Polypeptide, immunogen, vaccine and application

Abstract

The invention belongs to the field of biochemistry, and discloses a polypeptide, which has: (I) an amino acid sequence as shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No SEQ.3 and SEQ ID No.4; or (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) and having the same function as the amino acid sequence shown in (I); or (III) an amino acid sequence which is more than 80% similar to the amino acid sequence shown in (I) or (II). By inducing tumor cells to generate immunogenic cell death (ICD), the tumor antigen release can be promoted, the uptake and start of antigen-presenting cells (APC) to antigens can be enhanced, and finally T cell response is started, so that the effect of systematic anti-tumor immunity is achieved.

Description

Technical Field

[0001] The present invention relates to the biochemical field, in particular to a polypeptide, an immunogen, a vaccine and uses. Background Art

[0002] Specific immune response can effectively inhibit tumor progression, promote tumor regression and form anti-tumor immune memory. However, existing tumor vaccines have many limitations in clinical applications. For example, predefined vaccines constructed with a single antigen are difficult to fully initiate anti-tumor immune responses, and the research and development costs are high and the process is complicated. In addition, the tumor's own immune escape mechanism also greatly limits the efficacy of the vaccine. The research and development of predefined vaccines faces many technical challenges. For example, the production of tumor vaccines is limited by the high cost of sequencing technology and epitope prediction technology, as well as the long time-consuming process of tumor lysate preparation, and the lack of understanding of tumor antigen epitopes that constitute immunogenicity and uncertainty about whether new antigens can be effectively presented by the major tissue compatibility complex (MHC). Therefore, it is an urgent need for the current development of tumor vaccines to make up for the shortcomings of existing tumor vaccines and explore tumor vaccine technologies with better efficacy and higher safety.

[0003] Based on this, in situ anonymous antigen vaccines are highly anticipated. By vaccinating at the tumor site, initiating or stimulating an immune response in situ in the tumor, and using a large number of potential tumor-associated antigens to initiate an anti-tumor immune response, this method is called in situ vaccination (ISV). In situ anonymous tumor vaccines are not limited to a single tumor-associated antigen or tumor-specific antigen, but utilize the entire antigen library of each patient's tumor to induce specific anti-tumor immune responses with a full spectrum of tumor antigens, which is patient-specific. In order to induce a strong memory anti-tumor immune response, in situ vaccines are often able to induce immunogenic cell death (ICD) in tumors to promote the release of TAAs and enhance the uptake and activation of antigens by APCs, thereby inducing T cell responses and initiating systemic anti-tumor immunity. ICD is a process in which tumor cells die under external stimuli and transform from non-immunogenic to immunogenic, mediating the body's anti-tumor immune response. The immunogenicity of ICD is mainly generated by damage-associated molecular patterns (DAMPs), including the exposure of calreticulin (CRT) on the cell surface, the secretion of ATP, and the release of high mobility group protein B1 (HMGB1). DAMPs can bind to pattern recognition receptors on the surface of DCs and generate a series of immune activation signals. However, there is currently a lack of specific technical means to effectively improve tumor immunogenicity and activate systemic anti-tumor immunity. Summary of the invention

[0004] The present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a polypeptide, an immunogen and a vaccine.

[0005] The present invention uses viral protein R (Vpr) to construct an in situ anonymous antigen tumor vaccine. Vpr is an important regulatory protein of HIV-1 virus, which has the functions of penetrating cell membrane, regulating cell cycle and inducing cell apoptosis, and meets the construction requirements of in situ anonymous vaccine. The Vpr molecule has a size of 14kDa, consists of 96 amino acids, and contains three alpha helices, an N-terminal flexible region and a C-terminal basic amino acid region. Its pro-apoptotic domain, nuclear localization signal, transmembrane domain and other functional domains are found to be located in different structural regions, respectively, and is a candidate molecule for in situ anonymous tumor vaccine with application prospects. The present invention designs and synthesizes multiple Vpr polypeptides, and systematically studies their biological functions in vitro and in vivo, and applies Vpr polypeptide in situ vaccine to anti-tumor treatment, avoiding the complex process of predefined epitope screening and antigen preparation, and can induce a stronger immune response. The present invention can promote the release of tumor antigens, enhance the uptake and activation of antigens by antigen-presenting cells (APC), and finally activate T cell responses to achieve the effect of systemic anti-tumor immunity, providing a new method for tumor vaccines.

[0006] According to one aspect of the present invention, a polypeptide is provided, which has: (I) an amino acid sequence as shown in any one of ID No.1, SEQ ID No.2, SEQ ID No SEQ.3, and SEQ ID No.4; or (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence as described in (I), and having the same function as the amino acid sequence as described in (I); or (III) an amino acid sequence having more than 80% similarity with the amino acid sequence as described in (I) or (II), or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity, or can be composed of the above amino acid sequence.

[0007] According to another aspect of the present invention, an immunogen is provided, comprising the polypeptide as described above.

[0008] According to yet another aspect of the present invention, there is provided a vaccine comprising the immunogen as described above, and preferably, further comprising an adjuvant.

[0009] According to another aspect of the present invention, there is provided a use of the above-mentioned immunogen as an active ingredient of an anti-tumor vaccine.

[0010] The present invention aims to design an in situ vaccine based on the Vpr polypeptide, utilize its membrane-penetrating and apoptosis-inducing functions, significantly improve tumor immunogenicity, initiate systemic anti-tumor immune response, and overcome the defects of the prior art. The Vpr polypeptide is derived from the auxiliary protein viral protein R (Vpr) of the HIV-1 virus, and has the functions of penetrating the cell membrane, inducing cell apoptosis, and regulating the cell cycle, which meets the needs of constructing an in situ vaccine. By inducing immunogenic cell death (ICD) in tumor cells, the present invention can promote the release of tumor antigens, enhance the uptake and activation of antigens by antigen presenting cells (APCs), and ultimately initiate T cell responses to achieve the effect of systemic anti-tumor immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0012] Figure 1 The data graphs of Vpr polypeptide-induced tumor cell apoptosis provided in the embodiments of the present invention. (A) After HT-29 cells were treated with different concentrations of Vpr polypeptide for 72 hours, the cell viability was detected by CCK8 method (n=3). (BD) After 40μM Vpr polypeptide was applied for 72 hours, HT-29, MC38 and CT-26 cells were stained with Annexin V / PI, and cell apoptosis was analyzed by flow cytometry (n=3). (ns: not statistically significant, *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001)

