate chloride was added dropwise. The molar ratio of sebacate chloride to p-nitrophenol was 2:1. The reaction temperature was 0℃, and the mixture was stirred for 2 hours. After the reaction was completed, the generated p-nitrophenyl sebacate was left at room temperature overnight, then precipitated and washed with distilled water, and then dried under vacuum. The obtained p-nitrophenyl sebacate was recrystallized three times at room temperature in an ethyl acetate / dimethylformamide mixture, wherein the volume ratio of ethyl acetate to dimethylformamide in the ethyl acetate / dimethylformamide mixture was 4:1. Finally, Class I monomers were obtained.

[0014] (2) Synthesis of Class II monomers: L-phenylalanine and butanediol were mixed in a molar ratio of 2:1, toluene and p-toluenesulfonic acid monohydrate were added, and the mixture was stirred and refluxed at 130°C for 24 hours. The reaction mixture was then cooled to room temperature. After toluene precipitated, the product was purified with isopropanol and finally dried under vacuum to obtain Class II monomers.

[0015] (3) In dimethyl sulfoxide, type I monomers and type II monomers are added in a molar ratio of 1:1, mixed well, and then triethylamine is added until the monomers are dissolved. The reaction is carried out at 75°C for 48 hours. The monomers are then precipitated and purified in pre-cooled ethyl acetate, dissolved in methanol, and precipitated and purified again. Finally, the monomers are dried under vacuum at 60°C to obtain the phenylalanine-based polyester amide polymer.

[0016] On the other hand, the present invention also provides the use of phenylalanine-based polyesteramide polymer as a delivery carrier for peptide vaccines, wherein the phenylalanine-based polyesteramide polymer is prepared by reacting triethylamine, p-nitrophenol, L-phenylalanine, and butanediol.

[0017] Preferably, the molar ratio of triethylamine to p-nitrophenol in a single reaction for synthesizing type I monomers is 32:31, and the molar ratio of L-phenylalanine to butanediol in a single reaction for synthesizing type II monomers is 2:1.

[0018] On the other hand, the present invention also provides a polypeptide vaccine comprising the polypeptide vaccine delivery vector described in the present invention, a polypeptide encoding a specific antigen, and a stabilizer.

[0019] Preferably, the mass ratio of the phenylalanine-based polyesteramide polymer to the polypeptide is 10:1, and the mass ratio of the phenylalanine-based polyesteramide polymer to the stabilizer is 5:1.

[0020] Preferably, the polypeptide is a fusion peptide composed of the short peptide FK-13 and a tumor antigen.

[0021] Preferably, the stabilizer includes, but is not limited to, distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

[0022] Preferably, the molar ratio of triethylamine to p-nitrophenol in a single reaction for synthesizing type I monomers is 32:31, and the molar ratio of L-phenylalanine to butanediol in a single reaction for synthesizing type II monomers is 2:1.

[0023] The present invention also provides a method for preparing the polypeptide vaccine, comprising the following steps:

[0024] (1) Dissolve the phenylalanine-based polyester amide polymer in dimethyl sulfoxide at a concentration of 20 mg / ml, and heat to 60°C until it is completely dissolved;

[0025] (2) Add phenylalanine-based polyester amide polymer, peptide and stabilizer in sequence, with dimethyl sulfoxide as solvent, and vortex mix.

[0026] (3) After mixing, add the mixture from step (2) dropwise to the corresponding volume of sterile water. The volume ratio of the mixture to the water is 1:10. After the addition is complete, vortex to mix.

[0027] (4) Let stand for 10 minutes, then remove dimethyl sulfoxide by ultrafiltration. Wash with sterile water during ultrafiltration. The remaining liquid after ultrafiltration is the polypeptide vaccine.

[0028] Preferably, an ultrafiltration tube with a pore size of 100 kDa is used for ultrafiltration.

[0029] On the other hand, the present invention also provides the use of the said polypeptide vaccine in the preparation of a medicament for the prevention or treatment of tumors.

[0030] On the other hand, the present invention also provides the use of the polypeptide vaccine in the preparation of a medicament that promotes the efficacy of immunotherapy. Preferably, the immunotherapy includes, but is not limited to, anti-PD-1 or anti-PD-L1 therapy.

