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 as well as a preparation method and application thereof, specifically, antigen peptide is subjected to lysine treatment, the free positive charge content of the antigen peptide is increased, the efficiency of loading the antigen peptide by combining adjuvant carrier AKK bacteria through simple stirring is increased, and the preparation and application of the tumor vaccine are successfully realized. The vaccine can significantly promote antigen cross presentation and immune activation efficiency of dendritic cells, induce potent antigen-specific CD8 + T cell immune response, and also has good tumor prevention, treatment and re-challenge protection effects. The invention provides a simple, effective and universal tumor vaccine construction strategy which is suitable for immunotherapy of various solid tumors and has a wide application prospect.

Description

Technical Field

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

[0002] Cancer is a major public health problem in our 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 a high human development index, including China, the cancer burden will increase by 50%. By then, the number of new cancer cases will exceed 30 million. How to reduce the burden of cancer is an urgent problem that needs 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. Currently, 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 CD8 + T cell-mediated efficient and specific anti-tumor immunity activation.

[0003] Enhancing the efficacy of tumor vaccines remains the core goal, and how to optimize the efficacy of tumor vaccines through scientific design is the key to current research. In order to achieve efficient tumor clearance, exogenous antigens need to be phagocytosed and processed by antigen-presenting cells (such as dendritic cells), and presented to the surface by MHC-1 molecules (cross-presentation). After migrating to the lymph nodes, they present the antigen information to CD8 + T cells; at the same time, antigen-presenting cells highly express costimulatory molecules after activation, promoting antigen-specific CD8 + T cell response ultimately achieves tumor killing. However, in the absence of inflammation or microbial stimulation, immature dendritic cells (DCs) present antigens in a stable state to induce immune tolerance rather than immune activation, so activation of DCs is an important part of vaccine design. Therefore, an ideal vaccine delivery system should have the following characteristics, including effective adsorption / loading of antigens, promotion of DCs' uptake and processing of antigens, and efficient activation of DCs, thereby activating antigen-specific CD8 + T cell response, ultimately achieving immune protection.

[0004] Bacteria provide revolutionary opportunities for cancer treatment due to their superior immune adjuvant capabilities. However, reducing toxic side effects remains the main challenge for bacterial preparations in immunotherapy. Probiotics can improve host health and have high biosafety. Since its discovery in the human intestine in 2004, Akkermansia muciniphila (AKK bacteria), as a mucosal symbiotic Gram-negative bacterium, has gradually demonstrated important scientific research and application value due to its unique performance in metabolic regulation, anti-tumor effects and immune intervention. Slightly different from other probiotics, the inactivated AKK bacteria still retains its immunoregulatory ability. At the same time, the inactivation of harmful substances in the inactivated AKK bacteria, while the key molecules are not affected (such as the membrane protein Amuc1100), can further reduce biological toxicity. In addition, AKK bacteria can not only reset the activation threshold of the strongest antigen-presenting cells DCs, induce DCs to produce IL-12, and enhance CD8 + The IFN-γ secretion capacity of the Tc1 subset of T cells, which possesses potent cytotoxicity, synergistically enhances the efficacy of immune checkpoint blockade. Despite its unique biological effects, the application of AKK bacteria in tumor immunity remains largely unstudied. Therefore, the development of a novel, highly effective, and safe tumor vaccine is of great significance. Summary of the Invention

[0005] In view of the key points of peptide vaccine in improving antigen cross-presentation and immunogenicity, as well as the potential and research needs of AKK bacteria in tumor immunity, the present invention designed a tumor vaccine with AKK bacteria as adjuvant and carrier and positively charged peptide segment as antigen. + This model antigen peptide (OVA257-264) is recognized by T cells and enhances contact and binding with AKK bacteria through lysine modification. Using pasteurized AKK bacteria as an adjuvant and carrier reduces toxic side effects while leveraging its pathogen-associated molecular patterns to activate and recruit antigen-presenting cells, thereby improving antigen utilization. This tumor vaccine is designed to enhance the efficiency of dendritic cell cross-presentation, thereby enhancing anti-tumor immunity. This invention aims to establish a broad-spectrum vaccine platform, deepen research on the interaction between AKK bacteria and immune cells, reveal the cellular mechanisms and effects of AKK bacteria in 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: S1, adding lysine during the synthesis of antigenic peptide to achieve lysination of antigenic peptide; S2, culture, amplify, and pasteurize AKK bacteria; S3, mixing the lysinated antigen peptide and pasteurized AKK bacteria in sterile water according to a certain ratio, removing unbound antigen peptide, and obtaining a tumor vaccine.

