Cancer vaccine loaded with lysate components of hapten substance modified cancer cells and / or tumor tissues and preparation method of cancer vaccine

Abstract

The invention relates to a preparation method and application for modifying cancer cells and / or tumor tissues by using hapten, or modifying whole-cell components / holoantigen components in the tumor tissues and / or lysates of the cancer cells by using hapten substances, and then loading the whole-cell components / holoantigen components into a nano vaccine or a micron vaccine. The preparation method comprises the following steps: firstly, modifying tumor tissues / cancer cells by using a hapten substance, then splitting the modified tumor tissues / cancer cells, dissolving by using a dissolving solution containing a dissolving agent, and then loading into a nano vaccine or a micron vaccine; or firstly splitting the tumor tissue / cancer cells to obtain a tumor tissue / cancer cell lysate, then modifying the tumor tissue / cancer cell lysate by using a hapten substance, then dissolving a whole-cell lysate component by using a dissolving solution containing a dissolving agent, and loading the whole-cell lysate component into a nano vaccine or a micron vaccine. The prepared nano vaccine or micron vaccine can be used for preventing or treating cancers. The vaccine system provided by the invention comprises nano particles and / or micro particles, and whole-cell components / whole-cell antigen components in cancer cells and / or tumor tissues modified by hapten substances, so that the specific immune response of the cancer cells can be efficiently activated, and the vaccine system can be used for preventing and treating diseases such as cancers.

Description

Technical Field

[0001] The present disclosure relates to the field of immunotherapy, and in particular to a cancer vaccine based on cancer cell and / or tumor tissue lysate components and synthetic cancer antigens and a preparation method thereof. Background Art

[0002] Cancer vaccine is one of the important methods of cancer immunotherapy. The main factors affecting cancer vaccines include tumor antigens, adjuvants and delivery dosage forms. Among them, antigens can induce activation of specific immune responses, adjuvants can inhibit specific immune responses, and dosage forms can affect the efficiency of antigen-presenting cells (APCs) phagocytosis of vaccines and subsequent activation of antigen-specific T cells. Among these three, antigens are the main components that can trigger specific immune responses and are therefore the most critical components. Cancer cells and / or tumor tissues contain all cancer cell-specific antigens and cancer cell-related antigens, and are the best raw materials for preparing cancer vaccines. However, sometimes the ability of some antigens to activate antigen-specific T cells is not strong enough, and haptens can enhance the immunogenicity of substances such as proteins. Therefore, the present invention uses haptens to modify components in cancer cells or tumor tissues, thereby enhancing the immunogenicity of tumor antigens and thereby enhancing the efficacy of cancer vaccines. Summary of the invention

[0003] Problem that the invention aims to solve

[0004] In order to enhance the immunogenicity of tumor antigens loaded into cancer vaccines, the present disclosure provides a method of using haptens to modify lysate components of cancer cells and / or tumor tissues, thereby enhancing the immunogenicity of antigens in the tumor lysate components, and loading the modified lysate components into nanoparticles or microparticles for use as cancer nanovaccines or microvaccines, thereby enhancing the efficacy of the vaccine.

[0005] Solutions for solving problems

[0006] The present invention provides a cancer vaccine, characterized in that the cancer vaccine comprises: (i) a nanoparticle and / or micron particle skeleton structure formed by a particle material, and (ii) a whole cell lysate component and / or a partial cell lysate component containing an antigen component from cancer cells and / or tumor tissues modified with a hapten substance (to enhance immunogenicity); wherein the whole cell lysate component and / or the partial cell lysate component containing an antigen component from cancer cells and / or tumor tissues with enhanced immunogenicity are loaded inside and / or on the surface of the skeleton structure;

[0007] Among them, the water-soluble components in the whole cell components in the lysate components from the cancer cell and / or tumor tissue and the water-insoluble components dissolved by the lysate containing a solvent act together with the hapten substance to enhance their immunogenicity; the antigen components contained in the partial cell components include protein and polypeptide components in the cancer cell and / or tumor tissue lysate and / or RNA components or mRNA components in the cell lysate, and the antigen components are separated and purified from the lysate using an appropriate method or are separated and purified from the water-soluble components and / or water-insoluble components respectively using an appropriate method, and the partial cell components act together with the hapten substance to enhance their immunogenicity; the lysate components and the hapten substance can act together before the cell or tumor tissue is lysed, or after the cell or tumor tissue is lysed.

[0008] The hapten substance is a substance that can increase the immunogenicity of a protein or polypeptide after co-acting with the protein or polypeptide.

[0009] The hapten substances include, but are not limited to, 2,4-dinitrofluorobenzene (DNFB), 2,4-dinitrochlorobenzene (DNCB), trinitrophenol (TNP), dinitrophenol (DNP), albumin, Ovalbumin (OVA), N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide (AED), substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, and other hapten substances containing aldehyde groups.

[0010] The hapten substance is used to interact with cells in cancer cells or tumor tissues for a certain period of time to modify the antigen components in the cancer cells or tumor tissues, and then the cancer cells and / or tumor tissues are lysed to obtain their lysates; or the cancer cells or tumor tissues are first lysed to obtain their lysates, and then the hapten substance is used to modify the antigen components in the cancer cell or tumor tissue lysates.

[0011] Before lysing cancer cells / tumor tissues or before allowing the hapten substance to act with cancer cells / tumor tissues, the cancer cells / tumor tissues can be irradiated with radiation to inactivate the cancer cells / tumor tissues. The radiation includes but is not limited to γ ​​rays, X-rays, β rays, α rays, etc.

[0012] The lysate components of cancer cells or tumor tissues modified with hapten substances can be directly loaded into nano- or micro-vaccines as antigen components, or loaded into nano- or micro-vaccines as antigen components after being properly treated, separated, purified, or further enhanced in immunogenicity.

[0013] The cancer vaccine disclosed in the present invention is characterized in that the preparation method of the whole cell component modified with a hapten comprises: collecting a single cell suspension of cancer cells or tumor tissues, then incubating the single cell suspension of cancer cells or tumor tissues with the hapten for 0.1-24 hours, then centrifuging to remove the supernatant, resuspending the cell precipitate with a solution containing protein, and then incubating for 0.1-24 hours, then centrifuging to remove the supernatant and washing the cells, then lysing the cancer cells and / or tumor tissues, then respectively collecting the water-soluble components and the water-insoluble components in the lysate, dissolving the water-insoluble components with a dissolving solution containing a dissolving agent, and then using them together with the water-soluble components;

[0014] or first using a lysing solution containing a lysing agent to lyse cancer cells and / or tumor tissues, and then using a lysing solution containing a lysing agent to dissolve the whole cell lysate before use;

[0015] Wherein, the dissolving agent is independently selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0016] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0017] Preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

[0018] The cancer vaccine disclosed in the present invention is characterized in that the preparation process of the partial cell component containing the antigen component modified by the hapten comprises: collecting a single cell suspension of cancer cells or tumor tissues, and then incubating the single cell suspension of cancer cells or tumor tissues with the hapten for 0.1-24 hours, and then centrifuging to remove the supernatant, resuspending the cell precipitate with a solution containing protein, and then incubating for 0.1-24 hours, and then centrifuging to remove the supernatant and washing the cells, and then lysing the cancer cells and / or tumor tissues to obtain a lysate, and then centrifuging the lysate and collecting the supernatant and precipitate in the lysate respectively, the supernatant is the water-soluble component, and the precipitate is dissolved with a dissolving solution containing a dissolving agent to obtain the dissolved water-insoluble component, and all water-soluble After the components are treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, mineralization, irradiation, etc., the precipitated part is redissolved using a dissolving solution containing a dissolving agent; the water-insoluble part is directly used after being dissolved using a dissolving solution containing a dissolving agent, or the precipitated component is treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation, etc. and then redissolved using a dissolving solution containing a dissolving agent; the protein polypeptide component in the water-soluble component is mixed with the water-insoluble component or the water-insoluble component is purified and then redissolved together with the antigen component to form a partial cell component containing the antigen component;

[0019] or lysing cancer cells and / or tumor tissues with a dissolving solution containing a dissolving agent, dissolving the lysate components with a dissolving solution containing a dissolving agent, and then subjecting the obtained dissolved lysate components to salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation and other appropriate methods, and then re-dissolving the obtained precipitated components with a dissolving solution containing a dissolving agent;

[0020] Wherein, the dissolving agent is independently selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0021] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0022] Preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

[0023] Preferably, the method for preparing the lysate of the whole cell component comprises: first lysing cancer cells and / or tumor tissues, then collecting water-soluble components and water-insoluble components in the lysate respectively, dissolving the water-insoluble components with a dissolving solution containing a dissolving agent and using them together with the water-soluble components;

[0024] or first using a lysing solution containing a lysing agent to lyse cancer cells and / or tumor tissues, and then using a lysing solution containing a lysing agent to dissolve the whole cell lysate before use;

[0025] or lysing cancer cells and / or tumor tissues with a dissolving solution containing a dissolving agent, dissolving the lysate components with a dissolving solution containing a dissolving agent, and then subjecting the obtained dissolved lysate components to salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation and other appropriate methods, and then re-dissolving the obtained precipitated components with a dissolving solution containing a dissolving agent;

[0026] Methods for isolating, purifying and / or enhancing immunogenicity of protein and polypeptide components in cells and / or RNA components (or mRNA components) in cells include, but are not limited to, one or more of salting out, heating, enzyme treatment, use of RNA isolation kits, use of mRNA isolation kits, oxidation, reduction, mineralization and the like.

[0027] The enzyme treatment method includes, but is not limited to, using one or more of nuclease, DNA enzyme, pepsin, chymotrypsin, trypsin, other protein digestive enzymes, protease inhibitors, and the like.

[0028] The enzymes used for enzymatic hydrolysis include, but are not limited to, one or more of nuclease, pepsin, trypsin, protease inhibitors, chymotrypsin, DNA enzyme, etc.

