Tumor antigenic polypeptide and application thereof in preparation of tumor vaccine

Abstract

The invention relates to the technical field of tumor vaccines, in particular to tumor antigenic polypeptide and application thereof in preparation of tumor vaccines. The amino acid sequence of the tumor antigenic polypeptide is PLLLMFITQCLLEEITGLLYLVGATLLLR, and the tumor antigenic polypeptide can stimulate the specific immune response of a patient body to tumors, prolong the survival time of the patient body and effectively induce stronger CTL killing activity, so that a more effective treatment effect is achieved. The tumor antigenic polypeptide provided by the invention can effectively stimulate the maturation of tumor specific DC, and the DC loaded with the antigen peptide can effectively process and present tumor antigen and activate tumor cell specific anti-tumor cell immune response, so that tumors can be effectively treated or prevented in animal bodies. The invention provides the application of the tumor polypeptide antigen in preparation of the tumor vaccine, and provides a new choice for tumor treatment scheme research and clinical application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor vaccine, in particular to a tumor antigenic polypeptide and its use in preparing tumor vaccine. BACKGROUND

[0002] In recent years, with the in-depth study of the molecular mechanism of tumor development and the rapid development of biotechnology, the tumor biological therapy based on immunotherapy is considered as the fourth tumor treatment mode after surgery, radiotherapy and chemotherapy. Unlike traditional treatment methods, tumor biological therapy is to achieve anti-tumor effect by mobilizing tumor host defense mechanism or giving natural (or genetically engineered) targeted substances with strong specificity without damaging the body's immune system and function. At present, biological therapy mainly includes cytokine therapy, immune cell therapy, gene therapy, molecular targeted therapy, antibody therapy and tumor vaccine technology. Among them, tumor vaccine therapy has developed into a more effective treatment method, which can effectively reduce the patient's condition and achieve the effect of preventing and treating high-risk individual cancer recurrence by injecting tumor vaccine into the body. Because the vaccine treatment of tumor has no obvious toxic and side effects, it has developed rapidly in recent years. The current tumor vaccine mainly includes cell-based tumor vaccine, monoclonal antibody tumor vaccine, tumor gene vaccine, tumor polypeptide vaccine, etc. Among them, the tumor antigen polypeptide vaccine is expected to overcome the shortcomings of current tumor vaccine therapy and achieve the purpose of effective treatment of tumor due to its strong specificity, safety, low cost, easy preservation and application. However, tumor antigen polypeptide is limited by major histocompatibility complex (MHC) molecules, and its immunogenicity is weak, which often cannot induce strong immune response. Therefore, how to effectively improve the immunogenicity of tumor antigen polypeptide and induce stronger cytotoxic T lymphocyte (CTL) activity has become an urgent problem in the research of tumor prevention and treatment. SUMMARY

[0003] In view of the deficiencies of the prior art, the present application provides a tumor antigenic polypeptide and its use in preparing a tumor vaccine. The tumor antigenic polypeptide provided by the present application can stimulate the specific immune response of the body to the tumor, prolong the survival time, and effectively induce stronger CTL killing activity. The addition of the immunoadjuvant prepared by the present application to the tumor antigenic polypeptide provided by the present application can induce stronger immune response, enhance the antigen processing and presentation performance of DC, effectively activate the cytotoxic T cell immune effect, and the combination of the immunoadjuvant and the tumor antigenic polypeptide can synergistically promote the maturation of dendritic cells, promote T cell proliferation, and induce enhanced anti-tumor effect in vitro. The tumor vaccine provided by the present application has good curative effect and can effectively treat various tumors, and has better effect in the treatment of tumors.

[0004] The present application is realized by the following technical solutions: A tumor antigenic polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1 in the sequence listing.

[0005] Further, the amino acid sequence of the tumor antigenic polypeptide is designed by arranging, adding, deleting and modifying the NY-ESO-1, HER-2 / neu and CEA amino acid sequences.

[0006] Further, the NY-ESO-1, HER-2 / neu and CEA amino acid sequences are shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0007] Further, the amino acid sequence of the present application is as follows: < Tumor antigenic polypeptide; AA; SEQ ID NO:1 >: PLLLMFITQCLLEEITGLLYLVGATLLLR; < NY-ESO-1; AA; SEQ ID NO:2 >: SLLMFITQC; < HER-2 / neu; AA; SEQ ID NO:3 >: YLEEITGYL; < CEA; AA; SEQ ID NO:4 >: YLSGADLNL.