[0007] The further antigenic peptide may be any antigenic peptide known in the art. Preferably, the antigenic peptide is an OVA257-264 model antigenic peptide.

[0008] Further lysination of the antigenic peptide can be performed at any position of the antigenic peptide; preferably, the lysination of the antigenic peptide is performed at the N-terminus of the antigenic peptide.

[0009] Furthermore, the antigen peptide carries a positive charge after lysine ligation.

[0010] Furthermore, the antigen peptide is combined with the pasteurized AKK bacteria through electrostatic interaction.

[0011] Furthermore, in step S1, lysine is first added during the synthesis of the antigen peptide. The specific steps for achieving lysination of the antigen peptide are as follows: S11, connecting the C-terminal amino acid residue in the target peptide to a solid phase support resin, wherein the solid phase support resin is Rink amide resin; S12, the remaining amino acid residues were introduced sequentially from the C-terminus to the N-terminus, wherein lysination was achieved by introducing Fmoc-L-Lys (Boc)-OH, wherein the Fmoc-L-Amino acid (Boc)-OH carries an Fmoc-protected α-amino group and a Boc-protected ε-amino group; S13, condensation reaction is carried out in N,N-dimethylformamide (DMF) or N-methylpyrrolidone (NMP) at room temperature (20–25°C) using O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU) as the condensation reagent and N,N-diisopropylethylamine (DIPEA) as the base. The condensation time for each amino acid is controlled within 30–60 minutes. The reaction system is run under alkaline conditions of approximately pH 8–9, without the need for external buffer. S14, in the final cleavage step, TFA is used to completely remove the Boc protecting group of lysine; after the synthesis is completed, the peptide chain is cleaved and the side chain protecting groups are removed using a mixture containing TFA, TIS and H2O, and the resulting cleavage solution is precipitated with anhydrous cold ether to obtain the crude peptide; S15, separating the target peptide segment from the impurity components of the crude peptide, and obtaining the purified lysine-modified peptide powder by freeze-drying.

[0012] In the further step S14, the mass ratio of TFA:TIS:H2O is 95:2.5:2.5.

[0013] Furthermore, in step S15, the purification step is to purify by reverse phase high performance liquid chromatography (RP-HPLC), using a C18 chromatographic column during the purification process, and the mobile phase consists of a 0.1-1% by mass trifluoroacetic acid (TFA) aqueous solution and a 0.1-1% by mass TFA acetonitrile solution, and linear gradient elution is used to separate the target peptide and impurity components.

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

[0015] Furthermore, the antigen peptide carries a positive charge, and the antigen peptide binds to 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: (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 and enhance antigen-specific CD8 + The number and activation level of T cells can effectively inhibit the occurrence and development of tumors. At the same time, this invention is the first to systematically study the immune activation ability of AKK bacteria in the "intradermal-lymph node-spleen" and its application in tumor vaccines, expanding the types of probiotics that can be used for tumor immunotherapy and providing a basis for the study of the mechanism of AKK bacteria in anti-tumor immunity.

[0019] (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, significantly improving the efficiency of cross-presentation by dendritic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :(a) Molecular simulation calculation of the effect of different amounts of lysine on the contact between the peptide and the bacterial membrane. (2) Visualization of molecular simulation results.

[0021] Figure 2Taking the six N-terminal lysine residues as an example, (a) Mass spectrum of the lysinated peptide. (b) Reversed-phase HPLC chromatography of the lysinated peptide.

[0022] Figure 3 (a) Efficiency of inactivated AKK bacteria loaded with different lysine-linked peptides. (b) Confocal microscopy image of the tumor vaccine. (c) Surface charge of the tumor vaccine. (d) Hydrated particle size of the tumor vaccine. (e) Transmission electron microscopy image of the tumor vaccine. (f) Efficiency of the tumor vaccine in releasing loaded antigen peptides in different environments.

[0023] Figure 4 :(a) Efficiency of tumor vaccines in inducing cross-presentation in various in vitro BMDC models.(b) Efficiency of tumor vaccines in inducing BMDC maturation.

