A cancer vaccine based on cancer cell and / or tumor tissue lysate components and synthetic cancer antigens and its preparation method

Abstract

The present invention relates to a vaccine system based on whole cell components / whole antigen components in cancer cells and / or tumor tissues and tumor-specific antigens or tumor-associated antigens, as well as its preparation method and application. First, the whole cell components or whole cell antigen components in cancer cells and / or tumor tissues are obtained, and then the above substances are mixed with known tumor-specific / associated antigen polypeptides or nucleic acids expressing them and co-loaded on nanoparticles or micron particles for the prevention or treatment of cancer. The vaccine system provided by the present invention comprises nanoparticles and / or micron particles, whole cell components / whole antigen components in cancer cells and / or tumor tissues, and prepared quantitative tumor-specific / associated antigen polypeptides / nucleic acids, which can efficiently activate cancer cell-specific immune responses and can be used for the prevention and treatment of diseases such as cancer.

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 vaccines are an important approach to cancer immunotherapy. They primarily consist of antigens and adjuvants, with antigens being the primary component that triggers a specific immune response and the most important component in cancer vaccines. Cancer cells and / or tumor tissues contain a full range of cancer cell-specific and cancer cell-associated antigens, making them the best raw materials for preparing cancer vaccines. Cancer cells and / or tumor tissues contain a wide variety of cancer antigens, the most important of which are proteins and peptides. Cancer antigens can be broadly categorized into two main groups: antigens that have been specifically studied and identified, and antigens that have not been clearly studied and identified. Specifically, antigens that have been specifically studied and identified can be further divided into cancer-specific antigens and cancer-associated antigens. Currently available cancer vaccines either exclusively use cancer-specific antigens (neoantigens), exclusively use cancer-associated antigens, or exclusively use a mixture of antigens that have not been clearly studied and identified. How to rationally use and optimize the combination of antigens that have been specifically studied and identified and antigens that have not been clearly studied and identified is a challenge facing cancer vaccines. Summary of the Invention

[0003] Problems to be solved by the invention

[0004] In order to solve the above technical problems, the present disclosure provides a cancer nano / micro vaccine that uses a method for simultaneously loading cancer cells and / or tumor tissue components with cancer-specific and / or related antigens and a method for preparing the same. The present disclosure loads whole-cell antigen components in cancer cells and / or tumor tissues, and cancer-specific antigens and / or cancer-related antigens separately or simultaneously on the interior and / or surface of a nanoparticle or microparticle skeleton to prepare a cancer nano vaccine or micro vaccine. In the present disclosure, the whole-cell antigen component is mixed with some cancer-specific / related antigens that have been clearly studied and identified, and then loaded on a nano vaccine or micro vaccine, thereby increasing the content of antigen components in a portion of cancer cells and / or tumor tissues, which can improve the overall efficacy of the cancer nano vaccine or micro 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 microparticle skeleton structure formed of a particle material, (ii) whole cell components of cancer cells and / or tumor tissues and / or partial cell components containing antigen components, and (iii) cancer-specific and / or related antigen polypeptides and / or nucleic acids capable of expressing cancer-specific and / or related antigen polypeptides; wherein the whole cell components of cancer cells and / or tumor tissues and / or partial cell components containing antigen components, and the cancer-specific and / or related antigen polypeptides and / or nucleic acids capable of expressing cancer-specific and / or related antigen polypeptides are simultaneously loaded inside and / or on the surface of the skeleton structure;

[0007] The whole cell component contains water-soluble components in the lysate components of cancer cells and / or tumor tissues and water-insoluble components dissolved using a dissolving solution containing a dissolving agent, the antigen components contained in the partial cell components include protein and polypeptide components in the lysate of cancer cells and / or tumor tissues and / or RNA components or mRNA components in the cell lysate, and the antigen components are obtained by separating and purifying from the lysate using an appropriate method or by separating and purifying from the water-soluble components and / or water-insoluble components respectively using an appropriate method.

[0008] The nucleic acid that can express cancer-specific and / or cancer-related antigen polypeptides is mRNA or DNA.

[0009] Preferably, the method for preparing the whole cell fraction 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 using a dissolving solution containing a dissolving agent and using the water-soluble components together;

[0010] Alternatively, cancer cells and / or tumor tissues may be first lysed using a lysis solution containing a lysis agent, and then the whole cell lysate may be dissolved using a lysis solution containing a lysis agent before use;

[0011] Wherein, the dissolving agent is independently selected from one or more of a compound containing a 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:

[0012] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0013] 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.

[0014] Preferably, the preparation process of the partial cell component containing the antigen component comprises: first lysing the cancer cells and / or tumor tissue, then collecting the water-soluble components and the water-insoluble components in the lysate separately, treating all the water-soluble components with appropriate methods such as salting out, heating, enzyme treatment (such as enzymatic hydrolysis and enzyme inhibition, etc.), oxidation, reduction, fixation, mineralization, irradiation, etc., and then re-dissolving the precipitated part with a dissolving solution containing a dissolving agent; the water-insoluble part is dissolved with a dissolving solution containing a dissolving agent, or after being dissolved with a dissolving solution containing a dissolving agent, it is treated with 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 re-dissolving the obtained precipitated component 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 re-dissolved after purification of the water-insoluble component to constitute the whole antigen component in the whole cell component;

[0015] Alternatively, the cancer cells and / or tumor tissues are lysed using a dissolving solution containing a dissolving agent, and the lysate components are dissolved using the dissolving solution containing the dissolving agent. The resulting dissolved lysate components are then treated by appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, and irradiation, and the resulting precipitated components are then redissolved a second time using the dissolving solution containing the dissolving agent.

[0016] 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 an RNA isolation kit, use of an mRNA isolation kit, oxidation, reduction, mineralization, and the like.

[0017] 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.

[0018] The enzymes used for enzymatic hydrolysis include, but are not limited to, one or more of nuclease, pepsin, trypsin, protease inhibitors, chymotrypsin, DNA enzyme, and the like.

[0019] 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 and other types of oxidants or one or more.

[0020] The oxidation can enhance the immunogenicity of some antigenic components.

[0021] Among them, the mineralization method includes but is not limited to using one or more mineralization methods such as silicification, calcification, magnesiumization, and biomineralization.

[0022] 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.

[0023] 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.

[0024] The irradiation is any commonly used irradiation method, including but not limited to one or more of radioactive material irradiation, ultraviolet irradiation, X-ray irradiation, α-ray irradiation, β-ray irradiation, and γ-ray irradiation.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Wherein, the dissolving agent is independently selected from one or more of a compound containing a 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:

[0029] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0030] 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.

[0031] 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 tissue 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.

[0032] Preferably, the cancer cells are derived from one or more organisms, or from one or more cancer cell lines; the tumor tissues are derived 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 / 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 the nanovaccine or microvaccine. The nucleic acid is mRNA or DNA.

[0033] Preferably, the cancer vaccine is further loaded with at least one component as shown below:

[0034] (iv) mRNA component or RNA component in the water-soluble component and / or the water-insoluble component;

[0035] (v) immune adjuvants;

[0036] (vi) positively charged substances,

[0037] 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 muramyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhancing reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn calf liver active peptide, imiquimod, polysaccharide, 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, active ingredients of Panax ginseng, active ingredients of Astragalus membranaceus;

[0038] Preferably, the immune adjuvant comprises at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist;

[0039] 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;

[0040] 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;

[0041] More preferably, the positively charged substance is selected from any one or more of melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

[0042] Preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-1:10;

[0043] More preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-1:2;

[0044] Most preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.05-1:1.

[0045] Preferably, the mass ratio of the particle skeleton structure to the RNA component or mRNA component is 1:0.001-1:10;

[0046] More preferably, the mass ratio of the particle skeleton structure to the RNA component or mRNA component is 1:0.01-1:2;

[0047] Most preferably, the mass ratio of the particle skeleton structure to the RNA component or mRNA component is 1:0.05-1:1.

[0048] Preferably, the protein and polypeptide components and RNA component / mRNA component are derived from lysate, and the cancer-specific and / or related antigen polypeptide / nucleic acid component is artificially synthesized;

[0049] Preferably, the mass ratio of the protein and polypeptide components and the RNA component / mRNA component to the cancer-specific and / or related antigen polypeptide / nucleic acid component is 1:0.001-1:10;

[0050] 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;

[0051] Most 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 1:0.05-1:1.

[0052] Preferably, the cancer vaccine further contains at least one component as shown below:

[0053] (a) cancer cell membrane components derived from tumor tissue and / or tumor cells;

[0054] (b) extracellular vesicle membrane fractions derived from extracellular vesicle lysates, wherein the extracellular vesicles are secreted by bacteria or tumor cells;

[0055] (c) bacterial membrane fractions derived from bacterial lysates;

[0056] (d) membrane fractions derived from antigen-presenting cells;

[0057] Preferably, the bacteria include at least one of the following: BCG, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus gestellii, Lactobacillus reuteri, and Lactobacillus rhamnosus.

[0058] Preferably, the process of separating and purifying the antigen component in the water-soluble component and / or the water-insoluble component or separating and purifying the antigen component in the lysate component 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.

[0059] Preferably, the antigen component contains protein and polypeptide components and / or mRNA components (or RNA components).

[0060] Preferably, the steps for preparing the protein and polypeptide components in the water-soluble components of the cancer cells and / or tumor tissues are as follows: first, lysing the cancer cells and / or tumor tissues to obtain a lysate thereof; then separating the water-soluble components and the water-insoluble components in the lysate using one or more methods selected from the group consisting of centrifugation, filtration, dialysis, and ultrafiltration to obtain the water-soluble components and the water-insoluble components in the lysate respectively; then subjecting the obtained water-soluble components in the lysate to salting-out, heating, enzyme treatment, oxidation, reduction, fixation, mineralization, and irradiation treatments to precipitate the protein and polypeptide components therein; and then dissolving 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.

[0061] Wherein, the dissolving agent is independently selected from one or more of a compound containing a 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:

[0062] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0063] 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.

[0064] Preferably, the steps for preparing the protein and polypeptide components in the water-insoluble components of the cancer cells and / or tumor tissues are as follows: first, lysing the cancer cells and / or tumor tissues to obtain a lysate thereof; then separating the water-soluble components and the water-insoluble components in the lysate using one or more methods selected from the group consisting of centrifugation, filtration, dialysis, and ultrafiltration to obtain the water-soluble components and the water-insoluble components in the lysate respectively; then subjecting the obtained water-insoluble components in the lysate to salting-out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation, and the like to precipitate the protein and polypeptide components therein; and then dissolving the precipitated protein and polypeptide components using a dissolving solution containing a dissolving agent to obtain the protein and polypeptide components in the water-insoluble components.

[0065] Wherein, the dissolving agent is independently selected from one or more of a compound containing a 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:

[0066] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0067] 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.

[0068] Preferably, the process of separating and purifying the antigen component in the water-soluble component and / or the water-insoluble component or separating and purifying the antigen component in the lysate component 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.

[0069] Preferably, the cations contained in the reagent used for salting out include: Al 3+ 、Fe 3+ 、Fe 2+ Mg 2+ 、Sn 2+ 、Zn 2 + , Ca 2+ 、Li + 、Na + NH4 + , K + 、Cu 2+ 、Ag + 、Ba 2+ etc.; the anions contained in the reagent used for the salting-out include: Cl - 、SO4 2- 、NO3 - 、CO3 2- 、SiO3 2- 、S2O7 2- 、B4O7 2- PO4 3- RCOO - 、NO2 - 、S2O8 2- 、S 2- 、CrO4 2- 、MnO4 - 、P2O74- wait.

[0070] Preferably, the bacterial lysate and / or extracellular vesicle lysate is obtained by lysing bacteria and / or extracellular vesicles with a lysis solution containing a lysis agent; preferably, the lysis agent is independently 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;

[0071] Wherein, structural formula 1 is as follows:

[0072] 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.

[0073] Preferably, the water-insoluble component, the bacterial lysate or the extracellular vesicle lysate are independently dissolved in a dissolving solution comprising at least one of the following dissolving agents: a compound containing the 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:

[0074] 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.

[0075] Preferably, the particle material is selected from natural polymer materials, synthetic polymer materials and / or inorganic materials.

[0076] 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.

[0077] Preferably, the cancer vaccine is a nanovaccine and / or a microvaccine;

[0078] Wherein, the particle size of the nano vaccine is 1nm-1000nm; preferably 50-500nm; more preferably 100-400nm;

[0079] Wherein, the particle size of the micron vaccine is 1 μm-1000 μm; preferably 1-10 μm; more preferably 1-5 μm.

[0080] Wherein, when a mixed vaccine of nano vaccine and micro vaccine is used, the particle size of the nano vaccine is 100-600 nm, and the particle size of the micro vaccine is 1.5-5 μm; preferably, the particle size of the nano vaccine is 150-500 nm, and the particle size of the micro vaccine is 2.0-3.5 μm.

[0081] 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 microvaccines 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%.

[0082] 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.

[0083] The present invention also provides a method for preparing the cancer vaccine, characterized in that the preparation method comprises the following steps:

[0084] (1) first lysing cancer cells and / or tumor tissues to obtain their lysates;

[0085] (2) then separating the water-soluble component and the water-insoluble component in the lysate by one or more methods such as centrifugation, filtration, dialysis, and ultrafiltration to obtain the water-soluble component and the water-insoluble component, respectively; or directly dissolving the cancer cells and / or tumor tissue using a lytic solution containing a lytic agent to obtain the lysate;

[0086] (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 using a dissolving solution containing a dissolving agent; the water-insoluble components in the lysate are dissolved using 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, After being treated by appropriate methods such as mineralization and irradiation, the lysate is dissolved in a solution containing a dissolving agent, or the water-insoluble components are directly dissolved in a solution containing a dissolving agent for a second time without treatment; or all lysate components dissolved in a solution containing a dissolving agent 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. to obtain a precipitate, and the precipitate is then dissolved in a solution containing a dissolving agent for a second time; wherein the dissolving agent in each step is selected separately;

[0087] (4) loading the protein polypeptide component and / or the water-insoluble component in the obtained water-soluble component or the protein polypeptide component therein into the interior and / or surface of nanoparticles or microparticles separately or simultaneously to obtain the nanovaccine or microvaccine.

[0088] The method of isolating and extracting mRNA components or RNA components from cancer cells and / or tumor tissues described in the present invention includes but is not limited to one or more methods such as using an mRNA separation and extraction reagent, using an RNA separation and extraction kit, using a DNA removal kit, and using enzymes to degrade DNA.

[0089] Optionally, the cancer cells or tumor tissues may be co-incubated with specific chemicals to stimulate the cancer cells or tumor tissues before lysis.

[0090] 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 of ginseng, plant root extracts, etc.), interferon, 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.

[0091] 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;

[0092] Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0093] 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.

[0094] 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) to (3):

[0095] (1) Preparation of drugs for preventing or treating diseases;

[0096] (2) preparing a medicament for inducing an immune response in a subject;

[0097] (3) Preparation of cancer vaccines;

[0098] Optionally, the disease is cancer or tumor;

[0099] Optionally, the cancer or tumor is a solid tumor or a hematological tumor.

[0100] Effects of the Invention

[0101] The present disclosure 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

[0102] Figure 1 Schematic diagrams of some feasible preparation processes and applications of nano- or micro-vaccines disclosed herein; among them, a and b are schematic diagrams of preparing nano- or micro-vaccines by collecting water-soluble antigen components and water-insoluble components and then mixing them with artificially synthesized polypeptides or mRNA / RNA; c and d are schematic diagrams of the process of preparing nano- or micro-vaccines by directly lysing whole-cell components of cancer cells and / or partial cell components containing antigen components using a lysis solution containing a dissolving agent and then mixing them with artificially synthesized polypeptides / mRNA (or RNA).

[0103] Figure 2 The structure is structural formula 1, wherein 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 and other groups.

[0104] Figure 3-Figure 19 These are the experimental results of tumor growth rate and survival when nanovaccines and / or micron vaccines are used to prevent or treat cancer in Examples 1-17; 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 when preventing or treating cancer (n≥8), and each data point is the mean ± standard error (mean±SEM); wherein, the significant difference in the tumor growth inhibition experiment in Figure a is analyzed by ANOVA, and the significant difference in Figure b is analyzed by Kaplan-Meier and log-rank test; *** indicates that there is a significant difference compared with the PBS blank control group, p<0.005; ** indicates that there is a significant difference compared with the PBS blank control group, p<0.01; ### indicates that there is a significant difference between the two groups, p<0.005; ## indicates that there is a significant difference between the two groups, p<0.01; # indicates that there is a significant difference between the two groups, p<0.05. DETAILED DESCRIPTION

[0105] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate 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.

[0106] The experimental techniques and methods used in this example are conventional unless otherwise specified. For example, in the following examples, where specific conditions are not specified, the experimental methods were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. The materials and reagents used in the examples were all obtained through regular commercial channels unless otherwise specified.

[0107] In this embodiment, mannose is used as an active targeting target to target dendritic cells. In practical applications, other targeting molecules can also be used, including but not limited to mannan, CD11c, CD103, CD32, CD11b, CD19, CD38, etc.

[0108] The amino acid sequences involved in the present disclosure are as follows:

[0109] R8 polypeptide, RRRRRRRR (SEQ ID NO: 1).

[0110] B16-M20 antigen polypeptide, FRRKAFLHWYTGEAMDEMEFTEAESNM (SEQ ID NO: 2).

[0111] B16-M24 antigen polypeptide, TAVITPPTTTTKKARVSTPKPATPSTD (SEQ ID NO: 3).

[0112] B16-M46 antigen polypeptide, NHSGLVTFQAFIDVMSRETTDTDTADQ (SEQ ID NO: 4).

[0113] TRP2:180-188 antigen polypeptide, SVYDFFVWL (SEQ ID NO: 5).

[0114] Melittin, GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 6).

[0115] KALA polypeptide, WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 7).

[0116] RALA polypeptide, WEARLARALARALARHLARARALALRACEA (SEQ ID NO: 8).

[0117] 10102 antigen polypeptide, DTLLKALLEIASCLEKALQVF (SEQ ID NO: 9).

[0118] 10103 antigen polypeptide, FMTYWHLLNAFTVTVPKDL (SEQ ID NO: 10).

[0119] 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".

[0120] 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.

[0121] The term "suffering from a disease" means that the body is experiencing symptoms of a disease.

[0122] The term "treatment" means that after a subject has contracted a disease, the subject is exposed to (e.g., administered) a delivery particle, delivery system, vaccine, antigen-loaded drug, or pharmaceutical composition, thereby alleviating the symptoms of the disease compared to the absence of such exposure, and does not necessarily mean that the symptoms of the disease are completely suppressed.

[0123] The term "prevention" means that before a subject develops a disease, the subject is exposed to (e.g., administered) the delivery particles, delivery systems, vaccines, antigen-loaded drugs, or pharmaceutical compositions of the present disclosure, thereby alleviating symptoms after developing the disease compared to when the subject has not developed the disease. It does not necessarily mean that the disease must be completely suppressed.

[0124] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to auxiliary materials widely used in the field of drug production. The main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and / or has specific functionality, and also to provide a method so that after the drug is administered to a subject, the active ingredient can be dissolved at a desired rate, or to promote the effective absorption of the active ingredient in the body of the subject receiving the drug. Pharmaceutically acceptable excipients can be inert fillers or functional ingredients that provide a certain function to the pharmaceutical composition (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.

[0125] The pharmaceutical compositions of the present disclosure can be prepared using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, pulverizing, encapsulating, embedding and / or lyophilizing processes.

[0126] In the present disclosure, the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the nature of the drug, the convenience of the patient and medical staff, and other relevant factors.

[0127] The terms "individual," "patient," or "subject" as used herein 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).

[0128] 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 pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0129] In the embodiments described in the present disclosure, some embodiments use cancer cells / tumor tissues obtained by various 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, to prepare antigen components. In actual applications, any other feasible approach can also be used to obtain cancer cells.

[0130] Due to space limitations, the cancers described in the examples of this disclosure are solid tumors. In practical applications, the nano- or micro-vaccines described herein can also be used to treat hematologic malignancies and lymphomas. Because the immune microenvironment of hematologic malignancies and lymphomas is less complex than that of solid tumors, the nano- or micro-vaccines described herein are more effective in treating hematologic malignancies and lymphomas.

[0131] The vaccine system disclosed herein is loaded with whole cell components of cancer cells / tumor tissues or partial cell components containing antigen components and in vitro customized synthesized polypeptide / RNA components (mRNA components). Some of its preparation processes and application fields are as follows: Figure 1 shown.

[0132] When preparing nano- or micro-vaccines, cells or tissues can be lysed, and then the protein polypeptides in the water-soluble components and the protein polypeptides in the water-insoluble components / the water-insoluble components can be collected separately, and the nano- or micro-vaccines can be prepared separately; or the cells or tissues can be directly lysed using a dissolving solution containing a dissolving agent, and the whole-cell antigens of the cancer cells can be dissolved, and then the protein and polypeptide components therein can be extracted and separated using an appropriate method, and the obtained antigen components can be loaded onto the nano- or micro-vaccines. 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 endolysis or degradation, nuclease treatment, etc. before or 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 endolysis or degradation, or nuclease treatment before or 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 both 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, treatment 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. can also be used. Those skilled in the art will understand that in actual application, technicians can make appropriate adjustments according to specific circumstances.

[0133] 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:

[0134] 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.

[0135] 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 sulfamate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0136] Urea, guanidine hydrochloride, and the like contain the structure of Structural Formula 1. The inventors have discovered that substances having the structure of 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 act as solvents to dissolve water-insoluble components.