[0008] Further, the preparation method of the antigenic polypeptide comprises the following steps: (1) According to the characteristics of the amino acid sequence, the peptide chain is extended from the C-terminal to the N-terminal. Take 50 g of Wang resin with a mesh size of 200, load it into the reaction kettle, wash it once with 500 mL of dimethylformamide (DMF), dry it, then swell the resin with 500 mL of dichloromethane (DCM) for 30 min, dry it, add the first protected amino acid, 1-hydroxybenzotriazole (HOBT), diisopropyl carbodiimide (DIC), 300 mL of DMF, 100 mL of DCM, mix well, and introduce nitrogen or inert gas into the reaction system to maintain the temperature at 20°C. Slowly add 4-dimethylaminopyridine (DMAP), stir at 24°C for 18 h, vacuum dry, wash with DMF twice, vacuum dry, add pyridine, acetic anhydride and 500 mL of DCM, stir at 27°C for 3 h, vacuum dry, wash the precipitate with DMF and methanol, and vacuum dry to constant weight; (2) Add 500 mL of 20 vol% piperidine / DMF solution to the reaction kettle containing the washed precipitate described in step (1), stir at 25°C for 10 min, vacuum dry, then add 500 mL of 20 vol% piperidine / DMF solution, stir at 25°C for 20 min, vacuum dry, wash with DMF once, methanol twice, DMF once, and DCM once, vacuum dry, add the second protected amino acid, HOBT, benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU) and 350 mL of DMF, 100 mL of DCM, introduce nitrogen or inert gas to quickly cool the temperature to 18°C, add N'N-diisopropylethylamine (DIPEA) dropwise, stir at 25°C for 3 h, and test with ninhydrin. The result is negative, vacuum dry, wash the precipitate with methanol once and DMF twice, and vacuum dry; (3) Repeat the operation of step (2), replace the protected amino acid according to the synthesis sequence, and couple according to the amino acid sequence. After all the amino acids are connected, washed, vacuum dried to constant weight, add the reaction liquid according to the ratio of 1 g:10 mL, stir at 26°C for 2.5 h, filter, concentrate the filtrate by vacuum distillation, precipitate with methyl tert-butyl ether, and centrifuge to collect the crude tumor antigenic polypeptide; (4) Mix the crude tumor antigenic polypeptide obtained in step (3) with 5 vol% propionitrile aqueous solution according to the ratio of 1 g:100 mL, purify it by high performance liquid chromatography, use 0.1 vol% trifluoroacetic acid (TFA) aqueous solution as the water phase and 0.1 vol% TFA propionitrile solution as the organic phase, concentrate by vacuum rotary evaporation, freeze-dry, and obtain the tumor antigenic polypeptide.

[0009] Further, the molar number of the first protected amino acid, HOBT and DIC in step (1) is 3 times of the Wang resin, the molar number of DMAP is 0.2 times of the Wang resin, and the molar number of pyridine and acetic anhydride is 10 times of the Wang resin.

[0010] Further, the molar number of the second protected amino acid, HOBT, HBTU and DIPEA in step (2) is 2 times of the Wang resin in step (1).

[0011] Further, the protected amino acid in steps (1)-(3) is an amino acid adopting Fmoc protection of alpha-amino group, except that the last one is an amino acid adopting Boc protection of functional group.

[0012] Further, the reaction solution in step (3) is a mixture of TFA, triisopropylsilane (TIS) and water in a volume ratio of 95:2.5:2.5.

[0013] The application also provides the use of the tumor antigenic polypeptide in the preparation of a tumor vaccine, comprising the following steps: S1: preparing a polypeptide vaccine: adding the tumor antigenic polypeptide into PBS to configure a solution to obtain a tumor antigenic polypeptide vaccine; S2: preparing a dendritic cell (DC) vaccine: separating mononuclear cells from peripheral blood, suspending the mononuclear cells in X-vivo20 medium, measuring the cell density by a blood cell counter, inoculating into a 25 cm 6 culture bottle at a concentration of 7×10 2 / mL, and culturing in a 37℃, 5% CO2 incubator for 4 h; taking out the culture solution and non-adherent cells in the culture bottle to obtain adherent cells and non-adherent cells; adding 10 mL of X-vivo20 culture solution containing 1000 U / mL of GM-CSF and 1000 U / mL of IL-4 to the obtained adherent cells per bottle to induce differentiation to DC, and culturing in a 37℃, 5% CO2 incubator; on the third day, replacing half of the liquid once, and supplementing 1000 U / ml of GM-CSF and 1000 U / ml of IL-4; on the fifth day of DC culture, mixing and culturing the tumor antigenic polypeptide with the DC cells, and the volume to mass ratio of DC to tumor antigenic polypeptide is 1 L:100 mg; on the seventh day of DC culture, adding 100 ng / mL of LPS to induce maturation of DC, and obtaining a DC vaccine after 72 h; S3: preparing antigen-specific cytotoxic T lymphocytes (CTLs): collecting the non-adherent cells obtained in step S2, resuspending in RPMI-1640 medium containing 3% serum, and adjusting the cell density to 1×10 6 / mL, the suspension is rich in T lymphocytes, the DC vaccine prepared in step S2 is mixed with T lymphocytes at a ratio of 1:10, and then fetal bovine serum 10% and IL-2 1000 U / mL are added in RPMI-1640 culture medium, the medium is replaced every 3 days, and the cell concentration is maintained at 1×10 6 / mL, and 14 days later, antigen-specific CTLs are obtained.