[0024] Figure 5 (a) Dendritic cell recruitment after intradermal injection of a tumor vaccine, including immunohistochemistry images of the skin at the injection site (CD11c) and statistical plots of CD11c-positive signals (a DC marker) in the dermis and fascia. (b) Effects of the tumor vaccine on lymph node migration of antigen peptides. (c) Efficiency of tumor vaccine-induced DCs in cross-presenting antigens in lymph nodes. Figure 6 : Experimental results on the generation and activation of antigen-specific CD8 T cells. (a) Demonstrates the experimental process of vaccination. (b) TNF-α and IFN-γ assays were used to assess vaccine activation of CD8 + (c) Evaluation of vaccine-induced antigen-specific CD8 T cell activity by Tetramer-MHCI assay + Generation of T cells.

[0025] Figure 7 In vivo efficacy studies of tumor vaccines in different models. (a) Experimental design scheme for the melanoma prevention study, including tumor growth curves and survival rates in mice. (b) Experimental design scheme for the melanoma treatment study, including tumor growth curves and survival rates in mice. (c) Experimental design scheme for the colon cancer study, including tumor growth curves and survival rates in mice.

[0026] Figure 8 Validation of the long-term protection and metastasis inhibition of the tumor vaccine in rechallenge and metastasis models. (a) Vaccine protection in cured mice, and its ability to protect against secondary melanomas 5 months later. (b) Vaccine protection in cured mice, and its ability to protect against secondary colon cancers 6 months later. (c) Vaccine inhibition of melanoma metastasis. DETAILED DESCRIPTION

[0027] 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. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0029] Example 1: (1) Simulation of bacterial-loaded peptides. Study on the binding mechanism between peptides and bacteria. The peptide structure was constructed using the Avogadro platform, and the planar membrane model of the Gram-negative bacterial outer membrane (72 Lipid A molecules) in equilibrium mode was constructed using the CHARMM-GUI platform. Molecular dynamics simulations were performed using the GROMACS software to optimize and relax the membrane conformation. Molecular dynamics simulations were performed using the GROMACS 2020.6 software package, the AMBER ff19SB+Lipid21 force field, and the SPC / E water model for water molecules. After the system was built, energy was minimized, and the system was optimized using Steepest descent to eliminate unreasonable geometric structures in the system. All hydrogen bonds were constrained under the LINCS algorithm to ensure the stability of the hydrogen bonds. During the ionization process, by adding appropriate ions (such as Ca 2+ and Cl - ) to avoid strong electric fields or other unnatural effects in the system. This process is also minimized using the Steepest descent algorithm. The system equilibrium process includes two processes: constant temperature (NVT) and constant pressure (NPT). During the NVT equilibrium process, the Berendsen temperature coupling method is used to control the temperature, and the reference temperature is set to 298 K. During the NPT equilibrium process, the Berendsen pressure coupling method is used to control the pressure, and a semi-isotropic pressure coupling method is used (the pressure coupling in different directions will be different). During the equilibrium and production processes, the LINCS algorithm is used to constrain hydrogen bonds to ensure the stability of hydrogen bonds and the rationality of the structure. The Verlet truncation scheme is used in the equilibrium simulation stage to improve computational efficiency, and the cut-off method is used to calculate the van der Waals force, and the particle mesh Ewald (PME) method is used to deal with long-range electrostatic interactions.

[0030] (2) After the simulation is completed, the contact between the peptide and different groups on the bacterial membrane is calculated using commands such as gmx mindist in Gromacs to analyze the potential of lysine of different lengths to adsorb to the bacterial membrane. PyMOL is used to visualize the system.

[0031] Example 2:

[0032] (1) Synthesis of lysine-modified peptides. Peptide synthesis is carried out using the standard Fmoc solid-phase peptide synthesis (Fmoc-SPPS) strategy, which is a service routinely provided by commercial peptide synthesis companies (such as GenScript, GL Biochem, etc.). The synthesis process is reproducible, controllable, and high-purity. It starts with the connection of the C-terminal amino acid residue in the target peptide to a solid-phase support resin (such as Rink amideresin), and the remaining amino acid residues are introduced sequentially from the C-terminus to the N-terminus. Lysine modification is achieved during the Fmoc-SPPS synthesis process by introducing Fmoc-L-Lys(Boc)-OH. Fmoc-L-Amino acid(Boc)-OH (amino acid derivative) carries an Fmoc-protected α-amino group and a Boc-protected ε-amino group, ensuring that the amino residues introduced sequentially do not undergo nonspecific reactions during the synthesis process. The condensation reaction was carried out at room temperature (20–25°C) in N,N-dimethylformamide (DMF) or N-methylpyrrolidone (NMP) using O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU) as the condensation reagent and N,N-diisopropylethylamine (DIPEA) as the base. The condensation time for each amino acid was controlled to be 30–60 minutes. The system was operated under alkaline conditions (pH approximately 8–9), without the need for external buffer. The Boc protecting group on lysine was completely removed by TFA in the final cleavage step. After completion of the synthesis, the peptide chain was cleaved and the side chain protecting groups were removed using TFA / TIS / H2O (95:2.5:2.5). The resulting cleavage solution was then precipitated with cold anhydrous ether to produce the crude peptide. The crude peptide was first purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The purification process used a C18 column with a mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in acetonitrile. A linear gradient elution was used to separate the target peptide from impurities. After the target peak was collected, it was freeze-dried to obtain a purified peptide powder.