[0013] Figure 2 : Figure 2 Vpr peptide can penetrate the tumor cell membrane and enter the cell. (A) After HT-29 cells were treated with 40μM Vpr peptide (FITC labeled) for 2h, they were observed by laser scanning confocal microscopy (CLSM). (BC) After HT-29 cells were treated with 40μM Vpr peptide (FITC labeled) for 2h, flow cytometry was used to analyze the MFI and the proportion of Vpr penetrating HT-29 cells (n=3, MFI: mean fluorescence intensity). (ns: not statistically significant, *p<0.0332, **p<0.0021, ****p<0.0001)

[0014] Figure 3:Vpr polypeptide releases tumor antigens and induces ICD in tumor cells. (A) ELISA was used to determine the amount of EGFR released (n=5). (BD) CLSM was used to observe the exposure of CRT on the surface of MC38, B16F10 and LLC cells. Blue: DAPI; Red: CRT. Scale bar, 50μm. (E) Flow cytometry was used to analyze the surface exposure of CRT (n=3). (F) ATP release was determined using an ATP detection kit (n=3). (G) ELISA was used to determine the amount of HMGB1 released (n=3). (HI) CLSM was used to observe the effect of Vpr polypeptide on promoting macrophage Ana-1 to phagocytose LLC cells (labeled with CFDA-SE). Green: LLC cells. Scale bar, 50μm. (**p<0.0021, ***p<0.0002, ****p<0.0001)

[0015] Figure 4 :Vpr peptide in situ vaccine inhibits MC38 tumor growth and initiates anti-tumor immunity. (A) Treatment plan. MC38 cells were inoculated into the right side of mice on day 0. Four days later, MC38 cells were implanted into the left side. Mice were injected intratumorally with P1 10 mg / kg or P4 15 mg / kg on days 8, 12, 15 and 19, respectively, and subcutaneously injected with Poly(I:C) 2.5 mg / kg (n=5). (B) Tumor growth on the right side. (C) Tumor growth on the left side. (DH) Immunohistochemical detection of CD8, F4 / 80 in the right tumor tissue and CD4, CD11C, and F4 / 80 in the spleen. Scale bar, 100 μm (n=3, three fields of view for each sample). (ns: not statistically significant, *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001)

[0016] Figure 5 :Vpr peptide vaccine inhibits LLC tumor growth in situ. (A) Treatment regimen. LLC cells were inoculated on the right and left sides of mice on day 0. Mice were given PBS, 10 mg / kg P1 (50 μL, intratumoral injection on the right side), 15 mg / kg P1 (50 μL, intratumoral injection on the right side), 10 mg / kg P1 (50 μL, intratumoral injection on the right side) + 2.5 mg / kg Poly (I: C) (subcutaneous injection) on days 15, 18, 23, and 26, respectively (n=5). (B) Tumor growth on the right side. (C) Tumor growth on the left side. (DE) Immunohistochemical staining of ki67 in right and left tumor tissues. Scale bar, 100 μm (n=5). (FG) Immunohistochemical staining of CRT and HMGB1 in right tumor tissues. Scale bar, 250 μm (n=5).

[0017] (*p<0.0332)

[0018] Figure 6 :Vpr peptide in situ vaccine activates DCs and T cells in dLN as well as macrophages in tumor and periphery. (AB) Flow cytometry was used to detect the percentage of cDC1 and cDC2 in dLN (n=5). (CD) Flow cytometry was used to detect the percentage of CD4 + or CD8 + The percentage of T cells (n=5). (EF) Flow cytometry was used to detect the percentage of macrophages in the spleen, right tumor, and left tumor (n=5). (ns: not statistically significant, *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001)

[0019] Figure 7 :Vpr peptide combined with SIRPαFc inhibits the growth of distant tumors. (AB) Immunohistochemical staining of CD47 in right and left tumor tissues. Scale bar, 100μm (n=5). (C) Treatment regimen. LLC cells were inoculated on the right and left sides of mice on day 0. Mice were injected with 10mg / kg P1 or / and 10mg / kg SIRPαFc (ip) on the right side of the tumor on days 8, 11, 15, and 19, respectively (n=5). (D) Tumor growth on the right side. (E) Tumor growth on the left side. (*p<0.0332, **p<0.0021)

[0020] Figure 8 : Proteomic analysis to explore the mechanism of Vpr peptide antitumor activity. (A) Volcano plot showing differentially expressed proteins between P1-treated and control groups. (BC) GO analysis and KEGG analysis of proteins significantly downregulated in P1 compared with the control group. (DE) ssGSEA analysis of immune cell infiltration in injected tumor and remote tumors. DETAILED DESCRIPTION

[0021] The following examples are provided to allow those skilled in the art to more clearly understand the present invention. It should be noted that the following examples do not limit the scope of protection claimed in the present invention and are only illustrative examples. The raw materials, reagents or devices mentioned in the following examples, unless otherwise specified, can be obtained from commercial sources or by known existing methods.

[0022] The present invention designs and synthesizes multiple Vpr polypeptides, and systematically studies their biological functions in vitro and in vivo, and applies Vpr polypeptide in situ vaccines to anti-tumor treatment, avoiding the complex process of predefined epitope screening and antigen preparation, and can induce a stronger immune response. The multiple Vpr polypeptide sequences are as follows:

[0023]

[0024]

[0025] Example 1: Vpr polypeptide induces tumor cell apoptosis

[0026] Vpr peptide has cytotoxic effect and inhibits the proliferation of colon cancer cells

[0027] Each Vpr sequence polypeptide was suspended and diluted with DMEM complete medium to prepare a series of suspensions with concentrations of 0, 0.1, 1, 10, 50, and 100 μM. 3 Colon cancer cells HT-29 were inoculated in the number of cells / well, and cultured overnight at 37°C and 5% CO2 until the cells adhered to the wall. The culture medium in the wells was discarded, and 100 μL of Vpr suspension was added to the wells after washing with PBS and incubated at 37°C and 5% CO2 for 72 hours. After aspirating the supernatant, 100 μL of fresh DMEM complete culture medium was added to each well, and then 10 μL of CCK-8 reagent was added to each well. After incubation in the dark for 1.5 hours, the absorbance at a wavelength of 450 nm was detected using an enzyme reader. Finally, Graphpad Prism 9 was used to perform statistical analysis on cell survival rate.