[0014] Preferably, the step S2 adds LPS, and at the same time, 100 mg / L of an immunoadjuvant can be added.

[0015] Further, the preparation method of the immunoadjuvant comprises the following steps: (a) 0.98 moL colchicine, 1.26 moL dimethyl sulfate 120 mL, and 300 mL of methanol are mixed together and stirred, heated to reflux for 30 min, cooled to room temperature to obtain a mixed solution A, the mixed solution A is added to 120 mL of a sodium hydroxide solution with a concentration of 4.5 moL / L at a speed of 15 mL / min, and stirring is continued for 30 min, 80 mL of dichloromethane is added for extraction, and 12 h of reduced pressure concentration is carried out at 70°C and 0.01 MPa, washed with anhydrous ethanol three times, washed with deionized water three times, and dried at 60°C until the weight is constant to obtain product A; (b) 9.75 mmol of glucosamine is mixed with 40 mL of DMF to prepare a glucosamine / DMF solution, 3.5 g of product A obtained in step (a) and camphor sulfonic acid 100 mg are added, connected to vacuum, heated in an oil bath at 50°C, 3 h later, 80°C rotary evaporation for 4 h, dissolved in a mixed solution B composed of 50 mL of diethyl ether and 2 mL of Et3N, and then 50 mL of saturated sodium bicarbonate is added, and the water layer is extracted with diethyl ether 3 times, each time with 50 mL of diethyl ether, the organic layers of the three extractions are combined, dried over sodium sulfate and filtered, 80°C rotary evaporation for 4 h, and purified by silica gel chromatography (120 g Redi Sep column, 50 min elution with 0% to 100% gradient of ethyl acetate / hexane, 85 mL / min), to obtain the immunoadjuvant.

[0016] Compared with the prior art, the present application has the following beneficial effects: The present application selects three polypeptide combinations matched with human leukocyte antigens, designs amino acid sequences through permutation, addition, deletion and modification replacement, and finally prepares a tumor antigenic polypeptide, which is superior to simple combination of polypeptides, can stimulate specific immune response of the patient's body to tumors, prolongs survival time, has unique advantages of bypassing immune diversity and tumor heterogeneity, can effectively induce stronger CTL killing activity, and thus achieves more effective treatment effect. On the basis of the tumor vaccine provided by the present application, stronger immune response can be induced by adding an immune adjuvant, the immune adjuvant prepared in the present application can further enhance the antigen processing and presentation performance of DCs, effectively activate cytotoxic T cell immune effect, and when used in combination with the tumor antigenic polypeptide, the two can synergistically promote maturation of dendritic cells, promote T cell proliferation, and induce enhanced in vitro anti-tumor effect. The tumor antigenic polypeptide provided by the present application can effectively stimulate maturation of tumor-specific DCs, and the DCs loaded with the antigenic polypeptide can effectively process and present tumor antigens, and activate tumor cell-specific anti-tumor cell immune response, and can effectively treat or prevent tumors in animals. Therefore, the present application provides application of the tumor polypeptide antigen as a cancer vaccine. Meanwhile, the present application provides a DC tumor vaccine obtained by sensitizing dendritic cells with the polypeptide, or an antigen-specific CTL prepared by activating T cells with the dendritic cells sensitized by the tumor polypeptide antigen, and a preparation method and use of the polypeptide as a tumor vaccine. The tumor vaccine provided by the present application has good curative effect, can effectively treat various tumors, has better effect in tumor treatment, and provides a new choice for research on treatment scheme of malignant tumors and clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only illustrate the present application, and those skilled in the art can also obtain other drawings according to these drawings without any creative effort.

[0018] Figure 1 Mass spectrum of the tumor antigenic polypeptide described in embodiment 1 of the present application; Figure 2 High performance liquid chromatogram of the tumor antigenic polypeptide described in embodiment 1 of the present application; Figure 3 Chemical structure diagram of the immune adjuvant described in embodiment 2 of the present application; Figure 4 Nuclear magnetic resonance spectrum of the immune adjuvant described in embodiment 2 of the present application, wherein a is 1 H spectrum, and b is 13 C spectrum; Figure 5Figure of killing effect of CTL specific to the antigen according to the present application embodiment 5-6 and comparative example 4-5 on tumor cells; Figure 6 Figure of proliferation ability of CTL specific to the antigen according to the present application embodiment 5-6 and comparative example 4-5; Figure 7 Figure of tumor induction effect of DC vaccine according to the present application embodiment 3-4 and comparative example 2-3 against 4T1 cells; Figure 8 Figure of tumor induction effect of DC vaccine according to the present application embodiment 3-4 and comparative example 2-3 against TC-1 cells; Figure 9 Figure of anti-tumor effect of tumor antigenic polypeptide according to the present application embodiment 1, immunoadjuvant according to the present application embodiment 2 and polypeptide according to comparative example 1. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is further described in detail below with specific examples, but the present application is not limited to the following examples.

