A tumor polypeptide vaccine for treating breast cancer and its preparation method

Abstract

This invention provides a tumor peptide vaccine for treating breast cancer and a method for preparing the same, wherein the tumor peptide vaccine comprises: a peptide mixture containing 8 CD8+ molecules. + T cell epitopes peptide fragments and 3 CD4+ + polypeptide fragments of T cell epitopes; the eight CD8... + The amino acid sequences of the polypeptide fragments of T cell epitopes are shown in SEQ ID NO: 1-8, and the three CD4 lines... + The amino acid sequences of the polypeptide fragments of T cell epitopes are shown in SEQ ID NO: 9-11; all of the polypeptide fragments are synthesized from mutant polypeptide sequences of the tumor-associated gene SWAP70; the present invention has the beneficial effect of effectively promoting the generation of T cell responses in the body and is applicable to the field of tumor immunology.

Description

Technical Field

[0001] This invention relates to the field of tumor immunology, and more specifically to a tumor polypeptide vaccine for the treatment of breast cancer. Background Technology

[0002] Tumor vaccines introduce tumor antigens into the patient's body in various forms, activating the body's immune system, thereby breaking the state of immunosuppression and inducing the body's own immune response. This transforms the body from passive to active cancer fighting, thereby inhibiting or eliminating tumors and achieving the goal of cancer treatment.

[0003] SWAP70 is a member of the guanine nucleotide exchange factor (GEF) family, with a molecular weight of 70 kDa. This protein has a relatively complex structure, composed of multiple domains, including an N-terminal EF-hand domain, a PH domain, a DH domain, a coiled-coil domain, and a C-terminal domain that binds F-actin. The EF-hand domain is an immune receptor tyrosine-activated motif, capable of binding Ca2+. 2+ The ability of the PH domain to bind phosphatidylinositol (3,4,5)-trisphosphate (PIP3) contributes to the activation of Rac1. The Coiled-coil domain contains a DH domain, which is arranged in the opposite order to other classic Rho-GEFs. This DH domain is the main catalytic domain of the SWAP70 protein and interacts with Rac-GTPases, promoting their GDP / GTP exchange. The 60 amino acid residues at the C-terminus can bind F-actin to participate in cell movement.

[0004] Most current therapeutic tumor vaccines can induce CD8+. + T cells respond to MHC I-restricted neoantigen fragments, but their anti-tumor immune effect is generally limited. Summary of the Invention

[0005] In view of the shortcomings of related technologies, the technical problem to be solved by the present invention is to provide a tumor polypeptide vaccine for the treatment of breast cancer that can effectively promote the body to produce T cell response and a method for preparing the same.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a tumor polypeptide vaccine for treating breast cancer, comprising: a polypeptide mixture: the polypeptide mixture comprising 8 CD8+ molecules. + T cell epitopes peptide fragments and 3 CD4+ +polypeptide fragments of T cell epitopes; the eight CD8... + The amino acid sequences of the polypeptide fragments of T cell epitopes are shown in SEQ ID NO: 1-8, and the three CD4 lines... + The amino acid sequences of the polypeptide fragments of T cell epitopes are shown in SEQ ID NO: 9-11; all of the polypeptide fragments are synthesized from mutant polypeptide sequences of the tumor-associated gene SWAP70.

[0007] Preferably, the tumor peptide vaccine further includes an adjuvant.

[0008] Preferably, the adjuvant is a polyinosinic acid-polycytosine nucleotide dilution.

[0009] This invention also provides a method for preparing a tumor polypeptide vaccine for treating breast cancer, comprising the following steps:

[0010] 1) 8 CD8s + Preparation of polypeptide fragments from T cell epitopes:

[0011] CD8 sequence analysis of the mutant polypeptide sequence of the tumor-associated gene SWAP70 + T-cell epitope prediction was used to select the top 20 mutant peptide sequences. These 20 sequences were then synthesized into peptide fragments. These fragments were mixed with an adjuvant, and eight fragments (eight CD8+) were selected. + The polypeptide fragments of T cell epitopes have amino acid sequences as shown in SEQ ID NO: 1-8.

[0012] 2) Preparation of polypeptide mixture:

[0013] The 8 selected CD8 + T cell epitope polypeptide fragments and 3 CD4 lines as shown in SEQ ID NO: 9-11 + The peptide fragments of T cell epitopes were prepared into a 50 mg / ml solution using sterile water. Then, the prepared solutions were mixed together in equal mass and diluted with PBS buffer to a concentration of 15 mg / ml to obtain the peptide mixture.