[0028] Vpr polypeptide induces apoptosis of tumor cells

[0029] HT-29 cells in the logarithmic growth phase were taken and the cell density was adjusted to 2×10 5 100 / well were inoculated into 6-well plates, and 40 μM Vpr peptides of each sequence were given and incubated at 37°C and 5% CO2 for 72 hours. The suspended cells were collected and the adherent cells were digested with trypsin. After merging, they were collected into a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. After resuspending with pre-cooled PBS and centrifuging to remove the supernatant, the cells were resuspended with 1 mL PBS and collected into a 1.5 mL EP tube. After centrifuging at 1000 rpm for 5 minutes and discarding the supernatant, the cells were resuspended with 1×Binding Buffer and the concentration was adjusted to 2×10 6 / mL, take 2.5μL of Annexin V-Alexa Fluor 647 and 5μL of PI solution and add them to 50μL of cell suspension in a dark place, mix them by pipetting, and incubate them in a dark place for 15 minutes. After incubation, add 200μL of PBS and mix them. Use the APC channel to detect Annexin-V signal and the PE channel to detect PI signal for apoptosis analysis in Beckman flow cytometer.

[0030] Figure 1 (A) is cell viability detection: Figure 1(A) shows the effect of different concentrations of Vpr peptide (P1 to P4) on the cell viability of HT-29 cells after 72 hours of treatment. As the concentration of Vpr peptide increases, the cell viability tends to decrease, especially under high concentration (such as P1), the cell viability is significantly reduced. This indicates that the growth inhibitory effect of Vpr peptide on HT-29 cells may be dose-related.

[0031] Figure 1 (BD) Cell apoptosis analysis: Annexin V / PI staining and flow cytometry. Figure 1 (B) Figure 1 (C) and Figure 1 (D) shows the apoptosis of HT-29, MC38 and CT-26 cells after treatment with 40 μM Vpr peptide for 72 hours. The four quadrants of each figure represent cell populations in different states: Q1 represents necrotic cells, Q2 represents late apoptotic cells, Q3 represents early apoptotic cells, and Q4 represents living cells. The results showed that after treatment with 40 μM Vpr peptide, the apoptotic cells of the three cell lines (especially the cells in the Q2 and Q3 quadrants) increased significantly, indicating that the Vpr peptide has the ability to induce cell apoptosis.

[0032] Statistical analysis: The bar graphs and p-values ​​in the figure illustrate the statistical significance of the experimental data. The places marked with "*" indicate significant differences between different concentrations or different treatment groups. The number of symbols "*" indicates the significance level, for example, "****" means p<0.0001, indicating that the data in this group have very significant statistical differences.

[0033] In summary, Figure 1 The results showed that Vpr polypeptide can significantly reduce the vitality of tumor cells (such as HT-29, MC38 and CT-26) and induce apoptosis at a certain concentration, and this effect is dose-dependent.

[0034] Example 2: Vpr polypeptides can penetrate tumor cell membranes

[0035] Laser confocal fluorescence microscopy observation of Vpr peptide penetrating tumor cell membrane: FITC-labeled Vpr peptides of various sequences were suspended and diluted with DMEM complete medium to prepare a suspension with a concentration of 40 μM. FITC-labeled Vpr peptides of various sequences were commissioned to Nanjing Yuanpeptide Company for synthesis. 4Colon cancer cells HT-29 were inoculated in the number of cells / well, cultured overnight at 37°C and 5% CO2 until the cells adhered to the wall, and then the medium in the wells was discarded. After washing with PBS, 1 mL of Vpr suspension was added to the wells and incubated at 37°C and 5% CO2 for 2 hours. After the supernatant was removed, the wells were washed with PBS three times, stained with Hoechst33342 for 20 minutes, the dye was discarded, washed with PBS, PBS was added to the dish, and the position of the FITC-labeled polypeptide was observed using a laser confocal microscope.

[0036] Flow cytometry detection of Vpr peptide penetrating tumor cell membrane: HT-29 cells in the logarithmic growth phase were taken and the cell density was adjusted to 1×10 6 / mL, take 1mL of cell suspension and add it to a 1.5mL EP tube, centrifuge at 1000rpm for 5min and remove the supernatant. Resuspend the cells in each tube with 200μL of 40μM FITC-labeled Vpr polypeptide suspension and incubate at 37℃ for 2h. After washing with PBS, centrifuge at 1000rpm for 5min, discard the supernatant and add 70μL of trypsin, incubate on ice for 30min, add 3 times the volume of culture medium to terminate digestion, centrifuge at 1000rpm for 5min, discard the supernatant and wash with PBS 2-3 times to remove the polypeptides attached to the cell membrane surface, resuspend with 200μL PBS and transfer to a 96-well plate, detect using the FITC channel in the Beckman flow cytometer, and analyze the FITC in each group. + The cell ratio was measured to compare the membrane penetration ability of different Vpr polypeptides.

[0037] Figure 2 (A) is the result of laser scanning confocal microscopy observation: Figure 2 (A) shows the microscope images of HT-29 cells treated with different types of Vpr peptides (P1 to P4, FITC labeled) for 2 hours. Hoechst33342 staining (blue) marks the cell nucleus, and FITC-labeled Vpr peptides (green) show their distribution in the cells. With the different types of Vpr peptides (P1 to P4), the intensity of the green fluorescence signal in the cells gradually increased, especially the P1 and P4 groups showed the strongest green fluorescence, indicating that they had the best effect in entering the cells. This shows that different types of Vpr peptides have different abilities to penetrate the cell membrane, among which the P1 and P4 groups have the strongest penetration.