[0031] This invention addresses the problems of low peptide antigen immunity and low antigen delivery efficiency in existing peptide tumor vaccines. Using the strength of CD8+ T cell activation as an indicator, a fusion peptide of the short peptide FK-13 and a tumor antigen was constructed. One polymer (code-named 8p4) was selected from six candidate phenylalanine-based polyesteramide polymers as the peptide vaccine delivery carrier. This phenylalanine-based polyesteramide polymer 8p4 was then combined with a peptide containing the tumor antigen (code-named FK-33) to form a peptide nanotumor vaccine. Experiments have demonstrated that this vaccine can efficiently deliver antigens in cells and animals, induce specific immune responses against tumor-specific antigens, and improve the immunosuppressed microenvironment within tumors, thereby achieving tumor killing. This invention confirms that the 8p4+FK-33 nanovaccine (code-named 8FNs) exhibits highly efficient antigen delivery capabilities, and the short peptide FK-13 can promote tumor antigen presentation and activate CD8+ T cells. + T cells play a role and can effectively inhibit the growth and metastasis of tumors in the body by inducing specific immune responses, alleviate the immunosuppressed microenvironment within tumors, and achieve better therapeutic effects when used in combination with anti-PD-1 therapy. Attached Figure Description

[0032] Figure 1 This is a schematic diagram illustrating the synthesis reaction principle of phenylalanine-based polyester amide polymer 8p4.

[0033] Figure 2 This is a schematic diagram and the specific amino acid sequence of the antigenic peptide FK-33.

[0034] Figure 3 This is a schematic diagram of the physicochemical characterization of the polypeptide nanovaccine 8FNs.

[0035] Figure 4 Based on CD8 + In vitro screening results of peptide vaccine vectors for T-cell activation experiments.

[0036] Figure 5 The therapeutic and preventive effects of the peptide nanovaccine 8FNs on a mouse B16-OVA tumor model were demonstrated.

[0037] Figure 6 The study demonstrated the preventive effect of the peptide nanovaccine 8FNs@Trp2 on a mouse B16-F10 tumor lung metastasis model and its therapeutic effect in combination with anti-PD-1 therapy. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following embodiments.

[0039] Unless otherwise specified, all reagents mentioned below are commercially available. For simplicity, some procedures have not been detailed in terms of parameters, steps, and instruments used. It should be understood that these are well-known and reproducible to those skilled in the art.

[0040] The phenylalanine-based polyester amide polymer 8p4 used in this invention is a neutral L-phenylalanine polymer with hydrophobic properties. When mixed with peptides and stabilizers for nanoprecipitation, it can self-assemble into nanoparticles that encapsulate the antigen peptides within. The raw materials for synthesizing phenylalanine-based polyester amide polymer 8p4 are triethylamine, p-nitrophenol, L-phenylalanine, and butanediol.

[0041] The preparation steps of phenylalanine-based polyesteramide polymer 8p4 are as follows:

[0042] (1) To synthesize type I monomers, 0.32 mol of triethylamine and 0.31 mol of p-nitrophenol (molar ratio 32:31) were mixed and added to a round-bottom flask containing 200 ml of acetone. The mixture was placed in an ice bath, and then 0.62 mol of sebacyl chloride was added dropwise. The reaction temperature was 0 °C, and the mixture was stirred for 2 hours. After the reaction was completed, the generated p-nitrophenyl sebacic acid was left at room temperature overnight, then precipitated with distilled water, washed, and dried under vacuum. At room temperature, the obtained p-nitrophenyl sebacic acid was recrystallized three times in a mixture of ethyl acetate / dimethylformamide (volume ratio of ethyl acetate to dimethylformamide 4:1) to finally obtain type I monomers.

[0043] (2) Synthesis of Class II monomers: 0.04 mol L-phenylalanine and 0.02 mol butanediol (molar ratio 2:1) were mixed in a three-necked flask, and 400 ml toluene and 0.082 mol p-toluenesulfonic acid monohydrate were added. The mixture was stirred and refluxed at 130 °C for 24 hours. The reaction mixture was then cooled to room temperature, and the precipitated toluene was poured off. 100 ml isopropanol was added, and the product was dissolved at 75 °C, then precipitated at 4 °C. The dissolution and precipitation steps were repeated three times for purification. Finally, the product was dried under vacuum to obtain the Class II monomers.