[0029] Among them, the oxidizing agent used to oxidize the antigen component includes but is not limited to hypochlorous acid, hydrogen peroxide (hydrogen peroxide), persulfate, KIO3, KBrO3, chlorine, dichromate, nitric acid, hydrogen peroxide, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, hydrogen peroxide, bromine, iodine, perchlorate, permanganate, dichromate, sodium peroxide, oxygen, chlorine, sodium dichromate, potassium dichromate, potassium permanganate, nitric acid, ClO3 - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, H2O2, NO3 - 、MnO4 -, F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3, etc. One or more of various oxidants.

[0030] The oxidation can enhance the immunogenicity of some antigenic components.

[0031] Among them, the mineralization method includes but is not limited to using one or more mineralization methods such as silicification, calcification, magnesiumization, and biomineralization.

[0032] The reduction is to reduce the antigen component using a component that can reduce the antigen. The reducing agent used to reduce the antigen component includes but is not limited to dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP) and other reducing agents.

[0033] The reduction is to reduce the antigen component using a reducing agent, and the reducing agent includes but is not limited to DTT and TCEP.

[0034] The irradiation is any commonly used irradiation method, including but not limited to one or more of irradiation methods such as radioactive material irradiation, ultraviolet irradiation, X-ray irradiation, α-ray irradiation, β-ray irradiation, and γ-ray irradiation.

[0035] The chromatography disclosed herein includes, but is not limited to, column chromatography, gas chromatography, high pressure liquid chromatography, adsorption chromatography, partition chromatography, thin layer chromatography, high performance liquid chromatography, ion exchange chromatography, thin film chromatography, affinity chromatography, gel chromatography, and the like.

[0036] The chromatography methods disclosed herein include, but are not limited to, column chromatography, thin layer chromatography, liquid chromatography, gas chromatography, supercritical fluid chromatography, and the like.

[0037] The electrophoresis method disclosed herein includes, but is not limited to, SDS electrophoresis, isoelectric focusing electrophoresis, isotachophoresis, immunoelectrophoresis, serum protein electrophoresis, nucleic acid electrophoresis, DNA sequencing electrophoresis, gel electrophoresis, preparative electrophoresis, and the like.

[0038] The source of cancer cells described in the present disclosure is any method that can obtain cancer cells, including but not limited to cancer cell lines, cancer cells isolated and extracted from tumor tissues and obtained by in vitro amplification, cancer cells isolated and extracted from blood and obtained by amplification, or cancer cells differentiated and cultured from stem cells, etc.

[0039] Preferably, the cancer cells are from one or more organisms, or from one or more cancer cell lines; the tumor tissues are from one or more organisms; the protein and polypeptide components / mRNA components (or RNA components) in the water-soluble components of the cancer cells and / or tumor tissues and the protein and polypeptide components / mRNA components (or RNA components) in the water-insoluble components / water-insoluble components of the cancer cells and / or tumor tissues contain antigen components; the cancer-specific and / or related antigen polypeptides and / or nucleic acids that can express cancer-specific and / or related antigen polypeptides are synthesized in vitro and mixed with components from cancer cell / tumor tissue lysates and then co-loaded in nano- or micro-vaccines. The nucleic acid is mRNA or DNA.

[0040] Preferably, the cancer vaccine is further loaded with at least one component as shown below:

[0041] (iv) mRNA component or RNA component in the water-soluble component and / or the water-insoluble component;

[0042] (v) immune adjuvants;

[0043] (vi) substances with a positive charge,

[0044] Preferably, the immune adjuvant comprises at least one of the following: pattern recognition receptor agonists, Toll-like receptor agonists, Bacillus Calmette-Guérin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG cell wall acyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhanced reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn calf liver active peptide, imiquimod, polysaccharides, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme QIO, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokine, mRNA, MF59, double-stranded RNA, double-stranded DNA, single-stranded DNA, aluminum adjuvant, manganese adjuvant, calcium adjuvant, STING agonist, endotoxin adjuvant, liposome adjuvant, CAF01, ginseng active ingredients, Astragalus membranaceus active ingredients;

[0045] Preferably, the immune adjuvant comprises at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist;

[0046] More preferably, the immune adjuvant comprises at least one of Poly(I:C), Poly ICLC, Class A CpG-OND, Class B CpG-OND and Class C CpG-OND;

[0047] Preferably, the positively charged substance is selected from positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and / or positively charged inorganic substances;

[0048] More preferably, the positively charged substance is selected from any one or more of bee venom peptide, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

[0049] Preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-1:10;

[0050] More preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-1:2;

[0051] Most preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.05-1:1.

[0052] Preferably, the mass ratio of the particle skeleton structure to the RNA component or mRNA component is 1:0.001-1:10;

[0053] More preferably, the mass ratio of the particle skeleton structure to the RNA component or mRNA component is 1:0.01-1:2;

[0054] Most preferably, the mass ratio of the particle skeleton structure to the RNA component or the mRNA component is 1:0.05-1:1.

[0055] Preferably, the protein and polypeptide components and RNA component / mRNA component are derived from lysate, and the cancer-specific and / or related antigenic polypeptide / nucleic acid component is derived from artificial synthesis;

[0056] Preferably, the mass ratio of the protein and polypeptide component and the RNA component / mRNA component to the cancer-specific and / or related antigen polypeptide / nucleic acid component is 1:0.001-1:10;

[0057] More preferably, the mass ratio of the protein and polypeptide component and the RNA component / mRNA component to the cancer-specific and / or related antigen polypeptide / nucleic acid component is 1:0.01-1:2;

[0058] Most preferably, the mass ratio of the protein and polypeptide components and the RNA component / mRNA component to the cancer-specific and / or related antigenic polypeptide / nucleic acid component is 1:0.05-1:1.

[0059] Preferably, the cancer vaccine further contains at least one component as shown below:

[0060] (a) cancer cell membrane components derived from tumor tissues and / or tumor cells;

[0061] (b) an extracellular vesicle membrane fraction derived from an extracellular vesicle lysate, wherein the extracellular vesicles are secreted by bacteria or tumor cells;

[0062] (c) bacterial membrane fractions derived from bacterial lysates;

[0063] (d) membrane fractions derived from antigen-presenting cells;

[0064] Preferably, the bacteria include at least one of the following: BCG, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus gestatum, Lactobacillus reuteri, and Lactobacillus rhamnosus.

[0065] Preferably, the process of separating and purifying the antigen components in the water-soluble components and / or the water-insoluble components or separating and purifying the antigen components in the lysate components can include one or more of salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, and irradiation.

[0066] Preferably, the antigen component contains protein and polypeptide components and / or mRNA components (or RNA components).

[0067] Preferably, the steps for preparing the protein and polypeptide components in the water-soluble components in the cancer cells and / or tumor tissues are as follows: firstly lyse the cancer cells and / or tumor tissues to obtain the lysates thereof; then separate the water-soluble components and the water-insoluble components in the lysates by one or more methods such as centrifugation, filtration, dialysis, ultrafiltration, etc., to obtain the water-soluble components and the water-insoluble components in the lysates respectively; then subject the obtained water-soluble components in the lysates to salting out, heating, enzyme treatment, oxidation, reduction, fixation, mineralization, and irradiation treatments to precipitate the protein and polypeptide components therein; then dissolve the precipitated protein and polypeptide components using a dissolving solution containing a dissolving agent to obtain the protein and polypeptide components in the water-soluble components;

[0068] Wherein, the dissolving agent is independently selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0069] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0070] Preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea, urea salt, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

[0071] Preferably, the particle material is selected from natural polymer materials, synthetic polymer materials and / or inorganic materials.

[0072] Preferably, the shape of the nanovaccine or microvaccine is any shape, including but not limited to sphere, ellipsoid, barrel, polygon, rod, sheet, line, worm, square, triangle, butterfly, disc, vesicle, etc.

[0073] Preferably, the cancer vaccine is a nanovaccine and / or a microvaccine;

[0074] Wherein, the particle size of the nano vaccine is 1nm-1000nm; preferably 50-500nm; more preferably 100-400nm;

[0075] Wherein, the particle size of the micron vaccine is 1 μm-1000 μm; preferably 1-10 μm; more preferably 1-5 μm.

[0076] Wherein, when a mixed vaccine of nano vaccine and micro vaccine is used, the particle size of the nano vaccine is 100-600nm, and the particle size of the micro vaccine is 1.5-5μm; preferably, the particle size of the nano vaccine is 150-500nm, and the particle size of the micro vaccine is 2.0-3.5μm.

[0077] In some embodiments, adding an appropriate amount of PEG-modified PLGA or PLA to the main materials such as PLGA or PLA for preparing nanovaccines or micron vaccines can better achieve the effects of long circulation and passive targeting; the mass ratio of PEG-modified PLGA or PLA to unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.

[0078] The nanoparticles / microparticles used to deliver antigens can also be bacteria and viruses. When the antigen delivery particles are bacteria and viruses, the bacteria and viruses can express tumor-specific antigens and / or tumor-associated antigens, or the bacteria and viruses contain DNA and / or mRNA that can express tumor-specific antigens and / or tumor-associated antigens.

[0079] The present invention also provides a method for preparing the cancer vaccine, characterized in that the preparation method comprises the following steps:

[0080] (1) First, the cancer cells or tumor tissues are allowed to react with the hapten substance for a certain period of time, and then the cancer cells and / or tumor tissues are lysed to obtain the lysate; or first, the cancer cells and / or tumor tissues are lysed to obtain the lysate, and then the lysate is allowed to react with the hapten substance for a certain period of time.

[0081] (2) The water-soluble components and water-insoluble components in the lysate are loaded as antigen components into the nanovaccine or micron vaccine; or the lysate obtained by dissolving cancer cells and / or tumor tissues using a dissolving solution containing a dissolving agent is loaded as antigen components into the nanovaccine or micron vaccine; or the lysate components are treated according to the method of step (3) and then loaded into the nanovaccine or micron vaccine.