[0137] Furthermore, the nano-vaccine or micro-vaccine prepared from the components in the cancer cells and / or tumor tissues can be one or two selected from the following: (1) protein and polypeptide components in the water-soluble component + water-insoluble component; (2) protein and polypeptide components in the water-soluble component + protein and polypeptide components in the water-insoluble component. In some preferred embodiments, the mass ratio of the protein and polypeptide in the water-soluble component to the protein and polypeptide in the water-insoluble component / water-insoluble component is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Exemplarily, the mass ratio of the protein and polypeptide component in the water-soluble component to the protein and polypeptide in the water-insoluble component / 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.

[0138] In some embodiments, the immunogenic protein and / or polypeptide is derived from a portion of the components in cancer cells / tumor tissue and an extracellular vesicle lysate. 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 the portion of the components in the cancer cells and / or tumor tissue 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.

[0139] In some embodiments, the immunogenic protein and / or polypeptide is derived from a portion of a component in a cancer cell / tumor tissue and a bacterial lysate. Furthermore, the mass ratio of the portion of the component 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.

[0140] 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 microvaccines 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%.

[0141] Furthermore, the particle material is PEG-modified or not. Preferably, when preparing the skeleton structure, the mass ratio of the particle material not modified with PEG to the particle material modified with PEG is 25-200:1.

[0142] The cancer vaccine disclosed herein, wherein, in the vaccine, the mass ratio of the skeleton material, protein and polypeptide components for particle preparation is 1:0.001-10; preferably, the mass ratio of the skeleton material, protein and polypeptide components for particle preparation is 1:0.01-2; most preferably, the mass ratio of the skeleton material, protein and polypeptide components for particle preparation is 1:0.05-1.

[0143] In some embodiments, the surface of the nanovaccine or microvaccine may also be loaded with membrane components, which may be derived from one or more of antigen-presenting cells, cancer cells, bacteria, or extracellular vesicles.

[0144] The antigen-presenting cells used to prepare the biofilm components loaded onto the surface of nano- or micro-vaccines can be autologous or allogeneic, or derived from cell lines or stem cells. These cells can be dendritic cells (DCs), B cells, macrophages, or any combination thereof, or other cells with antigen-presenting capabilities. These cells can be activated by antigen-loaded nanoparticles or microparticles.

[0145] When the biofilm components carried on the surface of cancer nano- or micro-vaccines are derived from extracellular vesicles, they can be one or more of the extracellular vesicles of cancer cells, extracellular vesicles of bacteria, or extracellular vesicles of antigen-presenting cells.

[0146] Any method for preparing nanoparticles or microparticles known to those skilled in the art can be used to prepare the nanovaccine or microvaccine described in the present disclosure, including but not limited to solvent evaporation, dialysis, phase separation, spray drying, emulsion polymerization, machine stirring and shearing, membrane emulsification, microfluidics, ultrafiltration, homogenization emulsification, dispersion, precipitation, and the like.

[0147] In some specific embodiments, the present disclosure takes the solvent volatilization method as an example and provides the following exemplary preparation methods:

[0148] Step 1: Lyse cancer cells and / or tumor tissue to obtain a lysate thereof. Ultrapure water, aqueous solutions, or the like can be used to lyse cancer cells and / or tumor tissue. The cancer cells can be one or more cancer cells or cancer cell lines, or cancer cells obtained by culturing cancer cells in tumor tissue, or cells obtained by amplifying circulating tumor cells; the tumor tissue can be tumor tissue derived from one or more organisms. The lysis method is a commonly used lysis method for cancer cells and / or tumor tissue, including but not limited to one or more of repeated freeze-thaw cycles, swelling, ultrasonic treatment, high-pressure treatment, homogenization, extrusion, homogenization, high-speed stirring, chemical treatment, high-shear treatment, ultrafiltration, shrinkage, and the like.

[0149] Alternatively, a lysis solution containing a lytic agent can be used to lyse cancer cells and / or tumor tissue. Cancer cells are one or more cancer cells or cancer cell lines; tumor tissue is tumor tissue derived from one or more organisms.

[0150] Step 2, after using ultrapure water or aqueous solution to lyse cancer cells and / or tumor tissues, use one or more methods such as centrifugation, filtration, dialysis, ultrafiltration, etc. to separate the water-soluble components and water-insoluble components in the lysate to obtain water-soluble components and water-insoluble components respectively. The water-insoluble component is dissolved using a dissolving solution containing a dissolving agent to obtain a dissolved water-insoluble component. The water-soluble component and the water-insoluble component are directly mixed with artificially synthesized polypeptides and / or nucleic acids and used as antigen components to load on nano-vaccines / micro-vaccines; or the obtained water-soluble component and the water-insoluble component are subjected to the treatment of step 3 and then mixed with artificially synthesized polypeptides and / or nucleic acids and used as antigen components to load on nano-vaccines / micro-vaccines. 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:

[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 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, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0152] Alternatively, after using a dissolving solution containing a dissolving agent to lyse cancer cells and / or tumor tissues, the dissolving solution containing a dissolving agent is directly used to dissolve cancer cells and / or tumor tissues to obtain a lysate thereof. The dissolved lysate components are directly mixed with artificially synthesized polypeptides and / or nucleic acids and used as antigen components to be loaded on nano-vaccines / micro-vaccines; or the dissolved lysate components are subjected to the treatment of step 3 and then mixed with artificially synthesized polypeptides and / or nucleic acids and used as antigen components to be loaded on nano-vaccines / micro-vaccines. 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 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, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0154] Step 3, the water-soluble components in the lysate are 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 are dissolved using a dissolving solution containing 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 to obtain a precipitate; 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 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 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, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0156] Alternatively, the water-soluble components of the lysate are treated with appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, mineralization, irradiation, and irradiation to obtain a precipitate and a supernatant. The precipitate is then dissolved in a solution containing a solvent, and the mRNA or RNA component in the supernatant is isolated and extracted using an appropriate method. The mRNA or RNA component in the supernatant is then mixed with the precipitate dissolved in the solution containing the solvent and used for later use. The water-insoluble components of the lysate are dissolved in a solvent and then treated with appropriate methods such as salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, mineralization, irradiation, and irradiation before being dissolved in a solution containing the solvent. Alternatively, the water-insoluble components of the lysate are dissolved in a solution containing the solvent and used directly without further treatment. The solvent is selected separately for each step. 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:

[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 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, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0158] Alternatively, the lysate components dissolved in a dissolving solution containing a dissolving agent are subjected to salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, mineralization, irradiation, radiation, or other appropriate methods to obtain a precipitate, and then the precipitate is re-dissolved in a dissolving solution containing a dissolving agent to obtain the antigen component. 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:

[0159] 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 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, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0160] Alternatively, the lysate component dissolved in a dissolving solution containing a dissolving agent is subjected to salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, mineralization, irradiation, radiation and other appropriate methods to obtain two parts, a precipitate and a supernatant, and then the precipitate part is dissolved using a dissolving solution containing a 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 by the dissolving solution containing a dissolving agent to obtain the antigen component. 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:

[0161] 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 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, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0162] Through research, the present disclosure has found 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 sulfamate, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, and the like.

[0163] Step 4: The lysate component obtained in step 2 is mixed with artificially synthesized polypeptides and / or nucleic acids and used as an antigen component to load on the nanovaccine / micro vaccine; or the protein and polypeptide components and / or water-insoluble components in the water-soluble components obtained in step 3 or the protein and polypeptide components therein are mixed separately or simultaneously with artificially synthesized polypeptides and / or nucleic acids and loaded as antigen components into the interior and / or surface of nanoparticles or microparticles to obtain the nanovaccine or micro vaccine; or the antigen component obtained in step 3 is mixed with artificially synthesized polypeptides and / or nucleic acids and loaded as antigen components into the interior and / or surface of nanoparticles or microparticles to obtain the nanovaccine or micro vaccine.

[0164] During preparation, the initial aqueous phase and the organic phase are mixed, specifically, a first predetermined volume of aqueous solution containing a first predetermined concentration of antigen components is added to a second predetermined volume of organic phase containing a second predetermined concentration of raw materials for preparing particles.

[0165] 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 part of the components thereof dissolved in a dissolving solution containing a dissolving agent such as urea or guanidine hydrochloride. The first predetermined concentration is the concentration of the protein and polypeptide contained in the aqueous solution, or the concentration of the water-soluble antigen and / or the concentration of the original water-insoluble antigen contained in the aqueous solution. The first predetermined concentration requires that the protein and polypeptide concentration be greater than 1 ng / mL so that sufficient antigen components can be loaded to activate relevant cells. The concentration of the immunoenhancing adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0166] In some embodiments, the organic solvent is dichloromethane. In addition, in some embodiments, the second predetermined concentration of the raw material for preparing the particles ranges from 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0167] In practice, the second predetermined volume of the organic phase is set based on 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 ranges from 1:1.1 to 1:5000, preferably 1:10. During specific implementations, 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.

[0168] In some embodiments, when the aqueous phase solution is a solution comprising cell and / or tissue lysate components, the concentration of proteins and polypeptides therein 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 comprising lysate components and immune adjuvants, the concentration of proteins and polypeptides therein 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.

[0169] In some embodiments, when the aqueous phase solution is a solution containing a lysate component, the concentration of the protein and polypeptide components therein 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 therein 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.

[0170] Step 5: subjecting the mixed solution obtained in step 4 to 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. 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 to 100 mL / min. Nano- and / or micron-size can be achieved through ultrasound, stirring, homogenization, or microfluidic processing. 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.

[0171] Step 6: Add the mixture obtained after the treatment in step 5 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of an emulsifier and perform any of the following treatments: i) ultrasonic treatment for greater than 2 seconds; ii) stirring for greater than 1 minute; iii) homogenization; or iv) microfluidic treatment. In this step, the mixture obtained in step 2 is added to the aqueous emulsifier solution and continues ultrasonication, stirring, homogenization, or mixing to perform nano- or micronization. In the present disclosure, the ultrasonication 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; and the stirring time is greater than 1 minute, such as 60 to 6000 seconds. Preferably, when stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, such as the stirring speed is 50 rpm to 1500 rpm and the stirring time is 0.5 hour to 5 hours; when ultrasonic treatment is performed, the ultrasonic power is 50 W to 500 W and the time is greater than 0.1 second, such as 2 to 200 seconds; when homogenizing, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and the pressure when using a high pressure / ultra-high pressure homogenizer is greater than 20 psi, such as 20 psi to 100 psi, and the speed when using a high shear homogenizer is greater than 1000 rpm, such as 1000 rpm to 5000 rpm; when using microfluidics, the flow rate is greater than 0.01 mL / min, such as 0.1 mL / min-100 mL / min. Ultrasonic or stirring or homogenization or microfluidics is used 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. Too large or too small will bring about changes in particle size.

[0172] 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 the 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 ​​​​all in order to obtain nanoparticles or micron particles of appropriate size.

[0173] Step 7: adding the liquid obtained after the treatment in step 6 to a fourth predetermined volume of an emulsifier aqueous solution with a fourth predetermined concentration, and stirring until a predetermined stirring condition is met.

[0174] In this step, the emulsifier aqueous solution is a PVA solution or other solutions.

[0175] The fourth predetermined concentration is 5 mg / mL. The fourth predetermined concentration is selected to obtain nanoparticles or microparticles of appropriate size. The fourth predetermined volume is selected 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 specific implementations, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted to control the size of the nanoparticles or microparticles.

[0176] 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.

[0177] Step 8, after the mixed solution that meets the predetermined stirring conditions obtained in step 7 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 normal saline); or ultrafiltration centrifugation or dialysis that can remove substances of specific molecular weight is used to remove free PVA and other substances, and 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 normal saline).

[0178] Step 9: freeze-dry the suspension containing the lyoprotectant obtained in step 8, and keep the lyophilized material for later use.

[0179] Step 10: The nanoparticle-containing suspension obtained in step 8 is resuspended in PBS (or physiological saline) with a sixth predetermined volume, or the freeze-dried material containing nanoparticles or microparticles and a lyoprotectant obtained in step 9 is resuspended in a sixth predetermined volume of PBS (or physiological saline) and then used directly; or the above sample is mixed with a seventh predetermined volume of a water-soluble antigen or a dissolved original water-insoluble antigen and then used.

[0180] 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.

[0181] In some embodiments, the nanovaccine or microvaccine is loaded with 0.001-2000 μg of protein or polypeptide components per 1 mg of particle material. In some embodiments, the nanovaccine or microvaccine is also loaded with an immune-enhancing adjuvant, wherein the amount of immune-enhancing adjuvant is 1-800 μg per 1 mg of particle material. In some embodiments, the nanovaccine or microvaccine is also loaded with positively charged molecules, wherein the amount of positively charged molecules is 1-800 μg per 1 mg of particle material. In actual applications, the content of antigenic components such as proteins and polypeptides loaded per 1 mg of particle material can be even higher.

[0182] In some embodiments, organic polymers 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.

[0183] In some embodiments of the present disclosure, hypochlorous acid is used as an oxidant to oxidize the antigen component. In practical applications, any other oxidant that can oxidize the antigen component can also be used, including but not limited to persulfate, 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.

[0184] The enzyme treatment methods in some embodiments of the present disclosure include, but are not limited to, the use of one or more of nucleases, DNA enzymes, pepsin, chymotrypsin, trypsin, other protein digestive enzymes, protease inhibitors, and the like.

[0185] In some embodiments of the present disclosure, the enzymatic hydrolysis method 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.

[0186] Due to space limitations, the embodiments of the present disclosure do not list the methods of mineralization. In practical applications, any mineralization method such as silicification, calcification, and magnesiumization may be used.

[0187] Due to space limitations, the examples of reducing antigen components using reducing agents are not listed in the examples disclosed herein. In practical applications, reducing agents can also be used to reduce antigen components before loading the antigen components into nano- or micro-vaccines. Reducing agents include, but are not limited to, dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP).

[0188] The irradiation is treatment using one or more of different irradiation methods such as ultraviolet rays, X-rays, gamma rays, alpha rays, beta rays, radioactive sources, etc.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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 root extracts, etc.), chemokines, 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.

[0193] In some embodiments, the interior and / or surface of the nanovaccine or microvaccine also contains membrane components, and the membrane components inside and / or on the surface of the nanovaccine or microvaccine are one or more selected from the cell membrane of antigen-presenting cells, extracellular vesicles of antigen-presenting cells, cell membranes of cancer cells, extracellular vesicles of cancer cells, cell membranes of bacteria, and extracellular vesicles of bacteria.

[0194] 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.

[0195] 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, dialysis, dispersion, microfluidics, high-pressure homogenization, stirring, spray drying, phase separation, electrostatic spraying, emulsion polymerization, machine stirring shearing, membrane emulsification, etc.

[0196] 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.

[0197] Step 11: Use the nano-vaccine or micro-vaccine prepared in step 10 to prevent or treat diseases such as cancer.

[0198] Example 1 Nanovaccine loaded with partially water-soluble components and water-insoluble components for the treatment of melanoma

[0199] In this example, ultrapure water was first used to lyse B16F10 melanoma tumor tissue to prepare protein and polypeptide components and water-insoluble antigens in the water-soluble components of the tumor tissue. Then, a nanovaccine was prepared using the organic polymer material PLGA as the nanoparticle skeleton material and Polyinosinic-polycytidylic acid (poly(I:C)) as the immune adjuvant by a solvent evaporation method.

[0200] (1) Preparation of antigen components

[0201] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to about 1000mm 3Mice were sacrificed and tumor tissue was removed. After preparing a tumor tissue cell suspension, an appropriate amount of ultrapure water was added and the cells were repeatedly frozen and thawed five times (with sonication) to lyse the cells. The lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble fraction. An 8M urea aqueous solution was added to the resulting precipitate to dissolve the precipitate, converting the water-insoluble fraction into a fraction soluble in 8M urea. Saturated ammonium sulfate was added dropwise to the water-soluble fraction in the lysate. After complete precipitation, the resulting sample was centrifuged at 3000g for 5 minutes. The precipitate was dissolved in 8M urea aqueous solution and set aside. The supernatant was heated at 95°C for 10 minutes and then centrifuged at 3000g for 5 minutes. The supernatant was discarded and the precipitate was dissolved in 8M urea aqueous solution. The precipitate after salting out and the heated precipitate dissolved in 8M urea were combined and used as part of the water-soluble fraction. The water-insoluble components in the lysate dissolved in the above 8M urea aqueous solution were mixed with four tumor-specific antigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) and two tumor-associated antigens (IO102: DTLLKALLEIASCLEKALQVF and IO103: FMTYWHLLNAFTVTVPKDL) at a mass ratio of 1:0.01:0.01:0.01:0.01:0.01:0.01 and used as the water-insoluble components; 8M urea was added. The components obtained by salting out and heating precipitation from the dissolved water-soluble components were mixed with four tumor-specific antigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) and two tumor-associated antigens (IO102:DTLLKALLEIASCLEKALQVF and IO103:FMTYWHLLNAFTVTVPKDL) in a mass ratio of 1:0.01:0.01:0.01:0.01:0.01:0.01 and used as part of the water-soluble components. Part of the water-soluble components and the water-insoluble components together constitute the antigen component 1 for preparing the cancer nanovaccine 1.

[0202] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 105 B16F10 cells, when the tumor grows to about 1000mm 3 Mice were sacrificed and tumor tissue was removed. After preparing a tumor tissue cell suspension, an appropriate amount of ultrapure water was added and the cells were repeatedly frozen and thawed five times (with sonication) to lyse the cells. The lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble fraction. The resulting precipitate was dissolved by adding an 8M urea aqueous solution to the resulting precipitate, converting the water-insoluble antigen into one that is soluble in 8M urea aqueous solution. Saturated ammonium carbonate aqueous solution was added dropwise to the water-soluble fraction in the lysate. After complete precipitation, the resulting sample was centrifuged at 3000g for 5 minutes. The precipitate was dissolved in 8M urea aqueous solution and set aside. The supernatant was heated at 95°C for 10 minutes and then centrifuged at 3000g for 5 minutes. The supernatant was discarded and the precipitate was dissolved in 8M urea aqueous solution. The precipitate after salting out and the heated precipitate were combined and used as part of the water-soluble fraction. The water-insoluble component in the lysate dissolved in the above 8M urea aqueous solution was mixed with four tumor-specific antigens B16-M20, B16-M24, B16-M46 and TRP2:180-188 and two tumor-associated antigens (IO102 and IO103) in a mass ratio of 1:0.01:0.01:0.01:0.01:0.01:0.01 to obtain the water-insoluble component; the component obtained by salting out and precipitating after heating in the water-soluble component dissolved in 8M urea was mixed with four tumor-specific antigens B16-M20, B16-M24, B16-M46 and TRP2:180-188 and two tumor-associated antigens (IO102 and IO103) in a mass ratio of 1:0.01:0.01:0.01:0.01:0.01:0.01 to obtain some components in the water-soluble component. The two together constitute the antigen component 2 for preparing the cancer nanovaccine 2.

[0203] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to a volume of approximately 1000 mm 3Mice were sacrificed and tumor tissue was removed. After preparing a tumor tissue cell suspension, an appropriate amount of ultrapure water was added and the cells were repeatedly frozen and thawed five times (with ultrasound) to lyse the cells. The lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble fraction soluble in pure water. The resulting precipitate was dissolved by adding 8M urea aqueous solution to the precipitate, converting the water-insoluble antigen into one soluble in 8M urea aqueous solution. The water-insoluble component in the lysate dissolved in the above 8M urea aqueous solution is mixed with four tumor-specific antigens B16-M20, B16-M24, B16-M46 and TRP2:180-188 and two tumor-associated antigens (IO102 and IO103) in a mass ratio of 1:0.01:0.01:0.01:0.01:0.01 to obtain a water-insoluble component; the water-soluble component is mixed with four tumor-specific antigens B16-M20, B16-M24, B16-M46 and TRP2:180-188 and two tumor-associated antigens (IO102 and IO103) in a mass ratio of 1:0.01:0.01:0.01:0.01:0.01 to obtain a water-soluble component; the water-soluble component and the water-insoluble component together are used to prepare cancer nanovaccine 3 antigen component 3.

[0204] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to a volume of approximately 1000 mm 3Mice were sacrificed and tumor tissue was removed. After preparing a tumor tissue cell suspension, an appropriate amount of ultrapure water was added and the cells were repeatedly frozen and thawed five times (with sonication) to lyse the cells. The lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble fraction. The resulting precipitate was dissolved by adding an 8M urea solution to the resulting precipitate, converting the water-insoluble fraction into a fraction soluble in 8M urea. Saturated ammonium sulfate was added dropwise to the water-soluble fraction in the lysate. After complete precipitation, the resulting sample was centrifuged at 3000g for 5 minutes. The precipitate was solubilized with a 1% Triton X100 solution and set aside. The supernatant was heated at 95°C for 10 minutes and then centrifuged at 3000g for 5 minutes. The supernatant was discarded, and the precipitate was solubilized with a 1% Triton X100 solution. The salting-out precipitate solubilized with the heated precipitate was then combined and used as part of the water-soluble fraction. The water-insoluble component in the lysate dissolved in the above 8M urea aqueous solution was mixed with four tumor-specific antigens B16-M20, B16-M24, B16-M46 and TRP2:180-188 and two tumor-associated antigens (IO102 and IO103) at a mass ratio of 1:0.01:0.01:0.01:0.01:0.01 to obtain the water-insoluble component; 1% Triton The components obtained by salting out and heating precipitation from the water-soluble components dissolved in the X100 aqueous solution are mixed with four tumor-specific antigens B16-M20, B16-M24, B16-M46 and TRP2:180-188 and two tumor-associated antigens (IO102 and IO103) in a mass ratio of 1:0.01:0.01:0.01:0.01:0.01 to form part of the water-soluble components; the two together form the antigen component 4 for preparing cancer nanovaccine 4.

