Tumor vaccine based on AKK bacteria as well as preparation method and application of tumor vaccine

Abstract

The invention provides a tumor vaccine based on AKK bacteria and a preparation method and application thereof.Specifically, antigen peptide is subjected to lysine, on one hand, the free positive charge content of the antigen peptide is increased, the antigen peptide is endowed with the cell penetrating capacity, the two combined cell entering modes of direct cell penetrating and endocytosis are achieved, the cell entering number and rate of the antigen peptide are successfully increased, and the cell entering efficiency of the antigen peptide is improved; on the other hand, the modified peptide fragment and a bacterial adjuvant carrier are combined through simple stirring, and preparation and application of the tumor vaccine are successfully achieved. Therefore, the invention provides a simple, effective and universal tumor vaccine construction means, and provides a new thought for development and application of the cell-penetrating peptide.

Description

Technical Field

[0001] The present invention relates to the field of tumor immunotherapy, and in particular to a tumor vaccine based on AKK bacteria and a preparation method and application thereof. Background Art

[0002] Cancer is a major public health problem in my country. The incidence of malignant tumors continues to rise, and the annual cost of cancer treatment is huge. It is estimated that in 2050, compared with 2022, in countries with high human development indexes, including China, the cancer burden will increase by 50%, and the number of new cancer cases will exceed 30 million. How to reduce the cancer burden is an urgent problem to be solved. Tumor vaccines, as an important tool for the prevention and treatment of tumors, are an effective means to reduce the burden of cancer. At present, the FDA has only approved two tumor vaccines, T-VEC for melanoma and Sipuleucel-T for prostate cancer, but their clinical efficacy is not ideal. Although many tumor vaccine clinical studies are in phase III clinical trials and have been granted fast track qualifications, further improving the efficacy is still the core pursuit of tumor vaccines. The key is the effective presentation of tumor antigens between antigen presenting cells and T cells, and the efficient and specific anti-tumor immune activation mediated by CD8T cells.

[0003] Protein / peptide vaccines are a type of tumor vaccine that can act quickly without transcription or translation, and there is no risk of changing the genome. However, due to low uptake, presentation efficiency and immunogenicity, its effect of inducing CD8T cell-mediated anti-tumor immunity is not good. Inducing antigen cross-presentation, that is, converting exogenous antigens into endogenous antigens, is one of the key strategies to improve CD8T cell-mediated cytotoxic T cell immunity. At present, there are two main means of inducing antigen cross-presentation, using cationic carriers to burst lysosomes to achieve antigen lysosomal escape, or using membrane-penetrating peptides to directly deliver antigens to the cytoplasm. Given that lysosomes are important organelles for cell life activities, using membrane-penetrating peptides to promote direct antigen penetration can avoid lysosomal degradation of antigens and ensure lysosomal integrity. However, the antigen loading rate, antigen utilization rate and immunogenicity of membrane-penetrating peptides still need to be improved, and they need to be combined with immune adjuvants and carriers to further improve cytotoxic T cell immunity and enhance the efficacy of tumor vaccines.

[0004] Bacteria provide revolutionary opportunities for tumor treatment due to their superior immune adjuvant ability. However, reducing toxic side effects is still the main challenge for bacterial preparations in immunotherapy. Probiotics can improve host health and have high biosafety. The probiotic Akkermansia muciniphila (AKK bacteria), discovered in 2004, has unique effects in metabolic diseases and cancer. Slightly different from other probiotics, the inactivated AKK bacteria still retains its immune regulation ability and can upregulate PD1 in the spleen. +The number of antigen-specific CD8T cells is reduced, and the activation threshold of dendritic cells is reset, significantly reducing the risk of immune overreaction. As a new generation of probiotics, AKK bacteria have unique efficacy in metabolic diseases and cancer, and the inactivated AKK bacteria still retains the ability to regulate immunity, but the application research in tumor immunity is still shallow. Therefore, it is of great significance to develop a new, efficient and safe tumor vaccine. Summary of the invention

[0005] In view of the key points of peptide vaccine improvement in antigen cross-presentation and immunogenicity, as well as the potential and research needs of AKK bacteria in tumor immunity, the present invention designs a tumor vaccine with AKK bacteria as adjuvant and carrier and positively charged peptide segment as antigen. The use of pasteurized AKK bacteria as adjuvant carrier not only reduces toxic side effects, but also uses its pathogen-associated molecular pattern to activate and recruit antigen-presenting cells, thereby improving antigen utilization. The model antigen peptide (OVA257-264) recognized by CD8T cells is selected, and its cell penetration is enhanced by lysine modification, aiming to improve the efficiency of dendritic cell cross-presentation, thereby enhancing anti-tumor immunity. The present invention is expected to build a broad-spectrum vaccine platform, deepen the study of the interaction between AKK bacteria and immune cells, reveal its cellular mechanism and effect of activating cytotoxic T cell immunity, and provide new strategies and scientific research support for tumor immunotherapy.