[0082] (3) the water-soluble components in the lysate are then subjected to salting out, heating, enzyme treatment (such as enzymolysis or enzyme inhibition), oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation and other appropriate methods to obtain a precipitate, and then the precipitate is dissolved in a dissolving solution containing a dissolving agent to obtain an antigen component; the water-insoluble components in the lysate are dissolved in a dissolving solution containing a dissolving agent and then subjected to salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, After being treated by appropriate methods such as irradiation, the antigen component is obtained by dissolving the lysate containing a solvent, or the non-water-soluble component is directly dissolved by a lysate containing a solvent without treatment, or the antigen component is obtained by secondary dissolution; or all lysate components dissolved by the lysate containing a solvent are treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation, etc., and then the precipitate is obtained by secondary dissolution of the lysate containing a solvent, and the antigen component is obtained; wherein the solvent in each step is selected separately. Then the obtained antigen component is loaded to the interior and / or surface of the nanoparticle or micron particle to obtain the nano vaccine or micron vaccine.

[0083] The antigen component can be a protein polypeptide component, a mixture of a protein polypeptide component and a total RNA component, or a mixture of a protein polypeptide component and a total mRNA component.

[0084] The method for separating and extracting mRNA components or RNA components in cancer cells and / or tumor tissues described in the present invention includes but is not limited to one or more methods such as using mRNA separation and extraction reagents, using RNA separation and extraction kits, using DNA removal kits, and using enzymes to degrade DNA.

[0085] Optionally, the cancer cells or tumor tissues may be co-incubated with specific chemicals to stimulate the cancer cells or tumor tissues before lysis.

[0086] Optionally, the specific substance that stimulates cancer cells includes, but is not limited to, small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, cytokines, chemokines, plant extracts (such as important extracts such as ginseng, plant rhizome extracts, etc.), interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate with cancer cells or tumor tissues to stimulate cancer cells or tumor tissues is to make the cancer cells produce more antigen components.

[0087] The present invention also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the cancer vaccine or the cancer vaccine prepared according to the method;

[0088] Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0089] The present invention also provides a method for preventing or treating a disease, wherein the method comprises administering to a subject a preventively or therapeutically effective amount of the cancer vaccine, or the cancer vaccine prepared according to the method, or the pharmaceutical composition.

[0090] The present invention also provides a use of the cancer vaccine, or the cancer vaccine prepared according to the method, or the pharmaceutical composition in at least one of the following (1)-(3):

[0091] (1) Preparation of drugs for preventing or treating diseases;

[0092] (2) preparing a medicament for inducing an immune response in a subject;

[0093] (3) Preparation of cancer vaccines;

[0094] Optionally, the disease is cancer or tumor;

[0095] Optionally, the cancer or tumor is a solid tumor or a hematological tumor.

[0096] Effects of the Invention

[0097] The present invention uses specific means to separate and purify antigen components in whole cell components of cancer cells and / or tumor tissues, which can enrich protein and polypeptide antigen components and avoid potential toxic side effects that may be caused by substances such as sugars and DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1The present invention is a schematic diagram of some feasible preparation processes and applications of nano- or micro-vaccines disclosed in the present invention; wherein, a and b are schematic diagrams of collecting water-soluble antigen components and water-insoluble components and then mixing them with artificially synthesized polypeptides or mRNA / RNA to prepare nano- / micro-vaccines; c and d are schematic diagrams of the process of preparing nano- / micro-vaccines by directly lysing the whole cell components of cancer cells and / or part of the cell components containing antigen components with a lysis solution containing a dissolving agent and then mixing them with artificially synthesized polypeptides / mRNA (or RNA).

[0099] Figure 2 It is a structure of formula 1, wherein R1 is C, S, P, N or O, and R2 to R5 are independently selected from any other groups such as hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine.

[0100] Figure 3-Figure 6 The experimental results of tumor growth rate and survival period when nano-vaccine and / or micro-vaccine are used to prevent or treat cancer in Examples 1-4; wherein, a is the experimental result of tumor growth rate when preventing or treating cancer (n≥8); b is the experimental result of mouse survival period when preventing or treating cancer (n≥8), and each data point is mean±standard error (mean±SEM); wherein, the significant difference of tumor growth inhibition experiment in Figure a is analyzed by ANOVA method, and the significant difference in Figure b is analyzed by Kaplan-Meier and log-ranktest; *** indicates that p<0.005 is significantly different compared with the PBS blank control group; ** indicates that p<0.01 is significantly different compared with the PBS blank control group; ### indicates that p<0.005 is significantly different when the two groups are compared; ## indicates that p<0.01 is significantly different when the two groups are compared; # indicates that p<0.05 is significantly different when the two groups are compared. DETAILED DESCRIPTION

[0101] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0102] The experimental techniques and methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0103] In the claims and / or description of the present invention, the term "a" or "an" or "the" may mean "one", but may also mean "one or more", "at least one" and "one or more than one".

[0104] As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0105] The term "suffering from a disease" means that the body is experiencing symptoms of a disease.

[0106] The term "treatment" means that after a subject suffers from a disease, the subject is contacted with (e.g., administered) a delivery particle, a delivery system, a vaccine, a drug loaded with an antigen, or a pharmaceutical composition, thereby alleviating the symptoms of the disease compared to when the subject has not been contacted, and does not necessarily mean that the symptoms of the disease must be completely suppressed.

[0107] The term "prevention" means that before a subject develops a disease, by contacting (e.g., administering) the delivery particles, delivery systems, vaccines, antigen-loaded drugs, and pharmaceutical compositions of the present disclosure, the symptoms after developing the disease are alleviated compared to when the subject has not been contacted. It does not mean that the disease must be completely suppressed.

[0108] The vaccines of the present disclosure can be prepared using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding and / or lyophilizing processes.

[0109] In the present disclosure, the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the properties of the drug, the convenience of the patient and the medical staff, and other relevant factors.

[0110] The terms "individual", "patient" or "subject" used in the context of this disclosure include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0111] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors. The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0112] When preparing the cancer vaccine disclosed in the present invention, antigen components can be prepared using cancer cells / tumor tissues obtained by several methods, such as tumor tissues, cancer cell lines, cancer cells isolated from tumor tissues and cultured and amplified, and cancer cells obtained by cultured and amplified circulating tumor cells isolated from peripheral blood. In actual applications, any other feasible approach can also be used to obtain cancer cells.

[0113] Due to space limitations, the cancer described in the embodiments of the present disclosure is a solid tumor. In practical applications, the nano-vaccine or micro-vaccine described in the present disclosure can also be used to treat blood tumors and lymphomas. Because the immune microenvironment of blood tumors and lymphomas is not as complex as that of solid tumors, the nano-vaccine or micro-vaccine described in the present disclosure is more effective in blood tumors and lymphomas.

[0114] The present invention provides a cancer vaccine, characterized in that the cancer vaccine comprises: (i) a nanoparticle and / or micron particle skeleton structure formed by a particle material, and (ii) a whole cell lysate component from cancer cells and / or tumor tissues and / or a partial cell lysate component containing an antigen component modified with a hapten substance to enhance immunogenicity; wherein the whole cell lysate component from cancer cells and / or tumor tissues and / or a partial cell lysate component containing an antigen component with enhanced immunogenicity is loaded inside and / or on the surface of the skeleton structure;

[0115] Among them, the water-soluble components in the whole cell components in the lysate components from the cancer cell and / or tumor tissue and the water-insoluble components dissolved by the lysate containing a solvent act together with the hapten substance to enhance their immunogenicity; the antigen components contained in the partial cell components include protein and polypeptide components in the cancer cell and / or tumor tissue lysate and / or RNA components or mRNA components in the cell lysate, and the antigen components are separated and purified from the lysate using an appropriate method or are separated and purified from the water-soluble components and / or water-insoluble components respectively using an appropriate method, and the partial cell components act together with the hapten substance to enhance their immunogenicity; the lysate components and the hapten substance can act together before the cell or tumor tissue is lysed, or after the cell or tumor tissue is lysed.

[0116] The hapten substance is a substance that can increase the immunogenicity of a protein or polypeptide after co-acting with the protein or polypeptide.

[0117] The hapten substances include but are not limited to 2,4-dinitrofluorobenzene (DNFB), 2,4-dinitrochlorobenzene (DNCB), trinitrophenol (TNP), dinitrophenol (DNP), albumin, Ovalbumin (OVA), N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide (AED), substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, and other hapten substances containing aldehyde groups.

[0118] When preparing a nano vaccine or a micro vaccine, the cancer cells or tumor tissues may be lysed, and then the water-soluble antigen components and the water-soluble antigen components may be collected separately, and then the haptens may be used to modify the above antigen components to enhance their immunogenicity, and then the nano or micro vaccines may be prepared respectively; or the hapten components may be used to modify the cancer cells or tumor tissues first, and then the water-soluble antigen components and the water-soluble antigen components may be collected separately after the cells or tissues are lysed, and then the nano or micro vaccines may be prepared respectively; or the cells or tissues may be directly lysed using a dissolving solution containing a dissolving agent, and the whole cell lysate antigens of the cancer cells may be dissolved using the dissolving solution containing a dissolving agent, and then the whole cell lysate antigens may be modified using a hapten substance to improve their immunogenicity, and then loaded onto the nano vaccine or the micro vaccine; or the hapten substance may be used to co-act with the cells in the cancer cells or tumor tissues to modify the antigen components therein, and then the cells or tissues may be directly lysed using a dissolving solution containing a dissolving agent, and the whole cell lysate antigens of the cancer cells may be dissolved using the dissolving solution containing a dissolving agent, and then loaded onto the nano vaccine or the micro vaccine. The cancer cells and / or tumor tissues disclosed herein can be subjected to treatments including but not limited to inactivation or (and) denaturation, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, heating, salting out, enzyme treatment, oxidation, reduction, mineralization, irradiation, radiation, ionization, chemical modification, nucleic acid separation and purification, protease endo-cut or degradation, nuclease treatment, etc. before or (and) after lysis, and then the protein and polypeptide components therein can be extracted and separated; or the protein and polypeptide components therein can be directly extracted and separated without any inactivation or (and) denaturation, fixation, heating, salting out, oxidation, reduction, mineralization, enzyme treatment, ionization, irradiation, radiation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, chemical modification, protease endo-cut or degradation, nuclease treatment before or (and) after cell lysis. In some embodiments of the present disclosure, tumor tissue cells are inactivated or (and) denatured before lysis. In actual use, they can also be inactivated or (and) denatured after cell lysis, or they can be inactivated or (and) denatured before and after cell lysis; in some embodiments of the present disclosure, the inactivation or (and) denaturation treatment methods before or after cell lysis are ultraviolet irradiation and high temperature heating. In actual use, they can also be treated by methods including but not limited to radiation irradiation, high pressure, nucleic acid separation and purification, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, irradiation, radiation, heating, salting out, oxidation, reduction, enzyme treatment, mineralization, ionization, chemical modification, nuclease treatment, protease endo- or degradation, collagenase treatment, freeze drying, etc. It can be understood by those skilled in the art that in actual application, the technician can make appropriate adjustments according to the specific circumstances.