[0205] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to a volume of approximately 1000 mm 3Mice were sacrificed and tumor tissue was removed. After preparing a tumor tissue cell suspension, an appropriate amount of ultrapure water was added and the cells were repeatedly frozen and thawed five times (with sonication) to lyse the cells. The lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble fraction. A 1% Triton X100 aqueous solution was added to the resulting precipitate to solubilize the precipitate, yielding the fraction soluble in 1% Triton X100. A saturated aqueous ammonium sulfate solution was added dropwise to the water-soluble component in the lysate. After complete precipitation, the resulting sample was centrifuged at 3000 g for 5 minutes. The precipitate was solubilized with a 1% Triton X100 aqueous solution and set aside. The supernatant was heated at 95° C. for 10 minutes, and the resulting sample was centrifuged at 3000 g for 5 minutes. After discarding the supernatant, the precipitate was solubilized with a 1% Triton X100 aqueous solution. The salted-out precipitate solubilized with the 1% Triton X100 aqueous solution and the heated precipitate were combined and used as part of the water-soluble component. The water-insoluble components in the lysate dissolved in the above 1% Triton X100 aqueous solution, the four tumor-specific antigens B16-M20, B16-M24, B16-M46 and TRP2:180-188 and the two tumor-associated antigens (IO102 and IO103) were mixed in a mass ratio of 1:0.01:0.01:0.01:0.01:0.01:0.01 to obtain the 1% Triton X100-solubilized water-insoluble components; 1% Triton The components obtained by salting out and heating precipitation from the water-soluble components dissolved in the X100 aqueous solution are mixed with four tumor-specific antigens B16-M20, B16-M24, B16-M46 and TRP2:180-188 and two tumor-associated antigens (IO102 and IO103) in a mass ratio of 1:0.01:0.01:0.01:0.01:0.01 to form part of the water-soluble components; the two together constitute the antigen component 5 for preparing cancer nanovaccine 5.

[0206] (2) Preparation of nanovaccines

[0207] In this example, nanovaccine 1 was prepared using the double emulsion method, a solvent evaporation method. During preparation, some of the water-soluble components of the tumor tissue lysate were purified by ammonium sulfate salting-out and heat separation, followed by dissolution in an 8M urea aqueous solution. Nanoparticles loaded with the water-soluble components of the cancer cell lysate (those dissolved in an 8M urea aqueous solution after ammonium sulfate salting-out and heat separation) and nanoparticles loaded with the water-insoluble antigens of whole cancer cell antigens (dissolved in an 8M urea aqueous solution) were prepared separately and then used together as nanovaccine 1. The antigen delivery nanoparticles used were made of PLGA with a molecular weight of 10-20 kDa, and the immunoadjuvant used was poly(I:C). The preparation method was as described above. First, the antigen components and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 18,000 g for 50 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The nanoparticles 1 have an average particle size of about 100 nm. Each 1 mg of PLGA nanoparticles is loaded with about 15 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.2 mg of poly(I:C).

[0208] Nanovaccine 2 was prepared using the same method and steps as Nanovaccine 1. During preparation, some of the water-soluble components of the tumor tissue lysate were purified by salting out with ammonium carbonate and heat-separated, then dissolved in an 8M urea solution. Nanoparticles loaded with the water-soluble components of the cancer cell lysate (the components dissolved in an 8M urea solution after salting out with ammonium carbonate and heat-separated) and nanoparticles loaded with the water-insoluble whole-cell antigens of the cancer cells (dissolved in an 8M urea solution) were prepared separately and then used together as Nanovaccine 2. The antigen delivery nanoparticles used were made of PLGA with a molecular weight of 10-20 kDa, and the immunoadjuvant used was poly(I:C). The preparation method was as described above. The antigen components and adjuvant were first loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 18,000 g for 50 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 2 is about 100 nm. Each 1 mg of PLGA nanoparticles is loaded with about 15 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.2 mg of poly(I:C).

[0209] The preparation method and preparation steps of Nanovaccine 3 are the same as those of Nanovaccine 1. During preparation, all water-soluble components in the tumor tissue lysate are used directly without any treatment. During preparation, nanoparticles loaded with water-soluble components in cancer cell lysates and nanoparticles loaded with water-insoluble antigens (dissolved in 8M urea aqueous solution) in whole-cell cancer cell antigens are prepared separately, and then used together as Nanovaccine 3. The PLGA molecular weight of the antigen delivery nanoparticle preparation material used is 10KDa-20KDa, and the immune adjuvant used is poly(I:C). The preparation method is as described above. First, the antigen components and adjuvants are loaded inside the nanoparticles by the double emulsion method. Then, 100mg of nanoparticles are centrifuged at 18000g for 50 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and freeze-dried for 48h. The nanoparticles 3 have an average particle size of about 100 nm. Each 1 mg of PLGA nanoparticles is loaded with about 15 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.2 mg of poly(I:C).

[0210] Nanovaccine 4 was prepared using the same method and steps as Nanovaccine 1. During preparation, some of the water-soluble components of the tumor tissue lysate were purified by ammonium sulfate salting-out and heat separation, followed by dissolution in a 1% Triton X100 aqueous solution. Nanoparticles loaded with the water-soluble components of the cancer cell lysate (the components dissolved in a 1% Triton X100 aqueous solution after ammonium sulfate salting-out and heat separation) and nanoparticles loaded with the water-insoluble whole-cell antigens of the cancer cells (dissolved in an 8M urea aqueous solution) were prepared separately and then used together as Nanovaccine 1. The antigen delivery nanoparticles used were made of PLGA with a molecular weight of 10-20 kDa, and the immunoadjuvant used was poly(I:C). The preparation method was as described above. The antigen components and adjuvant were first loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 18,000 g for 50 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 4 is about 100 nm. Each 1 mg of PLGA nanoparticles is loaded with about 15 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.2 mg of poly(I:C).

[0211] The preparation method and steps of Nanovaccine 5 are the same as those of Nanovaccine 1. During preparation, some of the water-soluble components in the tumor tissue lysate are purified by ammonium sulfate salting-out and heat separation and purification, followed by dissolution in a 1% Triton X100 aqueous solution. During preparation, nanoparticles loaded with the water-soluble components of the cancer cell lysate (components dissolved in a 1% Triton X100 aqueous solution after ammonium sulfate salting-out and heat separation and purification) and nanoparticles loaded with the water-insoluble antigens of the whole-cell cancer cell antigens (dissolved in a 1% Triton X100 aqueous solution) are prepared separately and then used together as Nanovaccine 1. The antigen delivery nanoparticle preparation material used, PLGA, has a molecular weight of 10KDa-20KDa, and the immune adjuvant used is poly(I:C). The preparation method is as described above. The antigen component and adjuvant are first loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles are centrifuged at 18,000 g for 50 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The nanoparticles have an average particle size of approximately 100 nm. Each mg of PLGA nanoparticles can be loaded with approximately 15 μg of protein or peptide component, and 0.2 mg of poly(I:C) per mg of PLGA nanoparticles.

[0212] (3) Nano-vaccines for cancer treatment

[0213] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells. On days 3, 6, 9, 14, and 20 after tumor inoculation, mice were subcutaneously injected with 2 mg of nanovaccine 1 (1 mg of nanoparticles loaded with a purified component from a water-soluble fraction + 1 mg of nanoparticles loaded with a water-insoluble fraction), 2 mg of nanovaccine 2 (1 mg of nanoparticles loaded with a purified component from a water-soluble fraction + 1 mg of nanoparticles loaded with a water-insoluble fraction), 2 mg of nanovaccine 3 (1 mg of nanoparticles loaded with a water-soluble fraction + 1 mg of nanoparticles loaded with a water-insoluble fraction), 2 mg of nanovaccine 4 (1 mg of nanoparticles loaded with a purified component from a water-soluble fraction + 1 mg of nanoparticles loaded with a water-insoluble fraction), or 2 mg of nanovaccine 5 (1 mg of nanoparticles loaded with a purified component from a water-soluble fraction + 1 mg of nanoparticles loaded with a water-insoluble fraction), or 100 μL of PBS. Tumor growth and survival were monitored. Tumor volume was recorded every three days starting on day 3. The tumor volume was calculated using the formula v = 0.52 × a × b 2Calculate, 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.

[0214] (4) Experimental results

[0215] like Figure 3 As shown, the tumors in the PBS group grew rapidly, and the mice died soon after. Mice treated with Nanovaccines 1, 2, 3, 4, and 5 all experienced significantly slower tumor growth and prolonged survival, with some mice recovering tumor-free. Nanovaccines 1 was more effective than Nanovaccines 2, 3, 4, and 5, indicating that using ammonium sulfate salting-out to separate and purify proteins and peptides from the water-soluble fraction is more effective than using ammonium carbonate. Furthermore, the separation and purification of a portion of the water-soluble fraction is more effective than using the water-soluble fraction directly. Furthermore, the precipitate produced by salting-out and heating the water-soluble fraction was more effectively dissolved using urea than using 1% Triton X100.

[0216] Example 2 Micronized vaccine for the treatment of melanoma

[0217] In this example, ultrapure water was first used to lyse cancer cells isolated from B16F10 melanoma tumor tissue. After culturing the isolated cancer cells, the protein and polypeptide components of the water-soluble components of the tumor tissue and the water-insoluble antigen dissolved in 6M guanidine hydrochloride were prepared. The two were then mixed and loaded onto the micron vaccine.

[0218] (1) Preparation of antigen components

[0219] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows 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 cancer cells in the tumor tissue were separated and 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 culture 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. After lysis, the lysate 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, and the water-insoluble antigen that was insoluble in pure water was converted into a soluble antigen in 6M guanidine hydrochloride aqueous solution.

[0220] Saturated magnesium sulfate aqueous solution was added dropwise to the water-soluble component or the water-insoluble component in the lysate. After the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes. The precipitate was dissolved in 6M guanidine hydrochloride aqueous solution and used as part of the antigen component in the water-soluble component. The obtained sample was mixed with the water-insoluble component in the lysate dissolved in 6M guanidine hydrochloride aqueous solution, four tumor-specific antigens B16-M20 (Tubb3), B16-M24 (Dag1), B16-M46 (Actn4) and TRP2:180-188 and two tumor-associated antigens (WT1 126-134 :RMFPNAPYL and WT1 35-52 :WAPVLDFAPPGASAYGSL) were mixed in a mass ratio of 1:5:0.001:0.001:0.001:0.001:0.001:0.001 to obtain the antigen component 1 for preparing micron vaccine 1 (Micronvaccine 1).

[0221] Saturated sodium silicate aqueous solution was added dropwise to the water-soluble component or the water-insoluble component in the lysate. After the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes. The precipitate was dissolved in 6M guanidine hydrochloride aqueous solution and used as part of the antigen component in the water-soluble component. The obtained sample was mixed with the water-insoluble component of the lysate dissolved in 6M guanidine hydrochloride aqueous solution, four tumor-specific antigens B16-M20 (Tubb3), B16-M24 (Dag1), B16-M46 (Actn4) and TRP2:180-188 (SVYDFFVWL) and two tumor-associated antigens (WT1 126-134 :RMFPNAPYL and WT1 35-52 :WAPVLDFAPPGASAYGSL) were mixed in a mass ratio of 1:5:0.001:0.001:0.001:0.001:0.001:0.001 to obtain antigen component 2 for preparing micron vaccine 2 (Micronvaccine 2).

[0222] Alternatively, the water-soluble components in the lysate were not treated, and the water-insoluble components in the lysate dissolved in 6M guanidine hydrochloride aqueous solution, four tumor-specific antigens B16-M20 (Tubb3), B16-M24 (Dag1), B16-M46 (Actn4) and TRP2:180-188 (SVYDFFVWL) and two tumor-associated antigens (WT1 126-134 :RMFPNAPYL and WT1 35-52 :WAPVLDFAPPGASAYGSL) were mixed in a mass ratio of 1:5:0.001:0.001:0.001:0.001:0.001:0.001 to obtain antigen component 3 for preparing micron vaccine 3 (Micronvaccine 3).

[0223] Alternatively, four melanoma cancer-specific antigen peptides B16-M20, B16-M24, B16-M46 and TRP2:180-188 and two cancer-associated antigens (WT1 126-134 :RMFPNAPYL and WT1 35-52 :WAPVLDFAPPGASAYGSL) were mixed in a mass ratio of 1:1:1:1:1:1 to prepare the antigen component 4 of micron vaccine 4 (Micronvaccine 4).

[0224] Alternatively, a saturated aqueous solution of magnesium sulfate is added dropwise to the water-soluble component in the lysate. After complete precipitation, the resulting sample is centrifuged at 3000 g for 5 minutes. The precipitate is dissolved in a 6M aqueous solution of guanidine hydrochloride and used as part of the antigen component in the water-soluble component; the resulting sample is mixed with the water-insoluble component in the lysate dissolved in a 6M aqueous solution of guanidine hydrochloride at a mass ratio of 1:5 to obtain antigen component 5 for preparing micron vaccine 5 (Micronvaccine 5).

[0225] (2) Preparation of micronized vaccines

[0226] In this example, micron vaccine 1 (Micronvaccine 1) was prepared using the solvent evaporation method, a double emulsion method. The micron particles used were prepared using PLGA, a material with a molecular weight of 38-54 kDa, and the immune adjuvants used were poly(I:C) and CpG ODN7909. The preparation method is as described above. During the preparation process, the micron particles were first loaded with antigen component 1 and adjuvant. Then, 100 mg of the micron particles were centrifuged at 8000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of micron vaccine 1 was approximately 1.2 μm. Each 1 mg of PLGA microparticles was loaded with approximately 10 μg of protein or polypeptide component, and each 1 mg of PLGA microparticles was loaded with 0.01 mg of poly(I:C) and 0.01 mg of CpG ODN.

[0227] In this embodiment, the preparation method and preparation steps of Micronvaccine 2 are the same as those of Micronvaccine 1. The micron particles are prepared using PLGA, a material with a molecular weight of 38-54 kDa, and the immunoadjuvants used are poly(I:C) and CpG ODN7909. The preparation method is as described above. First, the antigen component 2 and adjuvant are loaded into the micron particles. Then, 100 mg of the micron particles are centrifuged at 8000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of Micronvaccine 2 is approximately 1.2 μm. Each 1 mg of PLGA microparticles is loaded with approximately 10 μg of protein or polypeptide component, and each 1 mg of PLGA microparticles is loaded with 0.01 mg of poly(I:C) and 0.01 mg of CpG ODN7909.

[0228] Micronvaccine 3 in this example was prepared using the same method and steps as Micronvaccine 1. The micron particles were prepared using PLGA, a material with a molecular weight of 38-54 kDa, and the immunoadjuvants used were poly(I:C) and CpG ODN 7909. The preparation method was as described above. The micron particles were first loaded with antigen component 3 and the adjuvant. Then, 100 mg of the micron particles were centrifuged at 8000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Micronvaccine 3 had an average particle size of approximately 1.2 μm. Each mg of PLGA microparticles contained approximately 10 μg of the protein or polypeptide component, and each mg of PLGA microparticles contained 0.01 mg of poly(I:C) and 0.01 mg of CpG ODN 7909.

[0229] The preparation method and steps for Micronvaccine 4 in this example are the same as those for Micronvaccine 1. The microparticles used are made of PLGA, a material with a molecular weight of 38-54 kDa, and the immune adjuvants used are poly(I:C) and CpG ODN 7909. During preparation, the antigen component 4 and adjuvant are first loaded onto micronparticles 4. Then, 100 mg of the micronparticles are centrifuged at 10,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Micronvaccine 4 has an average particle size of approximately 1.2 μm. Each mg of PLGA microparticles is loaded with approximately 10 μg of the polypeptide component, and each mg of PLGA microparticles is loaded with 0.01 mg of poly(I:C) and 0.01 mg of CpG ODN.

[0230] Micronvaccine 5 in this example was prepared using the same method and steps as Micronvaccine 1. During preparation, the antigen component 5 and adjuvant were first loaded onto micron particles 5. Then, 100 mg of the micron particles were centrifuged at 8000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Micronvaccine 5 had an average particle size of approximately 1.2 μm. Each mg of PLGA microparticles was loaded with approximately 10 μg of the protein / peptide component, and each mg of PLGA microparticles was loaded with 0.01 mg of poly(I:C) and 0.01 mg of CpG ODN.

[0231] (3) Micron 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 5 B16F10 cells. 4 mg of micro vaccine (micro vaccine 1 or micro vaccine 2 or micro vaccine 3 or micro vaccine 4 or micro vaccine 5) or 100 μL PBS was injected subcutaneously into the mice on the 3rd, 6th, 9th, 14th and 20th days after tumor inoculation. The tumor growth rate and survival of the mice were monitored. In the experiment, the size of the mouse tumor was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculate, where v is the tumor volume, a is the tumor length, and b is the tumor width.

[0233] (9) Experimental results

[0234] like Figure 4As shown, tumors in mice in the PBS group rapidly expanded and soon died. Mice treated with Micronvaccine 1, 2, 3, 4, and 5 all showed significantly slower tumor growth and prolonged survival, with some mice recovering completely. Micronvaccine 1 was more effective than Micronvaccines 2, 3, 4, and 5, demonstrating that using magnesium sulfate salting-out to separate and purify proteins and peptides from the water-soluble fraction is more effective than using sodium silicate. Furthermore, separating and purifying a portion of the water-soluble fraction is more effective than using the water-soluble fraction directly. Furthermore, vaccines co-loading tumor tissue lysate fractions with cancer-specific and related antigen peptides are more effective than vaccines loaded solely with cancer-specific / related antigen peptides. Furthermore, vaccines co-loading tumor tissue lysate fractions with cancer-specific and related antigen peptides are more effective than vaccines using cancer cell lysate alone.

[0235] Example 3 Nano-vaccine for the treatment of melanoma

[0236] In this embodiment, circulating tumor cells in peripheral blood are first isolated, then cultured and expanded in vitro, the expanded circulating tumor cells are collected and lysed, and then antigen components from circulating tumor cells are prepared and loaded into nanovaccines for the prevention and treatment of cancer.

[0237] (1) Lysis of cancer cells and collection of components

[0238] The cultured B16F10 cells were collected and then inoculated subcutaneously on the back of each mouse at a rate of 1.5 × 10 5 The mice were then sacrificed 15 days after cancer cell inoculation to collect peripheral blood. Circulating tumor cells were isolated from the peripheral blood and then cultured and expanded in vitro. After collecting the expanded circulating tumor cells, an appropriate amount of ultrapure water was added and the cells were repeatedly frozen and thawed five times to lyse the circulating tumor cells. The lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble fraction. The resulting precipitate was dissolved in 10% sodium deoxycholate solution, converting the water-insoluble antigen into a soluble antigen in 10% sodium deoxycholate solution.

[0239] The water-soluble fraction in the lysate was heated at 100°C for 10 minutes, and the resulting sample was centrifuged at 3000g for 5 minutes. The supernatant was discarded and the precipitate was redissolved in a 10% sodium deoxycholate aqueous solution. The fraction obtained by heating the precipitate from the water-soluble fraction dissolved in 10% sodium deoxycholate, the water-insoluble fraction in the lysate dissolved in 10% sodium deoxycholate aqueous solution, and the cancer-associated antigen WT1 were mixed. 126-134 :RMFPNAPYL, cancer-associated antigen WT1 35-52 :WAPVLDFAPPGASAYGSL, and mRNA encoding four cancer-specific antigens (B16-M20, B16-M24, B16-M46 and TRP2:180-188) were mixed in a mass ratio of 10:1:22:22:22 to prepare the antigen component 1 of nanovaccine 1.

[0240] Alternatively, the water-soluble component in the lysate is used directly without any treatment. The water-soluble component and the water-insoluble component in the lysate dissolved in 10% sodium deoxycholate aqueous solution are mixed at a mass ratio of 10:1 to prepare the antigen component 2 of the nanovaccine 2.

[0241] Alternatively, the water-soluble fraction in the lysate can be used directly without any treatment. The water-soluble fraction, the water-insoluble fraction in the lysate dissolved in 10% sodium deoxycholate aqueous solution, and the cancer-associated antigen WT1 126-134 , cancer-associated antigen WT1 35-52 , and mRNA encoding four cancer-specific antigens (B16-M20, B16-M24, B16-M46 and TRP2:180-188) are mixed in a mass ratio of 10:1:22:22:22 to prepare antigen component 3 of nanovaccine 3.

[0242] Or directly transfect cancer-associated antigen WT1 126-134 , cancer-associated antigen WT1 35-52 , and mRNA encoding four cancer-specific antigens (B16-M20, B16-M24, B16-M46 and TRP2:180-188) were mixed in a mass ratio of 22:22:22 as antigen component 4 for preparing nanovaccine 4.

[0243] (2) Preparation of nanovaccines

[0244] In this embodiment, nano vaccine 1 (Nanovaccine 1) was prepared using the multiple emulsion method in the solvent evaporation method. The nano vaccine preparation material PLA used has a molecular weight of 10KDa-20KDa, and the immune adjuvants used are poly(I:C), CpG SL01 (Class B), and CpG SL03 (Class C). The preparation method is as described above. During preparation, the cell antigen component 1 and adjuvant are first loaded inside the nanoparticles. Then, 100mg of nanoparticles are centrifuged at 14000g for 20 minutes and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours. The average particle size of the nanoparticles 1 is about 500nm. Every 1mg of PLA nanoparticles is loaded with approximately 50μg of protein or polypeptide components, 20μg of mRNA, and 0.2mg each of poly(I:C), CpG SL01 (Class B), and CpG SL03 (Class C).

[0245] Nanovaccine 2 in this example was prepared using the same methods and materials as Nanovaccine 1. As previously described, the nanoparticles were loaded with the cell antigen component 2 and adjuvant. 100 mg of nanoparticles were then centrifuged at 14,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Nanoparticles 2 had an average particle size of approximately 500 nm. Each 1 mg of PLA (10-20 kDa) nanoparticles was loaded with approximately 70 μg of protein or peptide components, including 0.2 mg each of poly(I:C), CpG SL01 (Class B), and CpG SL03 (Class C).