[0014] 3) Preparation of tumor polypeptide vaccines:

[0015] A tumor peptide vaccine can be obtained by mixing the peptide mixture with an adjuvant in equal volumes.

[0016] Preferably, the adjuvant is a polyinosinic acid-polycytosine nucleotide dilution.

[0017] Preferably, the concentration of the polyinosinic acid-polycytosine nucleotide diluent in step 2) is 0.5 mg / ml.

[0018] Preferably, the preparation method of the polyinosinic acid-polycytosine nucleotide diluent is as follows: polyinosinic acid-polycytosine nucleotide powder with an average molecular weight of 1.5-8kb is prepared into a 1mg / ml stock solution with pyrogen-free deionized water; the stock solution is dissolved in a water bath at a temperature of 65-70℃ for 10 minutes, and then cooled at room temperature for 1 hour; then it is diluted with PBS buffer to a concentration of 0.5mg / ml to obtain the polyinosinic acid-polycytosine nucleotide diluent.

[0019] The beneficial technical effects of this invention are as follows:

[0020] 1. The tumor polypeptide vaccine provided by this invention has a high tumor inhibition rate, reaching 57.8%; and can effectively promote the body to produce T cell responses, generating CD4+. + T cells and CD8 + T cells actively participate in anti-tumor immune responses.

[0021] This invention will be able to induce CD8 + T cell responses to MHC I-restricted neoantigen fragments and CD4+-mediated responses + T-cell responses and MHC II-restricted neoantigen fragments were used to prepare a tumor peptide vaccine, which was generated from CD4 by MHC II-restricted neoantigens. + T cells can enhance CD8 + The anti-tumor capabilities of T cells are used in cancer treatment. This provides a reliable basis for the combination therapy of drugs such as tumor vaccines and immune checkpoint inhibitors in clinical practice.

[0022] 2. In this invention, polyinosinic acid-polycytosine nucleotide dilution is used as an adjuvant to prepare tumor peptide vaccines together with peptide mixtures. The adjuvant is easy to obtain and can better prepare tumor peptide vaccines.

[0023] 3. This invention will select 8 CD8 lines. + T-cell epitope peptide fragments and 3 designed CD4 lines + Equal masses of peptide fragments from T-cell epitopes are mixed together, and then combined with an adjuvant to obtain a tumor peptide vaccine. The preparation method is simple and easy to operate, and does not require special conditions.

[0024] In step 2), the 8 selected CD8 entries + T cell epitope polypeptide fragments and 3 CD4 lines as shown in SEQ ID NO: 9-11 + The polypeptide fragments of T cell epitopes were all prepared into 50 mg / ml solutions using sterile water. The use of sterile water ensures good solubility and is non-toxic, making it safer. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the results of ELISPOT screening for immunogenic mutant antigen polypeptide fragments provided by the present invention;

[0026] Figure 2 This is the tumor growth curve of the breast cancer xenograft model mice provided by the present invention after being given the tumor polypeptide vaccines prepared in Example 1, Comparative Example 2 and Comparative Example 3;

[0027] Figure 3 The tumor growth curves of tumor-bearing mice in the breast cancer xenograft model provided by the present invention after administration of the tumor polypeptide vaccines prepared in Example 1 and Comparative Example 1;

[0028] Figure 4 This is an immunoassay flow cytogram of spleen cells from tumor-bearing mice in this invention;

[0029] Figure 5 This is an immunoassay flow cytogram of tumor tissue cells from tumor-bearing mice in this invention;

[0030] Figure 6 This is a schematic diagram showing the proportion of lymphocyte subsets in the spleen of tumor-bearing mice in this invention;

[0031] Figure 7 This is a schematic diagram showing the proportion of immune cell subsets in tumor-infiltrating lymphocytes of tumor-bearing mice in this invention;

[0032] In the figure: G1 is the tumor polypeptide vaccine prepared in Example 1; G2 is the tumor polypeptide vaccine prepared in Comparative Example 1; G3 is the tumor polypeptide vaccine prepared in Comparative Example 2; and G4 is the tumor polypeptide vaccine prepared in Comparative Example 3. 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 some embodiments of the present invention, not all embodiments; 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. Furthermore, the present invention will be described in detail with reference to the schematic diagrams.

[0034] This application provides a tumor peptide vaccine for treating breast cancer, comprising: a peptide mixture: the peptide mixture includes 8 CD8 molecules. + T cell epitopes peptide fragments and 3 CD4+ + Peptide fragments of T-cell epitopes.