[0020] It should be noted that, unless otherwise specified, the chemical reagents involved in the present application are purchased through commercial channels.

[0021] Embodiment 1: The present embodiment provides a tumor antigenic polypeptide, and the sequence is shown as SEQ NO. 1.

[0022] The present embodiment also provides a preparation method of the tumor antigenic polypeptide, comprising the following steps: (1) According to the amino acid sequence characteristics, the peptide chain is extended from the C-terminal to the N-terminal one by one, 50 g of Wang resin with a 200 mesh sieve is weighed and loaded into a reaction kettle, washed once with 500 mL of dimethylformamide (DMF), dried, and then the resin is swelled with 500 mL of dichloromethane (DCM) for 30 min, dried, and then the first protected amino acid, 1-hydroxybenzotriazole (HOBT), diisopropyl carbodiimide (DIC), 300 mL of DMF and 100 mL of DCM are added and mixed uniformly, nitrogen or inert gas is introduced into the reaction system to make the temperature of the reaction system at 20℃, 4-dimethylaminopyridine (DMAP) is slowly added, stirred at 24℃ for 18 h, vacuum dried, washed twice with DMF, vacuum dried, added pyridine, acetic anhydride and 500 mL of DCM, stirred at 27℃ for 3 h, vacuum dried, and then the precipitate is washed twice with DMF and methanol in cross, and vacuum dried to constant weight; the molar number of the first protected amino acid, HOBT and DIC is 3 times of the Wang resin, the molar number of DMAP is 0.2 times of the Wang resin, and the molar number of pyridine and acetic anhydride is 10 times of the Wang resin; (2) to the reactor containing the precipitate after washing in step (1), 500 mL of a piperidine / DMF solution with a concentration of 20 vol% was added, stirring at 25°C for 10 min, vacuum drying, then adding 500 mL of a piperidine / DMF solution with a concentration of 20 vol%, stirring at 25°C for 20 min, vacuum drying, washing once with DMF, washing twice with methanol, washing once with DMF, washing once with DCM, vacuum drying, adding a second protected amino acid, HOBT, benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU) and 350 mL of DMF, 100 mL of DCM, rapidly cooling the temperature to 18°C by introducing nitrogen or inert gas, adding N'N-diisopropylethylamine (DIPEA) dropwise, stirring at 25°C for 3 h, ninhydrin detection being negative, vacuum drying, washing the precipitate once with methanol, washing twice with DMF, and vacuum drying; the molar amounts of the second protected amino acid, HOBT, HBTU and DIPEA in step (2) are all 2 times the amount of the Wang resin in step (1); (3) repeating the operation in step (2), replacing the protected amino acid with the sequence of amino acids, coupling according to the sequence of amino acids, after all the amino acids are connected, washed, vacuum dried to constant weight, adding the reaction liquid at a ratio of 1 g:10 mL, stirring at 26°C for 2.5 h, filtering, concentrating the filtrate by reduced pressure distillation, precipitating with methyl tert-butyl ether, and centrifuging to collect the crude tumor antigenic polypeptide; the reaction liquid is a mixture of TFA, triisopropylsilane (TIS) and water at a volume ratio of 95:2.5:2.5; the protected amino acid in steps (1)-(3) is a Boc-protected functional group amino acid except for the last one, which is an Fmoc-protected alpha-amino acid; (4) mixing the crude tumor antigenic polypeptide obtained in step (3) with a 5 vol% propionitrile aqueous solution at a ratio of 1 g:100 mL, purifying by high performance liquid chromatography, using 0.1 vol% trifluoroacetic acid (TFA) aqueous solution as the water phase and 0.1 vol% TFA propionitrile solution as the organic phase, concentrating by reduced pressure distillation, freeze-drying to obtain the tumor antigenic polypeptide; Figure 1 is the mass spectrum of the tumor antigenic polypeptide, with a molecular weight of 3353.19, which is consistent with the expected value, Figure 2 is the high performance liquid chromatogram, and the purity of the obtained tumor antigenic polypeptide is 99.7%.

[0023] Example 2: The present example provides an immunoadjuvant and a preparation method thereof. The preparation method of the immunoadjuvant comprises the following steps: (a) 0.98 moL colchicine, 1.26 moL dimethyl sulfate 120 mL, and 300 mL of methanol were mixed together and stirred, heated to reflux for 30 min, and then cooled to room temperature to obtain a mixed solution A. The mixed solution A was added to 120 mL of a sodium hydroxide solution with a concentration of 4.5 moL / L at a speed of 15 mL / min, and stirring was continued for 30 min. 80 mL of dichloromethane was added for extraction, and the mixture was concentrated under reduced pressure at 70°C and 0.01 MPa for 12 h. The mixture was washed with anhydrous ethanol three times, deionized water three times, and dried at 60°C until the weight was constant to obtain product A; (b) 9.75 mmol of glucosamine was mixed with 40 mL of DMF to prepare a glucosamine / DMF solution, 3.5 g of product A obtained in step (a) and camphorsulfonic acid 100 mg were added thereto, and the mixture was connected to a vacuum and heated in an oil bath at 50°C. After 3 h, the mixture was rotary evaporated at 80°C for 4 h, dissolved in a mixed solution B consisting of 50 mL of diethyl ether and 2 mL of Et3N, and then 50 mL of saturated sodium bicarbonate was added. The water layer was extracted with 50 mL of diethyl ether three times, and the organic layers were combined, dried over sodium sulfate, and filtered. The mixture was rotary evaporated at 80°C for 4 h, and purified by silica gel chromatography (120 g Redi Sep column, eluted with a gradient of 0% to 100% ethyl acetate / hexane for 50 min at 85 mL / min) to obtain an immunoadjuvant. The chemical structure is shown in Figure 3 , and the nuclear magnetic resonance spectrum is shown in Figure 4 .