[0246] Nanovaccine 3 in this example was prepared using the same methods and materials as Nanovaccine 1. As previously described, the nanoparticles were loaded with the cell antigen component 3 and adjuvant. 100 mg of the nanoparticles were then centrifuged at 14,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Nanoparticles 3 had an average particle size of approximately 500 nm. Each 1 mg of PLA (10-20 kDa) nanoparticles was loaded with approximately 50 μg of protein or polypeptide components, 20 μg of mRNA, and 0.2 mg each of poly(I:C), CpG SL01 (Class B), and CpG SL03 (Class C).

[0247] Nanovaccine 4 in this example was prepared using the same methods and materials as Nanovaccine 1. As previously described, nanoparticles were loaded with antigen component 4 and adjuvant. 100 mg of nanoparticles were then centrifuged at 14,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Nanoparticles 4 had an average particle size of approximately 500 nm. Each 1 mg of PLA (10-20 kDa) nanoparticles was loaded with approximately 50 μg of protein or polypeptide components, 20 μg of mRNA, and 0.2 mg each of poly(I:C), CpG SL01 (Class B), and CpG SL03 (Class C).

[0248] (3) Nano-vaccines for cancer treatment

[0249] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells were injected subcutaneously with 100 μL of 15 mg of nanovaccine (Nanovaccin 1, Nanovaccin 2, Nanovaccin 3, or Nanovaccin 4) or 100 μL of PBS on days 3, 6, 9, 14, and 20 after tumor inoculation. Tumor growth rate and survival of mice were monitored as described above.

[0250] (4) Experimental results

[0251] like Figure 5 As shown, the tumors in the PBS group grew rapidly and the mice died soon after. Mice treated with Nanovaccines 1 (Nanovaccine 1), 2 (Nanovaccine 2), 3 (Nanovaccine 3), or 4 (Nanovaccine 4) all experienced significantly slower tumor growth and prolonged survival, with some mice recovering without tumors. Nanovaccines 1 and 3 were more effective than Nanovaccines 2, and Nanovaccines 1 was more effective than Nanovaccines 3. Furthermore, Nanovaccines 1, 2, and 3 were all more effective than Nanovaccines 4. This suggests that using cell lysates is significantly more effective than using only antigenic peptides and mRNA encoding them; that heating to separate and purify the protein and peptide components from the water-soluble fraction can enhance vaccine efficacy; and that adding cancer-specific antigenic peptides and mRNA encoding cancer-related peptides significantly enhances efficacy.

[0252] Example 4 Nano-vaccine for the treatment of lung cancer

[0253] In this example, LLC mouse lung cancer cells were first lysed using ultrapure water to prepare protein and polypeptide components and water-insoluble components in the water-soluble components of the cancer cells, and then the nanovaccine was prepared using PLGA as the nanoparticle skeleton material.

[0254] In this embodiment, the cell membrane fraction of antigen-presenting cells was used. In practical applications, the membrane fraction of extracellular vesicles secreted by antigen-presenting cells may also be used; or the cell membrane fraction of bacteria or cancer cells may also be used at the same time.

[0255] In this embodiment, the antigen-presenting cell membrane component is located on the surface of the nanovaccine or microvaccine. In practical applications, the membrane component can also be located inside the nanovaccine or microvaccine. Furthermore, when the nanovaccine / microvaccine is loaded with membrane components of bacteria and cancer cells, the membrane component can be located on the surface of the nanovaccine or microvaccine or can also be loaded inside the nanovaccine or microvaccine.

[0256] (1) Preparation of antigen components

[0257] Cultured LLC cancer cells were harvested and enzymatically digested with pepsin (0.5 μg / mL) and trypsin (0.5 μg / mL) for 15 minutes. The cells were then resuspended in an appropriate amount of ultrapure water and repeatedly frozen and thawed five times. The lysate was then centrifuged at 5000 g for 5 minutes, and the supernatant, representing the water-soluble fraction, was collected. The resulting precipitate was dissolved in a 1 M guanidine sulfate and 0.1 M arginine aqueous solution, converting the water-insoluble antigen into a soluble antigen in the 1 M guanidine sulfate and 0.1 M arginine aqueous solution. Saturated sodium chloride aqueous solution was added dropwise to the water-soluble component in the lysate. After the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes, and the precipitate was dissolved in 1M guanidine sulfate and 0.1M arginine aqueous solution for later use. The supernatant was heated at 100°C for 10 minutes and the obtained sample was centrifuged at 3000g for 5 minutes. After the supernatant was discarded, the precipitate was dissolved in 1M guanidine sulfate and 0.1M arginine aqueous solution; the precipitate after salting out dissolved in 1M guanidine sulfate and 0.1M arginine aqueous solution and the precipitate after heating dissolved in 1M guanidine sulfate and 0.1M arginine aqueous solution were combined and used as part of the water-soluble component. The water-insoluble component in the lysate dissolved in 1M guanidine sulfate and 0.1M arginine aqueous solution, the component obtained by salting out and heating precipitate dissolved in 1M guanidine sulfate and 0.1M arginine in the water-soluble component, and three cancer-related antigen polypeptides (WT1 235-243 :CYTWNQMNL、WT1 86-102 :EQCLSAFTLHFSGQFTG and WT1 294-312:FRGIQDVRRVSGVAPTLVR) are mixed in a mass ratio of 10:1:1:1:1 to prepare antigen component 1 for cancer nanovaccine 1; the water-insoluble component in the lysate dissolved in the above 1M guanidine sulfate and 0.1M arginine aqueous solution and the component obtained by salting out and heating precipitation in the water-soluble component dissolved in 1M guanidine sulfate and 0.1M arginine are mixed in a mass ratio of 10:1 to prepare antigen component 2 for cancer nanovaccine 2.

[0258] (2) Preparation of nanovaccines loaded with antigen components and adjuvants

[0259] Nano vaccine 1 (Nanovaccine 1) in this embodiment is prepared by the double emulsion method in the solvent evaporation method. During preparation, the nano vaccine preparation material PLGA used has a molecular weight of 7KDa-17KDa, and the immune adjuvants used are poly(I:C), CpG ODN 1018 (Class B), and CpG ODN M362 (Class C). The preparation method is as described above. First, the cell antigen component 1 and adjuvant are loaded into the nanoparticles by the double emulsion method. Then, 100mg of nanoparticles are centrifuged at 12000g for 20 minutes and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours. The average particle size of this nano vaccine 1 is about 280nm. Every 1mg of PLGA nanoparticles is loaded with approximately 200μg of protein and polypeptide components, and 0.02mg each of poly(I:C), CpG ODN 1018 (Class B), and CpG ODN M362 (Class C).

[0260] Nanovaccine 2 was prepared using the same methods and materials as Nanovaccine 1. Cell antigen component 2 and adjuvant were first loaded into the nanoparticles using a double emulsion method. 100 mg of nanoparticles were then centrifuged at 12,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Nanovaccine 2 had an average particle size of approximately 280 nm. Each 1 mg of PLGA (7-17 kDa) nanoparticles was loaded with approximately 200 μg of protein or peptide components, including 0.02 mg each of poly(I:C), CpG ODN 1018 (Class B), and CpG ODN M362 (Class C).

[0261] Nanovaccine 3 was prepared using the same materials and methods as Nanovaccine 1. The average particle size of Nanovaccine 3 was approximately 280 nm. 1 mg of PLGA (7 KDa-17 KDa) nanoparticles was loaded with 0.02 mg each of poly(I:C), CpG ODN 1018 (Class B), and CpG ODN M362 (Class C), without any antigenic component.

[0262] (3) Preparation and activation of antigen-presenting cells

[0263] Bone marrow-derived dendritic cells (BMDC) and B cells from C57BL / 6 mice were used as antigen presenting cells. The BMDC preparation method used was the conventional mouse BMDC preparation method. The B cell extraction process was as follows: the mouse was sacrificed and the spleen was removed, and then a single cell suspension of mouse spleen cells was prepared. Then, CD19 was sorted from the single cell suspension of spleen cells using magnetic bead sorting. + B cells. BMDCs and B cells are mixed at a 1:1 ratio and used as mixed antigen-presenting cells.

[0264] Nanovaccine 1 (500 μg) or nanovaccine 2 (500 μg) or nanovaccine 3 (500 μg) + an equal amount of free lysate were co-incubated with 20 million mixed antigen-presenting cells (10 million BMDCs + 10 million B cells) in 15 mL RPMI1640 complete culture medium for 48 hours (37°C, 5% CO2); the incubation system contained a cytokine combination: IL-15 (10 ng / mL), IL-7 (20 ng / mL), and IL-21 (10 ng / mL).

[0265] Alternatively, nanovaccine 1 (500 μg) was co-incubated with 20 million BMDCs in 15 mL RPMI1640 complete culture medium for 48 hours (37°C, 5% CO2); the incubation system contained a cytokine combination: IL-15 (10 ng / mL), IL-7 (20 ng / mL), and IL-21 (10 ng / mL).

[0266] (4) Preparation of nanovaccines with antigens and adjuvants loaded internally and antigen-presenting cell membrane components loaded on the surface

[0267] After incubation, 20 million mixed antigen-presenting cells (10 million BMDCs + 10 million B cells) were collected by centrifugation at 400g for 5 minutes, and then washed twice with normal saline. After the cells were resuspended in normal saline, low-power 7.5W ultrasound was used at 4°C for 10 minutes to destroy the cells and prepare a sample containing cell membrane components. The sample was then filtered through a filter membrane with a pore size of 50μm, 10μm, 5μm, 1μm, 0.45μm, and 0.22μm. The filtrate was collected and incubated with the corresponding nanovaccine (50mg) prepared in step (2) for 10 minutes, and then repeatedly co-extruded using a 400nm filter membrane. The extrudate was centrifuged at 15000g for 60 minutes, the supernatant was discarded, and the resulting precipitate was resuspended in normal saline to obtain nanoparticles. Among them, the nanoparticles prepared by the co-action of the mixed antigen-presenting cell membrane components activated by nanovaccine 1 and nanovaccine 1 are nanovaccine 4 (Nanovaccine 4), with a particle size of 300nm. Each 1mg of PLGA nanoparticles is loaded with approximately 200μg of protein or polypeptide components, and 0.02mg each of poly(I:C), CpG ODN 1018 (type B) and CpG ODN M362 (type C); each 1mg of PLGA nanoparticles is loaded with approximately 100μg of cell membrane components. Nanoparticles produced by co-administering nanovaccine 2 with mixed antigen-presenting cell membrane fractions activated by nanovaccine 2 are called nanovaccine 5. They have a particle size of 300 nm and are loaded with approximately 200 μg of protein or peptide components per 1 mg of PLGA nanoparticles, including 0.02 mg each of poly(I:C), CpG ODN 1018 (Class B), and CpG ODN M362 (Class C). Approximately 100 μg of cell membrane components are loaded per 1 mg of PLGA nanoparticles. Nanoparticles produced by co-administering nanovaccine 3 with mixed antigen-presenting cell membrane fractions are called nanovaccine 6. They have a particle size of 300 nm and are loaded with 0.02 mg each of poly(I:C), CpG ODN 1018 (Class B), and CpG ODN M362 (Class C). Approximately 100 μg of cell membrane components are loaded per 1 mg of PLGA nanoparticles.

[0268] Alternatively, 20 million mixed antigen-presenting cells (10 million BMDCs + 10 million B cells) co-incubated with nanovaccine 1 were collected by centrifugation at 400g for 5 minutes, and then washed twice with saline. The cells were resuspended in saline and then subjected to low-power 7.5W ultrasound at 4°C for 10 minutes to disrupt the cells and prepare a sample containing cell membrane components. The sample was then filtered once through a filter membrane with pore sizes of 50μm, 10μm, 5μm, 1μm, 0.45μm, and 0.22μm. The resulting filtrate was collected to obtain nanovaccine 7 (Nanovaccine 7) prepared from antigen-presenting cell membranes with a particle size of 200nm.

[0269] Alternatively, 20 million BMDCs incubated with nanovaccine 1 were collected by centrifugation at 400 g for 5 minutes, and then the BMDCs were washed twice with physiological saline. After the cells were resuspended in physiological saline, low-power 7.5 W ultrasound was used at 4 ° C for 10 minutes to destroy the cells and prepare a sample containing cell membrane components. The sample was then filtered through a filter membrane with a pore size of 50 μm, 10 μm, 5 μm, 1 μm, 0.45 μm, and 0.22 μm. The resulting filtrate was collected and incubated with the nanovaccine 1 (50 mg) prepared in step (2) for 10 minutes, and then repeatedly co-extruded using a 400 nm filter membrane. The extrudate was centrifuged at 15000 g for 60 minutes, the supernatant was discarded, and the supernatant was resuspended in ultrapure water containing 4% trehalose and freeze-dried for 48 hours. That is Nanovaccine 8, with a particle size of 300nm. Each 1mg of PLGA nanoparticles is loaded with approximately 200μg of protein and peptide components, 0.02mg each of poly(I:C), CpGODN 1018 (Class B), and CpG ODN M362 (Class C); each 1mg of PLGA nanoparticles is loaded with approximately 100μg of cell membrane components.

[0270] 20 million mixed antigen-presenting cells (10 million BMDCs + 10 million B cells) without incubation and stimulation were directly collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice with saline. After the cells were resuspended in saline, low-power 7.5W ultrasound was used at 4°C for 10 minutes to destroy the cells and prepare a sample containing cell membrane components. The sample was then filtered once through filter membranes with pore sizes of 50 μm, 10 μm, 5 μm, 1 μm, 0.45 μm, and 0.22 μm. The resulting filtrate was collected and incubated with the corresponding nanovaccine 1 (50 mg) prepared in step (2) for 10 minutes. The sample was then repeatedly co-extruded using a 400 nm filter membrane. The extrudate was centrifuged at 15,000 g for 60 minutes. The supernatant was discarded and the resulting precipitate was resuspended in physiological saline to obtain nanovaccine 9 with a particle size of 300 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 200 μg of protein or polypeptide components, and 0.02 mg each of poly(I:C), CpG ODN 1018 (class B), and CpG ODN M362 (class C); and each 1 mg of PLGA nanoparticles was loaded with approximately 100 μg of cell membrane components.

[0271] (5) Nanovaccines for cancer treatment

[0272] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare lung cancer tumor-bearing mice. On day 0, 1.5×10 6 LLC lung cancer cells were inoculated. Mice were subcutaneously injected with 100 μL of 0.02 mg of the nanovaccine (Nanovaccin 4, 5, 6, 7, 8, or 9) or 100 μL of PBS on days 3, 6, 9, 14, and 20 after tumor inoculation. Tumor growth and survival were monitored as described above.

[0273] (6) Experimental results

[0274] like Figure 6 As shown, the tumors in mice in the PBS control group grew rapidly, and their survival was short. Compared with the control group, the tumor growth rate in mice in the vaccine group was significantly slowed, and some mice experienced tumor disappearance and recovery. Among them, nanovaccine 4 was the most effective. Furthermore, nanovaccines 4 and 5 were more effective than nanovaccine 9; and nanovaccines 5 and 8 were significantly better than nanovaccines 6 and 7. This suggests that membrane fractions derived from mixed antigen-presenting cells (DCs) and B cells are more effective than membrane fractions derived from single antigen-presenting cells (DCs). Furthermore, membrane fractions derived from antigen-presenting cells activated by nanoparticles loaded with cancer cell antigens are more effective than membrane fractions derived from antigen-presenting cells that have not been activated by nanoparticles.

[0275] In this example, pepsin and trypsin were used for enzymatic hydrolysis. In practical applications, other enzymes may be used for enzymatic hydrolysis, and oxidation, reduction, mineralization and other methods may be used to improve the immunogenicity of the antigen.

[0276] Example 5 Vaccine for the treatment of colorectal cancer

[0277] In this embodiment, ultrapure water is first used to lyse MC38 mouse colon cancer cells to prepare protein and polypeptide components and water-insoluble components in the water-soluble components of the cancer cells, and then a nano vaccine is prepared to treat colon cancer. In this embodiment, the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells are loaded on the surface of the nano vaccine. In actual application, the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells can also be loaded inside the nano vaccine as needed; or they can be loaded on the surface of the nano vaccine and inside the nano vaccine at the same time. The extracellular vesicles of bacteria and the extracellular vesicles of cancer cells used in this embodiment can also use the outer membrane components of bacteria or the membrane components of cancer cells in actual applications; or the membrane components (cell membrane or extracellular vesicle membrane) of antigen-presenting cells loaded with cancer cell antigens and any mixed membrane components derived from membrane components of cancer cells and / or membrane components derived from bacteria can also be used.

[0278] In this embodiment, ultrasound, co-incubation and co-extrusion methods were used to load the membrane components onto the surface of the nanovaccine. In actual applications, any method that can load the membrane components onto the surface of the nanovaccine or micron vaccine through co-action can also be used, including but not limited to one or more of ultrasound, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bond connection, stirring, dialysis, homogenization and homogenization.

[0279] (1) Preparation of antigen components

[0280] After collecting the cultured MC38 mouse colon cancer cells, discard the supernatant and add an appropriate amount of ultrapure water to the cell pellet to resuspend the cells and freeze-thaw repeatedly 5 times. The lysate was then centrifuged at 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, and the non-water-soluble antigen insoluble in pure water was converted into a soluble antigen in 6M guanidine hydrochloride aqueous solution. The non-water-soluble components in the lysate dissolved in the above 6M guanidine hydrochloride aqueous solution, the water-soluble components in the lysate, and the cancer-related antigen WT1 235-243 -IO102 (CYTWNQMNLDTLLKALLEIASCLEKALQVF) and cancer-associated antigen WT1 294-312-IO103 (FRGIQDVRRVSGVAPTLVR-FMTYWHLLNAFTVTVPKDL) was mixed at a mass ratio of 1:1:1:0.1 to prepare the antigen component 1 for cancer nanovaccine 1 and nanovaccine 2. Among them, the cancer-related antigen WT1 235-243 -IO102's sequence is WT1 294-312 and IO103, two cancer-associated antigen sequences, WT1 294-312 -IO103 by WT1 294-312 It is composed of a chimeric combination of two cancer-associated antigen sequences, IO103 and IO103, and does not contain any other redundant amino acid sequences.

[0281] (2) Preparation of nanovaccines loaded with antigens and adjuvants

[0282] In this embodiment, nano vaccine 1 (Nanovaccine 1) was prepared using the double emulsion method in the solvent evaporation method. During preparation, the loaded antigen component was antigen component 1. The nano vaccine preparation material PLGA has a molecular weight of 7KDa-17KDa, the immune adjuvants are poly(I:C), CpG ODN 7909 (Class B) and CpG ODN 2395 (Class C), and the substance used to promote lysosomal escape is melittin. The preparation method is as described above. First, the double emulsion method is used to load the cell antigen component 1, adjuvant and melittin inside the nanoparticles. Then, 100mg of nanoparticles are centrifuged at 12000g for 20 minutes and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h. The nano vaccine 1 has an average particle size of about 380 nm. Each 1 mg of PLGA nanoparticles is loaded with about 800 μg of antigen protein or polypeptide component, 0.05 mg each of poly(I:C), CpG ODN 7909 (class B) and CpG ODN 2395 (class C), and 0.1 mg of melittin.

[0283] Nanovaccine 2 was prepared using the same materials and methods as Nanovaccine 1. The average particle size of Nanovaccine 2 was approximately 380 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 800 μg of antigen protein or polypeptide component and 0.1 mg of melittin, without any adjuvant.

[0284] (3) Preparation of bacterial extracellular vesicles (OMVs) and cancer cell extracellular vesicles

[0285] Bifidobacterium longum was centrifuged at 5000 g for 30 minutes, and the precipitate was discarded and the supernatant was collected. The supernatant was filtered using a 1 μm filter membrane, ultrasonically treated at 20 W for 5 minutes at 4°C, and then centrifuged at 16000 g for 90 minutes. The precipitate was resuspended in PBS to obtain the collected bacterial extracellular vesicle membrane component.

[0286] Alternatively, the cancer cells were centrifuged at 5000 g for 30 minutes, and the precipitate was discarded and the supernatant was collected. The supernatant was filtered using a 1 μm filter membrane, ultrasonically treated at 20 W for 5 minutes at 4°C, and then centrifuged at 16000 g for 90 minutes. The precipitate was resuspended in PBS to obtain the extracellular vesicle membrane component of the collected cancer cells.

[0287] The bacterial extracellular vesicle membrane fraction and the cancer cell extracellular vesicle membrane fraction are mixed in a mass ratio of 1:1 and set aside.

[0288] (4) Preparation of nanovaccines with internal antigen loading and surface loading of bacteria and cancer cell extracellular vesicle membrane components

[0289] 30 mg of the nano vaccine (nano vaccine 1 or nano vaccine 2) prepared in step (2) was mixed with 5 mg of the mixed membrane component of cancer cells and bacterial extracellular vesicles prepared in step (3) and allowed to stand for 1 minute. The mixture was then subjected to low-power 7.5 W ultrasound at 4°C for 1 minute and then repeatedly co-extruded through a filter membrane with a pore size of 0.45 μm. The resulting filtrate was collected and freeze-dried to obtain the nano vaccine. Among them, the nano vaccine 3 (Nanovaccine 3) obtained by co-acting the nano vaccine 1 with the mixed membrane component of cancer cells and bacterial extracellular vesicles had a particle size of 400 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 800 μg of antigen protein or polypeptide component, 0.05 mg each of poly(I:C), CpG ODN 7909 (class B) and CpG ODN 2395 (class C), 0.1 mg of melittin, and 20 μg of membrane component. Nanovaccine 4 was prepared by using nanovaccine 2 to interact with the mixed membrane components of cancer cells and bacterial extracellular vesicles. It has a particle size of 400 nm and each 1 mg of PLGA nanoparticles is loaded with approximately 800 μg of antigen protein or polypeptide components, 0.1 mg of bee venom peptide, and 20 μg of membrane components, without any adjuvant.