[0119] The cancer vaccine disclosed in the present invention is characterized in that the preparation method of the whole cell component modified with a hapten comprises: collecting a single cell suspension of cancer cells or tumor tissues, then incubating the single cell suspension of cancer cells or tumor tissues with the hapten for 0.1-24 hours, then centrifuging to remove the supernatant, resuspending the cell precipitate with a solution containing protein, and then incubating for 0.1-24 hours, then centrifuging to remove the supernatant and washing the cells, then lysing the cancer cells and / or tumor tissues, then respectively collecting the water-soluble components and the water-insoluble components in the lysate, dissolving the water-insoluble components with a dissolving solution containing a dissolving agent, and then using them together with the water-soluble components;

[0120] or first using a lysing solution containing a lysing agent to lyse cancer cells and / or tumor tissues, and then using a lysing solution containing a lysing agent to dissolve the whole cell lysate before use;

[0121] Wherein, the dissolving agent is independently selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0122] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0123] Preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

[0124] The cancer vaccine disclosed in the present invention is characterized in that the method for preparing the whole cell component modified with a hapten comprises: collecting cancer cells or tumor tissues, then lysing the cancer cells or tumor tissues, then allowing the lysate to act with the hapten for 0.1-24 hours, then collecting the water-soluble component and the water-insoluble component in the lysate respectively, dissolving the water-insoluble component with a dissolving solution containing a dissolving agent and using it together with the water-soluble component;

[0125] or first use a lysing solution containing a lytic agent for cancer cells or tumor tissues and then use it;

[0126] Wherein, the dissolving agent is independently selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0127] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0128] Preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

[0129] The cancer vaccine disclosed in the present invention is characterized in that the preparation process of the partial cell component containing the antigen component modified by the hapten comprises: collecting a single cell suspension of cancer cells or tumor tissues, and then incubating the single cell suspension of cancer cells or tumor tissues with the hapten for 0.1-24 hours, and then centrifuging to remove the supernatant, resuspending the cell precipitate with a solution containing protein, and then incubating for 0.1-24 hours, and then centrifuging to remove the supernatant and washing the cells, and then lysing the cancer cells and / or tumor tissues to obtain a lysate, and then centrifuging the lysate and collecting the supernatant and precipitate in the lysate respectively, the supernatant is the water-soluble component, and the precipitate is dissolved with a dissolving solution containing a dissolving agent to obtain the dissolved water-insoluble component, and all water-soluble After the components are treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, mineralization, irradiation, etc., the precipitated part is redissolved using a dissolving solution containing a dissolving agent; the water-insoluble part is directly used after being dissolved using a dissolving solution containing a dissolving agent, or the precipitated component is treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation, etc. and then redissolved using a dissolving solution containing a dissolving agent; the protein polypeptide component in the water-soluble component is mixed with the water-insoluble component or the water-insoluble component is purified and then redissolved together with the antigen component to form a partial cell component containing the antigen component;

[0130] or lysing cancer cells and / or tumor tissues with a dissolving solution containing a dissolving agent, dissolving the lysate components with a dissolving solution containing a dissolving agent, and then subjecting the obtained dissolved lysate components to salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation and other appropriate methods, and then re-dissolving the obtained precipitated components with a dissolving solution containing a dissolving agent;

[0131] Wherein, the dissolving agent is independently selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0132] R1 is C, S, P, N or O, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine.

[0133] Compounds containing structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, urea salts, urea, other compounds containing guanidine groups, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0134] Urea and guanidine hydrochloride contain the structure in Structural Formula 1. The inventors have found that substances having the structure in Structural Formula 1 can be used as solvents in a dissolving solution to dissolve water-insoluble components in cells or tumor tissues. Therefore, in addition to common compounds containing a guanidine structure such as urea, guanidine hydrochloride, and metformin, other compounds containing this structure also have the ability to be used as solvents to dissolve water-insoluble components.

[0135] Furthermore, the nano-vaccine or micro-vaccine prepared from the components (hapten-modified) in the cancer cells and / or tumor tissues can be one or two selected from the following: (1) water-soluble components + water-insoluble components modified with hapten; (2) protein polypeptide components + water-insoluble components in the water-soluble components modified with hapten; (3) protein polypeptide components in the water-soluble components modified with hapten + protein polypeptide components in the water-insoluble components; (4) protein polypeptide components and RNA components (or mRNA components) in the water-soluble components modified with hapten + protein polypeptide components and RNA components (or mRNA components) in the water-insoluble components. In some preferred embodiments, the mass ratio of protein polypeptides in the water-soluble components to protein polypeptides in the water-insoluble components / water-insoluble components is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Exemplarily, the mass ratio of protein and polypeptide components in the water-soluble component to the water-insoluble component / protein and polypeptide in the water-insoluble component is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.

[0136] The immunogenic protein and / or polypeptide may be derived from a portion of the components in cancer cells / tumor tissues and extracellular vesicle lysates. Furthermore, the extracellular vesicle lysate is selected from extracellular vesicle lysates of cancer cells and / or extracellular vesicle lysates of bacteria. The mass ratio of a portion of the components in the cancer cells and / or tumor tissues to the extracellular vesicle lysate components is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Exemplarily, the mass ratio is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.

[0137] The immunogenic protein and / or polypeptide may be derived from a portion of the components in the cancer cell / tumor tissue and a bacterial lysate. Further, the mass ratio of the portion of the components in the cancer cell / tumor tissue to the bacterial lysate is (0.1-10):(0.1-10); preferably (0.5-2):(0.5-2). Exemplarily, the mass ratio is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.

[0138] Furthermore, the particle material is PEG-modified or not PEG-modified. Preferably, when preparing the skeleton structure, the mass ratio of the particle material not modified by PEG to the particle material modified by PEG is 25-200:1. In the preparation materials of nano-vaccine or micro-vaccine, an appropriate amount of PEG-modified PLGA or PLA can be added to the main materials such as PLGA or PLA to better achieve the effects of long circulation and passive targeting; wherein the mass ratio of PEG-modified PLGA or PLA to unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.

[0139] The cancer vaccine disclosed herein, wherein, in the vaccine, the mass ratio of the skeleton material, protein and polypeptide components prepared by the particles is 1:0.001-10; preferably, the mass ratio of the skeleton material, protein and polypeptide components prepared by the particles is 1:0.01-2; most preferably, the mass ratio of the skeleton material, protein and polypeptide components prepared by the particles is 1:0.05-1.

[0140] The surface of the nano-vaccine or micro-vaccine can also be loaded with membrane components, which can come from one or more of antigen presenting cells, cancer cells, bacteria or extracellular vesicles.

[0141] Antigen presenting cells used to prepare the biofilm components loaded on the surface of nano- or micro-vaccines can be derived from autologous or allogeneic, or from cell lines or stem cells. Antigen presenting cells can be DC cells, B cells, macrophages, or any mixture of the above three, or other cells with antigen presenting function. Antigen presenting cells can be activated by nanoparticles or microparticles loaded with antigens.

[0142] When the biomembrane components carried on the surface of cancer nano- or micro-vaccines are derived from extracellular vesicles, they may be one or more of the extracellular vesicles of cancer cells, extracellular vesicles of bacteria, or extracellular vesicles of antigen-presenting cells.

[0143] Any method for preparing nanoparticles or micron particles known to those skilled in the art can be used to prepare the nanovaccine or micron vaccine described in the present disclosure, including but not limited to solvent evaporation method, dialysis method, phase separation method, spray drying method, emulsion polymerization method, machine stirring shear method, membrane emulsification method, microfluidics method, ultrafiltration method, homogenization emulsification method, dispersion method, precipitation method, etc.

[0144] In some specific embodiments, the present disclosure takes the solvent volatilization method as an example and provides the following exemplary preparation method:

[0145] Step 1, using a hapten substance to interact with cells in cancer cells or tumor tissues for a certain period of time to modify the antigen components in the cancer cells or tumor tissues, and then lysing the cancer cells and / or tumor tissues to obtain their lysates; or first lysing the cancer cells or tumor tissues to obtain their lysates, and then using a hapten substance to modify the antigen components in the cancer cell or tumor tissue lysates.

[0146] The lysate components of cancer cells or tumor tissues modified with hapten substances can be directly loaded into nano- or micro-vaccines as antigen components, or loaded into nano- or micro-vaccines as antigen components after being properly treated, separated, purified, or further enhanced in immunogenicity.

[0147] When lysing cancer cells or tumor tissues, ultrapure water, aqueous solution, or a dissolving solution containing a dissolving agent can be used to lyse cancer cells and / or tumor tissues; after the cancer cells or tumor tissues are lysed, all the lysate components or the water-insoluble components in the lysate can be dissolved and solubilized using a dissolving solution that also contains a dissolving agent. Cancer cells can be one or more cancer cells or cancer cell lines, or cancer cells obtained by culturing cancer cells in tumor tissues, or cells obtained from circulating tumor cells after amplification; tumor tissues are tumor tissues derived from one or more organisms. The lysis method is a commonly used lysis method for cancer cells and / or tumor tissues, including but not limited to repeated freezing and thawing, swelling, ultrasonic treatment, high pressure treatment, homogenization, extrusion, homogenization, high-speed stirring, chemical treatment, high shear force treatment, ultrafiltration treatment, shrinkage and other treatment methods. One or more.