[0006] In order to achieve the above object, the present invention also provides a method for preparing a tumor vaccine based on AKK bacteria, characterized in that it comprises the following steps:

[0007] S1, first lysine the antigen peptide;

[0008] S2, culture, amplify and pasteurize AKK bacteria;

[0009] S3, mixing the lysine-modified antigen peptide and heat-killed AKK bacteria in sterile water according to a certain ratio, removing unbound antigen peptide, and obtaining a tumor vaccine.

[0010] The further antigenic peptide may be any antigenic peptide known in the art. Preferably, the antigenic peptide is an OVA257-264 antigenic peptide pattern or a GP70-AH1A5 antigenic peptide.

[0011] Further lysination of the antigenic peptide can be performed at any position of the antigenic peptide; further, it can be performed at the N-terminus, C-terminus and side chain of the antigenic peptide; preferably, lysination of the antigenic peptide is performed at the N-terminus of the antigenic peptide.

[0012] Furthermore, the antigen peptide carries a positive charge after lysine ligation.

[0013] Furthermore, the antigen peptide is combined with the pasteurized AKK bacteria through electrostatic interaction.

[0014] Also provided is a tumor vaccine based on AKK bacteria prepared by the above method.

[0015] Furthermore, the antigen peptide carries a positive charge, and the antigen peptide is combined with the pasteurized AKK bacteria through electrostatic interaction.

[0016] The present invention also provides a use of the above-mentioned tumor vaccine or the tumor vaccine prepared by the above-mentioned preparation method in drugs for preventing and treating tumors.

[0017] Furthermore, the tumor is one or more of melanoma, colon cancer, T lymphoma, breast cancer, bladder cancer, liver cancer, and gastric cancer.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention uses pasteurized AKK bacteria (pAKK) as an adjuvant carrier. The inactivated AKK bacteria can not only improve the efficiency of antigen uptake in dendritic cells, but also can be used as an adjuvant to amplify subsequent anti-tumor immunity, increase the number and activation level of antigen-specific CD8 T cells, and effectively inhibit the occurrence and development of tumors. At the same time, the present invention systematically studies the immune activation ability of AKK bacteria in the skin and its application in tumor vaccines for the first time, expands the types of probiotics that can be used for tumor immunotherapy, and provides a basis for the study of the mechanism of AKK bacteria in anti-tumor immunity.

[0020] (2) The tumor vaccine of the present invention comprises a lysine-modified antigen peptide, specifically a lysine-modified OVA257-264 model antigen peptide. Lysine modification increases the free positive charge content of the antigen peptide, giving it the ability to penetrate the membrane, achieving a combination of direct penetration and endocytosis, increasing the number and rate of antigen peptide entry, and thus significantly improving the efficiency of cross-presentation of dendritic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :(a) The ability of modified peptides to induce antigen cross-presentation; (b) The ability of modified antigen peptides to enter the cell under various endocytosis inhibitors; (c) The efficiency of modified peptides to enter the cell at different temperatures.

[0022] Figure 2 :(a) Transmission electron microscopy image of tumor vaccine. (b) Surface charge of tumor vaccine. (c) Hydration particle size of tumor vaccine. (d) Confocal microscopy image of tumor vaccine.

[0023] Figure 3:(a) Recruitment of dendritic cells after intradermal injection of tumor vaccines, including immunohistochemistry images of the skin at the injection site (CD11c), and statistical graphs of the area of ​​CD11c positive signals in the dermis and fascia. (b) Effect of tumor vaccines on lymph node migration of antigenic peptides.

[0024] Figure 4 :Experimental results on the generation and activation of antigen-specific CD8 T cells; (a) shows the experimental process of vaccination; (b) shows the ability of vaccine to induce antigen-specific CD8 T cell generation and activation through Tetramer-MHC I-OVA and TNF-α, IFN-γ detection; (c) flow cytometry analysis of Tetramer in different treatment groups + CD8 + The percentage of T cells.