[0044] (3) In 1.5 ml of dimethyl sulfoxide (DMSO), 1.0 mmol of type I monomer and 1.0 mmol of type II monomer (molar ratio 1:1) were added, stirred and mixed, and heated to 75 °C. Then, triethylamine (2.2 mmol) was added dropwise, and the mixture was stirred vigorously until the monomers were completely dissolved. The reaction was then carried out at 75 °C for 48 hours. The product was then precipitated in 10 ml of pre-cooled ethyl acetate, decanted, dried, and dissolved in 10 ml of methanol. The precipitation and purification steps were repeated. Finally, the product was dried under vacuum at 60 °C to obtain phenylalanine-based polyesteramide polymer 8p4 (pale yellow powder).

[0045] The obtained phenylalanine-based polyester amide polymer 8p4 was identified as a neutral and hydrophobic L-phenylalanine polymer. Figure 1 The synthesis reaction principle of phenylalanine-based polyesteramide polymer 8p4 is shown. In this diagram, A represents a type I monomer prepared by reacting triethylamine with p-nitrophenol, B represents a type II monomer prepared by reacting L-phenylalanine with butanediol, and C represents the phenylalanine-based polyesteramide polymer obtained by reacting type I and type II monomers.

[0046] The short peptide FK-13 (amino acid sequence FKRIVQRIKDFLR (SEQ ID NO:1)) and the fusion peptide FK-33 of the tumor antigen peptide in this invention were synthesized by solid-phase method, and the peptide purity was verified to be above 95% by high-performance liquid chromatography. In the specific experimental process, commonly used antigenic epitope peptides in immunoassays, such as the short peptide OVA composed of amino acids at positions 257-264 of ovalbumin (OVA), were selected. 257-264 And the short peptide Trp2, composed of amino acids 181-188 of the TRP2 protein (tumor antigen of mouse melanoma cells B16-10). 181-188 Different antigen fusion peptides, FK-33 or FK-33@Trp2, were synthesized by combining FK-13 with the short peptide FK-13 for experimental purposes. Taking the antigen fusion peptide FK-33 as an example, FK-33 is composed of FK-13, a cathepsin hydrolysis sequence (amino acid sequence SLVR), and an antigenic epitope peptide OVA. 257-264 A 33-amino acid polypeptide formed by linking 4 lysine (K) amino acids (amino acid sequence FKRIVQRIKDFLRSLVRSIINFEKLSLVRKKKK; SEQ ID NO:2). Figure 2 A schematic diagram of the antigen fusion peptide FK-33 and its corresponding amino acid sequence are shown. Furthermore, the included antigenic epitope sequences can be replaced as needed to adapt to the production of vaccines targeting different tumor antigens.

[0047] The preparation process of peptide tumor vaccines is as follows:

[0048] First, calculate the required amounts of nanomaterials and peptides based on the experimental calculations. Before use, heat phenylalanine-based polyesteramide polymer 8p4 (concentration 20 mg / ml, solvent: dimethyl sulfoxide) to 60°C until completely dissolved. Then, add phenylalanine-based polyesteramide polymer 8p4, fusion peptide FK-33, and stabilizer DSPE-PEG2000 (solvent: dimethyl sulfoxide, concentration 10 mg / ml) in the following order according to the mass ratio (phenylalanine-based polyesteramide polymer 8p4: fusion peptide FK-33: stabilizer DSPE-PEG2000 = 10:1:2). After the reagents are added, use a shaker (Vort) to... Mix thoroughly; after mixing, add it dropwise to the corresponding volume of sterile water at a volume ratio of 1:10. After the addition is complete, vortex to mix. Note that the sterile water should be added dropwise while vortexing with Vortex. After all the liquid has been added, mix it completely with Vortex. After thorough mixing, let it stand for 10 minutes, and then use a 100kDa ultrafiltration tube to remove reagents that have not formed nanoparticles. During ultrafiltration, use sterile water to wash the nanoparticle vaccine particles. The remaining liquid after ultrafiltration is the polypeptide tumor vaccine (8p4+FK-33 nanoparticle vaccine, i.e., 8FNs) with 8p4 as the delivery carrier.