[0148] The dissolving agent is selected from one or more of a compound containing structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0149] R1 is C, S, P, N or O, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine. Compounds containing the structure of structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, other compounds containing guanidine, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine aminosulfonate, guanidinosuccinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazole, etc.), nitrosoureas, etc.

[0150] The water-soluble components in the lysate modified by the hapten substance can also be treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, mineralization, irradiation, and radiation to obtain a precipitate, and then the precipitate is dissolved using a dissolving solution containing a dissolving agent; the water-insoluble components in the lysate modified by the hapten substance can also be treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, mineralization, irradiation, and radiation to obtain a precipitate, and then the precipitate is dissolved using a dissolving solution containing a dissolving agent; or the water-insoluble components in the lysate are directly dissolved using a dissolving solution containing a dissolving agent without treatment. The dissolving agent in each step is selected separately. The dissolving agent is selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0151] R1 is C, S, P, N or O, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine. Compounds containing the structure of structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, other compounds containing guanidine, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine aminosulfonate, guanidinosuccinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazole, etc.), nitrosoureas, etc.

[0152] Alternatively, the water-soluble components in the lysate modified by the hapten substance can also be treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, mineralization, irradiation, and radiation to obtain two parts, namely, a precipitate and a supernatant, and then the precipitate is dissolved using a dissolving solution containing a dissolving agent, and the mRNA component or RNA component in the supernatant is separated and extracted using an appropriate method, and then the mRNA component or RNA component in the supernatant is mixed with the precipitate dissolved by the dissolving solution containing a dissolving agent for standby use; the water-insoluble components in the lysate are dissolved using a dissolving agent and then treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, mineralization, irradiation, and radiation, and then dissolved using a dissolving solution containing a dissolving agent; or the water-insoluble components in the lysate are dissolved using a dissolving solution containing a dissolving agent and used directly without other treatment. The dissolving agent in each step is selected separately. The dissolving agent is selected from one or more of a compound containing structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0153] R1 is C, S, P, N or O, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine. Compounds containing the structure of structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, other compounds containing guanidine, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine aminosulfonate, guanidinosuccinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazole, etc.), nitrosoureas, etc.

[0154] Alternatively, the lysate component modified by the hapten substance dissolved in the dissolving solution containing the dissolving agent is treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, mineralization, irradiation, and radiation to obtain a precipitate, and then the precipitate is secondary dissolved in the dissolving solution containing the dissolving agent to obtain the antigen component. The dissolving agent is selected from one or more of the compounds containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0155] R1 is C, S, P, N or O, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine. Compounds containing the structure of structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, other compounds containing guanidine, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine aminosulfonate, guanidinosuccinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazole, etc.), nitrosoureas, etc.

[0156] Alternatively, the lysate component modified by the hapten substance dissolved in the dissolving solution containing the dissolving agent is treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, mineralization, irradiation, and radiation to obtain two parts, namely, a precipitate and a supernatant, and then the precipitate part is dissolved in the dissolving solution containing the dissolving agent; an mRNA component or an RNA component is separated from the supernatant using an appropriate method, and then the mRNA component or the RNA component is mixed with the precipitate part dissolved in the dissolving solution containing the dissolving agent to obtain the antigen component. The dissolving agent is selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0157] R1 is C, S, P, N or O, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine. Compounds containing the structure of structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, other compounds containing guanidine, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine aminosulfonate, guanidinosuccinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazole, etc.), nitrosoureas, etc.

[0158] The present disclosure has found through research that substances such as urea, guanidine hydrochloride, and metformin hydrochloride having the structure listed in Structural Formula 1 can be used as dissolving agents to effectively dissolve antigen components. Due to space limitations, only some dissolving agents are listed in the present disclosure. In practical applications, various other substances containing Structural Formula 1 can also be used as dissolving agents, including but not limited to guanidine salts, urea salts, urea, other compounds containing guanidine groups, guanidine aminosulfonates, sulfonylurea compounds (glibenclamide, gliclazide, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, and the like.

[0159] Step 2: Add the antigen component obtained in step 1 as the initial aqueous phase into the organic phase to prepare a colostrum sample.

[0160] During preparation, the initial aqueous phase and the organic phase are mixed, specifically, a first predetermined volume of an aqueous solution containing a first predetermined concentration of an antigen component is added to a second predetermined volume of an organic phase containing a second predetermined concentration of a raw material for preparing particles.

[0161] In some embodiments, the aqueous solution may contain at least one of the following i) to ii): i) each component in the lysate; ii) each component in the lysate and an immunoenhancing adjuvant. The components in the lysate are respectively the protein and polypeptide components in the water-soluble antigen during preparation, or the original water-insoluble antigen or some of its components dissolved in a dissolving solution containing a dissolving agent such as urea or guanidine hydrochloride. The first predetermined concentration is the concentration of proteins and polypeptides contained in the aqueous solution, or the concentration of water-soluble antigens and / or the concentration of original water-insoluble antigens contained in the aqueous solution. The first predetermined concentration requires that the protein and polypeptide concentration content be greater than 1 ng / mL so that sufficient antigen components can be loaded to activate related cells. The concentration of the immunoenhancing adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0162] In some embodiments, the organic solvent is dichloromethane. In addition, in some embodiments, the second predetermined concentration of the raw material for preparing particles ranges from 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0163] In practice, the second predetermined volume of the organic phase is set according to the ratio of the second predetermined volume of the organic phase to the first predetermined volume of the aqueous phase. In the present disclosure, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is in the range of 1:1.1-1:5000, preferably 1:10. In the specific implementation process, the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.

[0164] In some embodiments, when the aqueous phase solution is a solution containing cell and / or tissue lysate components, the concentration of proteins and polypeptides is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL. In some embodiments, when the aqueous phase solution is a solution containing lysate components and immune adjuvants, the concentration of proteins and polypeptides is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of immune adjuvants is greater than 0.01 ng / mL, preferably 0.001 mg / mL to 20 mg / mL. In some embodiments, in the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0165] In some embodiments, when the aqueous phase solution is a solution containing a lysate component, the concentration of the protein and polypeptide components is greater than 0.01 ng / mL, preferably 1 μg / mL to 1 mg / mL. In some embodiments, when the aqueous phase solution is a solution containing protein and polypeptide components and an immune adjuvant, the concentration of the protein and polypeptide components is greater than 1 ng / mL, preferably 1 μg / mL to 1 mg / mL, and the concentration of the immune adjuvant is greater than 0.01 ng / mL, preferably 0.001 mg / mL to 20 mg / mL. In some embodiments, in the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0166] Step 3, subjecting the mixed solution obtained in step 2 to any of the following treatments: i) ultrasonic treatment for more than 2 seconds; ii) stirring for more than 1 minute; iii) homogenization treatment; iv) microfluidic treatment. Preferably, during mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm, and the stirring time is greater than 1 minute, such as a stirring speed of 50 rpm to 1500 rpm, and a stirring time of 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5 W, and the time is greater than 0.1 second, such as 2 to 200 seconds; during homogenization, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and when a high pressure / ultra-high pressure homogenizer is used, the pressure is greater than 5 psi, such as 20 psi to 100 psi, and when a high shear homogenizer is used, the speed is greater than 100 rpm, such as 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, such as 0.1 mL / min-100 mL / min. Nano- and / or micron-size can be achieved by ultrasound, stirring, homogenization or microfluidics. The size of the prepared nanoparticles or micron particles can be controlled by the length of ultrasound time, stirring speed, or homogenization pressure and time. Too large or too small a particle size will result in a change in particle size.

[0167] Step 4, adding the mixture obtained after the treatment in step 3 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of an emulsifier and performing any of the following treatments: i) ultrasonic treatment for more than 2 seconds; ii) stirring for more than 1 minute; iii) homogenization; iv) microfluidic treatment. In this step, the mixture obtained in step 2 is added to the emulsifier aqueous solution and continues to be ultrasonicated or stirred or homogenized or mixed for nano- or micron-ization. In the present disclosure, the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds; the stirring speed is greater than 50 rpm, such as 50 rpm to 500 rpm; the stirring time is greater than 1 minute, such as 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, such as the stirring speed is 50rpm~1500rpm, and the stirring time is 0.5 hour~5 hours; during ultrasonic treatment, the ultrasonic power is 50W~500W, and the time is greater than 0.1 second, such as 2~200 seconds; during homogenization, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and the pressure is greater than 20psi when using a high pressure / ultra-high pressure homogenizer, such as 20psi~100psi, and the speed is greater than 1000rpm when using a high shear homogenizer, such as 1000rpm~5000rpm; the flow rate of microfluidics treatment is greater than 0.01mL / min, such as 0.1mL / min-100mL / min. Ultrasound or stirring or homogenization or microfluidics treatment is carried out for nano- or micronization, and the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared nanoparticles or micron particles, and too large or too small will bring about changes in particle size.

[0168] In certain embodiments, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg / mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present disclosure, the range of the ratio of the second predetermined volume to the third predetermined volume is 1:1.1-1:1000, preferably 2:5. In order to control the size of nanoparticles or micron particles during specific implementation, the ratio of the second predetermined volume to the third predetermined volume can be adjusted. Similarly, the ultrasonic time or stirring time or homogenization time of this step, the volume of the emulsifier aqueous solution and the concentration are based on the values ​​of obtaining nanoparticles or micron particles of suitable size.

[0169] Step 5, adding the liquid obtained after the treatment in step 4 to a fourth predetermined volume of an aqueous emulsifier solution of a fourth predetermined concentration, and stirring until a predetermined stirring condition is met.

[0170] In this step, the emulsifier aqueous solution is a PVA solution or other solutions.

[0171] The fourth predetermined concentration is 5 mg / mL, and nanoparticles or micron particles of suitable size are obtained by selecting the fourth predetermined concentration. The selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume. In the present disclosure, the ratio of the fourth predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. In the specific implementation process, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or micron particles.