[0025] Figure 5 :(a&b) The design plan, timetable and treatment methods of cancer prevention experiment, the preventive (a) and therapeutic (b) effects of vaccine on melanoma B16-OVA; (c) The design plan, timetable and treatment methods of cancer prevention experiment, the preventive effect of vaccine on colon cancer CT26; (d) The protective ability of vaccine on cured mice and the defense ability against secondary tumors 6 months later. DETAILED DESCRIPTION

[0026] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0027] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0028] Example 1

[0029] Study on the properties and cross-presentation ability of novel transmembrane antigen peptides:

[0030] (1) Extraction and culture of bone marrow-derived dendritic cells. Myeloid dendritic cells (BMDCs) were extracted from 6-8 week old C57BL / 6 mice. The tibia and femur were separated from the muscle and connective tissue in sterile phosphate buffer, and the bone marrow was flushed out with complete culture medium. The red blood cells in the precipitate were removed with red blood cell lysis buffer. The remaining cells were fixed to a volume of 3 mL of culture medium containing 100,000 cells, 20 ng / mL granulocyte-macrophage colony stimulating factor and 50 μM beta-mercaptoethanol. On the third day, an equal amount of RPMI complete culture medium containing granulocyte-macrophage colony stimulating factor and 50 μM beta-mercaptoethanol was added. On the sixth day, half of the medium was replaced (the same culture medium as on the third day), and the cells were cultured again in the original culture medium. The degree of differentiation of BMDCs was evaluated on the seventh day.

[0031] (2) Antigen cross-presentation assay. BMDCs were exposed to the original antigen peptide OVA257-264 and the N-terminal lysine-modified antigen peptide K-pep OVA257-264 (50nM) for 12 hours. After collecting and washing the cells, add blocking antibody anti-CD16 / 32 to reduce nonspecific adsorption of the Fc end, add flow cytometry antibodies anti-CD11c and anti-MHCI-SIINFEKL, and stain for 30 minutes in the dark. After staining, remove the unbound antibodies, and detect the cross-presentation efficiency of BMDCs by flow cytometry.

[0032] (3) Study on the properties of penetrating peptides. The endocytosis inhibitors and energy supply control were used to study the cellular entry mechanism of penetrating peptides. BMDCs were treated with three endocytosis inhibitors half an hour in advance, including actin polymerization inhibitor cytochalasin D, clathrin-dependent endocytosis inhibitor chlorpromazine hydrochloride, and caveolin-mediated endocytosis inhibitor genistein. K-pep was then added OVA257-264 (1μg / mL) for 12 hours, and the antigen cross-presentation detection steps were followed. The modified peptides were incubated with BMDCs at 4°C and 37°C. At 4°C, the lack of energy supply inhibited cell endocytosis and cell life activities including protein processing and presentation. After 12 hours, the antigen cross-presentation efficiency of BMDCs and the intracellular peptide fluorescence were studied.

[0033] Embodiment 2:

[0034] Tumor vaccine pAKK / K-pep OVA257-264 Preparation and characterization studies:

[0035] (1) Bacterial culture and inactivation. AKK bacteria (ATCC BAA-835) were cultured in brain heart infusion broth containing 0.5% porcine gastric mucin and 0.05% cysteine. After dilution, they were evenly dispersed on BHI agar plates and cultured at 37°C in highly anaerobic culture bags to obtain dispersed single colonies. Single colonies were isolated and inoculated into liquid culture medium, and the AKK bacterial solution was amplified statically until the stationary phase. The AKK bacterial solution was separated by centrifugation, and the residual BHI liquid culture medium was washed off with sterile saline, then resuspended in sterile water and heat-inactivated in a 70°C water bath for 15 minutes.

[0036] (2) Tumor vaccine pAKK / K-pep OVA257-264 Preparation and characterization of the modified K-pep OVA257-264 Mix with heat-killed AKK bacteria (pAKK) in sterile water in a certain proportion. After a certain period of time, the unbound K-pep OVA257-264 The loading efficiency of pAKK was quantitatively studied using a multifunctional microplate reader. The peptides with N-terminal FITC were used to visualize the loading results of pAKK using a confocal fluorescence microscope. OVA257-264 Dilute with ultrapure water and measure the hydrodynamic diameter and surface charge of the vaccine using a laser particle size analyzer; OVA257-264 Dilute with ultrapure water, scoop out the sample with a copper mesh, and visually detect the particle size of the complex using a transmission electron microscope. OVA257-264 Resuspend with ultrapure water, dilute and drop 20 μl of the suspension onto the silicon wafer, wait for the material to settle naturally and dry, then observe the surface morphology using a scanning electron microscope.