[0290] (5) Nanovaccines for cancer treatment

[0291] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer-bearing mice. On day 0, 1.0×10 6 MC38 colon cancer cells were inoculated. Mice were subcutaneously injected with 100 μL of 0.1 mg of either nanovaccine (Nanovaccine 3 or Nanovaccine 4) or 100 μL of PBS on days 3, 6, 9, 14, and 20 after tumor inoculation. Tumor growth and survival were monitored. Tumor volume was recorded every three days starting on day 3.

[0292] (6) Experimental results

[0293] The results show that Figure 7 As shown in the figure, the tumors in the PBS control group grew rapidly, resulting in a short survival period. However, the tumor growth rate of mice treated with the various nanovaccines was significantly slowed, and some mice even recovered from their tumors. Furthermore, nanovaccine 3 was more effective than nanovaccine 4. This suggests that the addition of an adjuvant can enhance the efficacy of nanovaccines.

[0294] In this embodiment, the extracellular vesicles of bacteria and cancer cells are loaded on the surface of the nanovaccine. In actual applications, the extracellular vesicles of bacteria and cancer cells can also be loaded inside the nanovaccine as needed; or they can be loaded on the surface of the nanovaccine and inside the nanovaccine at the same time. The extracellular vesicles of bacteria and cancer cells used in this embodiment can also use the outer membrane components of bacteria or the membrane components of cancer cells in actual applications; or a membrane component (cell membrane or extracellular vesicle membrane) of an antigen-presenting cell loaded with a cancer cell antigen and any mixed membrane component derived from a membrane component of a cancer cell and / or a membrane component derived from bacteria can also be used.

[0295] In this embodiment, ultrasound, co-incubation and co-extrusion methods were used to load the membrane components onto the surface of the nanovaccine. In actual applications, any method that can load the membrane components onto the surface of the nanovaccine or micron vaccine through co-action can also be used, including but not limited to one or more of ultrasound, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bond connection, stirring, dialysis, homogenization and homogenization.

[0296] In this embodiment, the membrane components of bacteria and cancer cells are located on the surface of the nano-vaccine or micro-vaccine. In practical applications, the membrane components of bacteria or cancer cells can also be loaded inside the nano-vaccine or micro-vaccine.

[0297] Example 6 Micron vaccine for the treatment of breast cancer

[0298] (1) Preparation of antigen components

[0299] The cultured 4T1 breast cancer cell line was collected and centrifuged at 350g for 5 minutes, and then the supernatant was discarded and washed twice with PBS. Then, ultrapure water containing 0.1% protease inhibitors was added to resuspend the cells and the cells were repeatedly frozen and thawed 5 times to lyse the cancer cells. The lysate components were then dissolved in a 3M guanidine sulfate aqueous solution. The cancer cell lysate components dissolved in the 3M guanidine sulfate aqueous solution and the cancer-associated antigen WT were added. 1235-243 -IO102( GASAYGSL CYTWNQMNLDTLLKALLEIASCLEKALQVF CYTWNQ ) and cancer-associated antigen WT1 294-312 IO103(FRGIQDVRRVSGVAPTLVR- EQCLSAFTL - FMTYWHLLNAFTVTVPKDL ) is the antigen component 1 for preparing the vaccine. Among them, the cancer-associated antigen WT1 235-243 -IO102's sequence is WT1 294-312 It is composed of two cancer-related antigen sequences, IO103 and some other additional amino acid sequences. The additional amino acid sequences GASAYGSL and CYTWNQ are located at both ends of the polypeptide sequence; WT1 294-312 -IO103 by WT1 294-312 The two cancer-associated antigen sequences IO103 and IO103 are chimeric with other additional amino acid sequences, and the additional sequence EQCLSAFTL is sandwiched between the other two cancer-associated antigen sequences.

[0300] (2) Preparation of micronized vaccines

[0301] In this example, micron vaccine 1 (Micronvaccine 1) was prepared using a double emulsion method. The PLGA material used in preparing micron vaccine 1 has a molecular weight of 24 kDa-38 kDa, and the immune adjuvants used are CpG ODN BW006, CpG ODN 2216, and PolyICLC. PolyICLC is a toll-like receptor 3 agonist, while various CpGs are toll-like receptor 9 agonists. Both toll-like receptor 3 and toll-like receptor 9 are located in the endosomal membrane structure within the cell. First, the antigen component 1 and the immune adjuvant were co-loaded into micron particles. The particles were then centrifuged at 10,000 g for 15 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Prior to use, the particles were resuspended in 7 mL of PBS, and 3 mL of the antigen component (protein concentration 50 mg / mL) was added and incubated at room temperature for 10 minutes to obtain micron vaccine 1 loaded with the antigen component both internally and externally. The average particle size of the micronized particles is about 2.50 μm; each 1 mg of PLGA micronized particles 1 is loaded with about 140 μg of protein or polypeptide components, and 0.03 mg of each of CpG BW006 (type B), CpG 2216 (type A), and Poly ICLC is loaded.

[0302] Micronvaccine 2 uses the same materials and methods, and is loaded with the following immune adjuvants: CpG2336 (Class A), CpG 2216 (Class A), and Poly-ICLC. Micronvaccine 2 has a particle size of approximately 2.50 μm, and each mg of PLGA microparticles carries approximately 140 μg of protein or peptide components. Each mg of PLGA microparticles contains 0.03 mg each of CpG2336 (Class A), CpG2216 (Class A), and Poly-ICLC.

[0303] Micronvaccine 3 uses the same materials and methods as the other vaccines, and is loaded with CpGBW006 (Class B) and CpG 2216 (Class A) as adjuvants. Each 1mg of PLGA microparticles in Micronvaccine 3 has a particle size of approximately 2.50μm and carries approximately 140μg of protein or peptide components. Each 1mg of PLGA microparticles contains 0.045mg each of CpG BW006 (Class B) and CpG 2216 (Class A).

[0304] (3) Treatment of cancer with micron vaccines

[0305] Female BALB / C mice aged 6-8 weeks were selected as model mice for the preparation of breast cancer tumor-bearing mice. On day 0, 1.0×10 6 4T1 cells were injected subcutaneously with 0.8 mg of the microvaccine (microvaccine 1, microvaccine 2, or microvaccine 3) or 100 μL of PBS on days 4, 7, 10, 15, 20, and 25 after cancer cell inoculation. Tumor growth and survival of mice were monitored as described above.

[0306] (4) Experimental results

[0307] like Figure 8 As shown, tumors in control mice grew, while tumor growth in mice treated with the microvaccine was significantly slowed and survival was significantly prolonged. Furthermore, the microvaccine loaded with a mixed adjuvant of CpG and Poly-ICLC was more effective than the microvaccine loaded with a mixed adjuvant of two CpGs. Furthermore, the microvaccine loaded with a mixed adjuvant of one class B CpG, one class A CpG, and Poly-ICLC was more effective than the microvaccine loaded with a mixed adjuvant of two class A CpGs and Poly-ICLC. This suggests that microvaccines loaded with mixed adjuvants of two different toll-like receptors are more effective, and that the microvaccine containing a mixed adjuvant of a class B CpG and a Toll-like receptor 3 agonist was even more effective.

[0308] Example 7 Micron Vaccine for Cancer Prevention

[0309] (1) Preparation of antigen components

[0310] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to about 1000mm 3Mice were sacrificed and tumor tissue was harvested. The tumor tissue was cut into pieces, passed through a cell strainer, and an appropriate amount of ultrapure water was added. The tumor tissue was then repeatedly frozen and thawed five times to lyse the tumor tissue. The lysate was then centrifuged at 5000g for 5 minutes. The supernatant was the water-soluble fraction soluble in pure water. A 0.3M aqueous semicarbazide hydrochloride solution was added to the resulting precipitate to dissolve the precipitate, converting the water-insoluble fraction into a fraction soluble in 0.3M semicarbazide hydrochloride. A saturated aqueous magnesium sulfate solution was added dropwise to the water-soluble fraction in the lysate. After complete precipitation, the resulting sample was centrifuged at 12000g for 5 minutes. The precipitate was dissolved in 0.3M semicarbazide hydrochloride and set aside. The supernatant was heated at 100°C for 2 minutes and then centrifuged at 3000g for 5 minutes. The supernatant was discarded and the precipitate was dissolved in 0.3M semicarbazide hydrochloride. The precipitate after salting out and the heated precipitate were combined and used as part of the water-soluble fraction. Saturated magnesium sulfate aqueous solution was added dropwise to the water-insoluble components of the lysate dissolved in 0.3 M hydrochloric acid semicarbazide aqueous solution. After complete precipitation, the resulting sample was centrifuged at 12,000 g for 5 minutes, and the precipitated components were redissolved in 0.3 M hydrochloric acid semicarbazide aqueous solution. The antigen component 1 for preparing the cancer micron vaccine was prepared by mixing a portion of the water-insoluble components of the lysate dissolved in 0.3 M hydrochloric acid semicarbazide aqueous solution, a portion of the water-soluble components dissolved in 0.3 M hydrochloric acid semicarbazide, and mRNA encoding two cancer-associated antigens (IO102: DTLLKALLEIASCLEKALQVF and IO103: FMTYWHLLNAFTVTVPKDL) in a mass ratio of 1:2:1.

[0311] Alternatively, 1.5 × 10 5 B16F10 cells, when the tumor grows to about 1000mm 3Mice were sacrificed and tumor tissue was harvested. The tumor tissue was cut into pieces, passed through a cell strainer, and added with an appropriate amount of ultrapure water. The tumor tissue was then repeatedly frozen and thawed five times to lyse the tumor tissue. The lysate was then centrifuged at 5000g for 5 minutes. The supernatant was collected as the water-soluble fraction soluble in pure water. A 5% PEG5000 aqueous solution was added to the resulting precipitate to solubilize the precipitate, thereby obtaining a fraction of the water-insoluble fraction dissolved in 5% PEG5000. A saturated magnesium sulfate aqueous solution was added dropwise to the water-soluble fraction in the lysate. After complete precipitation, the resulting sample was centrifuged at 12000g for 5 minutes. The precipitate was dissolved in 5% PEG5000 aqueous solution and set aside. The supernatant was heated at 100°C for 2 minutes and then centrifuged at 3000g for 5 minutes. The supernatant was discarded and the precipitate was solubilized in 5% PEG5000 aqueous solution. The precipitate after salting out and the heated precipitate dissolved in 5% PEG5000 aqueous solution were combined and used as part of the water-soluble fraction. Saturated magnesium sulfate solution was added dropwise to the water-insoluble fraction of the lysate dissolved in 5% PEG5000 aqueous solution. After complete precipitation, the resulting sample was centrifuged at 12,000 g for 5 minutes. The precipitated fraction was then solubilized a second time with 5% PEG5000 aqueous solution. Antigen component 2 for preparing the cancer micron vaccine was prepared by combining a portion of the water-insoluble fraction of the lysate dissolved in 5% PEG5000 aqueous solution, a portion of the water-soluble fraction dissolved in 5% PEG5000 aqueous solution, and mRNA encoding two cancer-associated antigens (IO102: DTLLKALLEIASCLEKALQVF and IO103: FMTYWHLLNAFTVTVPKDL) in a mass ratio of 1:2:1.

[0312] Alternatively, mRNA encoding two cancer-associated antigens (10102: DTLLKALLEIASCLEKALQVF and 10103: FMTYWHLLNAFTVTVPKDL) was used as antigen component 3 for preparing the control micro vaccine.

[0313] (2) Preparation of micrometers

[0314] In this example, micron vaccine 1 (Micronvaccine 1) was prepared using a solvent evaporation method. The PLGA material used in Micron Vaccine 1 has a molecular weight of 38-54 kDa, and the immunoadjuvants used are Poly(I:C), CpG2006 (Class B), and CpGSL01 (Class B). The preparation method is as described above. After loading the antigen component 1 and adjuvants into the micron particles, 100 mg of the micron particles were centrifuged at 10,000 g for 15 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours to obtain a lyophilized powder for later use. Micron Vaccine 1 has an average particle size of approximately 5.0 μm. Each 1 mg of PLGA micron particle is loaded with approximately 60 μg of tumor tissue protein and peptide components, 0.02 mg each of Poly(I:C), CpG2006, and CpGSL01, and 20 μg of mRNA.

[0315] Micronvaccine 2 was prepared using the same method as Micronvaccine 1. Antigen component 2 and adjuvant were co-loaded within the micron particles. Micronvaccine 2 had an average particle size of approximately 5.0 μm, and each 1 mg of PLGA microparticles was loaded with approximately 60 μg of tumor tissue protein and peptide components. Each 1 mg of PLGA microparticles contained 0.02 mg each of Poly(I:C), CpG2006, and CpGSL01, along with 20 μg of mRNA.

[0316] The preparation materials and methods of Micronvaccine 3 are the same as those of Micronvaccine 1. The particle size is about 5.0 μm, and it is loaded with an equal amount of antigen component 1. The loaded immune adjuvant is Poly(I:C), with 0.06 mg of Poly(I:C) and 20 μg of mRNA loaded per 1 mg of PLGA.

[0317] Micronvaccine 4 was prepared using the same materials and methods as Micronvaccine 1. It was loaded with antigen component 1 and adjuvant, and had a particle size of approximately 5.0 μm. Each 1 mg of PLGA microparticles was loaded with approximately 0.05 μg of tumor tissue protein and peptide components, 0.02 mg each of Poly(I:C), CpG2006, and CpGSL01 peptides, and 0.02 μg of mRNA.

[0318] Micronvaccine 5 was prepared similarly to Micronvaccine 1 and loaded with antigen component 3 and adjuvant. Micronvaccine 5 had an average particle size of approximately 5.0 μm. Each 1 mg of PLGA microparticles contained approximately 60 μg of tumor tissue protein and peptide components, approximately 0.02 mg each of Poly(I:C), CpG2006, and CpGSL01, and 20 μg of mRNA.

[0319] (3) Micron vaccines for cancer prevention

[0320] Melanoma-bearing mice were prepared by selecting 6-8 week old female C57BL / 6 mice as model mice. 6 mg of micro vaccine (micro vaccine 1, micro vaccine 2, micro vaccine 3, micro vaccine 4, or micro vaccine 5) or 100 μL of PBS were injected subcutaneously on days -35, -28, -21, and -14 before tumor inoculation. On day 0, 1.5×10 5 The methods for monitoring tumor growth rate and survival of mice were the same as above.

[0321] (4) Experimental results

[0322] The results show that Figure 9 As shown, tumors in mice in the PBS control group grew rapidly, while tumor growth in mice in the microvaccine-treated group slowed significantly, and tumors disappeared in most mice after cancer cell inoculation. Microvaccine 1 was more effective than microvaccines 2, 3, 4, and 5. This suggests that vaccines prepared using both lysate fractions and mRNA encoding cancer-associated antigens as antigenic components are more effective than vaccines prepared using only mRNA encoding the antigens; microvaccines loaded with two B-class CpGs and Poly(I:C) as mixed adjuvants are more effective than microvaccines loaded with Poly(I:C) alone; microvaccines prepared using an antigenic component derived from the precipitate of the water-insoluble fraction of the lysate dissolved in 0.3M hydrochloric acid semicarbazide and the water-soluble fraction, followed by salting-out, are more effective than PEG5000; and microvaccines with higher concentrations of antigenic components are more effective.

[0323] Example 8 Nano-vaccine for the treatment of T lymphoma

[0324] (1) Preparation of antigen components

[0325] After the cultured E.G7-OVA cell line is collected and centrifuged at 350g for 5 minutes, the supernatant is discarded and the cells are washed twice with PBS. The cells are then resuspended in ultrapure water and repeatedly frozen and thawed 5 times, optionally accompanied by sonication to disrupt the lysed cells. After the cells are lysed, trypsin (0.5mg / mL) and chymotrypsin (0.5mg / mL) are added and incubated for 10 minutes, followed by heating at 95°C for 10 minutes. The lysate is then centrifuged at 10,000g for 5 minutes and the supernatant is obtained as the water-soluble fraction soluble in pure water; 0.1M metformin hydrochloride aqueous solution is added to the resulting precipitate to dissolve the precipitate, thereby converting the water-insoluble fraction in pure water into a fraction soluble in 0.1M metformin hydrochloride aqueous solution.

[0326] A saturated aqueous ammonium sulfate solution was added dropwise to the water-soluble component in the lysate. After complete precipitation, the resulting sample was centrifuged at 12,000 g for 5 minutes, and the precipitate was dissolved in a 0.1 M metformin hydrochloride aqueous solution for later use. The supernatant was heated at 100° C. for 2 minutes, and the resulting sample was centrifuged at 3,000 g for 5 minutes. After discarding the supernatant, the precipitate was dissolved in a 0.1 M metformin hydrochloride aqueous solution. The salted-out precipitate dissolved in 0.1 M metformin hydrochloride and the heated precipitate were then combined and used as part of the water-soluble component.

[0327] All water-insoluble components in the lysate dissolved in the above 0.1M metformin hydrochloride solution, some components in the water-soluble components dissolved in the 0.1M metformin hydrochloride solution, cancer-related antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF) and cancer-related antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) were mixed in a mass ratio of 5:1:0.1:0.2, and then the above components were oxidized using hypochlorous acid for 15 minutes to prepare the antigen component 1 of the nanovaccine.

[0328] (2) Lysis and dissolution of BCG

[0329] BCG was collected, and the BCG was lysed using a metformin hydrochloride aqueous solution, and then the lysed components of the BCG were dissolved using the metformin hydrochloride aqueous solution for later use.

[0330] (3) Preparation of nanovaccines

[0331] Nano vaccine 1 (Nanovaccine 1) adopts solvent evaporation method to prepare in the present embodiment. Nano vaccine 1 preparation material PLA molecular weight is 20KDa, nanoparticle internal load antigen component 1, bacterial lysate and immune adjuvant, surface antigen component 1. The immune adjuvant adopted is CpG2395 (C class), CpGM362 (C class) and poly ICLC, and adjuvant is loaded in nanoparticle inside, and the mass ratio of antigen component 1 and bacterial lysate component used when preparing nanoparticle is 1:1. Preparation method is as mentioned above, first in nanoparticle internal load antigen component 1, bacterial lysate component and adjuvant, then 100mg nanoparticles are centrifuged 20 minutes at 10000g, and use 10mL to contain 4% trehalose ultrapure water resuspended back freeze drying 48h. Before use, 20mg nanoparticles are resuspended among the 0.9mL PBS, and can be used after 0.1mL contains the sample room temperature mixing of antigen component 1 (80mg / mL) and incubation 5 minutes. The average particle size of nanovaccine 1 is about 400 nm. Each 1 mg of PLA nanovaccine 1 is loaded with approximately 400 μg of protein or polypeptide components. Each 1 mg of PLA nanoparticles is loaded with 0.005 mg each of CpG2395 (type C), CpGM362 (type C) and Poly ICLC immune adjuvant.

[0332] Nanovaccine 2 Preparation Materials and Preparation Methods Nanovaccine 1 has a particle size of about 400 nm. Each 1 mg of PLA nanovaccine 2 is loaded with approximately 400 μg of protein or polypeptide components, and each 1 mg of PLA is loaded with 0.005 mg each of CpG1585 (class A), CpG2336 (class A), and Poly ICLC.

[0333] (4) Nanovaccines for cancer treatment

[0334] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. On day 0, each mouse was subcutaneously inoculated with 5×10 5 The mice were subcutaneously injected with 100 μL PBS or 0.1 mg of nanovaccine (nanovaccine 1 or nanovaccine 2) on days 5, 8, 13, and 20. The tumor volume and survival of the mice were monitored as above.

[0335] (5) Experimental results

[0336] like Figure 9 As shown, the tumors of mice in the PBS control group grew rapidly and the mice had a short survival period. Compared with the control group, the tumor growth rate of mice treated with both nanovaccines was significantly slowed down and the survival period was significantly prolonged, and the effect of nanovaccine 1 was better.

[0337] This embodiment uses hypochlorous acid as an oxidant to oxidize the antigen component. In practical applications, natural gas and other oxidants can also be used to oxidize the antigen.

[0338] Example 9 Vaccine for the treatment of melanoma

[0339] (1) Preparation of antigen components

[0340] Two cultured melanoma cell lines, B16F10 and S91, were harvested and mixed at a 1:1 ratio. The cells were then centrifuged at 500g for 5 minutes, the supernatant discarded, and the cells were washed twice with PBS. The cells were then resuspended in ultrapure water and repeatedly freeze-thawed five times with sonication to lyse the cells. Following cell lysis, the lysate was centrifuged at 10,000g for 5 minutes, and the supernatant was collected as the water-soluble fraction. The resulting precipitate was dissolved in a 0.2M arginine and 0.2M polyhexamethyleneguanidine hydrochloride aqueous solution, converting the water-insoluble fraction into a fraction soluble in the 0.2M arginine and 0.2M polyhexamethyleneguanidine hydrochloride aqueous solution.

[0341] Saturated ammonium sulfate was added dropwise to the water-soluble fraction in the lysate. After complete precipitation, the resulting sample was centrifuged at 12,000 g for 5 minutes. The precipitate was dissolved in a 0.2 M arginine and 0.2 M polyhexamethyleneguanidine hydrochloride aqueous solution for later use. The supernatant was treated with DNA digestion enzyme for 5 minutes, heated at 100°C for 2 minutes, and then centrifuged at 3,000 g for 5 minutes. The supernatant was discarded and the precipitate was dissolved in a 0.2 M arginine and 0.2 M polyhexamethyleneguanidine hydrochloride aqueous solution. The salted-out precipitate dissolved in 0.2 M arginine and the heated precipitate were then combined and used as part of the water-soluble fraction. Saturated ammonium sulfate was added dropwise to the water-insoluble fraction in the lysate. After complete precipitation, the resulting sample was centrifuged at 12,000 g for 5 minutes. The precipitate was redissolved in a 0.2 M arginine and 0.2 M polyhexamethyleneguanidine hydrochloride aqueous solution to form part of the water-insoluble fraction. The above 0.2M arginine and 0.2M polyhexamethyleneguanidine hydrochloride solution secondary dissolution of some components of the water-insoluble components in the lysate, 0.2M arginine and 0.2M polyhexamethyleneguanidine hydrochloride solution dissolved some components of the water-soluble components, cancer-related antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF) and cancer-related antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) were mixed in a mass ratio of 1:5:0.5:0.5 to prepare the antigen component 1 of the nanovaccine.

