Polypeptide vaccine based on top2a protein and uses thereof

Abstract

The application discloses a polypeptide vaccine based on TOP2A protein and use, and belongs to the technical field of medical immunology, and aims to solve the problem that there is no polypeptide vaccine based on TOP2A for primary liver cancer. + CD8 T cell epitopes of TOP2A are predicted by two tools IEDB and NetMHCpan, and new antigen mutation sequences are scored, then 9 mutation sequences with high scores are selected, 9 polypeptides are synthesized by a chemical synthesis method, and a polypeptide composition formed by the 9 polypeptides is used to prepare a tumor vaccine. The application verifies that TOP2A epitope peptides (polypeptide fragments shown in SEQ ID NO: 1-9) can enhance BMDC (dendritic cell) maturation and promote T cell proliferation, thereby significantly enhancing immune response, significantly inhibiting growth of a liver cancer subcutaneous tumor model in a mouse, and having good safety and immunogenicity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical immunology, and particularly relates to a polypeptide vaccine based on TOP2A protein and use. BACKGROUND

[0002] Although immunotherapy represented by PD-1 / L1 antibodies has achieved certain curative effect in some patients, more than 70% of HCC patients have no significant response to existing immunotherapy. This is because HCC is in a relatively obvious immunosuppressive microenvironment, and the anti-tumor function of immune cells is impaired or even exhausted, thus greatly limiting the effect of PD-1 / L1 blockade therapy. Tumor polypeptide vaccine is a tumor antigen-based immunotherapy, which can activate antigen-presenting cells, promote T cell proliferation and differentiation into cytotoxic T cells (CTLs), infiltrate into the tumor microenvironment to kill tumor cells, and thus enhance the curative effect of PD-1 / L1 blockade therapy. Previous clinical studies have selected HCC antigens such as AFP and GPC3, but the immunotherapy effect is not ideal. Therefore, it is crucial to screen more effective antigens for HCC treatment.

[0003] Topoisomerase II A (TOP2A) can catalyze the breakage and ligation of two double-stranded DNAs, and is one of the essential enzymes for cell proliferation. In HCC, the expression level of TOP2A is significantly increased, and is associated with high degree of malignancy, poor prognosis and low survival rate of HCC. Previous studies by the research group have shown that TOP2A leads to centrosome amplification and an increase in the proportion of abnormal karyotype cells in hepatoma cells, and promotes the proliferation, migration and invasion of hepatoma cells. Antigenic epitope polypeptides of TOP2A have been used for breast cancer vaccine research, but there is no CD8 + T cell epitope based on TOP2A and research on hepatoma polypeptide vaccine. SUMMARY

[0004] In view of the above problems, the purpose of the present application is to provide a polypeptide vaccine based on TOP2A protein and use, which predicts the CD8 + T cell epitope of TOP2A by two tools IEDB and NetMHCpan, scores the neoantigen mutation sequences, and then selects the top 9 mutation sequences with the highest scores, synthesizes the 9 polypeptides by chemical synthesis, and prepares a tumor vaccine by using the polypeptide composition formed by the 9 polypeptides.

[0005] To achieve the above purpose, the present application provides the following technical solutions:

[0006] In a first aspect, the present application provides a polypeptide composition consisting of an isolated polypeptide with a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0007] The sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are shown in the following table:

[0008] ;

[0009] The screening process of the polypeptide composition is as follows:

[0010] The amino acid sequence of TOP2A is determined by Uniprot database; 9 CD8 + T cell epitopes are screened by using two epitope prediction databases of IEDB and NETMHCpan; the amino acid sequences of the 9 CD8 + T cell epitopes are as shown in SEQ ID NO: 1-9; the polypeptide fragments are all synthesized from the polypeptide sequence of tumor associated antigen TOP2A.

[0011] In a second aspect, the present application provides a pharmaceutical composition comprising the polypeptide fragments as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 in the above table. The pharmaceutical composition further comprises an adjuvant and / or a pharmaceutically acceptable salt.

[0012] In a third aspect, the present application provides a tumor vaccine comprising the polypeptide fragments as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 in the above table.