[0024] Example 3: This example provides a DC vaccine and a preparation method thereof. The preparation method of the DC vaccine comprises the following steps: mononuclear cells are separated from peripheral blood, suspended with X-vivo20 medium, the cell density is measured by a blood cell counter, and inoculated into a 25 cm 6 petri dish at a concentration of 7×10 210 mL in a culture flask, 4 h in a 37℃, 5% CO2 incubator; remove the culture solution and non-adherent cells from the culture flask to obtain adherent cells and non-adherent cells; add 10 mL of X-vivo20 culture solution containing 1000 U / mL of GM-CSF and 1000 U / mL of IL-4 to the obtained adherent cells per flask to induce differentiation into DCs, and culture in a 37℃, 5% CO2 incubator; on the third day, replace half of the solution once, and supplement with 1000 U / mL of GM-CSF and 1000 U / mL of IL-4; on the fifth day of DC culture, mix the tumor antigenic polypeptide prepared in Example 1 with the DC cells, and the volume-to-mass ratio of DCs to tumor antigenic polypeptide is 1 L:100 mg; on the seventh day of DC culture, add 100 ng / mL of LPS and 100 mg / L of the immunoadjuvant prepared in Example 2, and obtain the DC vaccine after 72 h.

[0025] Example 4: This example provides a DC vaccine and a preparation method thereof. The preparation method of the DC vaccine is only different from that of Example 3 in that no immunoadjuvant is added.

[0026] Example 5: This example provides an antigen-specific CTL and a preparation method thereof. The preparation method of the antigen-specific CTL comprises the following steps: collect the non-adherent cells obtained in Example 3, resuspend them in RPMI-1640 culture medium containing 3% serum, and adjust the cell density to 1×10 6 / mL, which is rich in T lymphocytes; mix the DC vaccine prepared in Example 3 with the T lymphocytes according to a ratio of 1:10, and add 10% fetal bovine serum and 1000 U / mL of IL-2 to the RPMI-1640 culture medium; replace half of the solution every 3 days to maintain the cell concentration at 1×10 6 / mL, and obtain the antigen-specific CTL after 14 days.

[0027] Example 6: This example provides an antigen-specific CTL and a preparation method thereof. The preparation method of the antigen-specific CTL is only different from that of Example 5 in that the DC vaccine prepared in Example 4 is used instead of the DC vaccine prepared in Example 3.

[0028] Comparative Example 1 is only different from Example 1 in that the amino acid sequences of SEQ ID NOs: 2-4 are arranged in order.

[0029] Comparative Example 2 is different from Example 3 in that the polypeptide prepared in Comparative Example 1 is used instead of the tumor antigenic polypeptide prepared in Example 1.

[0030] Comparative Example 3 is different from Example 4 in that the polypeptide prepared in Comparative Example 1 is used instead of the tumor antigenic polypeptide prepared in Example 1.

[0031] Comparative Example 4 differs from Example 5 in that the DC vaccine prepared in Comparative Example 2 is co-incubated with T cells instead of the DC vaccine of Example 3.

[0032] Comparative Example 5 differs from Example 6 in that the DC vaccine prepared in Comparative Example 3 is co-incubated with T cells instead of the DC vaccine of Example 4.

[0033] Example 1: The antigen-specific CTL prepared in Examples 5-6 and Comparative Examples 4-5 were tested for their ability to secrete IFN-γ using an enzyme-linked immunoassay. An ELISPOT assay kit was used, and 100 μL of IFN-γ capture antibody diluted 1:100 with PBS was added to a 96-well plate and incubated at 4°C overnight. After washing with PBS, 100 μL of effector cells and 100 μL of target cells (human hepatoma cell line HepG2) were added, with the effector-to-target cell ratio (E / T) being 10:1, and the mixture was incubated at 37°C, 5% CO2for 24 h. After washing with PBS containing 0.1% Tween 20, 100 μL of biotin-labeled anti-IFN-γ antibody diluted 1:100 with PBS containing 1% BSA was added, and the mixture was incubated at 37°C, 5% CO2for 2 h. 100 μL of alkaline phosphatase labeled with streptavidin diluted 1:5000 with PBS containing 1% BSA was added, and the mixture was incubated for 1 h. After washing and drying the plate, the reaction was terminated with distilled water, and the spots were read after drying. The results are shown in Table 1.