[0172] In the present disclosure, the predetermined stirring condition of this step is until the organic solvent is completely volatilized, that is, the dichloromethane in step 1 is completely volatilized.

[0173] Step 6, after the mixed solution that meets the predetermined stirring conditions obtained in step 5 is centrifuged at a speed greater than 100 RPM for more than 1 minute, the supernatant is removed, and the remaining precipitate is re-suspended in a fifth predetermined volume of an aqueous solution containing a fifth predetermined concentration of a lyoprotectant or a sixth predetermined volume of PBS (or saline); or ultrafiltration centrifugation or dialysis that can remove substances with a specific molecular weight is used to remove free PVA and other substances, and at the same time, the solution in the system is replaced with a fifth predetermined volume of an aqueous solution containing a fifth predetermined concentration of a lyoprotectant or a sixth predetermined volume of PBS (or saline).

[0174] Step 7, freeze-drying the suspension containing the lyophilization protectant obtained in step 6, and then keeping the lyophilized material for later use.

[0175] Step 8, resuspending the nanoparticle-containing suspension obtained in step 6 in PBS (or physiological saline) with a sixth predetermined volume, or resuspending the freeze-dried material containing nanoparticles or micron particles and lyoprotectant obtained in step 7 with a sixth predetermined volume of PBS (or physiological saline), and then using it directly; or mixing the above sample with a seventh predetermined volume of water-soluble antigen or dissolved original water-insoluble antigen and then using it.

[0176] In the present disclosure, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1; preferably, the volume ratio is 1:100 to 100:1; most preferably, the volume ratio is 1:30 to 30:1.

[0177] In the present disclosure, in the nano vaccine or micro vaccine, 0.001-2000 μg of protein or polypeptide components are loaded per 1 mg of particle material. In some embodiments, the nano vaccine or micro vaccine also loads an immunopotentiating adjuvant, wherein 1-800 μg of immunopotentiating adjuvant is loaded per 1 mg of particle material. In some embodiments, the nano vaccine or micro vaccine also loads positively charged molecules, wherein 1-800 μg of positively charged molecules are loaded per 1 mg of particle material. In practical applications, the content of antigen components such as proteins and polypeptides loaded per 1 mg of particle material can be even higher.

[0178] In the present disclosure, organic polymer substances are used as particle preparation materials. In practical applications, any other preparation materials that can load antigens and prepare nano- or micron-sized particles can also be used, including but not limited to inorganic materials, viruses, bacteria, and materials from other biological sources (exosomes, extracellular vesicles, bacterial membrane components), etc.

[0179] Any oxidizing agent that can oxidize the antigen component can be used in the present disclosure, including but not limited to hypochlorous acid, sulfates, hydrogen peroxide (hydrogen peroxide), KIO3, KBrO3, chlorine, dichromate, nitric acid, hydrogen peroxide, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, bromine, iodine, perchlorate, permanganate, dichromate, sodium peroxide, oxygen, chlorine, sodium dichromate, potassium dichromate, potassium permanganate, nitric acid, ClO3. - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, H2O2, NO3 - 、MnO4 - , F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3 and other oxidants.

[0180] The enzyme treatment method in the present disclosure includes, but is not limited to, using one or more of nuclease, DNase, pepsin, chymotrypsin, trypsin, other protein digesting enzymes, protease inhibitors, and the like.

[0181] The enzymatic hydrolysis method disclosed in the present invention uses nuclease, pepsin, trypsin, protease inhibitors, etc. In actual use, any other feasible enzymatic hydrolysis method such as chymotrypsin, DNA enzyme, etc. can also be used.

[0182] Due to space limitations, the methods of mineralization are not listed in the embodiments of the present disclosure. In practical applications, any mineralization method such as silicification, calcification, and magnesiumization may be used.

[0183] Due to space limitations, the examples of reducing antigen components using reducing agents are not listed in the embodiments of the present disclosure. In practical applications, reducing agents can also be used to reduce antigen components and then load the antigen components into nano- or micro-vaccines. Reducing agents include, but are not limited to, dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), and the like.

[0184] The irradiation is one or more treatments using different irradiation methods such as ultraviolet rays, X-rays, gamma rays, alpha rays, beta rays, radioactive sources, etc.

[0185] The chromatography disclosed herein includes, but is not limited to, column chromatography, gas chromatography, high pressure liquid chromatography, adsorption chromatography, partition chromatography, thin layer chromatography, high performance liquid chromatography, ion exchange chromatography, thin film chromatography, affinity chromatography, gel chromatography, and the like.

[0186] The chromatography methods disclosed herein include, but are not limited to, column chromatography, thin layer chromatography, liquid chromatography, gas chromatography, supercritical fluid chromatography, and the like.

[0187] The electrophoresis method disclosed herein includes, but is not limited to, SDS electrophoresis, isoelectric focusing electrophoresis, isotachophoresis, immunoelectrophoresis, serum protein electrophoresis, nucleic acid electrophoresis, DNA sequencing electrophoresis, gel electrophoresis, preparative electrophoresis, and the like.

[0188] The cancer cells or tumor tissues can be co-incubated with specific chemical substances to stimulate the cancer cells or tumor tissues before lysis. The specific substances that stimulate cancer cells include but are not limited to small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, cytokines, plant extracts (such as important extracts such as ginseng, plant rhizome extracts, etc.), chemokines, interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate cancer cells or tumor tissues to stimulate cancer cells or tumor tissues is to make the cancer cells produce more antigen components.

[0189] In the present disclosure, the interior and / or surface of the nanovaccine or microvaccine may also contain membrane components, and the membrane components inside and / or on the surface of the nanovaccine or microvaccine are one or more selected from the group consisting of the cell membrane of antigen-presenting cells, the extracellular vesicles of antigen-presenting cells, the cell membrane of cancer cells, the extracellular vesicles of cancer cells, the cell membrane of bacteria, and the extracellular vesicles of bacteria.

[0190] When the membrane component is located on the surface of the nanovaccine or microvaccine, the method of loading the membrane component onto the surface of the nanovaccine or microvaccine includes but is not limited to one or more of ultrasound, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bond connection, stirring, dialysis, homogenization and homogenization.

[0191] In some exemplary embodiments of the present disclosure, a solvent volatilization method is used to prepare antigen delivery particles. In practical applications, any other method for preparing antigen delivery particles may also be used, including but not limited to precipitation method, dialysis method, dispersion method, microfluidics method, high-pressure homogenization method, stirring method, spray drying method, phase separation method, electrostatic spraying method, emulsion polymerization method, machine stirring shear method, membrane emulsification method, etc.

[0192] Nanoparticles / microparticles can also be bacteria and viruses. When the particles are bacteria and viruses, the bacteria and viruses can express tumor-specific antigens and / or tumor-associated antigens, or the bacteria and viruses contain DNA and / or mRNA that can express tumor-specific antigens and / or tumor-associated antigens.

[0193] Step 9, using the nano vaccine or micro vaccine prepared in step 8 to prevent or treat diseases such as cancer.

[0194] Example 1 Nano-vaccine for the treatment of pancreatic cancer

[0195] In this embodiment, the antigen component comes from the Pan02 mouse pancreatic cancer cell line, and the organic polymer material PLGA is used as the nanoparticle skeleton material, and Polyinosinic-polycytidylic acid (poly(I:C)), CpG7909 (i.e., CpG2006) and CpG2395 are used as immune adjuvants to prepare the nanovaccine using the solvent evaporation method.

[0196] (1) Preparation of antigen components

[0197] Collect 1.5×10 7Pan 02 mouse pancreatic cancer cells were resuspended in 1 mL PBS and irradiated with gamma rays for 2 hours, and then 100 μL of PBS containing 0.2 mM 2,4-dinitrofluorobenzene (DNFB) was added and acted for 1 hour. Then centrifuged at 400g for 5 minutes, the supernatant was removed, the cell pellet was resuspended in PBS with 5% BSA and allowed to stand for 30 minutes, and then centrifuged at 400g for 5 minutes. After removing the supernatant, the cell pellet was washed twice with PBS, an appropriate amount of ultrapure water was added, and the cells were repeatedly frozen and thawed 5 times (with ultrasound) to lyse the cancer cells. Then the lysate was centrifuged at 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a soluble component in 8M urea aqueous solution. The above-mentioned water-soluble component and water-insoluble component were mixed in a mass ratio of 1:2 and used as antigen component 1 for preparing nanovaccine 1.

[0198] Collect 1.5×10 7 Pan 02 mouse pancreatic cancer cells were resuspended in 1 mL PBS and irradiated with gamma rays for 2 hours, and then 100 μL PBS was added and acted for 1 hour. Then centrifuged at 400g for 5 minutes, the supernatant was removed, the cell pellet was resuspended in 5% BSA PBS and allowed to stand for 30 minutes, and then centrifuged at 400g for 5 minutes. After removing the supernatant, the cell pellet was washed twice with PBS, an appropriate amount of ultrapure water was added, and the cells were repeatedly frozen and thawed 5 times (with ultrasound) to lyse the cancer cells. Then the lysate was centrifuged at 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the insoluble component in pure water was converted into soluble in 8M urea aqueous solution. The above-mentioned water-soluble component and the water-insoluble component were mixed in a mass ratio of 1:2 and used as antigen component 2 for preparing nanovaccine 2.

[0199] (2) Preparation of Nanovaccines

[0200] Nano vaccine 1 (Nanovaccine 1) in the present embodiment adopts the emulsion method in the solvent evaporation method to prepare. The vaccine preparation material PLGA molecular weight adopted is 10KDa-20KDa, and the immunoadjuvant adopted is poly (I: C), CpG7909 and CpG2395. Preparation method As mentioned above, in the preparation process, first adopt the emulsion method to load antigen component 1 and adjuvant in nano particle inside, then 100mg nano particle is centrifuged at 12000g for 30 minutes, and uses 10mL to contain 4% trehalose ultrapure water resuspending back freeze drying 48h. This nano particle 1 mean particle size is about 250nm, and every 1mg PLGA nano particle is about loaded with 1.5mg protein and polypeptide components, and each 0.05mg of poly (I: C), CpG7909 and CpG2395 is loaded.