[0013] In some preferred embodiments of this aspect, the tumor vaccine further comprises an adjuvant, and the adjuvant is a polyinosinic-polycytidylic acid diluent.

[0014] In a fourth aspect, the present application provides a method for preparing a tumor vaccine, comprising the following steps:

[0015] Screening polypeptide fragments: 9 CD8 + T cell epitopes are screened from the TOP2A protein.

[0016] Preparing a polypeptide mixture: the 9 CD8 + T cell epitope polypeptide fragments are prepared to a concentration of 18 mg / ml using a buffer.

[0017] Preparing a tumor vaccine: the polypeptide mixture is mixed with an equal volume of adjuvant.

[0018] In some preferred embodiments of this aspect, the polypeptide fragment screening process: the amino acid sequence of TOP2A is determined through the Uniprot database; 9 CD8 + T cell epitopes are screened using two epitope prediction databases, IEDB and NETMHCpan. + The amino acid sequences of the 9 CD8

[0019] In some preferred embodiments of this aspect, the polypeptide mixture preparation process: the 9 CD8 + T cell epitope polypeptide fragments are prepared to a concentration of 18 mg / ml using a PBS buffer, and the polypeptide mixture is obtained.

[0020] In some preferred embodiments of this aspect, the tumor vaccine preparation process: the polypeptide mixture is mixed with an equal volume of adjuvant, and the tumor polypeptide vaccine is obtained; wherein the adjuvant is a polyinosinic-polycytidylic acid dilution solution with a concentration of 0.5 mg / ml.

[0021] In some preferred embodiments of this aspect, the concentration of the polypeptide mixture in the tumor vaccine is 9 mg / ml, and the concentration of the adjuvant is 0.25 mg / ml.

[0022] In a fifth aspect, the present application provides the use of a polypeptide composition, a pharmaceutical composition, and a tumor vaccine in the preparation of a medicament for preventing and / or treating cancer. Here, the cancer is primary liver cancer, especially hepatocellular carcinoma.

[0023] Compared with the prior art, the present application has the following advantages:

[0024] The present application verifies that the TOP2A epitope peptide (polypeptide fragments shown in SEQ ID NO: 1-9) enhances the maturation of BMDC (dendritic cells) and promotes the proliferation of T cells, thereby significantly enhancing the immune response, significantly inhibiting the growth of a subcutaneous tumor model of liver cancer in mice, and having good safety and immunogenicity. All selected polypeptides have 100% amino acid sequence homology between humans and mice, which is beneficial for realizing potential clinical transformation. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Cell gating strategy for TOP2A polypeptide-induced BMDC maturation, from left to right, total cell population, single cell population, and CD11c + cell population.

[0026] Figure 2 Flow cytometry of CD80 and CD86 expression in CD11c + cells in different groups.

[0027] Figure 3 Flow cytometry of CD80 and CD86 expression in CD11c + CD86 + cells in different groups. + Quantitative analysis of the proportion of CD11c

[0028] Figure 4 Flow cytometry of MHC-I expression in CD11c + cells in different groups.

[0029] Figure 5 Flow cytometry of MHC-I expression in CD11c + CD11c + cells in different groups.

[0030] Figure 6 Cell gating strategy for TOP2A polypeptide-induced BMDC maturation, from left to right, total cell population, single cell population, and CD3 + cell population.

[0031] Figure 7 Flow cytometry of CFDA-SE intensity distribution in CD3 + cells in different groups.

[0032] Figure 8 Quantitative analysis of T cell proliferation rate for control group, TOP2A-1 group, TOP2A-2 group, TOP2A-3 group, TOP2A-4 group, TOP2A-5 group, TOP2A-6 group, TOP2A-7 group, TOP2A-8 group and TOP2A-9 group.

[0033] Figure 9 Bioluminescence image of a liver cancer subcutaneous tumor model treated with a TOP2A polypeptide vaccine. DETAILED DESCRIPTION

[0034] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If the specific conditions are not specified in the examples, they are carried out under the conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained by purchase on the market.