[0035] 8 CDs +The amino acid sequence of the polypeptide fragment of the T cell epitope is shown in SEQ ID NO: 1-8, with 3 CD4 lines. + The amino acid sequences of the polypeptide fragments of T cell epitopes are shown in SEQ ID NO: 9-11.

[0036] The aforementioned polypeptide fragments were all synthesized from mutant polypeptide sequences of the tumor-associated gene SWAP70.

[0037] The tumor peptide vaccine provided by this invention exhibits a high tumor inhibition rate, reaching 57.8%; and it can effectively promote the body's T-cell response, producing CD4+. + T cells and CD8 + T cells actively participate in anti-tumor immune responses.

[0038] This invention will be able to induce CD8 + T cell responses to MHC I-restricted neoantigen fragments and CD4+-mediated responses + T-cell responses and MHC II-restricted neoantigen fragments were used to prepare a tumor peptide vaccine, which was generated from CD4 by MHC II-restricted neoantigens. + T cells can enhance CD8 + The anti-tumor capabilities of T cells are used in cancer treatment. This provides a reliable basis for the combination therapy of drugs such as tumor vaccines and immune checkpoint inhibitors in clinical practice.

[0039] Furthermore, tumor peptide vaccines also include adjuvants.

[0040] Furthermore, the adjuvant is a polyinosinic acid-polycytosine nucleotide dilution.

[0041] In this invention, polyinosinic acid-polycytosine nucleotide dilution is used as an adjuvant to prepare tumor peptide vaccines together with peptide mixtures. The adjuvant is easy to obtain and can better prepare tumor peptide vaccines.

[0042] Example 1

[0043] This application provides a method for preparing a tumor polypeptide vaccine for treating breast cancer, comprising the following steps:

[0044] 1) 8 CD8s + Preparation of polypeptide fragments from T cell epitopes

[0045] Whole-exome sequencing and mRNA gene expression sequencing: DNA from breast cancer cells was randomly fragmented into 150-200 bp segments using an ultrasonic disruptor. After purification with magnetic beads, the fragments underwent end-repair and A-base addition to the 3' ends to form sticky ends. These were then ligated with adapters containing specific barcode sequences. Magnetic beads were used to screen and remove incompletely ligated products and adapter self-ligation products. PCR amplification was performed using universal primers complementary to the adapter sequences to form the total sequencing library. Hybridization was then performed using Agilent probes in hybridization buffer, and the target fragments were captured and purified using magnetic beads. PCR amplification of the captured DNA fragments and purification of the PCR products yielded the target sequencing library. The library concentration was detected using a Qubit real-time fluorescence analyzer, and the fragment lengths were determined using an Agilent 2100 Bioanalyzer. Finally, next-generation sequencing was performed using Illumina.

[0046] The data obtained from whole-exome sequencing were analyzed. Then, the CD8 sequence of the mutant peptide of the tumor-associated gene SWAP70 was analyzed using NetMHC and NetMHCpan software. + T-cell epitope prediction was performed, and neoantigen mutation sequences were scored. The top 20 mutant polypeptide sequences were selected, and then polypeptide fragments were synthesized from the top 20 mutant polypeptide sequences using chemical synthesis methods.

[0047] Twenty chemically synthesized polypeptide fragments were mixed together and mixed with an adjuvant polyinosinic acid-polycytosine nucleotide dilution to prepare solution 1. BALB / c mice were immunized with solution 1 on the right hind limb back, with a booster immunization on day 7. Fourteen days after immunization, the spleens of the mice were harvested, and a single-cell suspension was prepared. The suspension was then analyzed using the ELISOPT enzyme-linked immunospot assay to generate IFN-γ spots greater than or equal to 10 / 5 × 10⁻⁶. 5 A single spleen cell count is considered a positive criterion. Figure 1 This is a schematic diagram illustrating the results of ELISPOT screening for immunogenic mutant antigen peptide fragments provided by the present invention, as shown in the figure. Figure 1 As shown, from 20 mutant polypeptide fragments, 8 polypeptide fragments with immunogenic mutant antigens, namely 8 CD8 peptides, were screened out. + The polypeptide fragments of T cell epitopes have amino acid sequences shown in Table 1 as SEQ ID NO: 1-8.