[0049] The prepared 8FNs nanovaccine was identified as a nanoparticle formed by encapsulating an antigenic peptide in a phenylalanine-based polyesteramide polymer. These nanoparticles have a diameter of approximately 120 nm, and monitoring over a certain period (7 days) revealed good stability. The nanoparticles encapsulate the antigenic peptide in the center of the phenylalanine-based polyesteramide polymer, thus protecting the peptide from nuclease degradation. The stabilizer DSPE-PEG2000 further enhances the stability of the nanoparticles, making them less susceptible to the influence of the internal physiological environment. Figure 3 The physicochemical characterization of the peptide nanovaccine 8FNs is shown. The prepared nanovaccine consists of particles with a diameter of approximately 120 nm. Figure 3 A). Nanoparticle stability testing showed that within a short period (7 days), the PDI (Polydispersity Index) of the nanoparticles remained around 0.25, and the particle size also remained basically at 120 nm. Figure 3 (B); This indicates that the mRNA nanovaccine of the present invention has good stability. Surface charge detection shows that the nanovaccine particles prepared by the present invention are molecules carrying negative charges (B). Figure 3 C). Figure 3 In the figure, D represents the morphology and size of the 8FNs nanoparticles used in the vaccine under a transmission electron microscope.

[0050] Figure 4This is the in vitro screening result of peptide vaccine vectors based on CD8+ T cell activation experiments. A is a schematic diagram of the in vitro antigen presentation experiment. The nanovaccine 8FNs encapsulating peptide antigens are taken up by dendritic cells, activating them and processing and presenting the antigen peptides to T cells. Activated T cells secrete the cytokine IL-2. Different peptide vaccines were prepared using the synthesized fusion peptide FK-33 with six different materials as delivery vectors (the raw materials and their proportions for the synthesis of the six different biomaterials used as delivery vectors are shown in Table 1). These six nanovaccines were co-incubated with mouse bone marrow-derived primary dendritic cells (BMDCs), and then used to stimulate B3Z cells (OVA-specific CD8+ T cells). + T cells can recognize the OVA antigen epitope presented by MHC. 257-264 (And is activated) to secrete the cytokine IL-2. The amount of IL-2 secreted is detected by ELISA. Figure 4 Results B showed that 8p4, as a delivery vector for the peptide vaccine, exhibited the highest level of T cell activation and stimulated higher levels of IL-2 secretion compared to five other phenylalanine-based polyesteramide polymer materials (8p2, 8p3, 8p5, 8p6, and 8p8) using different diols as vectors. Full-length OVA protein (ovalbumin) was used as a control in this experiment. The raw materials and formulations of the six different biomaterials used as delivery vectors are shown in Table 1 below.

[0051] Table 1. Raw materials and their proportions for the synthesis of six different biomaterials used as delivery carriers.

[0052]

[0053] To verify that the 8FN nanovaccine can induce tumor antigen-specific CD8 in mice... + T-cell immune responses are studied, and therefore their function is verified through animal experiments.

[0054] First, the experiment was conducted in four groups (control group, nanocarrier 8p4 group, Alum+FK-33 control group, and nanovaccine 8FNs group). In the Alum+FK-33 group, Alum is aluminum hydroxide, an immune adjuvant that is widely used in clinical vaccines. It can promote the body's immune response to antigens and improve the immune effect of the vaccine.

[0055] First, subcutaneously inject mouse melanoma cells B16-OVA (B16-OVA cells are subcutaneously injected into the back of each mouse, 2 × 10⁶ cells per mouse). 5 (each cell), and then subcutaneously inject the control group material or vaccine into the groin area of ​​each group of mice, immunizing them every 4 days for a total of 2 immunizations, as follows. Figure 5As shown in Figure A, it can be seen that tumor growth in mice was inhibited after immunization with the peptide nanovaccine 8FNs, and it achieved better therapeutic effects than the traditional adjuvant Alum+FK-33 vaccine. Figure 5 B).

[0056] To further investigate whether the 8FN nanovaccine could prevent tumor development, mice were divided into a control group and an 8FN nanovaccine group. Mice were immunized subcutaneously in the groin area every 7 days for a total of two immunizations, followed by subcutaneous inoculation with tumor cells B16-OVA (2 × 10⁶ B16-OVA cells were injected subcutaneously in the back of each mouse). 5 (cells), steps as follows Figure 5 As shown in C. (As illustrated in Figure 1) Figure 5 As shown in Figure D, 8FNs can significantly prevent tumor development, and after vaccination, the CD8+ of tumor antigen-specific (OVA tetramer, SIINFEKL-tetramer) in mice is reduced. + The proportion of T cells increased significantly. Figure 5 (E). Therefore, the nano-vaccine 8FNs of the present invention can activate the presentation of tumor antigens and promote antigen-specific CD8. + The production of T cells can treat and prevent tumor progression.