[0033] (2) After freeze-drying, the peptide was dissolved in a 50% water / 50% methanol (v / v) mixed solution and injected into an LCMS-IT-TOF mass spectrometer at a flow rate of 0.2 mL / min. Molecular weight determination was performed using electrospray ionization (ESI-MS) in positive ion mode. The instrument parameters included: DL temperature of 200°C, Block temperature of 220°C, nebulizing gas flow rate of 1.5 L / min, and drying gas flow rate of 10.0 L / min. At the same time, its purity was evaluated by reversed-phase high-performance liquid chromatography (RP-HPLC) at a wavelength of 220 nm. The chromatographic conditions were: using an Inertsil ODS-SP (4.6×250 mm) column, a flow rate of 1.0 mL / min, and an injection volume of 10 μL. Mobile phase A was 0.05% trifluoroacetic acid in water, and mobile phase B was 0.05% trifluoroacetic acid / acetonitrile solution. The following gradient elution program was used: initial 5% B, increased to 65% B in 25 min, increased to 95% B in 27 min, returned to 5% B in 35 min, and stopped at 35.01 min.

[0034] Example 3:

[0035] Tumor vaccine pAKK / K-pep OVA257-264 Preparation and characterization studies: (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, the bacteria were evenly dispersed on BHI agar plates and cultured in highly anaerobic culture bags at 37°C to obtain dispersed single colonies. Single colonies were isolated and inoculated into liquid culture medium. The AKK bacterial solution was allowed to amplify to the stationary phase. The AKK bacterial solution was centrifuged and the residual BHI liquid culture medium was washed off with sterile saline. The bacteria were then resuspended in sterile water and heat-inactivated in a 70°C water bath for 15 minutes (pasteurization).

[0036] (2) Tumor vaccine pAKK / K6-pep OVA257-264 Preparation and characterization of the heat-killed bacteria stock solution was quantified using Nanodrop, and the modified Kn-pep with fluorescence was prepared using the method in Example 1. OVA257-264 With K6-pep OVA257 -264 , the modified Kn-pep with fluorescence OVA257-264 Mix with heat-killed AKK bacteria (pAKK) at a ratio of 25:1 (nmole:OD value) in sterile water and dilute to 200 μl. After 15 to 30 minutes, remove unbound Kn-pep OVA257-264The loading efficiency of pAKK was quantitatively studied using a multifunctional microplate reader; FITC-modified peptides were used to visualize the results of pAKK loading peptides using a confocal fluorescence microscope; pAKK and pAKK / K6-pep 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 and pick up the sample with a copper mesh. Use transmission electron microscopy to visually detect the particle size of the complex. OVA257-264 Resuspend with ultrapure water, dilute and take 20 microliters of suspension and drop it on the silicon wafer. After the material is naturally settled and dried, the surface morphology is observed by scanning electron microscopy. The tumor vaccine pAKK / K6-pep constructed with fluorescently labeled peptides OVA257 -264 Different liquid environments were used to simulate different environments - commercial 1640 culture medium simulated in vitro cell culture environment, commercial HBSS buffer simulated extracellular fluid and living body fluid, lysosome simulation liquid (110mM KCl, 30mM NaCl, 1mMMgCl2·6H2O and 10mM MES pH adjusted to 4.5), and HCL was used to adjust the environmental pH at 6 hours to simulate the situation of the vaccine entering the lysosome.