[0038] Figure 2 (B) is the result of flow cytometry fluorescence intensity (MFI) analysis: Figure 2 (B) shows the mean fluorescence intensity (MFI) measured by flow cytometry, reflecting the accumulation of different types of Vpr polypeptides in HT-29 cells. It can be seen that the MFI value of the P4 group is significantly higher than that of other groups, indicating that the P4 polypeptide accumulates the most in the cells.

[0039] Figure 2 (C) is the result of the ratio of Vpr penetrating HT-29 cells: Figure 2 (C) shows the ratio of different types of Vpr polypeptides penetrating HT-29 cells. The bar graph shows the difference in penetration rate of different Vpr polypeptides in cells. The results show that the cell penetration rate of P1 polypeptide is significantly higher than that of other types, with statistical significance (the symbol "*" marks the significance level). This further indicates that the delivery effect of P1 polypeptide in cells is the most significant.

[0040] Figure 2 The results showed that there were significant differences in the penetration effects of different types of Vpr peptides in tumor cells. P1 peptide had the best cell penetration and showed the highest penetration ratio, indicating that it was the most effective intracellular delivery peptide.

[0041] Example 3: Vpr polypeptide releases tumor antigens and induces ICD in tumor cells

[0042] Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of tumor cell antigens induced by Vpr polypeptide: MC38, B16F10 and LLC cells in the logarithmic growth phase were taken and the cell density was adjusted to 5×10 3 Each well was inoculated into a 96-well plate, and 10, 20, and 40 μM Vpr peptides of each sequence were added and incubated for 48 h at 37° C. and 5% CO 2 . The cell culture supernatant was collected, centrifuged at 300 g for 10 min, and detected using an EGFR ELISA detection kit.

[0043] Laser confocal fluorescence microscopy was used to observe the exposure of CRT on the surface of tumor cells induced by Vpr peptide: MC38, B16F10 and LLC cells in the logarithmic growth phase were taken and the cell density was adjusted to 5×10 4Each well was inoculated into a confocal dish, and 40 μM Vpr peptides of each sequence were given and incubated at 37°C and 5% CO2 for 24 hours. The culture medium was discarded, and 200 μL 4% paraformaldehyde was added to the confocal dish for fixation at room temperature for 10 minutes after washing with PBS three times. After washing with PBS three times, 1 mL 0.1% Triton X-100 PBS was added to the confocal dish for permeabilization for 10 minutes. After washing with PBS three times, 1 mL of blocking buffer containing 5% BSA was added to the confocal dish for blocking on a shaker at room temperature for 1 hour. After washing with PBS three times, 50 μL CRT antibody was added to the confocal dish and incubated overnight at 4°C. After washing with PBS, 50 μL secondary antibody (goat anti-rabbit IgG AF 594) was added to the confocal dish and incubated at room temperature for 1 hour. After washing with PBS, the cells were stained with Hoechst 33342 for 20 minutes, washed with PBS three times, and the cell CRT exposure was observed using Confocal.

[0044] Flow cytometry was used to detect the exposure of CRT on the surface of tumor cells induced by Vpr peptide: MC38, B16F10 and LLC cells in the logarithmic growth phase were taken and the cell density was adjusted to 5×10 4 Each well was inoculated into a 6-well plate, and 40 μM Vpr peptides of each sequence were given and incubated at 37°C, 5% CO2 for 48 hours. The cells were collected, washed 3 times with PBS, and incubated with CRT antibody at room temperature for 1 hour. After washing 3 times with PBS, secondary antibody (goat anti-rabbit IgG AF 594) was added and incubated at room temperature in the dark for 1 hour. After washing 3 times with PBS, the cells were resuspended in 200 μL PBS and added to a 96-well plate, and placed in a Beckman flow cytometer for detection. The test results were analyzed in CytExpert software.

[0045] Colorimetric detection of Vpr polypeptide-induced ATP release in tumor cells: MC38, B16F10 and LLC cells in the logarithmic growth phase were taken and the cell density was adjusted to 5×10 3 Each well was inoculated into a 96-well plate, and 40 μM Vpr polypeptides of each sequence were added and incubated for 48 h at 37°C and 5% CO2. The cell culture supernatant was collected, centrifuged at 300 g for 10 min, and tested using an ATP detection kit.

[0046] ELISA detection of Vpr polypeptide-induced HMGB1 release in tumor cells: MC38, B16F10 and LLC cells in the logarithmic growth phase were taken and the cell density was adjusted to 5×10 3Each well was inoculated into a 96-well plate, and 40 μM Vpr peptides of each sequence were added and incubated for 48 h at 37°C and 5% CO2. The cell culture supernatant was collected, centrifuged at 300 g for 10 min, and detected using HMGB1 ELISA Kit.

[0047] Laser confocal fluorescence microscopy observation of Vpr peptide promoting macrophage phagocytosis: 1×10 6 LLC cells were resuspended in 1 mL of cell labeling solution (1×). 2 μL of CFDA-SE was added to 1 mL of cell labeling solution (1×), mixed and added to the cell suspension, incubated at 37°C for 5 min, and 5 mL of DMEM complete medium was added to wash the cells. After three washes, the CFDA-SE labeling of LLC tumor cells was completed. The labeled tumor cells were treated with 40 μM of Vpr peptides P1 and P4 for 24 h. 1×10 5 Ana-1 macrophages were inoculated into confocal microplates and cultured for 12 h to allow them to adhere to the wall. Ana-1 was starved for 2 h and drug-treated LLC was co-incubated with Ana-1. 2×10 5 LLC treated with CFDA SE-labeled drugs were incubated for 2 h, free tumor cells were washed away, and the cells were observed using a laser confocal microscope. The phagocytic index (the number of macrophages phagocytosing tumor cells per 100 macrophages) was counted and calculated.

[0048] Figure 3 (A) is the detection of EGFR release: Figure 3 (A) The release of EGFR after treatment with different Vpr peptides was measured by ELISA. The results showed that the release of EGFR increased under different peptide treatments, especially P1 showed a significant release effect. This indicates that P1 Vpr peptide treatment can promote the release of EGFR from tumor cells, suggesting the potential for tumor antigen release.