[0048] Based on the amino acid sequence of the SWAP70 protein and 8 CD8 sequences screened... + Mutation sites were designed in peptide fragments targeting T cell epitopes, with 19 amino acids covering the mutation sites. Three CD4 mutations were then designed. +The polypeptide fragments of T cell epitopes have amino acid sequences as shown in Table 1, SEQ ID NO: 9-11.

[0049] Table 1: Amino Acid Sequences

[0050] Serial Number Serial Number Related tumor genes amino acid sequence SEQ ID NO:1 P1 SWAP70 FLYEDKYPL SEQ ID NO:2 P4 SWAP70 STFYMRTSI SEQ ID NO:3 P7 SWAP70 YGLFSTFYM SEQ ID NO:4 P8 SWAP70 YEDKYPLII SEQ ID NO:5 P11 SWAP70 GLFSTFYMR SEQ ID NO:6 P15 SWAP70 TFYMRTSIH SEQ ID NO:7 P17 SWAP70 IWVIFNFLY SEQ ID NO:8 P20 SWAP70 IFNFLYEDK SEQ ID NO:9 C1 SWAP70 EDAFKIWVIFNFLYEDKYP SEQ ID NO:10 C2 SWAP70 IFNFLYEDKYPLIIVSEEI SEQ ID NO:11 C3 SWAP70 KMHLKYGLFSTFYMRTSIH

[0051] 2) Preparation of polypeptide mixture

[0052] The 8 selected CD8 + T cell epitopes peptide fragments and 3 CD4+ + The peptide fragments of T cell epitopes were prepared into a 50 mg / ml solution using sterile water. After mixing equal masses of each prepared solution 2 together, the solution was diluted with PBS buffer to a concentration of 15 mg / ml to obtain the peptide mixture.

[0053] 3) Preparation of tumor polypeptide vaccines:

[0054] A tumor peptide vaccine can be obtained by mixing an equal volume of a 15 mg / ml peptide mixture with an 0.5 mg / ml adjuvant polyinosinic acid-polycytosine nucleotide dilution.

[0055] Specifically, the concentration of the polypeptide mixture in the prepared tumor polypeptide vaccine is 7.5 mg / ml, and the concentration of the polyinosinic acid-polycytosine nucleotide diluent is 0.25 mg / ml.

[0056] This invention will select 8 CD8 lines + T-cell epitope peptide fragments and 3 designed CD4 lines + Equal masses of peptide fragments from T-cell epitopes are mixed together, and then combined with an adjuvant to obtain a tumor peptide vaccine. The preparation method is simple and easy to operate, and does not require special conditions.

[0057] In step 2), the 8 selected CD8 entries + T cell epitope polypeptide fragments and 3 CD4 lines as shown in SEQ ID NO: 9-11 + The polypeptide fragments of T cell epitopes were all prepared into 50 mg / ml solutions using sterile water. The use of sterile water ensures good solubility and is non-toxic, making it safer.

[0058] Further, the preparation method of the polyinosinic acid-polycytosine nucleotide diluent is as follows: polyinosinic acid-polycytosine nucleotide powder with an average molecular weight of 1.5-8kb is prepared into a 1mg / ml stock solution with pyrogen-free deionized water; the stock solution is dissolved in a water bath at a temperature of 65-70℃ for 10 minutes, and then cooled at room temperature for 1 hour; then it is diluted with PBS buffer to a concentration of 0.5mg / ml to obtain the polyinosinic acid-polycytosine nucleotide diluent.

[0059] This invention utilizes second-generation genome sequencing technology to perform whole-exome sequencing and mRNA gene expression sequencing on tumor cells from breast cancer patients, detect mutation sites in tumor cells, and perform CD8 sequencing on the mutant polypeptide sequence of the tumor-related gene SWAP70. + T-cell epitope prediction was used to select the top 20 mutated sequences. These top 20 mutated polypeptide sequences were then chemically synthesized into polypeptide fragments. Finally, the ELISPOT in vitro assay method was established to verify the immunogenicity of the SWAP70 tumor gene mutation sequence, and eight polypeptide fragments (eight CD8) of the immunogenic mutated antigen were screened out. + T cell epitope peptide fragments. Eight CD8 peptides were selected. + T-cell epitope peptide fragments and 3 designed CD4 lines + Equal masses of peptide fragments of T-cell epitopes are mixed together, and then mixed with adjuvants to obtain a tumor peptide vaccine.

[0060] Comparative Example 1

[0061] Example 2 of this application provides a method for preparing a tumor polypeptide vaccine for treating breast cancer. The difference between this method and Example 1 is that the adjuvant used in step 3) is Freund's complete adjuvant.