[0037] Embodiment 3:

[0038] Tumor vaccine pAKK / K-pep OVA257-264 Study on the effect on dendritic cells in vivo

[0039] (1) The recruitment effect of tumor vaccines on dendritic cells in vivo. Four hours after intradermal immunization of mice, the mouse skin was collected, the mouse skin tissue was fixed in a paraformaldehyde solution, and after being embedded in paraffin, it was cut into 4-5 μm thick sections and placed on slides. The sections were treated with xylene and gradient alcohol solutions to remove paraffin and hydrated in PBS. The sections were treated with an appropriate repair buffer (such as 0.01 M sodium citrate buffer, pH 6.0) at high temperature to remove cross-linked substances in the tissue and reveal antigens. To reduce nonspecific binding, the sections were then incubated in 5% bovine serum albumin (BSA) or normal goat serum at room temperature for 20-30 minutes. After adding diluted CD11c antibody and incubating overnight at 4°C, the excess antibody was washed off, and the labeled secondary antibody corresponding to the CD11c antibody was added and incubated at room temperature for 1 hour. Subsequently, DAB was used as a color substrate, and the sections were observed under a microscope after staining. Finally, the sections were sealed with a sealing agent for further analysis and recording.

[0040] (2) The effect of tumor vaccines on promoting the migration of antigens to lymph nodes. A peptide with N-terminal modification of FITC was used to construct a tumor vaccine according to the previous steps. After intradermal vaccination, the lymph nodes of mice were fluorescently imaged at 24 h using a small animal in vivo imaging system to analyze the lymph node migration effect of tumor antigens.

[0041] Embodiment 4:

[0042] Study on the in vivo immune and biological effects of tumor vaccine pAKK / K-pepOVA257-264

[0043] Tumor vaccines activate adaptive immunity dominated by CD8 T cells. Fluorescently labeled tumor vaccines were used, and mice were imaged with fluorescence at different time points using small animal live imaging to analyze the lymph node migration effect of tumor vaccines. Mouse lymph nodes were collected and dispersed into single cells using physical and enzymatic methods. Immunofluorescence, flow cytometry, and immunohistochemistry were used to study the number, maturation, presentation, B cell status, and T cell status of dendritic cells in the lymph nodes. After continuous immunization of mice, mouse spleens were collected, and the content of T cells that can specifically recognize OVA was detected using TetramerH-2Kb-OVA antibody. In order to explore the degree of activation of CD8 T cells by tumor vaccines, spleen cells were treated with BFA half an hour in advance, and then OVA257-264 peptide was added to stimulate spleen cells for secondary stimulation. Anti-CD8 and anti-CD3 antibodies were used for membrane surface staining, and anti-TNF- and anti-IFN-γ antibodies were added for intracellular staining. The production of TNF- and IFN-γ in CD8 T cells was analyzed by flow cytometry. In order to explore the degree of activation of CD8 T cells by tumor vaccines, spleen cells were stained with CFSE, and BMDCs activated by OVA257-264 were added and co-incubated with spleen cells. After 3-5 days, the proliferation of CD8 T cells was detected by flow cytometry.

[0044] Embodiment 5:

[0045] In vivo efficacy study of tumor vaccine pAKK / K-pep in different models

[0046] (1) To investigate the effect of tumor vaccine pAKK / K-pep in different conditions in an orthotopic melanoma B16-OVA model OVA257 -264 In vivo efficacy. Case 1: Mice were immunized continuously for three weeks (once a week), and an orthotopic melanoma B16-OVA model was established on the fourth week to monitor the survival rate and tumor size of the mice. Case 2: An orthotopic melanoma B16-OVA model was established on day zero to simulate postoperative tumor metastasis. Mice were immunized on the second, fifth, eighth and eleventh days to monitor the survival rate and tumor size of the mice. Case 3: Lung metastasis model. Mice were immunized continuously for three weeks (once a week), and a melanoma B16-OVA lung metastasis model was established on the fourth week to monitor the survival rate and tumor size of the mice.

[0047] (2) In vivo efficacy study of the tumor vaccine pAKK / K-pep in multiple tumor models. The mutant antigen peptide GP70-AH1A5 of colon cancer CT26 cells was modified with lysine. After optimizing the number of lysine modifications using flow cytometry, a personalized tumor vaccine with AKK bacteria as a carrier was constructed. The tumor vaccine was inoculated in the mouse model corresponding to the cell line once a week for three consecutive weeks. The corresponding tumor model was constructed in the fourth week, and the tumor growth and mouse survival rate were monitored.

[0048] Experimental results analysis:

[0049] See also Figure 1 a, showing the potential to induce efficient CD8 T cell immune response. Figure 1 b. After using a variety of endocytosis inhibitors, the cell entry efficiency of the modified cell-penetrating peptide was less affected, indicating that it has the ability to directly penetrate the cell membrane. At the same time, the slight decrease in cell entry efficiency means that the modified cell-penetrating peptide enters the cell through a combination of direct membrane penetration and endocytosis, which is consistent with the cell entry mechanism of the cell-penetrating peptide that has been developed. Figure 1 c In the absence of energy supply, that is, when the cell endocytosis activity is greatly inhibited, the lysine-modified peptide can still enter the dendritic cells, which once again proves that the modified antigen peptide has the endocytic property similar to the membrane-penetrating peptide.