[0037] Example 4:

[0038] Dendritic cell-based tumor vaccines activate CD8 + T cell potential research: (1) Extraction and culture of bone marrow-derived dendritic cells. Bone marrow-derived 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 buffered saline, and the bone marrow was flushed out with complete culture medium. The erythrocytes in the precipitate were removed with erythrocyte lysis buffer. The remaining cells were fixed to a volume of 3 mL of culture medium containing 100,000 cells, 50 μM mercaptoethanol, and 20 ng / mL granulocyte-macrophage colony-stimulating factor (for differentiation into moDCs) or 200 ng / mL FMS-like tyrosine kinase 3 ligand (for differentiation into cDC1 and cDC2). On the third day, an equal amount of RPMI complete culture medium containing granulocyte-macrophage colony-stimulating factor and 50 μM mercaptoethanol was added. On the sixth day, half of the medium was changed (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 assessed on the seventh day.

[0039] (2) Antigen cross-presentation and maturation detection. BMDCs were respectively exposed to inactivated bacteria, the original antigen peptide OVA257-264, and the lysine-modified antigen peptide K-pep OVA257-264Cells were incubated with the tumor vaccine (peptide concentration 50 nM) for 12 hours. After harvesting and washing, the blocking antibody anti-CD16 / 32 was added to reduce nonspecific adsorption to the Fc end. Flow cytometry antibodies anti-CD11c, anti-B220, anti-CD24, anti-CD172a, and anti-MHCI-SIINFEKL (cross-presentation assay) or anti-CD11c, anti-CD80, and anti-CD86 (maturation assay) were added and stained for 30 minutes in the dark. After staining, unbound antibodies were removed, and the cross-presentation efficiency or maturation efficiency of BMDCs was assessed by flow cytometry.

[0040] Example 5:

[0041] Study on the effect of tumor vaccines on dendritic cells in vivo: (1) The effect of tumor vaccines on dendritic cell recruitment 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. Diluted CD11c antibody was added and incubated overnight at 4°C. The excess antibody was washed off and the corresponding labeled secondary antibody for CD11c antibody was added and incubated at room temperature for 1 hour. Subsequently, DAB was used as a color substrate. After staining, the sections were observed under a microscope and finally sealed with a sealing agent for further analysis and recording.

[0042] (2) Tumor vaccine promotes antigen lymph node migration. A tumor vaccine was constructed using FITC-modified peptides according to the previous steps. After intradermal vaccination, the lymph nodes of mice were fluorescently imaged using a small animal in vivo imaging system at 24 hours to analyze the lymph node migration effect of tumor antigens.

[0043] (3) Tumor vaccine promotes antigen presentation by DCs in lymph nodes. 24 hours after intradermal vaccination, the inguinal lymph nodes of mice were collected and ground with a 300-mesh mesh, filtered, and the lymph node suspension was collected. After washing the cells, blocking antibodies anti-CD16 / 32 were added to reduce nonspecific adsorption of the Fc end. Flow cytometry antibodies anti-CD11c and anti-MHCI-SIINFEKL were added, and the cells were stained in the dark for 30 minutes. After staining, unbound antibodies were removed, and the cross-presentation efficiency of DCs in the lymph nodes was detected by flow cytometry.

[0044] Example 6:

[0045] Tumor vaccines based on CD8 + Research on the in vivo immune function and biological effects of T cells: Tumor vaccines against CD8 + The activation effect of adaptive immunity dominated by T cells. Fluorescently labeled tumor vaccines were used, and small animal live imaging was used to perform fluorescence imaging on mice at different time points to analyze the lymph node migration effect of tumor vaccines. Mouse lymph nodes were collected and dispersed into single cells by physical and enzymatic methods. Immunofluorescence technology, 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, the spleens of mice were collected, and the Tetramer H-2Kb-OVA antibody was used to detect the content of T cells that can specifically recognize OVA. In order to explore the effect of tumor vaccines on CD8 + To investigate the activation degree of T cells, spleen cells were treated with BFA for half an hour in advance, and then OVA257-264 peptide was added to stimulate spleen cells for the second time. Anti-CD8 and anti-CD3 antibodies were used for surface staining, and anti-TNF-⍺ and anti-IFN-γ antibodies were added for intracellular staining. Flow cytometry was used to analyze the production of TNF-⍺ and IFN-γ in CD8 T cells. + To determine the degree of T cell activation, spleen cells were stained with CFSE and BMDCs activated by OVA257-264 were added to co-incubate with spleen cells. CD8 T cells were detected by flow cytometry after 3-5 days. + T cell proliferation.