[0201] Nano vaccine 2 (Nanovaccine 2) adopts the emulsion method in the solvent evaporation method to prepare in the present embodiment. The vaccine preparation material PLGA molecular weight adopted is 10KDa-20KDa, and the immunoadjuvant adopted is poly (I: C), CpG7909 and CpG2395. The preparation method is as previously mentioned, in the preparation process, first adopts the emulsion method to load antigen component 2 and adjuvant in nano particle inside, then 100mg nano particle is centrifuged at 12000g for 30 minutes, and uses 10mL to contain the ultrapure water resuspending of 4% trehalose and freeze drying 48h. This nano particle 2 mean particle diameter is about 250nm, and every 1mg PLGA nano particle is about loaded with 1.5mg protein and polypeptide components, and each 0.05mg of load poly (I: C), CpG7909 and CpG2395.

[0202] (3) Nano-vaccines for cancer treatment

[0203] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare pancreatic cancer tumor-bearing mice. On day 0, 1.5×10 6 Pan 02 cells. On the 3rd, 6th, 9th, 14th, 20th and 27th days after the mice were inoculated with tumors, 2 mg of nanovaccine 1 or 2 mg of nanovaccine 2 or 100 μL PBS were injected subcutaneously into the mice. The tumor growth rate and survival of the mice were monitored. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0204] (4) Experimental results

[0205] like Figure 3 As shown in the figure, the tumor volume of mice in the PBS group grew rapidly and the mice died soon. The tumor growth rate of mice using Nanovaccines 1 (Nanovaccine 1) and 2 (Nanovaccine 2) was significantly slowed down, the survival period was significantly prolonged, and most mice recovered without tumors. Moreover, Nanovaccines 1 was more effective than Nanovaccines 2, indicating that the whole cell lysate components of cancer cells modified with hapten were more effective after being loaded into Nanovaccines.

[0206] Example 2 Micron vaccine for preventing brain cancer

[0207] (1) Preparation of antigen components

[0208] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 6 GL261 cells, and when the tumors grew to a volume of approximately 1000 mm 3 The mice were killed and the tumor tissue was removed. A single cell suspension of the tumor tissue was prepared, and then the cells were cultured in RPMI1640 (containing 10% FBS) complete medium for 7 days (37°C, 5% CO2). After the culture was completed, the cancer cells were collected and centrifuged at 400g for 5 minutes to remove the medium. The precipitated cancer cells were resuspended in an appropriate amount of ultrapure water and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy and lyse the cancer cells. 100 μL of PBS containing 0.2mM 2,4-dinitrochlorobenzene (DNCB) was added to 5mL of lysate and acted for 0.2 hours. Then, 5mL of the same amount of unmodified cancer cell lysate was added to the modified cancer cell lysate and allowed to stand for 0.5 hours. Then, the above mixture was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 6M guanidine hydrochloride aqueous solution was added to the obtained precipitate to dissolve the precipitate to convert the insoluble antigen in pure water into a soluble antigen in 6M guanidine hydrochloride aqueous solution. The water-soluble component and the water-insoluble component were mixed at a mass ratio of 3:1 and used as antigen component 1.

[0209] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 6 GL261 cells, and when the tumors grew to a volume of approximately 1000 mm 3The mice were killed and the tumor tissue was removed. A single cell suspension of the tumor tissue was prepared, and then the cells were cultured in RPMI1640 (containing 10% FBS) complete medium for 7 days (37°C, 5% CO2). After the culture was completed, the cancer cells were collected and centrifuged at 400g for 5 minutes to remove the medium. The precipitated cancer cells were resuspended in an appropriate amount of ultrapure water and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy and lyse the cancer cells. 100μL of PBS was added to 5mL of lysate and acted for 0.2 hours, and then 5mL of the same amount of cancer cell lysate was added and allowed to stand for 0.5 hours. The above mixture was then centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 6M guanidine hydrochloride aqueous solution was added to the obtained precipitate to dissolve the precipitate to convert the insoluble antigen in pure water into a soluble antigen in 6M guanidine hydrochloride aqueous solution. The water-soluble component and the water-insoluble component were mixed at a mass ratio of 3:1 and used as antigen component 2.

[0210] (2) Preparation of micronized vaccines

[0211] In this embodiment, micron vaccine 1 (Micronvaccine 1) is prepared by the emulsion method in the solvent evaporation method. The molecular weight of the micron particle preparation material PLA used is 30KDa-50KDa, and the immune adjuvants used are poly(I:C), CpG1018 and CpG2395. The preparation method is as described above. In the preparation process, the antigen component 1 and adjuvant are first loaded inside the micron particle, and then 100mg of micron particles are centrifuged at 8000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is used to resuspend and freeze-dry for 48h. The average particle size of the micron vaccine 1 is about 2.5μm, and each 1mg PLGA micron particle is loaded with about 20μg of protein or polypeptide components, and each 1mg PLGA micron particle is loaded with 0.01mg of poly(I:C), CpG 1018 and CpG2395.

[0212] In this embodiment, micron vaccine 2 (Micronvaccine 2) is prepared by the emulsion method in the solvent evaporation method. The molecular weight of the micron particle preparation material PLA used is 30KDa-50KDa, and the immune adjuvants used are poly (I: C), CpG1018 and CpG2395. The preparation method is as described above. In the preparation process, the antigen component 2 and adjuvant are first loaded inside the micron particle, and then 100mg of micron particles are centrifuged at 8000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is used to resuspend and freeze-dry for 48h. The average particle size of the micron vaccine 2 is about 2.5μm, and each 1mg PLA micron particle is loaded with about 20μg of protein or polypeptide components, and each 1mg PLA micron particle is loaded with 0.01mg of poly (I: C), CpG 1018 and CpG2395.

[0213] (3) Micron vaccines for cancer prevention

[0214] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. 4 mg of micron vaccine 1 or 4 mg of micron vaccine 2 or 100 μL PBS were subcutaneously injected into the mice on days -35, -28, -21, -14 and -7 before the mice were inoculated with tumors. On day 0, 1.5×10 6 The methods for monitoring the tumor growth rate and survival of mice were the same as above.

[0215] (9) Experimental results

[0216] like Figure 4 As shown, the tumor volume of mice in the PBS group grew rapidly and the mice died soon. The tumor growth rate of mice using Micronvaccine 1 and Micronvaccine 2 was significantly slowed down, the survival period was significantly prolonged, and most mice were tumor-free. Among them, Micronvaccine 1 was more effective than Micronvaccine 2, indicating that the effect of the whole cell lysate component of cancer cells modified with hapten was better after being loaded into the nanovaccine. .

[0217] Example 3 Nano-vaccine for the treatment of liver cancer

[0218] (1) Preparation of antigen components

[0219] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 Hepa 1-6 liver cancer cells, when the tumor grows to a volume of about 1000mm 3 The mice were killed and the tumor tissues were removed and irradiated under X-ray for 20 minutes, then a single cell suspension of the tumor tissue was prepared, and then 1 mL of 4% paraformaldehyde was added to 2 mL of the single cell suspension and acted for 1 hour, then centrifuged at 400 g for 5 minutes, the supernatant was discarded and the cell pellet was resuspended in 50 mL of PBS containing 20% ​​bovine serum albumin (BSA), then allowed to stand for 40 minutes, and then centrifuged at 400 g for 5 minutes, the supernatant was removed and the cell pellet was washed twice with PBS, and then 5 mL of 8M urea aqueous solution was added to lyse the cancer cells and then the cancer cell lysate component was dissolved. The lysate component dissolved in the 8M urea aqueous solution was then added to 95 mL of saturated ammonium sulfate aqueous solution to salt out the protein polypeptide component, and the protein polypeptide component precipitated after salting out was dissolved again with 8M urea to prepare the antigen component 1 of the nano vaccine 1.

[0220] (2) Preparation of Nanovaccines

[0221] In the present embodiment, nano vaccine 1 (Nanovaccine 1) is prepared by the emulsion method in the solvent evaporation method. The molecular weight of the nano vaccine preparation material PLGA used is 10KDa-20KDa, the molecular weight of PEG5000-PLGA is 15KDa-25KDa, and the mass ratio of PLGA and PEG5000-PLGA used is 99:1. The immunoadjuvants used are poly(I:C), CpG 2395 and CpG SL03. The preparation method is as described above. During the preparation, the cell antigen component 1 and adjuvant are first loaded inside the nanoparticles, and then 100mg of nanoparticles are centrifuged at 12000g for 30 minutes, and 10mL of ultrapure water containing 4% trehalose is resuspended and freeze-dried for 48h. The average particle size of the nanoparticle 1 is about 300nm, and each 1mg of PLA nanoparticles is about loaded with 600μg of protein or polypeptide components, and each of poly(I:C), CpG 2395 and CpG SL03 is loaded with 0.1mg.

[0222] (3) Nano-vaccines for cancer treatment

[0223] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 6 Hepa1-6 liver cancer cells were grown in the mice. On the 3rd, 6th, 9th, 14th, 19th and 25th days after tumor inoculation, 100 μL of 2 mg nanovaccine 1 or 100 μL of PBS was injected subcutaneously into the mice. The tumor growth rate and survival period of mice were monitored in the same way as above.

[0224] (4) Experimental results

[0225] like Figure 5 As shown, the tumor volume of mice in the PBS group grew rapidly and the mice died soon. The survival time of mice using Nanovaccin 1 was significantly prolonged, and most of the mice recovered without tumor.

[0226] Example 4 Nano-vaccine for the treatment of colon cancer

[0227] (1) Preparation of antigen components

[0228] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 MC38 colon cancer cells, when the tumor grows to a volume of about 1000mm 3The mice were killed and the tumor tissues were removed, and then a single-cell suspension of the tumor tissue was prepared. Then, 1 mL of 10% formaldehyde was added to 2 mL of the single-cell suspension and allowed to act for 1 hour. Then, the suspension was centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 50 mL of PBS containing 20% ​​BSA, and then allowed to stand for 40 minutes. Then, the supernatant was removed and the cell pellet was washed twice with PBS. Then, 5 mL of 8 M urea aqueous solution was added to lyse the cancer cells and the cancer cell lysate component was dissolved to prepare the antigen component 1 of the nanovaccine 1.