[0050] Since the modified antigen peptide has more free amino groups and more positive charges, after simple stirring, the modified peptide can bind to the pasteurized AKK bacteria through electrostatic interaction. After adding the antigen peptide, the contrast of the outer bacterial wall of AKK bacteria under the transmission electron microscope changed from low to high ( Figure 2 a), indicating that the antigen peptide binds to the surface of AKK bacteria. The addition of the antigen peptide makes the surface of AKK bacteria, which was originally negatively charged, tend to be neutrally charged ( Figure 2 b) from Figure 2 c It can be seen that the overall hydrated particle size increases slightly. Figure 2 It can be seen that the fluorescence images of the confocal microscope once again verified that AKK bacteria have the ability to load antigen peptides ( Figure 2 d).

[0051] After intradermal injection, tumor vaccines need to be processed by dendritic cells and presented to CD8 T cells in the lymph nodes to induce antigen-specific CD8 T cell immune responses. Immunohistochemistry results showed ( Figure 3 a), after intradermal injection of the tumor vaccine, the CD11c positive signal (dendritic cells) in the dermis and fascia of the skin was significantly enhanced, and a large number of inflammatory cells infiltrated the inoculation site, proving that the tumor vaccine has the ability to recruit dendritic cells. 24 hours after vaccine injection, the signal of the antigen peptide in the lymph node was significantly higher than that in the control group and the single antigen peptide group ( Figure 3b), indicating the important role of AKK bacteria vector in helping antigen lymph node migration. Therefore, the above experimental results prove that tumor vaccines with AKK bacteria as vectors have the potential to activate CD8 T cells.

[0052] pass Figure 4 The experimental results show that the pAKK / K-pepOVA treatment group induced the production of OVA-specific CD8 + T cells (Tetramer + CD8 + When exposed to antigens, the CD8 + T cells can be rapidly activated to produce cytokines (TNF-α and IFN-γ) related to CTL activation. Therefore, after vaccination, it can induce an antigen-specific immune response in the living body and provide a faster immune activation time when exposed to the antigen for the second time, which helps to enhance the body's immune defense capabilities.

[0053] pass Figure 5 From the experimental results, we can see that after using the same strategy to construct a vaccine platform (pAKK / K-pep) carrying different antigens, the tumor vaccine showed significant preventive effects in both melanoma B16-OVA and colon cancer CT26 ( Figure 5 a and c), and showed significant tumor therapeutic ability in melanoma B16-OVA ( Figure 5 b). At the same time, when the cured mice were inoculated with the same tumor cells again 6 months later, the growth of the tumor was completely suppressed ( Figure 5 d), demonstrating the superior long-term protection of the vaccine platform (the average lifespan of mice is 2 years). This treatment can effectively inhibit tumor growth in multiple cancer tumors and disease conditions, demonstrating the application value of vaccines in tumor prevention and treatment and prevention of recurrence.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a tumor vaccine based on AKK bacteria, characterized in that: The steps include: S1, first lysine the antigen peptide; S2, culture, amplify and pasteurize AKK bacteria; S3, mixing the lysine-modified antigen peptide and heat-killed AKK bacteria in sterile water according to a certain ratio, removing unbound antigen peptide, and obtaining a tumor vaccine.

2. The preparation method according to claim 1, characterized in that: In step S1, the antigen peptide is an OVA257-264 model antigen peptide or a GP70-AH1A5 model antigen peptide.

3. The preparation method according to claim 1 or 2, characterized in that: In step S1, lysination of the antigen peptide is performed at the N-terminus, C-terminus and side chain of the antigen peptide.

4. The preparation method according to claim 1, characterized in that: In step S1, the antigen peptide carries a positive charge after lysine ligation.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In step S3, the antigen peptide is combined with the pasteurized AKK bacteria through electrostatic interaction.

6. A tumor vaccine based on AKK bacteria prepared by the method according to any one of claims 1 to 5.

7. Use of the tumor vaccine according to claim 6 or the tumor vaccine prepared by the preparation method according to any one of claims 1 to 5 in drugs for preventing and treating tumors.

8. The use according to claim 7, characterized in that: The tumor is one or more of melanoma, colon cancer, T lymphoma, breast cancer, bladder cancer, liver cancer, and gastric cancer.

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