[0342] Alternatively, without separating and purifying the water-soluble and water-insoluble components, all water-insoluble components, water-soluble components, cancer-associated antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF) and cancer-associated antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) in the lysate dissolved in 0.2 M arginine and 0.2 M polyhexamethyleneguanidine hydrochloride solution are directly mixed in a mass ratio of 1:5:0.5:0.5 to prepare the antigen component 2 of the nanovaccine.

[0343] Alternatively, the cancer-associated antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF) and the cancer-associated antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) are mixed at a mass ratio of 1:1 to obtain antigen component 3.

[0344] (2) Preparation of nanovaccines

[0345] In this example, nanovaccine 1 was prepared using a double emulsion method. Nanovaccine 1 is made of PLGA (with a molecular weight of 24-38 kDa), with poly(I:C) and CpG1018 as immune adjuvants, and the substance that enhances lysosomal immune escape is the KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA). The preparation method is as described above. First, the antigen component 1, adjuvant, and KALA polypeptide are loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles are centrifuged at 12,000 g for 25 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The nanoparticles have an average particle size of approximately 250 nm. Each 1 mg of PLGA nanoparticles carries approximately 100 μg of the antigen protein or polypeptide component, 0.04 mg each of poly(I:C) and CpG1018, and 0.3 mg of the KALA polypeptide.

[0346] Nanovaccine 2 was prepared using the same materials and methods, but the loaded antigen component was antigen component 2, which had a particle size of approximately 250 nm. Each 1 mg of PLGA nanoparticles loaded approximately 100 μg of antigen protein or polypeptide component, 0.04 mg each of poly(I:C) and CpG1018, and 0.3 mg of KALA polypeptide.

[0347] The preparation materials and preparation methods of Nanovaccine 3 are the same, but the loaded antigen component is antigen component 3, whose particle size is about 250nm. Each 1mg of PLGA nanoparticles is loaded with approximately 100μg of antigen polypeptide component, 0.04mg each of poly(I:C) and CpG1018, and 0.3mg of KALA polypeptide.

[0348] (3) Nanovaccines for cancer treatment

[0349] Melanoma-bearing mice were prepared by selecting 6-8 week old female C57BL / 6 mice as model mice. On day 0, 1.5×10 5 B16F10 cells were inoculated. 100 μL of PBS or 0.4 mg of the corresponding nanovaccine were subcutaneously injected on days 4, 7, 10, 15, and 20 after melanoma inoculation. Tumor volume and survival of mice were monitored as described above.

[0350] (4) Experimental results

[0351] like Figure 11 As shown, tumors in the PBS control group grew rapidly. Compared to the control group, mice treated with the nanovaccines showed significantly slower tumor growth and prolonged survival. Furthermore, nanovaccine 1 was more effective than nanovaccines 2 and 3, demonstrating that isolating and purifying protein and peptide components from cell lysates can enhance vaccine efficacy. Furthermore, vaccines loaded with both cell lysate components and synthetic antigen peptide components were more effective than those loaded with only the antigen peptide component.

[0352] Example 10 Micron Vaccine for Cancer Prevention

[0353] (1) Preparation of antigen components

[0354] Cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, washed twice with PBS, and resuspended in ultrapure water. The resulting cancer cells were inactivated and denatured by ultraviolet light and high-temperature heating (55°C), respectively. The cancer cells were then repeatedly frozen and thawed five times to lyse the cancer cells. 0.01 mg / mL nuclease was then added for 5 minutes to digest and degrade the nucleic acids in the lysate. The nuclease was then inactivated by heating at 95°C for 5 minutes. The cancer cell lysate components were then dissolved using 0.3 M methylguanidine hydrochloride and 0.1 M tetramethylguanidine hydrochloride aqueous solutions.

[0355] Lactobacillus acidophilus was centrifuged at 5000g for 30 minutes, and the precipitate was discarded and the supernatant was collected. The supernatant was filtered using a 1 μm filter membrane and then centrifuged at 16000g for 90 minutes. The supernatant was discarded and the resulting precipitate was the bacterial extracellular vesicle component. The precipitate was lysed using 0.3M methylguanidine hydrochloride and 0.1M tetramethylguanidine hydrochloride aqueous solution to dissolve the bacterial extracellular vesicle component.

[0356] A cancer cell lysate component dissolved in a 0.3M methylguanidine hydrochloride and 0.1M tetramethylguanidine hydrochloride aqueous solution and a bacterial extracellular vesicle component dissolved in a 0.3M methylguanidine hydrochloride and 0.1M tetramethylguanidine hydrochloride aqueous solution were mixed at a mass ratio of 8:1. Saturated ammonium sulfate aqueous solution was then added dropwise to the mixed sample for salting out. After complete precipitation, the resulting sample was centrifuged at 12,000 g for 5 minutes. The supernatant was discarded, and the salted-out precipitate was redissolved in a 0.3M methylguanidine hydrochloride and 0.1M tetramethylguanidine hydrochloride aqueous solution. The protein and polypeptide components of the cancer cells and bacteria, which were redissolved in the 0.3M methylguanidine hydrochloride and 0.1M tetramethylguanidine hydrochloride aqueous solution, and the cancer-associated antigen polypeptides 10102 (DTLLKALLEIASCLEKALQVF) and 10103 (FMTYWHLLNAFTVTVPKDL) were mixed at a mass ratio of 1:1:1 to obtain antigen component 1 for preparing the micron vaccine.

[0357] (2) Preparation of micronized vaccines

[0358] In this example, micron vaccine 1 (Micronvaccine 1) was prepared using a double emulsion method. The PLGA backbone material of Micronvaccine 1 has a molecular weight of 20-40 kDa, and the immune adjuvants are CpG2006 (Class B), CpG2216 (Class A), and Poly-ICLC. During preparation, micron particles loaded with the antigen component and adjuvant were prepared using the double emulsion method. Subsequently, 100 mg of the micron particles were centrifuged at 8000 g for 15 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours to obtain Micronvaccine 1. The average particle size was approximately 3.0 μm. Each 1 mg of PLGA Micronvaccine 1 was loaded with approximately 2 mg of protein or peptide components, and 0.07 mg each of CpG2006, CpG2216, and Poly-ICLC.

[0359] The preparation materials and preparation methods of Micronvaccine 2 are the same as those of Micronvaccine 1. The particle size is about 3.0 μm. It is loaded with the same amount of antigen component 1, but does not load CpG2006, CpG2216 and Poly ICLC.

[0360] (3) Micron vaccines for cancer prevention

[0361] Tumor-bearing mice were prepared by selecting female C57BL / 6 mice aged 6-8 weeks. 100 μL PBS or 1.5 mg of micro-vaccine (micro-vaccine 1 or micro-vaccine 2) were subcutaneously injected into the mice on days -35, -28, -21, -14, and -7 before tumor inoculation. 5×10 5The tumor volume and survival of mice were monitored using the same method as above.

[0362] (4) Experimental results

[0363] like Figure 12 As shown, compared with the PBS control group, the tumor growth rate of mice treated with micro-vaccine was significantly slowed and the survival time of mice was significantly prolonged. Moreover, micro-vaccine 1 was significantly better than micro-vaccine 2, indicating that the use of mixed adjuvants can help improve the effectiveness of vaccines.

[0364] Example 11 Nano-vaccine for the treatment of colon cancer

[0365] (1) Preparation of antigen components based on cancer cell lysate components and cancer cell extracellular vesicle components

[0366] The MC38 cancer cell line and its culture medium were collected and centrifuged at 500g for 5 minutes. The cell pellet was collected and lysed with a 6M guanidine hydrochloride aqueous solution until completely dissolved, thereby obtaining the collected cancer cell lysate component. The culture supernatant was collected and filtered using a 1μm filter membrane, then centrifuged at 160,000g for 90 minutes. The supernatant was discarded and the pellet was lysed with a 6M guanidine hydrochloride aqueous solution until completely dissolved, thereby obtaining the collected cancer cell extracellular vesicle lysate component. The above two components were combined, and then a saturated magnesium sulfate aqueous solution was added until the precipitation was complete. The supernatant was discarded and the pellet was redissolved in an 8M urea aqueous solution. The protein and peptide components of the cancer cells dissolved in the 8M urea aqueous solution, the cancer-associated antigen peptide IO102 (DTLLKALLEIASCLEKALQVF), and the cancer-associated antigen peptide IO103 (FMTYWHLLNAFTVTVPKDL) were mixed in a mass ratio of 1:1:1 to obtain the antigen component 1 for preparing nanoparticles 1 and nanovaccine 1.

[0367] The MC38 cancer cell line and its culture medium were collected and centrifuged at 500g for 5 minutes. The cell pellet was collected and lysed with a 6M guanidine hydrochloride solution until completely dissolved, thereby obtaining the collected cancer cell lysate component. The culture supernatant was collected and filtered through a 1μm filter membrane, then centrifuged at 160,000g for 90 minutes. The supernatant was discarded, and the pellet was lysed with a 6M guanidine hydrochloride solution until completely dissolved, thereby obtaining the collected cancer cell extracellular vesicle lysate component. The two components were combined, and then saturated magnesium sulfate solution was added until complete precipitation was completed. The supernatant was discarded, and the pellet was solubilized with a 5% PEG5000 solution. The protein and peptide components of the cancer cells dissolved in the 5% PEG5000 solution, the cancer-associated antigen peptide IO102 (DTLLKALLEIASCLEKALQVF), and the cancer-associated antigen peptide IO103 (FMTYWHLLNAFTVTVPKDL) were mixed in a mass ratio of 1:1:1 to obtain antigen component 2 for preparing nanoparticles 2 and nanovaccine 2.

[0368] The MC38 cancer cell line and its culture medium were collected and centrifuged at 500g for 5 minutes. The cell pellet was collected, lysed with a 5% PEG5000 aqueous solution, and solubilized with a 5% PEG5000 aqueous solution to obtain the collected cancer cell lysate component. The culture supernatant was collected, filtered through a 1 μm filter membrane, and then centrifuged at 160,000g for 90 minutes. The supernatant was discarded, and the pellet was lysed with a 5% PEG5000 aqueous solution and completely dissolved to obtain the collected cancer cell extracellular vesicle lysate component. The two components were combined, and then saturated magnesium sulfate aqueous solution was added until the pellet was completely precipitated. The supernatant was discarded and the pellet was dissolved with an 8M urea aqueous solution. The protein and peptide components of the cancer cells dissolved in the 8M urea aqueous solution, the cancer-associated antigen peptide IO102 (DTLLKALLEIASCLEKALQVF), and the cancer-associated antigen peptide IO103 (FMTYWHLLNAFTVTVPKDL) were mixed in a mass ratio of 1:1:1 to obtain antigen component 3 for preparing nanoparticles 3 and nanovaccine 3.

[0369] (2) Preparation of nanovaccines

[0370] In this example, nanovaccine 1 (Nanovaccine 1) was prepared using a double emulsion method. The material used in preparing nanovaccine 1 is PLGA, with a molecular weight of 7-17 kDa. Poly(I:C), CpG2336, and CpG2006 are used as adjuvants. Both the antigen component 1 and the immune adjuvant are loaded into the nanoparticles. The preparation method is as described above. First, the antigen component 1 and the adjuvant are loaded into the nanoparticles. Then, 100 mg of the nanoparticles are centrifuged at 15,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours before use. The nanoparticles 1, or nanovaccine 1, have an average particle size of approximately 110 nm. Each 1 mg of PLGA nanoparticles is loaded with approximately 2 μg of protein and polypeptide components, and 0.01 mg each of poly(I:C), CpG2336, and CpG2006.

[0371] The preparation materials and preparation methods of nanovaccine 2 are the same as those of nanovaccine 1, but the internal load is antigen component 2 and adjuvant. The particle size is about 110 nm. Each 1 mg of PLGA nanoparticles 2 or nanovaccine 2 is loaded with approximately 2 μg of protein and polypeptide components, and 0.01 mg each of poly(I:C), CpG2336 and CpG2006.

[0372] The preparation materials and preparation methods of nanovaccine 3 are the same as those of nanovaccine 1, but the internal load is antigen component 3 and adjuvant. The particle size is about 110nm. Each 1mg of PLGA nanoparticles 3 or nanovaccine 3 is loaded with approximately 2μg of protein and polypeptide components, and 0.01mg each of poly(I:C), CpG2336 and CpG2006.

[0373] (3) Preparation and activation of antigen-presenting cells

[0374] This example uses BMDC and B as antigen-presenting cells. The BMDC preparation method is the same as above. B cells are obtained from mouse peripheral blood PBMCs using magnetic bead sorting. BMDC and B cells are mixed in a 1:1 ratio to form mixed antigen-presenting cells. 1 mg of nanoparticles 1 or nanoparticles 2 or nanoparticles 3 were co-incubated with BMDC (10 million) and B cells (10 million) in 15 mL of high-glucose DMEM complete culture medium for 48 hours (37°C, 5% CO2); the incubation system contained vinblastine (10 ng / mL) and IL-15 (50 ng / mL).

[0375] (4) Preparation of nanovaccines loaded with membrane components of antigen-presenting cells and extracellular vesicle membrane components of cancer cells

[0376] After incubation, 20 million mixed antigen-presenting cells were collected and centrifuged at 400 g for 5 minutes. The precipitate was discarded and the supernatant was collected. The supernatant was centrifuged at 16,000 g for 60 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS to obtain the extracellular vesicles of activated antigen-presenting cells.

[0377] 20 million cultured MC38 cells were collected and centrifuged at 400g for 5 minutes. The precipitate was discarded and the supernatant was collected. The supernatant was centrifuged at 15000g for 60 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS to obtain extracellular vesicles of cancer cells.

[0378] The collected activated antigen-presenting cell extracellular vesicles were mixed with cancer cell extracellular vesicles, and then sonicated at 4°C for 2 minutes at low power (20W), and then repeatedly co-extruded using a 0.22μm filter membrane. The extrudate was mixed with the nanoparticles 1 or 2 prepared in step (2) and treated with a high-pressure homogenizer (10000 bar) for 1 minute. Then, the mixture was repeatedly co-extruded using a 0.22μm filter membrane. After centrifugation at 15000g for 30 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in a 4% trehalose aqueous solution and freeze-dried for 48 hours to obtain a nanovaccine. Among them, the nanovaccine prepared by co-acting the antigen-presenting cell extracellular vesicle membrane component activated by nanoparticle 1 with nanoparticle 1 is nanovaccine 4, which has a particle size of 120nm and is loaded with 10μg each of poly(I:C), CpG2336, and CpG2006 immune adjuvants per 1mg of PLGA, with approximately 80μg of membrane component loaded per 1mg of PLGA. Nanovaccine 5 was prepared by co-administering the extracellular vesicle membrane fraction of antigen-presenting cells activated by nanoparticle 2 with nanoparticle 2. Its particle size was 120 nm, and 10 μg each of poly(I:C), CpG2336, and CpG2006 immune adjuvants were loaded per 1 mg of PLGA, resulting in approximately 80 μg of membrane fraction per 1 mg of PLGA. Nanovaccine 6 was prepared by co-administering the extracellular vesicle membrane fraction of antigen-presenting cells activated by nanoparticle 3 with nanoparticle 3. Its particle size was 120 nm, and 10 μg each of poly(I:C), CpG2336, and CpG2006 immune adjuvants were loaded per 1 mg of PLGA, resulting in approximately 80 μg of membrane fraction per 1 mg of PLGA.

[0379] (5) Nanovaccines for cancer treatment

[0380] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice for colon cancer. On day 0, 2×10 6MC38 cells were inoculated. 0.4 mg of the nanovaccine (Nanovaccine 4, Nanovaccine 5, or Nanovaccine 6) or 100 μL of PBS was subcutaneously injected on days 6, 9, 12, 15, 20, and 25 after colon cancer cell inoculation. Tumor growth and survival of the mice were monitored as described above.

[0381] (6) Experimental results

[0382] like Figure 13 As shown, compared with the PBS control group, mice treated with nanovaccines showed significantly slower tumor growth and prolonged survival. Furthermore, nanovaccine 4 was more effective than nanovaccines 5 and 6. This demonstrates that vaccines prepared from precipitates treated with salting-out methods must be dissolved or solubilized with appropriate solvents to achieve effective therapeutic effects.

[0383] In this embodiment, a nano-vaccine or micro-vaccine is used to load the membrane components of activated dendritic cells and B cells on its surface. In actual applications, only the membrane components of activated dendritic cells can be loaded, or a mixed membrane component of activated dendritic cells, B cells and macrophages can be loaded, or a membrane component of cancer cells can be added to the membrane component of the above-mentioned antigen-presenting cells, or a membrane component of bacteria can be added to the membrane component of the above-mentioned antigen-presenting cells.

[0384] Example 12 Nano-vaccines for cancer prevention

[0385] (1) Preparation of cancer cell-related components

[0386] Cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, washed twice with PBS, and resuspended in ultrapure water. A 6M guanidine hydrochloride aqueous solution was then used to lyse the cancer cells and dissolve the lysate components. A saturated ammonium sulfate aqueous solution was then added until complete precipitation, after which the supernatant was discarded. The precipitate was redissolved in a 6M guanidine hydrochloride aqueous solution to obtain protein and polypeptide components from the cancer cells dissolved in a 6M guanidine hydrochloride aqueous solution. The protein and polypeptide components from the cancer cells dissolved in a 6M guanidine hydrochloride aqueous solution, the cancer-associated antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF), and the cancer-associated antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) were mixed in a mass ratio of 1:1:1 to form antigen component 1.

[0387] Cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, washed twice with PBS, and resuspended in ultrapure water. The cancer cells were then lysed with a 3% Tween 80 aqueous solution, and the lysate components were solubilized. Saturated ammonium sulfate aqueous solution was then added until complete precipitation, after which the supernatant was discarded. The precipitate was again solubilized with a 3% Tween 80 aqueous solution to obtain a protein and polypeptide component of the cancer cells dissolved in a 3% Tween 80 aqueous solution. The protein and polypeptide component of the cancer cells dissolved in a 3% Tween 80 aqueous solution, the cancer-associated antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF), and the cancer-associated antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) were mixed in a mass ratio of 1:1:1 to form antigen component 2.

[0388] Cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, washed twice with PBS, and resuspended in ultrapure water. The cancer cells were then lysed with a 6M guanidine hydrochloride aqueous solution, and the lysate components were solubilized. Saturated ammonium sulfate aqueous solution was then added until precipitation was complete, after which the supernatant was discarded. The precipitate was again solubilized with a 3% Tween 80 aqueous solution to obtain a protein and polypeptide component of the cancer cells dissolved in a 3% Tween 80 aqueous solution. The protein and polypeptide component of the cancer cells dissolved in a 3% Tween 80 aqueous solution, the cancer-associated antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF), and the cancer-associated antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) were mixed in a mass ratio of 1:1:1 to form antigen component 3.

[0389] Cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, washed twice with PBS, and resuspended in ultrapure water. The cancer cells were then lysed with a 3% Tween 80 aqueous solution, and the lysate fraction was solubilized with a 3% Tween 80 aqueous solution. Saturated ammonium sulfate aqueous solution was then added until complete precipitation, after which the supernatant was discarded. The precipitate was again dissolved with a 6M guanidine hydrochloride aqueous solution to obtain a protein and polypeptide fraction from the cancer cells dissolved in a 6M guanidine hydrochloride aqueous solution. The protein and polypeptide fraction from the cancer cells dissolved in a 6M guanidine hydrochloride aqueous solution, the cancer-associated antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF), and the cancer-associated antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) were mixed in a mass ratio of 1:1:1 to obtain antigen fraction 4.

[0390] (2) Preparation of nanovaccines

[0391] In this example, nanovaccine 1 was prepared using a double emulsion method. The framework materials for nanovaccine 1 were PLA (molecular weight 40 kDa) and mannose-PEG2000-PLA (PLA molecular weight 10-20 kDa), with a mass ratio of PLGA (molecular weight 10-20 kDa) to mannose-PEG2000-PLGA (PLGA molecular weight 40 kDa) of 9:1. The immunoadjuvants used were CpG2006 (Class B), CpG2216 (Class A), and Poly ICLC, and the positively charged substance used was the R8 polypeptide (RRRRRRRR). During preparation, the double emulsion method is first used to prepare micron particles loaded with antigen component 1, adjuvant and R8 polypeptide. Then, 100 mg of nanoparticles are centrifuged at 12000g for 25 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours to obtain nanovaccine 1 with an average particle size of about 200 nm. Each 1 mg of PLGA nanoparticles 1 is loaded with approximately 5 μg of protein and polypeptide components of cancer cells, 0.02 mg each of CpG2006, CpG2216 and Poly ICLC, and 0.05 mg of R8 polypeptide.

[0392] Nanovaccine 2 was prepared similarly to Nanovaccine 1. However, it contained antigen component 2, adjuvant, and R8 peptide. Nanovaccine 2 had an average particle size of approximately 200 nm. Each 1 mg of PLGA nanovaccine 2 was loaded with approximately 5 μg of cancer cell protein and peptide components, including 0.02 mg each of CpG2006, CpG2216, and Poly ICLC, and 0.05 mg of R8 peptide.