[0034] Table 1: Determination of the ability of antigen-specific CTL to produce IFN-γ

[0035] The results in Table 1 show that the number of spots in Examples 5-6 is significantly higher than that in Comparative Examples 4-5, and the number of spots in Comparative Example 4 is significantly higher than that in Comparative Example 5, indicating that the tumor antigenic polypeptide of Example 1 has a significantly higher ability to induce antigen-specific CTL to secrete IFN-γ than the polypeptide of Comparative Example 1, and the addition of an immunoadjuvant can synergize with the tumor antigenic polypeptide of the present application to effectively induce stronger CTL killing activity, achieving a more effective therapeutic effect.

[0036] Example 2: The DC vaccines prepared in Examples 3-4 and Comparative Examples 2-3 were centrifuged at 1700 rpm for 5 min, and the precipitate was resuspended in PBS to prepare 1.5 x 10 6The 6 tubes were each dispensed with 100 μL, and each tube was added with 10 μL of fluorescein-labeled CD80, CD83, HLA-DR antibody and its isotype control, respectively, mixed well, incubated at 4°C for 30 min, centrifuged at 1500 rpm for 5 min to remove the supernatant, washed twice with 2 mL of PBS, added with 200 μL of 1wt% paraformaldehyde per tube, mixed well, placed at 4°C in the dark, and detected by flow cytometry within 24 h. The results are shown in Table 2.

[0037] Table 2: Results of flow cytometry detection of surface markers of DC vaccine

[0038] The results in Table 2 show that the expression amounts of CD80 and CD83 of the DC vaccines prepared in Examples 5-6 are significantly higher than those of Comparative Examples 4-5, and the expression amount of Comparative Example 4 is significantly higher than that of Comparative Example 5, indicating that the tumor antigenic polypeptide and the immune adjuvant can effectively promote the maturation of DC and immune response and enhance the immune response.

[0039] Example 3: The antigen-specific CTL prepared in Examples 5-6 and Comparative Examples 4-5 were added into MKN45 cells at a ratio of 10:1, 25:1, 50:1 and 100:1 as effector cells, and cultured in DMEM medium at 37°C and 5% CO2. After 5 h, the supernatant was collected, and the LDH release test was performed using an LDH release kit to observe the killing effect of CTL. The results are shown in Table 3. Figure 5

[0040] Figure 5 The results show that the killing effect of the antigen-specific CTL of Examples 5-6 is significantly higher than that of Comparative Examples 4-5, indicating that the DC cells induced by the tumor antigenic polypeptide can stimulate the mouse to produce specific cytotoxic T cells, enhance the tumor toxicity, kill tumor cells, and induce the body to produce a strong cellular immune response. The immune adjuvant can enhance this effect. The tumor antigenic polypeptide is successfully processed and presented to T cells by DC, thereby effectively activating tumor cell-specific CTL. One tumor antigenic polypeptide of the present application can stimulate specific immune response and can be developed into a vaccine or drug for treating tumors.

[0041] Example 4: The antigen-specific CTL was cultured by the method of Examples 5-6 and Comparative Examples 4-5, and the T cell proliferation was detected by CCK-8 method when the DC cells and T cells were mixed and cultured for 72 h. The results are shown in Table 4. Figure 6

[0042] Figure 6 ​​The results show that the cell proliferation index of the group of examples 5-6 is significantly higher than that of the comparative examples 4-5, and the comparative example 4 is higher than the comparative example 5, indicating that the DC cells induced by the tumor antigenic polypeptide of the present application have a strong ability to induce T cell proliferation, and the immunoadjuvant of the present application can enhance the ability of DC cells to induce T cell proliferation.

[0043] Example 5: 4T1 cells 5x10 4 were implanted subcutaneously in the mammary pad of female Balb / C mice (4 groups, 12 mice in each group) to establish a mouse tumor model. The mice in the 4 groups were injected with 100 μL of the DC vaccine prepared in examples 3-4 and comparative examples 2-3, respectively. The mice were immunized 7 days, 14 days and 21 days after the tumor cells were inoculated. At the same time, the tumor volume was measured at different times (as shown in the table) after the tumor cells were inoculated in the animals. Figure 7

[0044] The tumor model was established by injecting 1x10 6 TC-1 cells into each mouse. After the tumor model was established, the mice were injected with 100 μL of the DC vaccine of examples 3-4 and comparative examples 2-3 on the 5th day and the 12th day, and the tumor volume was measured every 5 days. The results are shown in the table. Figure 8

[0045] Figure 7 and Figure 8 The results show that the tumors of examples 3-4 are significantly smaller than those of comparative examples 2-3, indicating that the DC vaccine of the present application can significantly inhibit tumor growth. The DC vaccine changes the number and activity state of T cells, inducing a tumor-specific cellular immune response. The tumor antigenic polypeptide and the immunoadjuvant of the present application can promote DC maturation, thereby promoting the immune response of the DC vaccine and improving the anti-tumor effect.