[0049] Figure 3 (BD) is CRT exposure observation: Figure 3 (BD) Confocal laser scanning microscopy (CLSM) was used to observe the exposure of CRT on the cell surface of MC38, B16F10, and LLC cells. CRT (calreticulin, red) is one of the markers of immunogenic cell death (ICD), which can activate the immune system when exposed on the cell surface. The results showed that after treatment with P1 peptide, the exposure of CRT on the surface of the three tumor cells increased significantly, indicating that Vpr peptides, especially P1 group, can induce ICD in tumor cells and expose CRT.

[0050] Figure 3 (E) Flow cytometry analysis of CRT exposure: Figure 3(E) Flow cytometry was used to further quantify the exposure of CRT on the tumor cell surface. Compared with the untreated control group, the exposure of CRT in the P1 group was significantly increased, with statistical significance. This further verified the ability of the P1 Vpr peptide to induce ICD, and the effect was more significant than that of the P4 peptide.

[0051] Figure 3 (F) is the detection of ATP release: Figure 3 (F) ATP release was measured by ATP detection kit. ATP release is another hallmark of ICD, which can attract and activate anti-tumor immune cells. The results showed that the ATP release in the P1 group was significantly higher than that in the other groups, indicating that the P1 peptide has a stronger effect in inducing ICD.

[0052] Figure 3 (G) Detection of HMGB1 release: Figure 3 (G) The release of HMGB1 was detected by ELISA. HMGB1 release is another hallmark of ICD and can further stimulate the immune system. Compared with the control group, P1 peptide significantly increased the release of HMGB1, further indicating the advantage of P1 peptide in inducing ICD.

[0053] Figure 3 (HI) is macrophage phagocytosis observation: Figure 3 (HI) CLSM was used to observe the promotion of macrophage phagocytosis of LLC cells by Vpr peptides. LLC cells labeled with CFDA-SE are shown in green. Under the treatment of P1 peptide, the phagocytosis of LLC cells by macrophages was significantly enhanced, indicating that Vpr peptides (especially P1) can induce tumor antigen exposure through ICD, thereby enhancing the phagocytic activity of macrophages.

[0054] Figure 3 The results showed that different types of Vpr peptides have the ability to induce immunogenic cell death (ICD) in tumor cells, and the P1 peptide has the most significant effect on the release of EGFR, CRT, ATP and HMGB1. The P1 peptide not only promotes the release and surface exposure of tumor antigens, but also enhances the phagocytic ability of macrophages, indicating that it has potential application value in tumor immunotherapy.

[0055] Example 4: In situ vaccines based on Vpr polypeptides have systemic anti-tumor effects and inhibit the growth of remote tumors in combination with SIRPαFc

[0056] (1) 2×10 6 MC38 mouse colon cancer cells or LLC cells were inoculated subcutaneously in the right flank of C57BL / 6 mice. 6The same type of cells were inoculated subcutaneously in the left rib to construct the MC38 or LLC bilateral subcutaneous transplant tumor model. When the tumor volume grew to about 100mm 3 At 4 pm, the mice were randomly divided into groups and started to be administrated twice a week. SIRPαFc was expressed and prepared by our laboratory. The specific grouping and dosing schedule were as follows:

[0057]

[0058]

[0059] The changes in tumor volume and body weight on both sides of the mice were recorded as the drug was administered. After the drug administration, all mice were euthanized and the tumor tissues, major organs (heart, liver, spleen, lungs, kidneys) and lymph nodes were removed. The tumor tissue and spleen were divided into two parts, one part was ground and passed through a cell sieve for flow analysis; the other part was fixed in a neutral tissue fixative and then embedded in wax blocks for immunohistochemical analysis. The heart, liver, lungs, and kidneys were fixed in a neutral tissue fixative and then embedded in wax blocks for immunohistochemical analysis. The lymph nodes were ground and passed through a cell sieve for flow analysis.

[0060] (2) Flow cytometry detection of immune cells: Fresh tumor tissue, spleen and lymph nodes of mice were obtained, ground and passed through a 70 μm cell sieve, washed with 1× PBS and resuspended to prepare single cell suspension; mouse CD45, CD3, CD4, CD8, and CD69 flow cytometry antibodies were added to some samples, incubated at room temperature in the dark for 30 min, washed twice with PBS, resuspended in 200 μL PBS and added to a 96-well plate, and mouse T cell activation, cytotoxic function and proliferation ability were detected in a Beckman flow cytometer; mouse CD45, CD11c, CD103, CD11b, MHCⅠ, and MHCⅡ flow cytometry antibodies were added to some samples, incubated at room temperature in the dark for 30 min, washed twice with PBS, and resuspended in 200 μL PBS was added to a 96-well plate, and the activation and antigen presentation function of mouse DCs were detected in a Beckman flow cytometer; mouse CD45, CD11b, F4 / 80, and MHCⅡ flow antibodies were added to some samples, incubated at room temperature in the dark for 30 minutes, washed twice with PBS, resuspended in 200 μL PBS and added to a 96-well plate, and the activation and polarization of mouse macrophages were detected in a Beckman flow cytometer. The test results were analyzed in Flowjo 10.6.2 software.

[0061] (3) Immunohistochemical staining: The tumor tissues and spleens removed in the efficacy experiment of the mouse transplanted tumor model were soaked in 4% paraformaldehyde tissue fixative for more than 24 hours, and then embedded in wax blocks and made into 5 μm slices for immunohistochemical staining to detect the expression of CD4, CD8, F4 / 80, CD11c, Ki67, CRT and HMGB1. The slices were scanned using a whole slide scanner, and the proportion of positive areas was calculated using ImageJ. One-way-ANOVA analysis was performed using GraphPad Prism 9 software to detect statistical differences.

[0062] (5) HE staining: The main organs used in the drug efficacy experiment of the mouse transplanted tumor model, including the heart, liver, spleen, lung, and kidney, were collected and embedded in wax blocks, and then made into 5 μm slices. HE staining was performed to detect tissue necrosis, and the slices were scanned using a whole slide scanner.

[0063] Figure 4 (A) A timeline of the experimental design is shown. MC38 tumor cells were inoculated into the right and left sides of mice on days 0 and 4, respectively, to establish a bilateral tumor model. On days 8, 12, 15, and 19, Vpr peptides (P1 or P4) were injected intratumorally into the right tumor of the mice, and Poly(I:C) was injected subcutaneously to enhance the immune response. This regimen mimics the therapeutic effect of Vpr peptides as an in situ vaccine, inducing systemic anti-tumor immune responses through local injection.