[0062] A tumor peptide vaccine can be obtained by mixing a 15 mg / ml peptide mixture with Freund's complete adjuvant in equal volumes.

[0063] Comparative Example 2

[0064] Comparative Example 1 of this application provides a method for preparing a tumor peptide vaccine for treating breast cancer. The difference between this method and Example 1 lies in the preparation method of the peptide mixture in step 2): the 8 selected CD8 peptides are... + The peptide fragments of T cell epitopes were prepared into a 50 mg / ml solution using sterile water. After mixing equal masses of each prepared solution 2 together, the solution was diluted with PBS buffer to a concentration of 15 mg / ml to obtain the peptide mixture.

[0065] Comparative Example 3

[0066] Comparative Example 2 of this application provides a method for preparing a tumor peptide vaccine for treating breast cancer. The difference between this method and Example 1 lies in the preparation method of the peptide mixture in step 2): three CD4 molecules as shown in SEQ ID NO: 9-11 are... + The peptide fragments of T cell epitopes were prepared into a 50 mg / ml solution using sterile water. After mixing equal masses of each prepared solution 2 together, the solution was diluted with PBS buffer to a concentration of 15 mg / ml to obtain the peptide mixture.

[0067] Table 2: Combinations of peptide fragments in the examples and comparative examples

[0068]

[0069]

[0070] To demonstrate the beneficial effects of the tumor peptide vaccines prepared in the embodiments of this application for treating breast cancer, the tumor peptide vaccines prepared in the examples and comparative examples were tested.

[0071] 1. A mouse xenograft model of breast cancer was established. Starting the day after tumor cell inoculation, tumor-bearing mice were treated with the tumor peptide vaccine prepared in the examples and comparative examples at a dose of 77.5 mg / kg (including 75 mg / kg peptide mixture and 2.5 mg / kg adjuvant). A blank control group was also established, and tumor-bearing mice were administered PBS extended-release formulation starting the day after tumor cell inoculation. Administration was twice weekly for 3 weeks. Tumor diameter was measured using calipers during the twice-weekly administration. The tumor-suppressive efficacy of the tumor peptide vaccine was evaluated using TGI (%), which reflects the tumor growth inhibition rate.

[0072] The formula for calculating tumor volume is: V = 0.5a × 2b, where a and b represent the long and short diameters of the tumor, respectively.

[0073] TGI(%) = [1 - (mean tumor volume at the end of treatment in a certain example - mean tumor volume at the beginning of treatment in that example) / (mean tumor volume at the end of treatment in the blank control - mean tumor volume at the beginning of treatment in the blank control)] × 100%.

[0074] Figure 2 This is a tumor growth curve of a breast cancer xenograft model mouse provided by the present invention after administration of the tumor polypeptide vaccines prepared in Example 1, Comparative Example 2, and Comparative Example 3. Figure 2 It is known that the peptide fragments in the most effective breast cancer tumor peptide vaccines include the aforementioned 8 CD8 sequences. + T cell epitopes peptide fragments and 3 CD4+ +The peptide fragments are T-cell epitopes, and in Example 1, the tumor inhibition rate was 57.8% on day 21 after the start of treatment.

[0075] Figure 3 This is a tumor growth curve of a breast cancer xenograft model mouse provided by the present invention after administration of the tumor polypeptide vaccine prepared in Example 1 and Comparative Example 1. Figure 3 It can be seen that when the adjuvant in Comparative Example 1 was the complete Freund's adjuvant, the prepared tumor polypeptide vaccine did not show a significant anti-tumor effect.

[0076] 2. Pharmacokinetic Tests

[0077] Three weeks into the treatment, tumor tissue and spleen cells from mice were taken, digested into single-cell suspensions, and flow cytometry (FCW) was used to detect the immune cell components and tumor-infiltrating lymphocytes (TILs) in the spleen of the mouse tumor tissue samples. Figure 4 This is an immunoassay flow cytogram of spleen cells from tumor-bearing mice in this invention. Figure 5 This is an immunoassay flow cytogram of tumor tissue cells from tumor-bearing mice in this invention, composed of... Figure 4 and Figure 5 It can be seen that after mice were treated with tumor peptide vaccines, the number of T cells and CD4+ in the spleen increased. + T cells and CD8 + The proportion of T cells was significantly increased compared to the blank control group; in tumor tissue, T cells and CD4+ were also significantly increased. + T cells and CD8 + The proportion of T cells was also significantly increased compared with the blank control group, indicating that the breast cancer tumor peptide vaccine produced a very effective T cell response in tumor-bearing mice. Figure 6 This is a schematic diagram illustrating the proportions of lymphocyte subsets in the spleen of tumor-bearing mice in this invention. Figure 7 This is a schematic diagram illustrating the proportions of immune cell subsets in tumor-infiltrating lymphocytes from tumor-bearing mice in this invention, as shown below. Figure 6 and Figure 7 As shown, CD4 produced in tumor-bearing mice + T cells and CD8 + T cells actively participate in anti-tumor immune responses.