[0057] Subsequently, to verify whether the nanovaccine 8FNs could adapt to different tumor antigens, treat tumor metastases, and improve the therapeutic effect of anti-PD-1 immunotherapy, a commonly used immunotherapy regimen in clinical tumor treatment, the antigenic epitope peptide Trp2 of the TRP2 protein in mouse melanoma cells B16-F10 was first used. 181-188 (Amino acid sequence VYDFFVWL; SEQ ID NO:3) replaced the model antigen OVA. 257-264 A novel antigenic peptide, FK-33@Trp2 (amino acid sequence FKRIVQRIKDFLRSLVRVYDFFVWLSLVRKKKK; SEQ ID NO:4), was synthesized. Figure 6 A) and synthesized a new nanovaccine 8FNs@Trp2.

[0058] To investigate whether 8FNs@Trp2 can prevent lung metastasis of B16-F10 cells, mice were divided into a control group and an 8FNs@Trp2 group. Mice were then immunized subcutaneously in the groin every 7 days for a total of two immunizations. Following this, B16-F10 cells (1.6 × 10⁶ cells per mouse) were injected via the tail vein. 5 (10 B16-F10 tumor cells), mice were sacrificed 17 days later, and lung tissue was taken to observe the number of tumor metastases. The procedure is as follows: Figure 6 As shown in Figure B. Experimental results show that the nano-vaccine 8FNs@Trp2 can effectively prevent lung metastasis of B16-F10 ( Figure 6 C).

[0059] To further investigate whether the combined use of the nanovaccine 8FNs@Trp2 could enhance the efficacy of anti-PD-1 immunotherapy, mice were first divided into four groups: a control group, an anti-PD-1 treatment group, an 8FNs@Trp2 nanovaccine group, and an 8FNs@Trp2 combined with anti-PD-1 treatment group. Mice were first subcutaneously injected with mouse melanoma cells B16-F10 (2 × 10⁶ cells per mouse, subcutaneous injection on the back). 5 (cells), and then subcutaneously injected the vaccine into the groin area of ​​mice in each group, immunizing them every 4 days for a total of 2 immunizations. Mice in the anti-PD-1 treatment group were injected with anti-PD-1 antibody (intraperitoneal injection, 150 μg per mouse) on days 9, 12, and 15 after tumor inoculation, as follows: Figure 6 As shown in Figure D. Tumor growth curve ( Figure 6 (E) and mouse survival curve ( Figure 6 The results showed that 8FNs@Trp2 could improve the efficacy of anti-PD-1 treatment, inhibit tumor growth, and prolong the survival time of tumor-bearing mice.

Claims

1. A polypeptide vaccine, characterized in that, The peptide vaccine comprises a peptide vaccine delivery carrier, a peptide, and a stabilizer; The mass ratio of the peptide vaccine delivery carrier to the peptide is 10:1, and the mass ratio of the peptide vaccine delivery carrier to the stabilizer is 5:

1. The polypeptide is a fusion peptide composed of the short peptide FK-13 and a tumor antigen; The sequence of the fusion peptide is shown in SEQ ID NO:2 or SEQ ID NO:4; The structure of the polypeptide vaccine delivery vector is as follows: 。 2. The polypeptide vaccine according to claim 1, characterized in that, The stabilizer is distearylphosphatidylethanolamine-polyethylene glycol 2000.

3. The method for preparing the polypeptide vaccine according to claim 1 or 2, comprising the following steps: (1) Dissolve the polypeptide vaccine delivery carrier in dimethyl sulfoxide at a concentration of 20 mg / ml and heat to 60°C until it is completely dissolved; (2) Add the polypeptide vaccine delivery carrier, the polypeptide and the stabilizer in sequence, with dimethyl sulfoxide as the solvent, and vortex mix. (3) After mixing, add the mixture from step (2) dropwise to the corresponding volume of sterile water. The volume ratio of the mixture to the water is 1:

10. After the addition is complete, vortex to mix. (4) Let stand for 10 minutes, then remove dimethyl sulfoxide by ultrafiltration. Wash with sterile water during ultrafiltration. The remaining liquid after ultrafiltration is the polypeptide vaccine.

4. The method according to claim 3, characterized in that, Ultrafiltration was performed using an ultrafiltration tube with a pore size of 100 kDa.

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