[0064] Figure 4 (B) shows the growth of right tumors (tumors injected with Vpr peptides) in different treatment groups at different time points. The growth of tumors in the P1 group was significantly inhibited, and the difference was statistically significant compared with the control group. This shows that the P1 group peptide has a significant effect in inhibiting in situ tumors, further supporting its potential as a tumor suppressor.

[0065] Figure 4 (C) shows the growth of left tumors (tumors without direct injection of Vpr peptide) in different treatment groups. The growth of left tumors in mice treated with P1 was also significantly inhibited, indicating that Vpr peptide injection triggered a systemic anti-tumor immune response in the right tumor. This phenomenon supports the effect of Vpr peptide as an in situ vaccine, that is, inducing a systemic immune response through local treatment to inhibit the growth of distant tumors.

[0066] Figure 4 (DH) shows the expression levels of different immune cell markers in the right tumor and spleen tissues, including CD8, F4 / 80 (macrophage marker) and other immune cell markers (CD4, CD11C, etc.). +The significant increase in T cells indicates that Vpr peptides (especially P1) can recruit and activate anti-tumor CD8 + T cells. The number of CD4 and CD11C-positive cells in the spleen also increased significantly in the P1 group, indicating systemic immune activation. The expression of these markers suggests that Vpr polypeptides may initiate anti-tumor immunity by activating specific immune cells (such as T cells and dendritic cells).

[0067] Figure 4 The results showed that Vpr peptides (especially P1) effectively inhibited the growth of both in situ and distal tumors through an in situ vaccine strategy. Immunohistochemical analysis showed that Vpr peptides induced the infiltration and activation of immune cells in tumor tissues and spleen, especially CD8 + The results support the potential application of Vpr peptides in tumor immunotherapy, which can inhibit tumor growth by activating systemic anti-tumor immunity.

[0068] Figure 5 (A) The experimental design and treatment scheme are shown. On day 0, mice were bilaterally inoculated with LLC cells for tumor model establishment. Mice received different treatments on days 15, 18, 23, and 26, including PBS control group, 10 mg / kg and 15 mg / kg P1 peptide intratumoral injection, and 10 mg / kg P1 plus 2.5 mg / kg Poly(I:C) combined treatment. This scheme aims to evaluate the inhibitory effect of Vpr peptide P1 alone or in combination with Poly(I:C) on LLC tumor growth.

[0069] Figure 5 (B) shows the growth of the right tumor (tumor injected with Vpr polypeptide). Compared with the control group, the tumor growth of the P1 and P1+Poly(I:C) treatment groups was inhibited to varying degrees, and the inhibitory effect of the combined treatment group was more significant. This shows that Vpr polypeptide has a certain tumor inhibitory effect when used alone, and this inhibitory effect can be enhanced when used in combination with Poly(I:C).

[0070] Figure 5 (C) shows the growth of the left tumor (the tumor that was not directly injected with Vpr peptide). The results showed that the growth of the left tumor in the P1+Poly(I:C) combined treatment group was significantly inhibited, indicating that the in situ treatment on the right side induced a systemic immunosuppressive effect on the left tumor. This further supports the possibility that Vpr peptide can be used as an in situ vaccine to induce a systemic anti-tumor immune response through local treatment.

[0071] Figure 5(DE) shows the immunohistochemical staining of ki67 in the right and left tumors. Ki67 is a cell proliferation marker, and its staining intensity can reflect the proliferation activity of tumor cells. The results showed that in the P1 and P1+Poly(I:C) treatment groups, the proportion of ki67 positive areas in the right and left tumors was lower than that in the control group, indicating that the proliferation activity of tumor cells was inhibited. Especially in the left tumor, the inhibitory effect of the P1+Poly(I:C) combined treatment group was more significant.

[0072] Figure 5 (FG) shows the immunohistochemical staining of CRT and HMGB1 in the right tumor tissue. Both CRT and HMGB1 are markers of immunogenic cell death (ICD), and their expression can reflect the immunogenicity induced by tumor cells. The results showed that in the P1 and P1+Poly(I:C) treatment groups, the expression levels of CRT and HMGB1 were higher than those in the control group, especially in the combined treatment group. This indicates that Vpr peptide treatment can induce ICD in tumor cells, thereby stimulating anti-tumor immune response.

[0073] Figure 5 The results showed that Vpr peptide P1 has an inhibitory effect on tumor growth in the LLC tumor model, and this effect can be further enhanced when used in combination with Poly(I:C). The proliferation marker ki67 was significantly reduced in both right and left tumors, showing inhibition of tumor cell proliferation activity. In addition, the expression of CRT and HMGB1 increased, indicating that Vpr peptide can induce immunogenic cell death (ICD) and further enhance anti-tumor immune response. These findings support Vpr peptide as a potential candidate for tumor immunotherapy.

[0074] Figure 6 (AB) show dendritic cell (DC) subsets in draining lymph nodes (dLN). Figure 6 (A) and 6(B) show the percentage of classical dendritic cells (cDC1 and cDC2) in dLN in different treatment groups. cDC1 and cDC2 are important subsets of antigen presenting cells that can effectively initiate immune responses. The results showed that different concentrations of Vpr peptide (P1) treatment groups can increase the proportion of cDC1 and cDC2 in dLN to a certain extent, indicating that Vpr peptide can activate dendritic cells in dLN, enhance antigen presentation function, and lay the foundation for subsequent immune responses.

[0075] Figure 6(C) and 6(D) show the percentages of CD4+ and CD8+ T cells in dLN. CD4+ T cells and CD8+ T cells are helper T cells and cytotoxic T cells, respectively, with different immunomodulatory and killing effects. The results showed that Vpr peptide (P1) can increase the proportion of CD4+ and CD8+ T cells in dLN, indicating that it can effectively initiate T cell responses and further enhance anti-tumor immune responses.