[0393] Nanovaccine 3 was prepared similarly to Nanovaccine 1. However, it contained antigen component 3, adjuvant, and R8 peptide. Nanovaccine 3 had an average particle size of approximately 200 nm. Each 1 mg of PLGA nanovaccine 3 was loaded with approximately 5 μg of cancer cell protein and peptide components, including 0.02 mg each of CpG2006, CpG2216, and Poly ICLC, and 0.05 mg of R8 peptide.

[0394] Nanovaccine 4 was prepared using the same method as Nanovaccine 1. However, it contained antigen component 4, adjuvant, and R8 peptide. Nanovaccine 4 had an average particle size of approximately 200 nm. Each 1 mg of PLGA nanovaccine 4 was loaded with approximately 5 μg of cancer cell protein and peptide components, including 0.02 mg each of CpG2006, CpG2216, and Poly ICLC, and 0.05 mg of R8 peptide.

[0395] (3) Nanovaccines for cancer prevention

[0396] Tumor-bearing mice were prepared by selecting female C57BL / 6 mice aged 6-8 weeks. 100 μL PBS or 1 mg of nanovaccine 1, 1 mg of nanovaccine 2, 1 mg of nanovaccine 3, or 1 mg of nanovaccine 4 were injected into the mice on days -35, -28, -21, -14, and -7 before tumor inoculation. 5×10 5 The tumor volume and survival of mice were monitored using the same method as above.

[0397] (4) Experimental results

[0398] like Figure 14 As shown, compared with the PBS control group, the tumor growth rate of mice treated with nanovaccines was significantly slowed and the survival time of mice was significantly prolonged. Moreover, nanovaccine 1 was more effective than nanovaccines 2, 3, and 4, indicating that an appropriate dissolving agent is necessary.

[0399] Example 13 Nano-vaccine for the treatment of melanoma

[0400] In this example, ultrapure water was first used to lyse B16F10 melanoma tumor tissue to prepare protein and polypeptide components and water-insoluble antigens in the water-soluble components of the tumor tissue. Then, a nanovaccine was prepared using the organic polymer material PLGA as the nanoparticle skeleton material and poly(I:C) as the immune adjuvant by a solvent evaporation method.

[0401] (1) Preparation of antigen components

[0402] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to a volume of approximately 1000 mm 3Mice were killed and tumor tissue was removed. The tumor tissue was cut into pieces and ground, then passed through a cell strainer, added with an appropriate amount of ultrapure water, and repeatedly frozen and thawed five times, accompanied by sonication to destroy and lyse the cells. After lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble component soluble in pure water. The resulting precipitate was dissolved by adding a 2M aqueous solution of semicarbazide hydrochloride and 0.2M agmatine sulfate to the precipitate, converting the water-insoluble component in pure water into a component soluble in a 2M aqueous solution of semicarbazide hydrochloride and 0.2M agmatine sulfate. A saturated aqueous ammonium sulfate solution was added dropwise to the water-soluble component in the lysate. After complete precipitation, the resulting sample was centrifuged at 3000 g for 5 minutes, and the precipitate was dissolved in a 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate aqueous solution for later use. The supernatant was heated at 100° C. for 5 minutes, and the resulting sample was centrifuged at 3000 g for 5 minutes. After discarding the supernatant, the precipitate was dissolved in a 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate aqueous solution; the salted-out precipitate dissolved with 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate and the heated precipitate were combined and used as part of the water-soluble component. The water-insoluble component of the lysate dissolved in the 2M hydrochloric acid semicarbazide aqueous solution was mixed with the cancer-associated antigen polypeptide 10102 (DTLLKALLEIASCLEKALQVF) and the cancer-associated antigen polypeptide 10103 (FMTYWHLLNAFTVTVPKDL) at a mass ratio of 1:1:1. The component obtained by salting out and heating precipitation from the water-soluble component dissolved in the 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate was mixed with the cancer-associated antigen polypeptide 10102 (DTLLKALLEIASCLEKALQVF) and the cancer-associated antigen polypeptide 10103 (FMTYWHLLNAFTVTVPKDL) at a mass ratio of 1:1:1. The two mixtures together constituted the antigen component 1 for preparing the cancer nanovaccine 1.

[0403] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to a volume of approximately 1000 mm 3The mice were killed and the tumor tissues were removed. The tumor tissue was cut into pieces and ground, and then an appropriate amount of ultrapure water was added through a cell strainer and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells. After lysis, the lysate was centrifuged at 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate aqueous solution were added to the resulting precipitate to dissolve the precipitate, and the insoluble component in pure water was converted into a component soluble in 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate aqueous solution. Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble component in the lysate. After complete precipitation, the resulting sample was centrifuged at 3000g for 5 minutes, and the precipitate was dissolved in 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate aqueous solution and used as part of the water-soluble component. The water-insoluble component of the lysate dissolved in the 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate aqueous solution and 2M hydrochloric acid semicarbazide were mixed with the cancer-associated antigen polypeptide 10102 (DTLLKALLEIASCLEKALQVF) and the cancer-associated antigen polypeptide 10103 (FMTYWHLLNAFTVTVPKDL) at a mass ratio of 1:1:1. The component obtained by salting out and precipitating from the water-soluble component dissolved in the 0.2M agmatine sulfate was mixed with the cancer-associated antigen polypeptide 10102 (DTLLKALLEIASCLEKALQVF) and the cancer-associated antigen polypeptide 10103 (FMTYWHLLNAFTVTVPKDL) at a mass ratio of 1:1:1. The two mixtures were used to prepare the antigen component 2 of the cancer nanovaccine 2.

[0404] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to a volume of approximately 1000 mm 3Mice were sacrificed and tumor tissue was removed. Tumor tissue was minced and ground, then passed through a cell strainer, added to an appropriate amount of ultrapure water, and repeatedly frozen and thawed five times, accompanied by sonication to disrupt and lyse cells. After lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected, representing the water-soluble fraction. The resulting precipitate was dissolved in a 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution, converting the water-insoluble fraction into a fraction soluble in the 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution. The water-soluble fraction in the lysate was heated at 100°C for 5 minutes, and the resulting sample was centrifuged at 3000g for 5 minutes. The supernatant was discarded, and the precipitate was dissolved in a 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution, representing a portion of the water-soluble fraction. The water-insoluble component of the lysate dissolved in the 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate aqueous solution was mixed with the cancer-associated antigen polypeptide 10102 (DTLLKALLEIASCLEKALQVF) and the cancer-associated antigen polypeptide 10103 (FMTYWHLLNAFTVTVPKDL) at a mass ratio of 1:1:1. The component obtained by heating and precipitating the water-soluble component dissolved in the 2M hydrochloric acid semicarbazide and 0.2M agmatine sulfate was mixed with the cancer-associated antigen polypeptide 10102 (DTLLKALLEIASCLEKALQVF) and the cancer-associated antigen polypeptide 10103 (FMTYWHLLNAFTVTVPKDL) at a mass ratio of 1:1:1. The two mixtures together constituted antigen component 3 for preparing cancer nanovaccine 3.

[0405] (2) Preparation of nanovaccines

[0406] In this example, nanovaccine 1 (Nanovaccine 1) was prepared using the double emulsion method, a solvent evaporation method. During preparation, some of the water-soluble components of the tumor tissue lysate were purified by ammonium sulfate salting-out and heat separation and purification, followed by dissolution in a lysate. During preparation, nanoparticles loaded with the water-soluble components of the cancer cell lysate (the components dissolved in a lysate after ammonium sulfate salting-out and heat separation and purification, plus synthetic antigen polypeptides) and nanoparticles loaded with the water-insoluble antigens of whole cancer cell antigens (the components dissolved in a lysate plus synthetic antigen polypeptides) were prepared separately and then used together as nanovaccine 1. The PLGA material used for nanoparticle preparation had a molecular weight of 10 kDa-20 kDa, and the immunoadjuvant used was poly(I:C). The preparation method was as described above. First, the antigen components and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 13,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The nanoparticles 1 have an average particle size of about 300 nm. Each 1 mg of PLGA nanoparticles is loaded with about 950 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.01 mg of poly(I:C).

[0407] In this example, Nanovaccine 2 was prepared using the double emulsion method, a solvent evaporation method. During preparation, some of the water-soluble components in the tumor tissue lysate were purified by ammonium sulfate precipitation and then dissolved in a lysing solution. Nanoparticles loaded with the water-soluble components of the cancer cell lysate (the components dissolved in a lysing solution after separation and purification by ammonium sulfate precipitation, plus synthetic antigen polypeptides) and nanoparticles loaded with the water-insoluble antigens of whole cancer cell antigens (the components dissolved in a lysing solution plus synthetic antigen polypeptides) were prepared separately and then used together as Nanovaccine 2. The PLGA material used for nanoparticle preparation had a molecular weight of 10 kDa-20 kDa, and the immunoadjuvant used was poly(I:C). The preparation method was as described above. First, the antigen components and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 13,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 2 is about 300 nm. Each 1 mg of PLGA nanoparticles is loaded with about 950 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.01 mg of poly(I:C).

[0408] In this example, Nanovaccine 3 was prepared using the double emulsion method, a solvent evaporation method. During preparation, some of the water-soluble components in the tumor tissue lysate were dissolved in a lysing solution after being heated, separated, and purified. Nanoparticles loaded with the water-soluble components of the cancer cell lysate (the components dissolved in the lysing solution after being heated, separated, and purified, plus the synthetic antigen polypeptide) and nanoparticles loaded with the water-insoluble antigens of the whole cancer cell antigens (the components dissolved in the lysing solution plus the synthetic antigen polypeptide) were prepared separately and then used together as Nanovaccine 3. The PLGA material used for nanoparticle preparation had a molecular weight of 10 kDa-20 kDa, and the immune adjuvant used was poly(I:C). The preparation method was as described above. First, the antigen components and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 13,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The nanoparticles 3 have an average particle size of about 300 nm. Each 1 mg of PLGA nanoparticles is loaded with about 950 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.01 mg of poly(I:C).

[0409] (3) Nano-vaccines for cancer treatment

[0410] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells. On days 3, 6, 9, 14, and 20 after tumor inoculation, mice were subcutaneously injected with 2 mg of nanovaccine 1 (1 mg of nanoparticles loaded with the purified component of the water-soluble component + 1 mg of nanoparticles loaded with the water-insoluble component), 2 mg of nanovaccine 2 (1 mg of nanoparticles loaded with the purified component of the water-soluble component + 1 mg of nanoparticles loaded with the water-insoluble component), or 2 mg of nanovaccine 3 (1 mg of nanoparticles loaded with the purified component of the water-soluble component + 1 mg of nanoparticles loaded with the water-insoluble component), or 100 μL of PBS. Tumor growth and survival of mice were monitored as described above.

[0411] (4) Experimental results

[0412] like Figure 15As shown, tumors in mice in the PBS group grew rapidly and soon died. Mice treated with Nanovaccine 1, Nanovaccine 2, and Nanovaccine 3 all experienced significantly slower tumor growth and prolonged survival, with some mice recovering tumor-free. Nanovaccine 1 was more effective than Nanovaccine 2 and Nanovaccine 3, indicating that the combined use of salting-out and heating for separation and purification is significantly more effective than either salting-out or heating alone.

[0413] Example 14 Nano-vaccine for the treatment of melanoma

[0414] In this example, ultrapure water was first used to lyse B16F10 melanoma cancer cells to prepare protein and polypeptide components and water-insoluble components in the water-soluble components of the cancer cells. Then, PLGA and PEG-modified PLGA were used as nanoparticle skeleton materials to prepare nanovaccines.

[0415] (1) Preparation of antigen components

[0416] After harvesting cultured B16F10 cells and discarding the supernatant, the cancer cell pellet was resuspended in an appropriate amount of ultrapure water and repeatedly frozen and thawed five times with sonication to lyse the cells. Following lysis, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was collected as the water-soluble fraction. The resulting precipitate was dissolved in 6M guanidine hydrochloride to convert the water-insoluble antigen into a 6M guanidine hydrochloride-soluble fraction.

[0417] The water-soluble fraction in the lysate was heated at 100°C for 10 minutes, and the resulting sample was centrifuged at 3000 g for 5 minutes. The supernatant was discarded and the precipitate was dissolved in a 6M guanidine hydrochloride aqueous solution. The water-soluble fraction dissolved in 6M guanidine hydrochloride was heated and precipitated, the water-insoluble fraction in the lysate dissolved in 6M guanidine hydrochloride aqueous solution, and WT1 were added. 235-243 WT1 86-102 and WT1 294-312 : Mix according to the mass ratio of 1:1:0.1:0.1:0.1 to prepare the antigen component 1 of nanovaccines 1, 2 and 3.

[0418] Alternatively, the water-soluble components in the lysate can be used directly without any treatment. 235-243 WT1 86-102 and WT1 294-312 : Mix according to the mass ratio of 1:1:0.1:0.1:0.1 to prepare the antigen component 2 of nanovaccine 4.

[0419] (2) Preparation of nanovaccines

[0420] In this example, nanovaccine 1 was prepared using the double emulsion method, a solvent evaporation method. The nanovaccine materials used were PGLA (molecular weight 10-20 kDa) and PEG2000-PLGA (PLGA molecular weight 10-20 kDa), with a mass ratio of PLGA to PEG2000-PLGA of 49:1. The immunoadjuvants used were poly(I:C) and CpG7909. The preparation method was as described above. First, the antigen component 1 and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 14,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Nanovaccine 1 had an average particle size of approximately 250 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 500 μg of protein or peptide component, and 0.05 mg each of poly(I:C) and CpG7909.

[0421] In this example, nanovaccine 2 was prepared using the double emulsion method, a solvent evaporation method. The nanovaccine materials used were PGLA (molecular weight 10-20 kDa) and PEG2000-PLGA (PLGA molecular weight 10-20 kDa), with a mass ratio of PLGA to PEG2000-PLGA of 500:1. The immunoadjuvants used were poly(I:C) and CpG7909. The preparation method was as described above. First, the antigen component 1 and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 14,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Nanovaccine 2 had an average particle size of approximately 250 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 500 μg of protein or peptide component, and 0.05 mg each of poly(I:C) and CpG7909.

[0422] In this example, nanovaccine 3 was prepared using the double emulsion method, a solvent evaporation method. The nanovaccine materials used were PGLA (molecular weight 10KDa-20KDa) and PEG2000-PLGA (PLGA molecular weight 10KDa-20KDa), with a mass ratio of PLGA to PEG2000-PLGA of 4:1. The immunoadjuvants used were poly(I:C) and CpG7909, and the adjuvants were loaded within the nanovaccine. The preparation method was as described above. First, the nanoparticles were loaded with antigen component 1 and adjuvant using the double emulsion method. Then, 100mg of the nanoparticles were centrifuged at 14,000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of nanovaccine 3 is about 250 nm. Each 1 mg of PLGA nanoparticles is loaded with approximately 500 μg of protein or polypeptide components, and 0.05 mg each of poly(I:C) and CpG7909.

[0423] Nanovaccine 4 in this example was prepared using the same methods and materials as Nanovaccine 1. The materials used were PGLA (molecular weight 10-20 kDa) and PEG2000-PLGA (PLGA molecular weight 10-20 kDa), with a mass ratio of 49:1 between PLGA and PEG2000-PLGA. The immunoadjuvants used were poly(I:C) and CpG7909. The preparation method was as described above. First, the antigen component 2 and adjuvant were loaded into the nanoparticles using a double emulsion method. Then, 100 mg of nanoparticles were centrifuged at 14,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. Nanovaccine 4 had an average particle size of approximately 250 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 500 μg of protein or peptide component, and 0.05 mg each of poly(I:C) and CpG7909.

[0424] (3) Nano-vaccines for cancer treatment

[0425] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells were injected subcutaneously with 100 μL of 0.2 mg of nanovaccine 1, 0.2 mg of nanovaccine 2, 0.2 mg of nanovaccine 3, 100 μL of 0.2 mg of nanovaccine 4, or 100 μL of PBS on days 3, 6, 9, 14, and 20 after tumor inoculation. Tumor growth and survival were monitored as described above.

[0426] (4) Experimental results

[0427] like Figure 16 As shown, the tumor volume of mice in the PBS group grew rapidly and the mice died soon after. The tumor growth rate of mice using nanovaccine 1 (Nanovaccine 1), nanovaccine 2 (Nanovaccine 2), nanovaccine 3 (Nanovaccine 3), and nanovaccine 4 (Nanovaccine 4) was significantly slowed, and their survival was significantly prolonged. Among them, nanovaccine 1 was more effective than nanovaccine 4, indicating that using heated purification of protein and peptide components in the water-soluble components can improve the effectiveness of the vaccine, and the effect of separating and purifying a portion of the water-soluble components is better than using the water-soluble components directly. Among them, nanovaccine 1 was more effective than nanovaccine 2 and nanovaccine 3, indicating that using a specific ratio of PEG to modify the particle surface can improve the efficacy of nanovaccines or micron vaccines.

[0428] Example 15 Nanovaccine for the treatment of melanoma

[0429] (1) Preparation of antigen components

[0430] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells, when the tumor grows to a volume of approximately 1000 mm 3 The mice were killed and the tumor tissues were removed. The tumor tissue was cut into pieces and ground, and then passed through a cell strainer to prepare a single cell suspension. Then, an appropriate amount of 8M urea aqueous solution was added to lyse the cells, and the lysate components were dissolved using an 8M urea aqueous solution. Then, a saturated ammonium sulfate aqueous solution was added dropwise. After the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes, and the precipitate was redissolved in an 8M urea aqueous solution; mRNA was isolated from the supernatant using an mRNA isolation kit. The components redissolved in the above 8M urea aqueous solution and the mRNA components isolated from the lysate were mixed with cancer-related antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF) and cancer-related antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) in a mass ratio of 2:1:1:1 to prepare antigen component 1 of cancer nanovaccine 1.

[0431] (2) Preparation of nanovaccines

[0432] In this embodiment, nanovaccine 1 (Nanovaccine 1) was prepared using the multiple emulsion method within the solvent evaporation method. The nanoparticle preparation material, PLGA, has a molecular weight of 10KDa-30KDa, and the immune adjuvant used is poly(I:C). The preparation method is as described above. First, the antigen component 1 and adjuvant are loaded into the nanoparticles. Then, 100mg of nanoparticles are centrifuged at 12000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 1 is approximately 350nm. Each 1mg of PLGA nanoparticles is loaded with approximately 500μg of protein or polypeptide components and 0.01mg of poly(I:C).

[0433] (3) Nano-vaccines for cancer treatment

[0434] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells were injected subcutaneously into the mice on days 3, 6, 9, 14, and 20 after tumor inoculation. Tumor growth and survival were monitored as described above.

[0435] (4) Experimental results

[0436] like Figure 17 As shown, the tumors of mice in the PBS group grew rapidly and the mice died soon after. The tumor growth rate of mice treated with nanovaccine 1 was significantly slowed down, and the survival time was significantly prolonged. Some mice recovered without tumors.

[0437] In this embodiment, after using a dissolving solution containing a dissolving agent to lyse and dissolve the whole cell components of the tumor tissue, the protein and polypeptide components and the mRNA component in the cell components are collected separately. In actual applications, the protein and polypeptide components and the mRNA component or RNA component in the water-soluble component and the water-insoluble component can also be collected separately.

[0438] Example 16 Nanovaccine for the treatment of melanoma

[0439] (1) Preparation of antigen components

[0440] After collecting the cultured B16F10 cells, an 8M urea (containing 0.1M arginine) aqueous solution was added to lyse the cancer cells, and then an 8M urea (containing 0.1M arginine) aqueous solution was used to dissolve the lysate components, heated at 55°C for 5 minutes, and then saturated ammonium sulfate was added dropwise to salt out the protein and polypeptide components. The sample was centrifuged at 5000g for 10 minutes, and then the precipitate was dissolved again using an 8M urea (containing 0.1M arginine) aqueous solution. The protein and polypeptide components dissolved in 8M urea (containing 0.1M arginine) for the second time, B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), and IO102 four antigen components were mixed at a mass ratio of 60:1:1:1 to prepare the antigen component 1 for the nanovaccine and microvaccine.

[0441] (2) Preparation of nano- and micro-vaccines

[0442] In this embodiment, nanovaccine 1 (Nanovaccine 1) was prepared using the double emulsion method in the solvent evaporation method. The nanoparticle preparation material used was PLGA with a molecular weight of 10KDa-30KDa, and the immune adjuvants were poly(I:C) and CpG7909. The preparation method was as described above. The antigen component 1 and adjuvant were first loaded into the nanoparticles using the double emulsion method. Then, 100mg of nanoparticles were centrifuged at 13000g for 20 minutes and resuspended in 10mL of ultrapure water containing 4% trehalose, followed by freeze-drying for 48 hours. The average particle size of the nanoparticles 1 was approximately 300nm. Each 1mg of PLGA nanoparticles was loaded with approximately 0.6mg of protein or polypeptide component, and 0.01mg of poly(I:C) and CpG7909 were loaded.

[0443] In this embodiment, nano vaccine 2 (Nanovaccine 2) was prepared by the double emulsion method in the solvent evaporation method. The nanoparticle preparation material used was PLGA with a molecular weight of 10KDa-30KDa, and the immune adjuvants were poly(I:C) and CpG7909. The preparation method was as described above. First, the antigen component 1 and adjuvant were loaded into the nanoparticles by the double emulsion method. Then, 100mg of nanoparticles were centrifuged at 13000g for 20 minutes and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours. The average particle size of the nanoparticles 2 was about 120nm. Every 1mg of PLGA nanoparticles was loaded with approximately 0.6mg of protein or polypeptide components, and 0.01mg of poly(I:C) and CpG7909 were loaded.

[0444] In this embodiment, nano vaccine 3 (Nanovaccine 3) was prepared using the double emulsion method in the solvent evaporation method. The nanoparticle preparation material used was PLGA with a molecular weight of 10KDa-30KDa, and the immune adjuvants were poly(I:C) and CpG7909. The preparation method was as described above. First, the antigen component 1 and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100mg of nanoparticles were centrifuged at 13000g for 20 minutes and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours. The average particle size of the nanoparticles 3 was about 700nm. Each 1mg of PLGA nanoparticles was loaded with approximately 0.6mg of protein or polypeptide component and 0.01mg of poly(I:C) and CpG7909.