[0035] Example 1: CD8 + Prediction of T cell epitopes:

[0036] The amino acid sequence of TOP2A was determined by Uniprot database (https: / / www.uniprot.org / ), and the CD8 + T cell epitopes of TOP2A were predicted by two tools IEDB (http: / / tools.iedb.org / main / tcell / ) and NetMHCpan (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ), and then the top 9 mutant sequences were selected according to the scores of the analysis tools (IEDB and NetMHCpan), as shown in Table 1 below, and 9 polypeptides were finally synthesized. The amino acid sequences of the 9 polypeptides are shown in Table 2 below.

[0037] Table 1: Score ranking of mutant sequences:

[0038] ;

[0039] Table 2: Amino acid sequences:

[0040] ;

[0041] Example 2: TOP2A polypeptide induces maturation of dendritic cells (BMDC):

[0042] To evaluate the immunogenicity of the TOP2A peptide, its effect on promoting BMDC maturation was first verified. Progenitor cells were isolated from the bone marrow of C57 mice and differentiated into BMDCs in vitro using specific cytokines GM-CSF and IL-4. In flow cytometry analysis, CD11c cells were selected... + The reason for using CD11c as the target cell population is that CD11c is an important marker for the differentiation of mouse monocytes into dendritic cells. Figure 1 As shown in the figure, when dendritic cells mature, the expression levels of their surface co-stimulatory molecules CD80 and CD86 are significantly upregulated. Therefore, the expression levels of CD80 in the control group, TOP2A-1 group, TOP2A-2 group, TOP2A-3 group, TOP2A-4 group, TOP2A-5 group, TOP2A-6 group, TOP2A-7 group, TOP2A-8 group, and TOP2A-9 group were observed. + and CD86 + Cells in CD11c + The percentage of cell populations was as follows: the control group contained only BMDCs; TOP2A-1, TOP2A-2, TOP2A-3, TOP2A-4, TOP2A-5, TOP2A-6, TOP2A-7, TOP2A-8, and TOP2A-9 groups had peptides with corresponding sequences added to BMDCs. It can be seen that, except for TOP2A-3 group, the other TOP2A groups had CD80... + and CD86 + The proportion of dendritic cells increased significantly. Figure 2 (As shown). Quantitative analysis showed that CD80 + and CD86 + Cells account for CD11c + The cell proportions were as follows: control group 22.20%, TOP2A-1 group 35.90%, TOP2A-2 group 30.90%, TOP2A-3 group 23.90%, TOP2A-4 group 31.30%, TOP2A-5 group 30.00%, TOP2A-6 group 28.60%, TOP2A-7 group 34.60%, TOP2A-8 group 35.90%, and TOP2A-9 group 41.30%. Figure 3 (As shown).

[0043] The establishment process of the control group and the nine experimental groups is as follows:

[0044] ① Control group: BMDC was directly dispersed in 1640 complete culture medium (1×10⁻⁶). 5① **TOP2A-1 Group:** Cells were dispersed in 1640 complete medium containing 10 μg / mL TOP2A-1 and added to 12-well plates, 1 mL per well, for a total of 3 wells. ② **TOP2A-2 Group:** Cells were dispersed in 1640 complete medium containing 10 μg / mL TOP2A-2 and added to 12-well plates, 1 mL per well, for a total of 3 wells. ③ **TOP2A-3 Group:** Cells were dispersed in 1640 complete medium containing 10 μg / mL TOP2A-3 and added to 12-well plates, 1 mL per well, for a total of 3 wells. ④ **TOP2A-4 Group:** Cells were dispersed in 1640 complete medium containing 10 μg / mL TOP2A-4 and added to 12-well plates, 1 mL per well, for a total of 3 wells. ⑥ TOP2A-5 group: Disperse cells in 1640 complete medium containing 10 μg / mL TOP2A-5, add 1 mL to each well of a 12-well plate, for a total of 3 wells. ⑦ TOP2A-6 group: Disperse cells in 1640 complete medium containing 10 μg / mL TOP2A-6, add 1 mL to each well of a 12-well plate, for a total of 3 wells. ⑧ TOP2A-7 group: Disperse cells in 1640 complete medium containing 10 μg / mL TOP2A-7, add 1 mL to each well of a 12-well plate, for a total of 3 wells. ⑨ TOP2A-8 group: Disperse cells in 1640 complete medium containing 10 μg / mL TOP2A-8, add 1 mL to each well of a 12-well plate, for a total of 3 wells. ⑩ TOP2A-9 group: Disperse cells in 1640 complete medium containing 10 μg / mL TOP2A-9, add 1 mL to each well of a 12-well plate, for a total of 3 wells.