[0076] Figure 6 (E) and 6(F) show the proportion of macrophages in the spleen, right tumor, and left tumor. Macrophages play an important role in the tumor microenvironment, and changes in their subtypes and functions can affect anti-tumor or pro-tumor immunity. The results showed that the proportion of macrophages in the Vpr peptide-treated group (P1) increased in both right and left tumor tissues, indicating that the Vpr peptide can promote the infiltration of macrophages in the tumor microenvironment and help regulate the immune activity in the tumor microenvironment.

[0077] Figure 6 The results showed that Vpr peptide (P1) can activate dendritic cells and T cells in dLN, while increasing the proportion of macrophages in the spleen and tumor tissue. The peptide can enhance anti-tumor immune response through multi-level immune regulation by initiating antigen presentation and T cell activation in draining lymph nodes, combined with macrophage regulation in the tumor microenvironment. These findings further support Vpr peptide as an effective candidate for tumor immunotherapy.

[0078] Figure 7 (A) and 7(B) show CD47 immunohistochemical staining in the right and left tumor tissues. CD47 is a "don't eat me" signal molecule that inhibits the phagocytic function of macrophages by interacting with SIRPα on the surface of macrophages, thereby helping tumor cells escape clearance by the immune system. From the staining results, the CD47 expression level in the Vpr peptide and SIRPαFc combined treatment group was reduced, indicating that the combined treatment may have a role in reducing the expression of CD47 in tumor cells, thereby enhancing the phagocytic activity of macrophages.

[0079] Figure 7 (C) shows the experimental design. LLC tumor cells were inoculated on both sides of the mouse on day 0 to establish a bilateral tumor model. Starting from day 8, 10 mg / kg of Vpr peptide (P1) or 10 mg / kg of SIRPαFc was injected intraperitoneally into the right tumor every few days. This scheme is designed to study the inhibitory effect of Vpr peptide combined with SIRPαFc on orthotopic tumors and distant tumors (left tumors that were not directly injected).

[0080] Figure 7(D) shows the growth curve of the right tumor (tumor directly injected with Vpr peptide) in different treatment groups. The results showed that compared with the control group, the growth of the right tumor in the P1 and P1+SIRPαFc combined treatment groups was significantly inhibited, especially the combined treatment group. This indicates that the combination of Vpr peptide and SIRPαFc can significantly inhibit the growth of in situ tumors.

[0081] Figure 7 (E) shows the growth of the left tumor (distant tumor without direct injection of Vpr peptide) in different treatment groups. The growth of the left tumor in the P1+SIRPαFc combined treatment group was significantly inhibited, indicating that the combined treatment of the right tumor may induce a systemic anti-tumor immune response, thereby inhibiting the growth of the distant tumor. This further supports the potential of combined treatment of Vpr peptide and SIRPαFc in initiating a systemic immune response.

[0082] Figure 7 The results showed that the combination of Vpr peptide (P1) and SIRPαFc had a significant inhibitory effect on both in situ and distant tumors. CD47 immunohistochemistry analysis showed that the combined treatment may reduce the expression of CD47 in tumor cells and enhance the phagocytic activity of macrophages on tumor cells. The effect of combined treatment was significantly better than that of Vpr peptide alone, indicating that the combined use of Vpr peptide and SIRPαFc can achieve the purpose of inhibiting tumor growth by reducing CD47 expression and initiating a systemic immune response. These results support the potential of Vpr peptide combined with SIRPαFc as a candidate for tumor immunotherapy.

[0083] Example 5: Proteomic Analysis of the Anti-tumor Mechanism of Vpr Peptide

[0084] Tumor samples collected from the LLC transplanted tumor model efficacy test in mice were subjected to proteomic analysis by Genentech. Proteins identified in the samples were screened by trypsin lysis and liquid chromatography-mass spectrometry analysis, and database search and bioinformatics analysis were performed on the proteins. The P1 / Control Ratio value was calculated, and Fold change>1.5 or Foldchange<0.67, P<0.05 were considered significant changes, and GO and KEGG enrichment analysis was performed on these phosphorylated proteins. (Foldchange=Log2 Ratio).

[0085] Figure 8(A) shows the differentially expressed proteins between the P1-treated group and the control group. The horizontal axis of the volcano plot represents Log2Fold Change (fold change in protein expression), and the vertical axis represents -log10 (P-value), which is used to evaluate the significance of differential expression. In the figure, red dots represent significantly upregulated proteins, blue dots represent significantly downregulated proteins, and gray dots represent proteins with no significant changes. After P1 treatment, some proteins were significantly upregulated or downregulated, indicating that the Vpr polypeptide had a significant effect on the protein expression pattern in the cell.

[0086] Figure 8 (B) shows the results of GO (Gene Ontology) analysis, showing the biological processes that were significantly enriched in the P1-treated group compared with the control group. GO analysis is used to describe the functions of genes and proteins in biological processes. The results showed that biological processes related to "skeletal muscle thin filament assembly", "superoxide free radical scavenging regulation", and "muscle contraction" were enriched in the P1-treated group, indicating that the Vpr polypeptide may affect these specific cellular physiological processes, thereby exerting its anti-tumor effect.

[0087] Figure 8 (C) KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis results are shown, highlighting the metabolic pathways that were significantly downregulated in the P1-treated group compared with the control group. Significantly enriched pathways in the results include "protein digestion and absorption" and "calcium signaling pathway", suggesting that these pathways were inhibited after P1 treatment. The effects of P1 on these pathways may be related to its anti-tumor activity.

[0088] Figure 8 (D) shows the analysis of immune cell infiltration in the injected tumor by ssGSEA (single sample gene set enrichment analysis). ssGSEA is used to evaluate the enrichment of different immune cell types in the sample. The results showed that compared with the control group, the infiltration of certain immune cells in the injected tumor of the P1 treatment group was significantly increased, such as activated CD8 + T cells, memory CD4 + This suggests that Vpr polypeptide P1 can enhance anti-tumor immune response by regulating the infiltration of immune cells in the tumor microenvironment.

[0089] Figure 8 (E) shows the analysis of immune cell infiltration in remote tumors (non-injected side) by ssGSEA analysis. The results showed that the P1-treated group had a significant increase in certain immune cell types in remote tumors (such as activated natural killer cells, CD8 + T cell infiltration was significantly increased, indicating that local injection of Vpr peptide can also induce systemic immune response in distant tumors.