[0046] Example 6: 48 BALB / c male mice of the same age and weight were selected and divided into groups A, B, C and D, 12 mice in each group. The tumor antigenic polypeptide prepared in example 1 and the immunoadjuvant prepared in example 2 were dissolved in PBS, both at a concentration of 1 μg / 10 μL. The mice in group A were injected with 100 μL per mouse. The tumor antigenic polypeptide prepared in example 1 was dissolved in PBS at a concentration of 1 μg / 10 μL, and the mice in group B were injected with 100 μL per mouse. The polypeptide prepared in example 3 and the immunoadjuvant prepared in example 2 were dissolved in PBS, both at a concentration of 1 μg / 10 μL, and the mice in group C were injected with 100 μL per mouse. The polypeptide prepared in example 3 was dissolved in PBS at a concentration of 1 μg / 10 μL, and the mice in group D were injected with 100 μL per mouse. The injection method was subcutaneous injection on the nape of the neck, and the injection was performed once a week. After the fourth injection, 1x10​​6 H22 hepatoma cells were inoculated into mice. The survival rates of mice in each group were recorded within three months, and the results are shown in Table 1. Figure 9

[0047] Figure 9 The results show that the survival rates of mice in groups A and B are significantly higher than those in groups C and D, indicating that the tumor antigen polypeptide vaccine obtained according to the present application has high anti-tumor activity, and the use of the immunoadjuvant described in the present application can further improve the anti-tumor effect, and can be used for preparing a tumor vaccine.

[0048] Those skilled in the art will understand that the discussion of any of the above embodiments is merely exemplary and is not intended to suggest that the scope of the application (including the claims) is limited to these examples; the embodiments or technical features among different embodiments can also be combined, and the steps can be implemented in any order, and there are many other changes of the different aspects of the present application as described above, which are not provided in detail for the sake of brevity.

[0049] The present application is intended to cover all such alternatives, modifications, and variations as fall within the broad scope of the appended claims. Accordingly, any and all such modifications, variations, and equivalents that fall within the spirit and scope of the application are intended to be included within the scope of the application.​

Claims

1. A tumor antigenic polypeptide, characterized in that, The amino acid sequence of the tumor antigenic polypeptide is shown in SEQ ID NO:1 in the sequence listing; <Tumor antigenic polypeptide; AA; SEQ ID NO:1>: PLLLMFITQCLLEEITGLLYLVGATLLLR; The method for preparing the tumor antigenic polypeptide includes the following steps: (1) According to the amino acid sequence characteristics, the peptide chain is extended from the C-terminus to the N-terminus one by one. Weigh 50g of Wang resin that has passed through a 200-mesh sieve, put it into a reaction vessel, wash it once with 500 mL DMF, dry it under vacuum, swell the resin with 500 mL DCM for 30 min, dry it under vacuum, add the first protected amino acid, HOBT, DIC, 300 mL DMF and 100 mL DCM, mix them evenly, introduce nitrogen or inert gas into the reaction system to keep the temperature of the reaction system at 20℃, slowly add DMAP, stir at 24℃ for 18 h, dry under vacuum, wash twice with DMF, dry under vacuum, add pyridine, acetic anhydride and 500 mL DCM, stir at 27℃ for 3 h, dry under vacuum, wash the precipitate twice with DMF and methanol, and dry under vacuum to constant weight; (2) Add 500 mL of 20 vol% piperidine / DMF solution to the reaction vessel containing the washed precipitate described in step (1), stir at 25°C for 10 min, vacuum dry, add another 500 mL of 20 vol% piperidine / DMF solution, stir at 25°C for 20 min, vacuum dry, wash once with DMF, wash twice with methanol, wash once with DMF, wash once with DCM, vacuum dry, add the second protected amino acid, HOBT, HBTU, and 350 mL DMF and 100 mL DCM, purge with nitrogen or inert gas to rapidly cool to 18°C, add DIPEA dropwise, stir at 25°C for 3 h, ninhydrin test is negative, vacuum dry, wash the precipitate once with methanol, wash twice with DMF, vacuum dry; (3) Repeat step (2) to protect the amino acids as they are replaced in the order of synthesis. Couple them according to the amino acid sequence. After all the amino acid linkages are completed, wash and vacuum dry to constant weight. Add the amino acid to the reaction solution at a ratio of 1 g: 10 mL. Stir at 26°C for 2.5 h. Filter. Concentrate the filtrate by vacuum distillation. Add methyl tert-butyl ether to precipitate. Centrifuge to collect the crude tumor antigenic polypeptide. (4) The crude tumor antigenic peptide obtained in step (3) is mixed with a 5 vol% propionitrile aqueous solution at a ratio of 1 g: 100 mL. The mixture is purified by high performance liquid chromatography. The aqueous phase is prepared by 0.1 vol% TFA aqueous solution and the organic phase is prepared by 0.1 vol% TFA propionitrile solution. The mixture is concentrated by rotary evaporation under reduced pressure and freeze-dried to obtain the tumor antigenic peptide.