[0045] As the most potent antigen-presenting cells, MHC molecules are crucial for the antigen-presenting function of dendritic cells. Therefore, in single-cell populations and CDI1c... + Based on the cell population, MHC-I was analyzed. + Cell ratio ( Figure 4 As shown in the figure, the quantification rates in the control group, TOP2A-1 group, TOP2A-2 group, TOP2A-3 group, TOP2A-4 group, TOP2A-5 group, TOP2A-6 group, TOP2A-7 group, TOP2A-8 group, and TOP2A-9 group were 75.1%, 91.70%, 92.60%, 90.90%, 92.50%, 87.40%, 89.40%, 91.60%, 92.10%, and 93.7%, respectively. Figure 5 (As shown in the image). The above experiments suggest that the TOP2A peptide has a good ability to induce dendritic cell maturation and promote the antigen presentation function of dendritic cells.

[0046] Example 3: TOP2A peptide promotes T cell proliferation:

[0047] Cell populations with low CFDA-SE signal intensity were selected for quantitative analysis and used as an indicator of T cell proliferation rate. After treating BMDCs with different drugs (control group, TOP2A-1 group, TOP2A-2 group, TOP2A-3 group, TOP2A-4 group, TOP2A-5 group, TOP2A-6 group, TOP2A-7 group, TOP2A-8 group, TOP2A-9 group), these BMDCs were co-cultured with CFDA-SE-labeled spleen cells. Finally, CD3+ was observed by flow cytometry. + The signal intensity of CFDA-SE in cells. In the analysis of flow cytometry results, CD3 was first analyzed. + Cell gating ( Figure 6 (As shown), further observe CD3. + The signal intensity of CFDA-SE in the cell population ( Figure 7 (As shown). Quantitative results showed that the T cell proliferation rates in the control group, TOP2A-1 group, TOP2A-2 group, TOP2A-3 group, TOP2A-4 group, TOP2A-5 group, TOP2A-6 group, TOP2A-7 group, TOP2A-8 group, and TOP2A-9 group were 29.50%, 36.90%, 36.40%, 39.00%, 35.90%, 34.20%, 35.30%, 36.30%, 27.50%, and 41.60%, respectively. Figure 8 (As shown in the figure). The above results suggest that TOP2A peptide has a significant promoting effect on T cell proliferation after activating BMDC.

[0048] Experimental Example 4: Preparation of TOP2A peptide vaccine:

[0049] The 9 selected CD8 + The peptide fragments of T-cell epitopes were prepared to a concentration of 18 mg / ml using PBS buffer to obtain a peptide mixture. The 18 mg / ml peptide mixture was then mixed in equal volumes with a 0.5 mg / ml adjuvant polyinosinic acid-polycytosine nucleotide diluent to obtain the TOP2A peptide vaccine. Specifically, the prepared TOP2A peptide vaccine contained a peptide mixture concentration of 9 mg / ml and a polyinosinic acid-polycytosine nucleotide diluent concentration of 0.25 mg / ml.