[0090] Figure 8 The results showed that the Vpr peptide P1 exerted its anti-tumor effect by regulating specific protein expression and biological pathways (such as muscle contraction, superoxide clearance, etc.). GO and KEGG analysis revealed the significant effects of P1 treatment on protein expression and cellular pathways. In addition, ssGSEA analysis showed that the P1 peptide could not only enhance immune cell infiltration in the injected tumor, but also activate immune responses in distant tumors, indicating that it has the potential to induce systemic immune effects. Overall, these results provide important mechanistic support for the application of Vpr peptides in anti-tumor immunotherapy.

[0091] Compositions and vaccines

[0092] On the other hand, the present invention provides a composition comprising a polypeptide or immunogen of the present disclosure, and at least one pharmaceutically acceptable carrier and / or excipient. In some preferred embodiments, the composition is provided as a pharmaceutical composition. According to another preferred embodiment, the composition can be provided as a vaccine. "Vaccine" is generally understood to provide at least one antigen. "Providing at least one antigen" means, for example, that the vaccine contains an antigen or the vaccine contains, for example, a molecule encoding an antigen. Therefore, the vaccine of the present disclosure may include at least one RNA nucleic acid molecule encoding at least one antigenic polypeptide as defined herein, which antigenic polypeptide or protein may be derived, for example, from a tumor antigen, a bacterial antigen, a viral antigen, a fungal antigen or a protozoan antigen, an autoantigen, an allergen or an alloantigen, and preferably induces an immune response to the corresponding antigen when it is expressed and presented to the immune system.

[0093] The composition or vaccine may also contain at least one additional active agent, preferably, the composition or vaccine according to the invention contains at least one pharmaceutically acceptable carrier and / or excipient. The term "pharmaceutically acceptable" refers to a compound or agent that is compatible with one or more than one active agent and does not interfere with and / or significantly reduce its drug effect. Pharmaceutically acceptable carriers and excipients preferably have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to the subject to be treated.

[0094] Pharmaceutically acceptable excipients can serve different functional roles and include, but are not limited to, diluents, fillers, expanders, carriers, disintegrants, binders, lubricants, glidants, coatings, solvents and co-solvents, buffers, preservatives, adjuvants, antioxidants, wetting agents, antifoaming agents, thickeners, sweeteners, flavoring agents, and humectants.

[0095] For compositions in liquid form, useful pharmaceutically acceptable carriers and excipients include solvents, diluents or vehicles such as (pyrogen-free) water, (isotonic) saline solutions such as phosphate or citrate buffered saline, fixed oils, vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.); lecithin; surfactants; preservatives such as benzyl alcohol, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; isotonic agents such as sugars, polyols such as mannitol, sorbitol or sodium chloride; aluminum monostearate or gelatin; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; and agents for adjusting tonicity such as sodium chloride or glucose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The buffer may be hypertonic, isotonic or hypotonic relative to a specific reference medium, i.e., it may have a higher, identical or lower salt content relative to a specific reference medium, wherein preferably such concentrations of the above salts may be used which do not cause cell damage due to osmotic or other concentration effects. The reference medium is, for example, a liquid produced in an "in vivo" method, such as blood, lymph, cytoplasmic fluid or other body fluids, or a liquid which may be used as a reference medium in an "in vitro" method, such as a conventional buffer or liquid. Such conventional buffers or liquids are known to the skilled person.

[0096] For compositions in (semi)solid form, useful pharmaceutically acceptable carriers and excipients include binders such as microcrystalline cellulose, tragacanth or gelatin; starch or lactose; sugars such as lactose, glucose and sucrose; starches such as corn starch or potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate; disintegrants such as alginic acid; lubricants such as magnesium stearate; glidants such as stearic acid, magnesium stearate; calcium sulfate, colloidal silicon dioxide, etc.; sweeteners such as sucrose or saccharin; and / or flavorings such as mint, methyl salicylate or orange flavor.

[0097] Suitable pharmaceutically acceptable carriers and excipients can generally be selected based on the desired formulation of the composition.

[0098] Liquid compositions administered by injection, especially intravenous injection, should be sterile and stable under the conditions of manufacture and storage. Such compositions are usually formulated as parenterally acceptable aqueous solutions that are pyrogen-free, have a suitable pH, are isotonic, and maintain the stability of the active ingredient. Particularly useful pharmaceutically acceptable carriers and excipients for liquid compositions according to the present invention include water, typically pyrogen-free water; isotonic saline or buffer solutions, such as phosphate, citrate, and other buffer solutions. In particular, for injection of the compositions of the present disclosure, water or preferably a buffer, more preferably an aqueous buffer, can be used, which contains a sodium salt, preferably at least 50 mM of a sodium salt; a calcium salt, preferably at least 0.01 mM of a calcium salt; and an optional potassium salt, preferably at least 3 mM of a potassium salt.

[0099] According to a preferred embodiment, the sodium salt, calcium salt and optionally the potassium salt may be present in the form of their halides, such as chloride, iodide or bromide, in the form of their hydroxides, carbonates, bicarbonates or sulfates, etc. In addition, organic anions of the above-mentioned cations may be contained in the buffer.

[0100] According to a preferred embodiment, a buffer suitable for injection purposes as defined above may comprise a salt selected from sodium chloride (NaCl), calcium chloride (CaCl ) and optional potassium chloride (KCl), wherein in addition to chloride ions, other anions may also be present. CaCl 2 may also be replaced with another salt such as KCl. Typically, the concentration of the salt in the injection buffer is at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl) and at least 0.01 mM calcium chloride (CaCl ).

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that The polypeptide has: (I) an amino acid sequence as shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No SEQ.3, and SEQ ID No.4; or (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or (III) an amino acid sequence having 80% or more similarity to the amino acid sequence described in (I) or (II).

2. An immunogen, characterized in that Comprising the polypeptide according to claim 1.

3. A vaccine, characterized in that Comprising the immunogen of claim 2.

4. The vaccine according to claim 4, characterized in that Also contains an adjuvant.

5. Use of the immunogen according to claim 2 as an active ingredient of an anti-tumor vaccine.

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