2. The tumor antigenic polypeptide according to claim 1, characterized in that, In step (1), the molar amounts of the first protected amino acid, HOBT, and DIC are all 3 times that of Wang resin, DMAP is 0.2 times that of Wang resin, and the molar amounts of pyridine and acetic anhydride are all 10 times that of Wang resin.

3. The tumor antigenic polypeptide according to claim 2, characterized in that, The molar amounts of the second protective amino acid, HOBT, HBTU, and DIPEA in step (2) are all twice that of the Wang resin in step (1).

4. The tumor antigenic polypeptide according to claim 3, characterized in that, The amino acids described in steps (1)-(3) are all amino acids with α-amino groups protected by Fmoc, except for the last one which is an amino acid with a Boc-protected functional group.

5. The tumor antigenic polypeptide according to claim 4, characterized in that, The reaction solution in step (3) is a mixture of TFA, TIS and water in a volume ratio of 95:2.5:2.

5.

6. The use of a tumor antigenic polypeptide according to any one of claims 1-5 in the preparation of a tumor vaccine, characterized in that, Includes the following steps: S1: Preparation of peptide vaccine: Tumor antigenic peptides are added to PBS to prepare a solution to obtain a tumor antigenic peptide vaccine; S2: Preparation of DC vaccine: Mononuclear cells were isolated from peripheral blood, suspended in X-vivo20 medium, and cell density was measured using a hematology analyzer at a rate of 7 × 10⁻⁶. 6 Inoculate at a concentration of / mL to 25 cm 2 Cultured in culture flasks at 37°C and 5% CO2 for 4 hours; culture medium and non-adherent cells were removed from the flasks to obtain adherent and non-adherent cells; each flask of adherent cells was added to 10 mL of X-vivo20 culture medium containing 1000 U / mL GM-CSF and 1000 U / mL IL-4 to induce differentiation into dendritic cells (DCs), and cultured at 37°C and 5% CO2; on day 3, half the medium was replaced, and GM-CSF and IL-4 were replenished to 1000 U / mL; on day 5 of DC culture, tumor antigenic peptides were mixed with DC cells at a volume-to-mass ratio of 1 L:100 mg; on day 7 of DC culture, LPS 100 ng / mL was added to induce DC maturation, and after 72 hours, the DC vaccine was obtained; S3: Preparation of antigen-specific CTLs: Collect the non-adherent cells obtained in step S2, resuspend them in RPMI-1640 medium containing 3% serum, and adjust the cell density to 1×10⁻⁶. 6 / mL, this suspension is rich in T lymphocytes. The DC vaccine prepared in step S2 is mixed with T lymphocytes at a ratio of 1:10, and then 10% fetal bovine serum and 1000 U / mL IL-2 are added to RPMI-1640 medium. The medium is changed by half every 3 days to maintain a cell concentration of 1×10 6 / mL, and after 14 days, antigen-specific CTLs were obtained.

7. The use of the tumor antigenic polypeptide according to claim 6 in the preparation of a tumor vaccine, characterized in that, In step S2, while adding LPS, an immune adjuvant of 100 mg / L may also be added.

8. The use of the tumor antigenic polypeptide according to claim 7 in the preparation of tumor vaccines, characterized in that, The method for preparing the immune adjuvant includes the following steps: (a) 0.98 mol of colchicine and 120 mL of dimethyl sulfate were mixed with 300 mL of methanol and stirred. The mixture was heated under reflux for 30 min and then cooled to room temperature to obtain mixed solution A. Mixed solution A was added to 120 mL of 4.5 mol / L sodium hydroxide solution at a rate of 15 mL / min and stirred for another 30 min. 80 mL of dichloromethane was added for extraction, and the mixture was concentrated under reduced pressure at 70 °C and 0.01 MPa for 12 h. The product was washed three times with anhydrous ethanol and three times with deionized water and dried at 60 °C to constant weight to obtain product A. (b) Prepare a glucosamine / DMF solution by mixing 9.75 mmol glucosamine with 40 mL DMF. Add 3.5 g of product A obtained in step (a) and 100 mg camphor sulfonic acid to the solution. Connect to a vacuum and heat in an oil bath at 50 °C for 3 h. Then, rotary evaporate at 80 °C for 4 h. Dissolve the solution in a mixed solution B consisting of 50 mL diethyl ether and 2 mL Et3N. Add 50 mL saturated sodium bicarbonate. Extract the aqueous layer three times with 50 mL diethyl ether each time. Combine the organic layers from the three extractions, dry on sodium sulfate and filter. Rotary evaporate at 80 °C for 4 h. Purify by silica gel chromatography to obtain the immunoadjuvant.

Images