[0050] Experimental Example 5: TOP2A peptide vaccine treatment model of subcutaneous hepatocellular carcinoma:

[0051] Mice in good condition and of similar weight were selected. The hair on the upper front of the right hind leg was shaved. 100 μL of a uniformly dispersed hep1-6 cell suspension was injected subcutaneously into the mice using a sterile insulin needle, forming a relatively obvious wheal. During treatment, the control group received a subcutaneous injection of sterile saline (100 μL / time) every 3 days for a total of 3 injections on the right dorsum of the thigh. The TOP2A peptide vaccine group received a 1 mg / mL concentration of Vac in sterile saline. 100 μL of the Vac solution was injected subcutaneously into the right dorsum of the thigh every 3 days for a total of 3 injections. Bioluminescence was used to quantify the growth of tumor cells in the mice, thus preliminarily assessing the immune effect. Bioluminescence images showed that the subcutaneous hepatocellular carcinoma tumors (HCC) in the TOP2A peptide group... Figure 9 The brightness of the TOP2A antigen peptide (as shown in the image) was significantly reduced, noticeably lower than that of the control group. There was no significant difference in body weight among the four groups, preliminarily highlighting the biocompatibility of the TOP2A antigen peptide. These results preliminarily suggest that TOP2A peptide inoculation activates the immune system in mice and significantly inhibits the growth of liver cancer cells in vivo.

[0052] It is understood that the above specific description of the present invention is only for illustrating the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention to achieve the same technical effect; as long as the use needs are met, they are all within the protection scope of the present invention.

Claims

1. A polypeptide composition, characterized in that, consists of the isolated polypeptide amino acid sequences shown in SEQ ID NO: 1-9: the amino acid sequence of SEQ ID NO: 1: RIYQKKTQL; the amino acid sequence of SEQ ID NO: 2: VLFDHVGCL; the amino acid sequence of SEQ ID NO: 3: HMWIFVNAL; the amino acid sequence of SEQ ID NO: 4: FIHHNWPSL; the amino acid sequence of SEQ ID NO: 5: TSYLTYNDF; the amino acid sequence of SEQ ID NO: 6: ASPRYIFTM; the amino acid sequence of SEQ ID NO: 7: AGPTFNYLL; the amino acid sequence of SEQ ID NO: 8: QNFVGSNNL; the amino acid sequence of SEQ ID NO: 9: VGLHKVFKL.

2. The polypeptide composition of claim 1, wherein each of the isolated polypeptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is selected from a fragment of a TOP2A protein.

3. The polypeptide composition of claim 2, wherein The fragment comprises CD8 + T cell epitopes.

4. A pharmaceutical composition, characterized by, comprising the polypeptide composition of claim 1.

5. A tumor vaccine, characterized by, comprising the polypeptide composition of claim 1.

6. The tumor vaccine as described in claim 5, characterized in that, further comprising an adjuvant.

7. A method for preparing a tumor vaccine, characterized by, comprising the following steps: Screening of polypeptide fragments: 9 CD8 + polypeptide fragments of T cell epitopes; Preparation of polypeptide mixture: 9 CD8 + The polypeptide fragments of T-cell epitopes were formulated using a buffer to a concentration of 18 mg / ml; preparing a tumor vaccine: mixing the polypeptide mixture with an adjuvant in equal volume; The tumor vaccine consists of 9 CD8 molecules + T cell epitopes are composed of polypeptide fragments, including 9 CD8 molecules. + The sequence of the polypeptide fragment of the T cell epitope is shown below: the amino acid sequence of SEQ ID NO: 1: RIYQKKTQL; the amino acid sequence of SEQ ID NO: 2: VLFDHVGCL; the amino acid sequence of SEQ ID NO: 3: HMWIFVNAL; the amino acid sequence of SEQ ID NO: 4: FIHHNWPSL; the amino acid sequence of SEQ ID NO: 5: TSYLTYNDF; the amino acid sequence of SEQ ID NO: 6: ASPRYIFTM; the amino acid sequence of SEQ ID NO: 7: AGPTFNYLL; the amino acid sequence of SEQ ID NO: 8: QNFVGSNNL; the amino acid sequence of SEQ ID NO: 9: VGLHKVFKL.

8. Use of the polypeptide composition of any one of claims 1-3, the pharmaceutical composition of claim 4, the tumor vaccine of claim 5 or 6 in the preparation of a medicament for preventing and / or treating cancer, the cancer being primary liver cancer.

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