[0046] Example 7:

[0047] In vivo efficacy studies of tumor vaccines in different models: (1) The in vivo efficacy of tumor vaccines under different conditions was investigated in an orthotopic melanoma B16-OVA model. Preventive model: Mice were immunized continuously for three weeks (once a week), and an orthotopic melanoma B16-OVA model was established on the fourth week. The survival rate and tumor size of the mice were monitored. Therapeutic model: An orthotopic melanoma B16-OVA model was established on day 0 to simulate postoperative tumor metastasis. Mice were immunized on days 2, 5, 8, and 11, and the survival rate and tumor size of the mice were monitored.

[0048] (2) In vivo efficacy of tumor vaccines in the CT26 colon cancer model. Using the same strategy, we lysinated the mutant antigen peptide GP70-AH1A5 of colon cancer CT26 cells (the same as the OVA antigen peptide) to construct a tumor vaccine with AKK bacteria as the carrier. The tumor vaccine was inoculated into the mouse model corresponding to the cell line once a week for three consecutive weeks. The corresponding tumor model was established in the fourth week, and the tumor growth and mouse survival rate were monitored.

[0049] Example 8:

[0050] Validation of long-term protection and metastasis inhibition of tumor vaccines in re-challenge and metastasis models: (1) Tumor re-challenge: In the B16-OVA or CT26 tumor model, mice were first vaccinated with the tumor vaccine and a subcutaneous tumor model was established. The tumor vaccine caused the tumor to clear naturally, and the tumor-free state was continuously monitored. Five to six months after the first tumor vaccination, the same number of B16-OVA or CT26 tumor cells were subcutaneously injected into the contralateral abdomen for a tumor re-challenge experiment. Tumor growth was continuously observed to evaluate the long-term immune protection effect induced by the vaccine.

[0051] (2) Tumor lung metastasis: After completing three tumor vaccinations, B16-OVA melanoma cells were injected through the tail vein on day 21 to establish a lung metastasis model. On day 30, the mouse lung tissues were collected, photographed, and fixed in 4% paraformaldehyde solution. The fixed lung tissues were dehydrated with 70%, 80%, 95%, and 100% ethanol gradients, then transparentized in xylene and embedded in paraffin. The embedded tissue sections (approximately 4–5 μm thick) were sliced ​​and dewaxed, rehydrated, and then stained with hematoxylin-eosin (H&E). Finally, the formation and number of lung metastases were observed under an optical microscope to evaluate the inhibitory effect of the vaccine on tumor metastasis.

[0052] Analysis of experimental results: Molecular simulation of bacterial loading of peptides. In the bacterial membrane model common to Gram-negative bacteria, see Figure 1a. As the number of N-terminal lysine modifications increases, the contact behavior of the peptide with various head groups in the bacterial membrane shows a differentiated trend. When the number of lysines is 6, the average number of contact pairs between the peptide and the sugar ring increases significantly, reaching a peak, which is significantly enhanced compared to other modification states, suggesting that this conformation is most conducive to binding to the membrane glycosyl region. At the same time, the contact between the peptide and the phosphate group also increases synchronously, indicating that the charge-rich N-terminal modification helps to enhance the electrostatic attraction with the negative charge of the phosphate. In contrast, the number of contact pairs of the ether bond is generally low and does not change significantly, indicating that it may not serve as the main recognition site in membrane binding. See also Figure 1 b, Simulation visualization shows that peptides modified with six lysine residues at the N-terminus are able to contact the bacterial membrane more quickly than unmodified peptides. The increased number of lysine residues at the N-terminus increases the peptide length, increasing the likelihood of self-binding (long chain aggregation) and reducing the number of sites for binding to the bacterial membrane. Therefore, peptides modified with six lysine residues at the N-terminus exhibit stronger membrane affinity in the Gram-negative bacterial membrane model, favoring membrane binding.

[0053] Evaluation of lysine peptide results. Take the peptide with 6 lysine residues added to the N-terminus as an example, see Figure 2 a. The test results show that [M+H] + 、[M+2H] 2+ and [M+3H] 3+ The peaks appeared at m / z = 1733.2, 867.0 and 578.3, respectively, corresponding to a molecular weight of 1732.0 Da, which is consistent with the theoretical value and verifies the accuracy of peptide synthesis. Figure 2 b. The chromatogram shows that the main peak retention time is 10.162 minutes and the area ratio is 95.368%, indicating that the lysination of the peptide was successfully completed and the target peptide is a high-purity product, suitable for subsequent immunology, cell biology and animal experimental research.