[0078] This application established a mouse xenograft model of breast cancer to verify the efficacy of the tumor peptide vaccine provided in this application. The results showed that the tumor peptide vaccine produced a very effective T cell response in the patient, generating CD4+. + T cells and CD8 + T cells actively participate in anti-tumor immune responses.

[0079] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0080] It is understood that the relevant features of the above methods, apparatus, and systems can be referenced interchangeably. Furthermore, the terms "first," "second," etc., in the above embodiments are used to distinguish between embodiments and do not represent the superiority or inferiority of any particular embodiment.

[0081] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process of the systems and modules described above can be referred to the corresponding process in the foregoing method embodiments, and will not be repeated here.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tumor polypeptide vaccine for treating breast cancer, characterized by: The application relates to a tumor polypeptide vaccine, a preparation method thereof and application thereof. polypeptide mixture: the polypeptide mixture comprises 8 CD8 + polypeptide fragments of T cell epitopes and 3 CD4 + polypeptide fragments of T cell epitopes; The 8 CD8 + The amino acid sequences of the polypeptide fragments of T cell epitopes are shown in SEQ ID NOs: 1~8, the 3 CD4 + The amino acid sequences of the polypeptide fragments of T cell epitopes are shown in SEQ ID NOs: 9~11; The polypeptide fragments are synthesized from a mutant polypeptide sequence of a tumor-related gene SWAP70; The preparation method comprises the following steps: 1) 8 CD8 + Preparation of polypeptide fragments of T cell epitopes: CD8 + T cell epitope prediction, the top 20 mutant polypeptide sequences were selected, and then the top 20 mutant polypeptide sequences were synthesized into polypeptide fragments, the 20 polypeptide fragments were mixed and then mixed with adjuvant, and then 8 polypeptide fragments were screened out, i.e. 8 CD8 + The polypeptide fragments of the T cell epitope, the amino acid sequences of which are shown in SEQ ID NO: 1~8; 2) preparation of a polypeptide mixture solution: The 8 selected CD8 + T-cell epitope polypeptide fragments and 3 CD4 molecules as shown in SEQ ID NO: 9~11 + The peptide fragments of T cell epitopes were all prepared into a 50 mg / ml solution using sterile water. Then, the prepared solutions were mixed together in equal mass and diluted with PBS buffer to a concentration of 15 mg / ml to obtain the peptide mixture. 3) preparation of a tumor polypeptide vaccine: The polypeptide mixture solution is mixed with an adjuvant in equal volume to obtain the tumor polypeptide vaccine.

2. The tumor polypeptide vaccine for treating breast cancer according to claim 1, wherein the tumor polypeptide vaccine is a vaccine comprising a polypeptide having an amino acid sequence of SEQ ID NO: 1 or 2. The adjuvant is a polyinosinic acid-polycytidylic acid diluent.

3. The tumor polypeptide vaccine for treating breast cancer according to claim 2, wherein the tumor polypeptide vaccine is a vaccine comprising a polypeptide having an amino acid sequence of SEQ ID NO: 1 or 2. The concentration of the polyinosinic acid-polycytidylic acid diluent is 0.5 mg / ml.

4. The tumor polypeptide vaccine for use in the treatment of breast cancer according to claim 2 or 3, characterized in that: The preparation method of the polyinosinic acid-polycytidylic acid diluent is as follows: Polyinosinic acid-polycytidylic acid powder with an average molecular weight of 1.5-8 kb is prepared into a 1 mg / ml mother liquor with non-thermal deionized water; The mother liquor is dissolved in a water bath at a temperature of 65-70 DEG C for 10 minutes, and then is cooled at room temperature for 1 hour; and then is diluted with a PBS buffer to a concentration of 0.5 mg / ml to obtain the polyinosinic acid-polycytidylic acid diluent.

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