[0445] In this example, micron vaccine 1 (Micronvaccine 1) was prepared using the double emulsion method, a solvent evaporation method. The nanoparticle preparation material used was PLGA with a molecular weight of 10 kDa to 30 kDa, and the immune adjuvants were poly(I:C) and CpG7909. The preparation method was as described above. First, the antigen component 1 and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 13,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the micron particles 1 was approximately 2.5 μm. Each 1 mg of PLGA nanoparticles was loaded with approximately 0.6 mg of the protein or polypeptide component, and 0.01 mg each of poly(I:C) and CpG7909.

[0446] In this example, micron vaccine 2 (Micronvaccine 2) was prepared using the double emulsion method, a solvent evaporation method. The nanoparticle preparation material used was PLGA with a molecular weight of 10 kDa to 30 kDa, and the immune adjuvants were poly(I:C) and CpG7909. The preparation method was as described above. First, the antigen component 1 and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 13,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the micron particles 2 was approximately 5.0 μm. Each 1 mg of PLGA nanoparticles was loaded with approximately 0.6 mg of the protein or polypeptide component, and 0.01 mg each of poly(I:C) and CpG7909.

[0447] In this example, micron vaccine 3 (Micronvaccine 3) was prepared using the double emulsion method, a solvent evaporation method. The nanoparticle preparation material used was PLGA with a molecular weight of 10 kDa to 30 kDa, and the immune adjuvants were poly(I:C) and CpG7909. The preparation method was as described above. First, the antigen component 1 and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 13,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the micron particles 3 was approximately 1.5 μm. Each 1 mg of PLGA nanoparticles was loaded with approximately 0.6 mg of the protein or polypeptide component, and 0.01 mg each of poly(I:C) and CpG7909.

[0448] Nano vaccine 1 and micro vaccine 1 are mixed in a mass ratio of 1:1 and used to obtain mixed vaccine 1; nano vaccine 1 and micro vaccine 2 are mixed in a mass ratio of 1:1 and used to obtain mixed vaccine 2; nano vaccine 1 and micro vaccine 3 are mixed in a mass ratio of 1:1 and used to obtain mixed vaccine 3; micro vaccine 1 and nano vaccine 2 are mixed in a mass ratio of 1:1 and used to obtain mixed vaccine 4; micro vaccine 1 and nano vaccine 3 are mixed in a mass ratio of 1:1 and used to obtain mixed vaccine 5.

[0449] (3) Cancer treatment with a hybrid vaccine consisting of nano- and micro-vaccines

[0450] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells were injected subcutaneously into mice on days 3, 6, 9, 14, and 20 after tumor inoculation. Mice received either 2 mg of the mixed vaccine (mixture 1, 2, 3, 4, or 5) or 100 μL of PBS. Tumor growth and survival were monitored using the same methods as above.

[0451] (4) Experimental results

[0452] like Figure 18 As shown, tumors in mice in the PBS group grew rapidly and the mice died soon after. Mice treated with the vaccine showed significantly slower tumor growth and prolonged survival, with some mice recovering tumor-free. Mixed vaccine 1 was more effective than mixed vaccines 2, 3, 4, and 5, indicating that a combination of nanoparticle-sized and microparticle-sized vaccines is more effective.

[0453] Example 17 Nano-vaccine for the treatment of melanoma

[0454] (1) Preparation of antigen components

[0455] The B16F10 cell line was co-incubated with a low concentration of doxorubicin (1nM) in RPMI1640 complete medium for 2h (37°C, 5% CO2). Doxorubicin can stimulate cancer cells during the co-incubation process. Then, the cultured B16F10 cells were collected, the supernatant was discarded, and an appropriate amount of ultrapure water was added to the cancer cell pellet to resuspend and repeatedly freeze-thawed 5 times to lyse the cells. Then, 2% hypochlorous acid was added to oxidize the lysate component, and then heated at 45°C for 5 minutes, and then 0.1M sodium chloride was used to salt out and precipitate the protein and polypeptide components, and then the sample was centrifuged at 3000g for 15 minutes, and then the precipitate was dissolved with a 6M guanidine hydrochloride aqueous solution. The protein and polypeptide components dissolved in the 6M guanidine hydrochloride aqueous solution, WT1 235-243 WT1 86-102 and WT1 294-312 : Mix them in a mass ratio of 20:1:1:1 to obtain antigen component 1.

[0456] (2) Preparation of nanovaccines

[0457] In this embodiment, nano vaccine 1 (Nanovaccine 1) was prepared using the double emulsion method in the solvent evaporation method. The nano vaccine preparation material used was PGLA (molecular weight 10KDa-20KDa), and the immune adjuvants were poly(I:C) and CpG7909. The preparation method was as described above. First, the antigen component 1 and adjuvant were loaded into the nanoparticles using the double emulsion method. Then, 100mg of nanoparticles were centrifuged at 14000g for 20 minutes and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours. The average particle size of nano vaccine 1 was about 280nm. Each 1mg of PLGA nanoparticles was loaded with approximately 300μg of protein or polypeptide components, and 0.005mg of poly(I:C) and CpG7909 were loaded.

[0458] (3) Nano-vaccines for cancer treatment

[0459] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells were placed in the mice. On days 3, 6, 9, 14, and 20 after tumor inoculation, mice were subcutaneously injected with 100 μL of 0.2 mg of nanovaccine 1 or 100 μL of PBS. Tumor growth and survival were monitored as described above.

[0460] (4) Experimental results

[0461] like Figure 19As shown, the tumors in the PBS group grew rapidly and the mice died soon after. Mice treated with Nanovaccine 1 experienced significantly slower tumor growth and prolonged survival. This demonstrates that the antigen components obtained by oxidizing, heating, and salting out, as well as by enhancing their immunogenicity, are effectively loaded into nanovaccines to treat cancer.

[0462] In this embodiment, hypochlorous acid was used to oxidize the antigen component to enhance the immunogenicity of the antigen component. In actual applications, hydrogen peroxide (hydrogen peroxide) or other oxidants may also be used to oxidize the antigen component. In this embodiment, oxidation was used to enhance the immunogenicity of the antigen component. In actual applications, a reducing agent may also be used to reduce the antigen component. The reducing agent that can be used includes but is not limited to DTT, TCEP, etc.

[0463] In summary, the vaccine disclosed herein has excellent therapeutic effects on cancer and can be used to prevent or treat cancer.

[0464] In this example, 1 nM doxorubicin was used. In practical applications, any other feasible doxorubicin concentration may also be used, such as 0.001 nM-2 μM doxorubicin.

[0465] In this embodiment, low concentrations of doxorubicin were used to stimulate cancer cells. In actual use, other substances such as paclitaxel, vincristine, bacterial secretions, bacterial membrane components, retinoic acid, arsenic trioxide, cytokines, plant extracts (such as extracts of traditional Chinese medicines such as ginseng), growth factors, chemokines, etc. can also be used to stimulate cancer cells.

[0466] In this disclosure, several methods for separating and purifying antigen components or enhancing the immunogenicity of antigen components are listed, such as salting out, heating, enzyme treatment (enzymatic hydrolysis or enzyme inhibition), oxidation, reduction, fixation, mineralization, radiation, and irradiation. In actual applications, other similar methods for separating and purifying antigen components or enhancing the immunogenicity of antigen components can also be used, and are also considered to be within the scope of coverage of this patent.

[0467] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A cancer vaccine, characterized in that The cancer vaccine comprises: (i) a nanoparticle and / or microparticle skeleton structure formed by a particle material, (ii) a whole cell component of a cancer cell and / or tumor tissue and / or a partial cell component containing an antigen component, and (iii) a cancer-specific and / or related antigen polypeptide and / or a nucleic acid expressing a cancer-specific and / or related antigen polypeptide; wherein the whole cell component of the cancer cell and / or tumor tissue and / or the partial cell component containing the antigen component, and the cancer-specific and / or related antigen polypeptide and / or the nucleic acid expressing the cancer-specific and / or related antigen polypeptide are simultaneously loaded inside and / or on the surface of the skeleton structure; The whole cell component contains a water-soluble component of a cancer cell and / or tumor tissue lysate component and a water-insoluble component dissolved in a lysate containing a dissolving agent; the antigen component contained in the partial cell component comprises a protein and polypeptide component of a cancer cell and / or tumor tissue lysate and / or an RNA component of a cell lysate; the antigen component is separated and purified from the lysate or separated and purified from the water-soluble component and / or the water-insoluble component; and the mass ratio of the protein and polypeptide component and the RNA component to the cancer-specific and / or related antigen polypeptide / nucleic acid component is 1:0.001-1:10; 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 in the water-soluble components of the cancer cells and / or tumor tissues and the water-insoluble components / protein and polypeptide components and RNA components in the 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 expressing 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 the nanovaccine or microvaccine; The mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-1:10; The mass ratio of the particle skeleton structure to the RNA component is 1:0.001-1:10; The cancer vaccine is prepared by a double emulsion method; The cancer-specific and / or cancer-related antigen polypeptide is selected from one or more of B16-M20, B16-M24, B16-M46, TRP2, IO102, IO103, and WT1.

2. The cancer vaccine according to claim 1, wherein The RNA component is an mRNA component.

3. The cancer vaccine according to claim 1, wherein The method for preparing the whole cell fraction comprises: first lysing cancer cells and / or tumor tissues, then collecting water-soluble components and water-insoluble components in the lysate, and dissolving the water-insoluble components in a dissolving solution containing a dissolving agent and using them together with the water-soluble components; Alternatively, cancer cells and / or tumor tissues may be first lysed using a lysis solution containing a lysis agent, and then the whole cell lysate may be dissolved using a lysis solution containing a lysis agent before use; Wherein, the dissolving agent is independently selected from one or more of a compound containing a 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, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl.

4. The cancer vaccine according to claim 3, characterized in that The dissolving agent is selected from one or more of urea, guanidine salt, urea salt, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

5. The cancer vaccine according to claim 4, characterized in that The guanidine salt is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, guanidine hydrochloride, guanidine sulfate, and guanidine sulfonate.

6. The cancer vaccine according to claim 1, wherein The preparation process of the partial cell component containing the antigen component includes: first lysing cancer cells and / or tumor tissues, then collecting water-soluble components and water-insoluble components in the lysate respectively, subjecting all water-soluble components to salting out, heating, enzyme treatment, oxidation, reduction, fixation, mineralization, and irradiation treatments, and then re-dissolving the precipitated component using a dissolving solution containing a dissolving agent; the water-insoluble component is directly used after being dissolved in a dissolving solution containing a dissolving agent, or is 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, and irradiation treatments, and then re-dissolving the obtained precipitated component using 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 re-dissolved after the water-insoluble component is purified to form the partial cell component containing the antigen component; Alternatively, the cancer cells and / or tumor tissues are lysed using a dissolving solution containing a dissolving agent, and the lysate components are dissolved using the dissolving solution containing the dissolving agent. The resulting dissolved lysate components are then subjected to salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, and irradiation treatments, and the resulting precipitated components are then redissolved using the dissolving solution containing the dissolving agent for a second time. Wherein, the dissolving agent is independently selected from one or more of a compound containing a 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, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl.

7. The cancer vaccine according to claim 6, characterized in that The dissolving agent is selected from one or more of urea, guanidine salt, urea salt, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

8. The cancer vaccine according to claim 5, characterized in that The guanidine salt is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, guanidine hydrochloride, guanidine sulfate, and guanidine sulfonate.

9. The cancer vaccine according to claim 1, wherein The cancer vaccine is further loaded with at least one component as shown below: (iv) RNA components in the water-soluble fraction and / or the water-insoluble fraction; (v) immune adjuvants; (vi) Positively charged substances.

10. The cancer vaccine according to claim 9, characterized in that The RNA component is an mRNA component.

11. The cancer vaccine according to claim 9, characterized in that 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 muramyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhancing reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn calf liver active peptide, imiquimod, polysaccharide, 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 ingredient, Astragalus membranaceus active ingredient.

12. The cancer vaccine according to claim 11, characterized in that The immune adjuvant includes at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist.

13. The cancer vaccine according to claim 12, characterized in that The immune adjuvant includes at least one of Poly(I:C), PolyICLC, class A CpG-OND, class B CpG-OND and class C CpG-OND.

14. The cancer vaccine according to claim 9, characterized in that 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.

15. The cancer vaccine according to claim 14, characterized in that The positively charged substance is selected from any one or more of melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

16. The cancer vaccine according to claim 1, characterized in that The mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-1:

2.

17. The cancer vaccine according to claim 16, characterized in that The mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.05-1:

1.

18. The cancer vaccine according to claim 1, wherein The mass ratio of the particle skeleton structure to the RNA component is 1:0.01-1:

2.

19. The cancer vaccine according to claim 18, characterized in that The mass ratio of the particle skeleton structure to the RNA component is 1:0.05-1:

1.

20. The cancer vaccine according to claim 19, characterized in that The RNA component is an mRNA component.

21. The cancer vaccine according to claim 1, wherein The protein, polypeptide and RNA components are derived from lysates, and the cancer-specific and / or related antigen polypeptide / nucleic acid components are artificially synthesized.

22. The cancer vaccine according to claim 21, characterized in that The mass ratio of the protein and polypeptide components and the RNA component to the cancer-specific and / or related antigen polypeptide / nucleic acid component is 1:0.01-1:

2.

23. The cancer vaccine according to claim 22, characterized in that The mass ratio of the protein and polypeptide components and the RNA component to the cancer-specific and / or related antigen polypeptide / nucleic acid component is 1:0.05-1:

1.

24. The cancer vaccine according to claim 23, characterized in that The RNA component is an mRNA component.

25. The cancer vaccine according to claim 1, wherein The cancer vaccine further contains at least one component as shown below: (a) cancer cell membrane components derived from tumor tissues and / or tumor cells; (b) extracellular vesicle membrane fractions derived from extracellular vesicle lysates secreted by bacteria or tumor cells; (c) bacterial membrane fractions derived from bacterial lysates; (d) Membrane fractions derived from antigen-presenting cells.

26. The cancer vaccine according to claim 25, characterized in that The bacteria include at least one of the following: BCG, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus gestell, Lactobacillus reuteri, and Lactobacillus rhamnosus.

27. The cancer vaccine according to claim 1, wherein The steps for preparing the protein and polypeptide components in the water-soluble components of the cancer cells and / or tumor tissues are as follows: firstly lysing the cancer cells and / or tumor tissues to obtain lysates thereof; Then, one or more of centrifugation, filtration, dialysis, and ultrafiltration are used to separate the water-soluble component and the water-insoluble component in the lysate, respectively obtaining the water-soluble component and the water-insoluble component in the lysate; then, the water-soluble component in the obtained lysate is subjected to one or more of salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, and irradiation to precipitate the protein and polypeptide components therein; then, the protein and polypeptide components in the precipitated portion are dissolved in a dissolving solution containing a dissolving agent to obtain the protein and polypeptide components in the water-soluble component; Wherein, the dissolving agent is independently selected from one or more of a compound containing a 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, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl.

28. The cancer vaccine according to claim 27, characterized in that The dissolving agent is selected from one or more of urea, guanidine salt, urea salt, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

29. The cancer vaccine according to claim 28, characterized in that The guanidine salt is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, guanidine hydrochloride, guanidine sulfate, and guanidine sulfonate.

30. The cancer vaccine according to claim 1, wherein The steps of preparing the protein and polypeptide components in the water-insoluble components of the cancer cells and / or tumor tissues are as follows: firstly lysing the cancer cells and / or tumor tissues to obtain lysates thereof; Then, the water-soluble component and the water-insoluble component in the lysate are separated by one or more of centrifugation, filtration, dialysis, and ultrafiltration to obtain the water-soluble component and the water-insoluble component in the lysate, respectively; then, the protein and polypeptide components in the obtained water-insoluble component in the lysate are precipitated after salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, and irradiation; then, the protein and polypeptide components in the precipitated portion are dissolved in a dissolving solution containing a dissolving agent to obtain the protein and polypeptide components in the water-insoluble component; Wherein, the dissolving agent is independently selected from one or more of a compound containing a 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, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl.

31. The cancer vaccine according to claim 30, characterized in that The dissolving agent is selected from one or more of urea, guanidine salt, urea salt, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

32. The cancer vaccine according to claim 31, characterized in that The guanidine salt is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, guanidine hydrochloride, guanidine sulfate, and guanidine sulfonate.

33. The cancer vaccine according to claim 1, wherein The separation and purification process includes one or more of salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, and irradiation.

34. The cancer vaccine according to claim 33, characterized in that The cations contained in the reagent used for the salting-out include: Al 3+ 、Fe 3+ 、Fe 2+ Mg 2+ 、Sn 2+ 、Zn 2+ , Ca 2+ 、Li + 、Na + NH4 + , K + 、Cu 2+ 、Ag + 、Ba 2+ ; The anions contained in the reagent used for salting out include: Cl - 、SO4 2- 、NO3 - 、CO3 2- 、SiO3 2- 、S2O7 2- 、B4O7 2- PO4 3- RCOO - 、NO2 - 、S2O8 2- 、S 2- 、CrO4 2- 、MnO4 - 、P2O7 4- .

35. The cancer vaccine according to claim 25, wherein The bacterial lysate and / or extracellular vesicle lysate is obtained by lysing bacteria and / or extracellular vesicles with a lysis solution containing a lysis agent.

36. The cancer vaccine according to claim 35, characterized in that The lysing agent is independently selected from one or more of a compound containing the structure of 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, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl.

37. The cancer vaccine according to claim 25, characterized in that The water-insoluble component, the bacterial lysate or the extracellular vesicle lysate are independently dissolved in a dissolving solution containing at least one of the following dissolving agents: one or more of a compound containing 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, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl.

38. The cancer vaccine according to claim 1, wherein The particle material is selected from natural polymer materials, synthetic polymer materials and / or inorganic materials.

39. The cancer vaccine according to claim 1, wherein The shape of the nano- or micro-vaccine is any shape, including but not limited to sphere, ellipsoid, barrel, polygon, rod, sheet, line, worm, square, triangle, butterfly, disc, and vesicle.

40. The cancer vaccine according to claim 1, wherein The cancer vaccine is a nano vaccine and / or a micro vaccine; Wherein, the particle size of the nano vaccine is 1nm-1000nm; Wherein, the particle size of the micron vaccine is 1 μm-1000 μm; Wherein, when a mixed vaccine of nano vaccine and micro vaccine is used, the particle size of the nano vaccine is 100-600 nm, and the particle size of the micro vaccine is 1.5-5 μm.

41. The cancer vaccine according to claim 40, characterized in that The particle size of the nano vaccine is 50-500 nm.

42. The cancer vaccine according to claim 41, characterized in that The particle size of the nano vaccine is 100-400 nm.

43. The cancer vaccine according to claim 40, characterized in that The particle size of the micron vaccine is 1-10 μm.

44. The cancer vaccine according to claim 43, characterized in that The particle size of the micron vaccine is 1-5 μm.

45. The cancer vaccine according to claim 40, characterized in that When a mixed vaccine of nano vaccine and micro vaccine is used, the particle size of the nano vaccine is 150-500 nm, and the particle size of the micro vaccine is 2.0-3.5 μm.

46. ​​A method for preparing the cancer vaccine according to any one of claims 1 to 45, characterized in that: The preparation method comprises the following steps: (1) First, lyse cancer cells and / or tumor tissues to obtain their lysates; (2) then using one or more of centrifugation, filtration, dialysis, and ultrafiltration to separate the water-soluble components and the water-insoluble components in the lysate to obtain the water-soluble components and the water-insoluble components, respectively; or directly using a lysis solution containing a lytic agent to dissolve the cancer cells and / or tumor tissue to obtain the lysate; (3) The water-soluble components in the lysate are then subjected to salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, and irradiation to obtain a precipitate, and the precipitate is then dissolved in a solution containing a solvent; the water-insoluble components in the lysate are dissolved in a solution containing a solvent and then subjected to salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, and irradiation to obtain a precipitate, or the water-insoluble components are dissolved in a solution containing a solvent and then used directly without other treatment; Alternatively, the lysate components dissolved in a dissolving solution containing a dissolving agent are subjected to salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, and irradiation to obtain a precipitate, and the precipitate is then redissolved in a dissolving solution containing a dissolving agent; wherein the dissolving agent in each step is selected independently; (4) Loading the protein polypeptide component and / or the water-insoluble component in the obtained water-soluble component or the protein polypeptide component therein separately or simultaneously into the interior and / or surface of nanoparticles or microparticles to obtain the nanovaccine or microvaccine.

47. The method according to claim 46, wherein The cancer cells or tumor tissues are co-incubated with specific chemicals to stimulate the cancer cells or tumor tissues before being lysed; The specific chemical substances are selected from one or more of small molecule compounds, growth factors, cytokines, chemokines, plant extracts, interferons, bacterial secretions, and bacterial extracellular vesicles.

48. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cancer vaccine according to any one of claims 1 to 45 or the cancer vaccine prepared by the method according to any one of claims 46 to 47.

49. The pharmaceutical composition according to claim 48, characterized in that The pharmaceutical composition further includes one or more pharmaceutically acceptable carriers.

50. Use of the cancer vaccine according to any one of claims 1 to 45, or the cancer vaccine prepared according to the method according to any one of claims 46 to 47, or the pharmaceutical composition according to any one of claims 48 to 49 in at least one of the following (1) to (2): (1) Preparation of drugs for preventing or treating diseases; (2) Preparation of cancer vaccines.

51. The use according to claim 50, characterized in that The disease is cancer or tumor.

52. The use according to claim 51, characterized in that The cancer or tumor is a solid tumor or a blood tumor.

Images