[0054] Characterization of tumor vaccines. 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. Figure 3 a. As the number of N-terminal lysine modifications increases, the peptide loading efficiency first increases and then decreases. The highest loading efficiency is achieved when the number of lysine modifications is 6. The results indicate that a moderate positive charge is beneficial for the binding efficiency between AKK bacteria and the peptide, but excessive lysination can negatively impact the loading efficiency. This result is consistent with the simulation results. Therefore, 6 N-terminal lysine modifications represent the optimal modification level, maximizing the loading efficiency. Figure 3 b, Fluorescence images from a confocal microscope once again confirmed that AKK bacteria have the ability to efficiently load antigenic peptides with 6 lysine residues modified at the N-terminus. Figure 3c and d, the addition of antigenic peptides makes the surface of AKK bacteria, which was originally negatively charged, tend to be neutral. It can be seen that the overall hydrated particle size increases slightly. Figure 3 e, Transmission electron microscopy shows that the contrast of the outer bacterial wall of AKK bacteria changes from low to high, indicating that the antigen peptide is bound to the surface of AKK bacteria. The above characterization proves that the heat-inactivated AKK bacteria have efficient carrier function.

[0055] Delivery characteristics of antigen-loaded peptides for tumor vaccines. Figure 3 f. The results showed that under neutral conditions (cell culture medium, extracellular fluid simulation, pH = 7.4), peptide release was relatively slow, while under acidic conditions, release was significantly accelerated, especially in the simulated lysosomal environment (pH = 4.5), where over 90% release was achieved within almost 1 hour. Notably, release also increased rapidly after acidification of the extracellular fluid and culture medium (pH = 4.5), indicating that the system has good pH responsiveness, which facilitates efficient release of antigens in lysosomes after endocytosis into antigen-presenting cells (such as DCs). This behavior is crucial for subsequent MHC-I antigen processing and presentation and is a key step in achieving an effective immune response. Therefore, these results clearly demonstrate the potential of using Akkermansia as an adjuvant and antigen carrier platform in the construction of effective tumor vaccines.

[0056] Tumor vaccines have been shown to induce efficient CD8 + The potential of T cell immune response. CD8 + T cell activation is a key step in the anti-tumor immune response of tumor vaccines. Effective activation requires at least two signals: the first is the specific recognition of antigen peptides by MHC-I and TCR, i.e., cross-presentation of exogenous antigens; the second signal comes from co-stimulatory molecules (such as CD80 / CD86) provided by dendritic cells. The lack of any one of these signals can lead to T cell anergy or functional tolerance, significantly impairing the effectiveness of the vaccine. Figure 4 a. In various BMDCs models (moDC, cDC1 and cDC2), tumor vaccines can effectively activate DCs cross-presentation and increase CD8 + The first signal for T cell activation. Although lysine-modified antigenic peptides can also enhance DC cross-presentation, see Figure 4 b. Only with the help of AKK bacteria can DCs enter a mature state, that is, highly express CD8 + The second signal for T cell activation. At the same time, in the absence of antigenic peptides, AKK bacteria alone cannot provide the first signal. Therefore, the tumor vaccine constructed in this project can synergistically enhance the antigen cross-presentation and co-stimulatory signal transmission of dendritic cells, and has the ability to fully activate CD8 + T cells, the potential to enhance anti-tumor immune efficacy.

[0057] After intradermal injection, tumor vaccines need to be processed by dendritic cells and presented to CD8 + T cells to induce antigen-specific CD8 + T cell immune response. Immunohistochemistry results showed ( Figure 5 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 5 b), indicating the important role of AKK bacterial carriers in helping antigens migrate to lymph nodes. At the same time, the cross-presentation efficiency of DCs in lymph nodes is also significantly improved ( Figure 5 c) Therefore, the above experiments preliminarily proved that tumor vaccines have the ability to activate CD8 + T cell potential.

[0058] CD8 + T cells play a core role in anti-tumor immunity, and their antigen-specific generation and activation status directly determine the therapeutic effect of vaccines. Figure 6 The experimental results show that the tumor vaccine treatment group induced the production of OVA-specific CD8 + T cells (Tetramer + CD8 + cells); at the same time, when exposed to antigens, the CD8 + T cells can be rapidly activated to produce cytotoxic CD8 + T cell activation-related cytokines (TNF-α and IFN-γ). 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, helping to enhance the body's immune defense capabilities.