[0229] (2) Preparation of Nanovaccines

[0230] Nano vaccine 1 (Nanovaccine 1) in the present embodiment is prepared by the multiple emulsion method in the solvent evaporation method. The molecular weight of the nano vaccine preparation material PLGA adopted is 20KDa-40KDa. The immunoadjuvant adopted is poly(I:C), CpG1018 and CpG 7909. The preparation method is as mentioned above. During the preparation, the cell antigen component 1 and adjuvant are first loaded inside the nano particle, and then 100mg nano particles are centrifuged at 12000g for 30 minutes, and 10mL ultrapure water containing 6% trehalose is resuspended and freeze-dried for 48h, and then irradiated with gamma rays for 16 hours. The average particle size of the nano particle 1 is about 230nm, and each 1mg PLA nano particle is loaded with about 200μg protein or polypeptide components, and each 0.02mg of poly(I:C), CpG 1018 and CpG 7909 is loaded.

[0231] (3) Nano-vaccines for cancer treatment

[0232] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 6 MC38 colon cancer cells were inoculated in the mice. On the 3rd, 6th, 9th, 14th, 19th and 25th days after the mice were inoculated with tumors, 100 μL of 4 mg nanovaccine 1 or 100 μL of PBS was injected subcutaneously into the mice. The tumor growth rate and survival period of the mice were monitored in the same way as above.

[0233] (4) Experimental results

[0234] like Figure 6 As shown, the tumor volume of mice in the PBS group grew rapidly and the mice died soon. The survival time of mice using Nanovaccin 1 was significantly prolonged, and most of the mice recovered without tumor.

Claims

1. A cancer vaccine, characterized in that The cancer vaccine comprises: (i) a nanoparticle and / or micron particle skeleton structure formed by a particle material, and (ii) a whole cell lysate component from cancer cells and / or tumor tissues and / or a partial cell lysate component containing an antigen component modified with a hapten substance; wherein the whole cell lysate component from cancer cells and / or tumor tissues and / or the partial cell lysate component containing an antigen component that enhances immunogenicity are loaded inside and / or on the surface of the skeleton structure; Among them, the water-soluble components in the whole cell components in the lysate components from the cancer cells and / or tumor tissues and the water-insoluble components dissolved by the lysate containing the solvent act together with the hapten substance to enhance their immunogenicity; the antigen components contained in the partial cell lysate components include the protein and polypeptide components in the cancer cell and / or tumor tissue lysate and / or the RNA component or mRNA component in the cell lysate, the antigen components are separated and purified from the lysate using an appropriate method or are separated and purified from the water-soluble components and / or the water-insoluble components respectively using an appropriate method, and the partial cell lysate components act together with the hapten substance to enhance their immunogenicity; the lysate components act together with the hapten substance before the cell or tumor tissue is lysed, and can also be after the cell or tumor tissue is lysed.

2. The hapten substance is a substance that can increase the immunogenicity of a protein or polypeptide after co-acting with the protein or polypeptide.

3. The hapten substances include but are not limited to 2,4-dinitrofluorobenzene, 2,4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, Ovalbumin (OVA), N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, and other hapten substances containing aldehyde groups.

4. The cancer vaccine according to claim 1, characterized in that The method for preparing the whole cell component modified with hapten comprises: collecting a single cell suspension of cancer cells or tumor tissues, then incubating the single cell suspension of cancer cells or tumor tissues with the hapten for 0.1-24 hours, then centrifuging to remove the supernatant, resuspending the cell precipitate with a solution containing protein, and then incubating for 0.1-24 hours, then centrifuging to remove the supernatant and washing the cells, then lysing the cancer cells and / or tumor tissues, then respectively collecting the water-soluble components and the water-insoluble components in the lysate, dissolving the water-insoluble components with a dissolving solution containing a dissolving agent, and then using them together with the water-soluble components; or first using a lysing solution containing a lysing agent to lyse cancer cells and / or tumor tissues, and then using a lysing solution containing a lysing agent to dissolve the whole cell lysate before use; Wherein, the dissolving agent is independently selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows: R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl; Preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

5. The cancer vaccine according to claim 1 or 2, characterized in that The preparation process of the hapten-modified partial cell component containing the antigen component comprises: collecting a single cell suspension of cancer cells or tumor tissues, then incubating the single cell suspension of cancer cells or tumor tissues with the hapten for 0.1-24 hours, then centrifuging to remove the supernatant, resuspending the cell precipitate with a solution containing protein, and then incubating for 0.1-24 hours, then centrifuging to remove the supernatant and washing the cells, then lysing the cancer cells and / or tumor tissues to obtain a lysate, then centrifuging the lysate and collecting the supernatant and precipitate in the lysate respectively, the supernatant is the water-soluble component, the precipitate is dissolved with a dissolving solution containing a dissolving agent to obtain the dissolved water-insoluble component, and all the water-soluble components are subjected to salting out, heating, and filtration. , enzyme treatment, oxidation, reduction, fixation, mineralization, irradiation and other appropriate methods, the precipitated part is redissolved with a dissolving solution containing a dissolving agent; the water-insoluble part is directly used after being dissolved with a dissolving solution containing a dissolving agent, or is dissolved with a dissolving solution containing a dissolving agent and then treated with salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation and other appropriate methods, and then the obtained precipitated component is redissolved with a dissolving solution containing a dissolving agent; the protein polypeptide component in the above-mentioned water-soluble component is mixed with the water-insoluble component or together with the antigen component that is redissolved after purification of the water-insoluble component to form a partial cell component containing the antigen component; or lysing cancer cells and / or tumor tissues with a dissolving solution containing a dissolving agent, dissolving the lysate components with a dissolving solution containing a dissolving agent, and then subjecting the obtained dissolved lysate components to salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation and other appropriate methods, and then re-dissolving the obtained precipitated components with a dissolving solution containing a dissolving agent; Wherein, the dissolving agent is independently selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows: R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl; Preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

6. The cancer vaccine according to any one of claims 1 to 3, characterized in that The cancer cells are from one or more organisms, or from one or more cancer cell lines; the tumor tissues are from one or more organisms; the protein and polypeptide components and RNA components / mRNA components in the water-soluble components of the cancer cells and / or tumor tissues and the protein and polypeptide components and RNA components / mRNA components in the water-insoluble components / water-insoluble components of the cancer cells and / or tumor tissues contain antigen components; the cancer-specific and / or related antigen polypeptides and / or nucleic acids that can express cancer-specific and / or related antigen polypeptides are custom-synthesized in vitro, mixed with components from cancer cell / tumor tissue lysates, and then co-loaded into a nanovaccine or micron vaccine.

7. The cancer vaccine according to any one of claims 1 to 4, characterized in that The cancer vaccine is further loaded with at least one component as shown below: (iv) RNA components or mRNA components in the water-soluble component and / or the water-insoluble component; (v) immune adjuvants; (vi) substances with a positive charge, Preferably, the immune adjuvant comprises at least one of the following: pattern recognition receptor agonists, Toll-like receptor agonists, Bacillus Calmette-Guérin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG cell wall acyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhanced reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn calf liver active peptide, imiquimod, polysaccharides, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme QIO, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokine, mRNA, MF59, double-stranded RNA, double-stranded DNA, single-stranded DNA, aluminum adjuvant, manganese adjuvant, calcium adjuvant, STING agonist, endotoxin adjuvant, liposome adjuvant, CAF01, ginseng active ingredients, Astragalus membranaceus active ingredients; Preferably, the immune adjuvant comprises at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist; More preferably, the immune adjuvant comprises at least one of Poly(I:C), Poly ICLC, Class A CpG-OND, Class B CpG-OND and Class C CpG-OND; Preferably, the positively charged substance is selected from positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and / or positively charged inorganic substances; Preferably, the positively charged substance is selected from any one or more of bee venom peptide, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

8. The cancer vaccine according to any one of claims 1 to 5, characterized in that The mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-1:10; Preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-1:2; Most preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.05-1:

1.

9. The cancer vaccine according to any one of claims 1 to 6, characterized in that The mass ratio of the particle skeleton structure to the RNA component or the mRNA component is 1:0.001-1:10; Preferably, the mass ratio of the particle skeleton structure to the RNA component or mRNA component is 1:0.01-1:2; Most preferably, the mass ratio of the particle skeleton structure to the RNA component or the mRNA component is 1:0.05-1:

1.

10. The cancer vaccine according to any one of claims 1 to 9, characterized in that The dissolving agent is independently selected from one or more of a compound containing a structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, a protein degrading enzyme, a polypeptide, an amino acid, a glycoside and choline; wherein structural formula 1 is as follows: R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl; Preferably, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

11. The cancer vaccine according to any one of claims 1 to 17, characterized in that The cancer vaccine is a nano vaccine and / or a micro vaccine; Wherein, the particle size of the nano vaccine is 1nm-1000nm; preferably 50-500nm; more preferably 100-400nm; Wherein, the particle size of the micron vaccine is 1 μm-1000 μm; preferably 1-10 μm; more preferably 1-5 μm; Wherein, when a mixed vaccine of nano vaccine and micro vaccine is used, the particle size of the nano vaccine is 100-600nm, and the particle size of the micro vaccine is 1.5-5μm; preferably, the particle size of the nano vaccine is 150-500nm, and the particle size of the micro vaccine is 2.0-3.5μm.

12. Use of the cancer vaccine according to any one of claims 1 to 9 in at least one of the following (1) to (3): (1) Preparation of drugs for preventing or treating diseases; (2) preparing a medicament for inducing an immune response in a subject; (3) Preparation of cancer vaccines; Optionally, the disease is cancer or tumor; Optionally, the cancer or tumor is a solid tumor or a hematological tumor.

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