[0059] Tumor vaccines can be used in two key application scenarios in tumor immunotherapy: one is the "prevention model", that is, the vaccine is given before tumor cell inoculation to evaluate its ability to induce an initial immune response and prevent tumor occurrence; the other is the "treatment model", that is, the vaccine is given after the tumor is established to verify its potential to reverse tumor progression and control the growth of existing tumors. These two types of models reflect the application value of vaccines in tumor prevention and control and intervention. Figure 7 From the experimental results, we can see that after using the same strategy to construct tumor vaccines carrying different antigens, the tumor vaccines showed significant preventive effects in both melanoma B16-OVA and colon cancer CT26 ( Figure 7 a and c), and showed significant tumor therapeutic ability in melanoma B16-OVA ( Figure 7 b).

[0060] At the same time, whether it is a melanoma model or a colon cancer model, when the cured mice are inoculated with the same tumor cells again 5 to 6 months later, the growth of the tumor is basically completely inhibited ( Figure 8 a and b), demonstrating the superior long-term immune protection afforded by this vaccine platform (over an average mouse lifespan of 2 years). Furthermore, in a melanoma lung metastasis model, the vaccine also effectively inhibited the spread and colonization of tumor cells in the lungs, significantly reducing the number of lung metastases, demonstrating that the vaccine not only controls primary tumor growth but also curbs distant metastasis. These results demonstrate that this vaccine strategy exhibits broad-spectrum, highly effective anti-tumor effects across a variety of tumor types and pathological settings, suggesting broad potential for application in tumor treatment and prevention of recurrence.

[0061] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modification, equivalent replacement and improvement made by any technician familiar with the profession to the above embodiment without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of protection 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, adding lysine during the synthesis of antigenic peptide to achieve lysination of antigenic peptide; S2, culture, amplify, and pasteurize AKK bacteria; S3, mixing the lysinated antigen peptide and pasteurized 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 the OVA257-264 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 of the antigen peptide; the antigen peptide carries a positive charge after lysination.

4. The preparation method according to claim 1, characterized in that In step S1, lysine is first added during the synthesis of the antigen peptide. The specific steps for achieving lysination of the antigen peptide are as follows: S11, connecting the C-terminal amino acid residue in the target peptide to a solid phase support resin, wherein the solid phase support resin is Rinkamide resin; S12, the remaining amino acid residues were introduced sequentially from the C-terminus to the N-terminus, wherein lysination was achieved by introducing Fmoc-L-Lys (Boc)-OH, wherein the Fmoc-L-Amino acid (Boc)-OH carries an Fmoc-protected α-amino group and a Boc-protected ε-amino group; S13, condensation reaction was carried out in N,N-dimethylformamide (DMF) or N-methylpyrrolidone (NMP) at room temperature (20–25°C) using O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU) as the condensation reagent and N,N-diisopropylethylamine (DIPEA) as the base. The condensation time for each amino acid was controlled at 30–60 minutes. The reaction system was run under alkaline conditions of approximately pH 8–9, without the need for additional buffer. S14, in the final cleavage step, TFA is used to completely remove the Boc protecting group of lysine; after the synthesis is completed, the peptide chain is cleaved and the side chain protecting groups are removed using a mixture containing TFA, TIS and H2O, and the resulting cleavage solution is precipitated with anhydrous cold ether to obtain the crude peptide; S15, separating the target peptide segment from the impurity components of the crude peptide, and obtaining the purified lysine-modified peptide powder by freeze-drying.

5. The preparation method according to claim 4, characterized in that In step S14, the mass ratio of TFA:TIS:H2O is 95:2.5:2.

5.

6. The preparation method according to claim 4, characterized in that In step S15, the purification step is to purify by reverse phase high performance liquid chromatography (RP-HPLC). A C18 chromatographic column is used in the purification process. The mobile phase consists of a 0.1-1% by mass trifluoroacetic acid (TFA) aqueous solution and a 0.1-1% by mass TFA acetonitrile solution. Linear gradient elution is used to separate the target peptide and impurity components.

7. The preparation method according to any one of claims 1 to 6, characterized in that In step S3, the antigen peptide is combined with the pasteurized AKK bacteria through electrostatic interaction.

8. A tumor vaccine based on AKK bacteria prepared by the method according to any one of claims 1 to 7.

9. Use of the tumor vaccine according to claim 8 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.

10. The use according to claim 9, 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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