Method for extracting antigen component, and delivery particle containing same and use

Abstract

The present invention relates to a method for extracting an antigen component, and a delivery particle containing same and the use. The method comprises the following steps: (1) lysing cells and / or tissues to obtain a cell lysate component; (2) dissolving the lysate component lysed in step (1) with water and / or a first solving solution containing a dissolving agent; and (3) mixing the solution containing the lysate component obtained in step (2) with an organic solvent to obtain the antigen component, wherein the organic solvent comprises one or more of alcohols, ketones, phenols, nitriles or acids. The preparation method is simple, rapid and low in consumption, the viscosity of the solution is low during centrifugation, and high-speed centrifugation is not required to obtain the antigen component. In addition, the obtained antigen component has a high immunogenicity.

Description

Method for extracting antigen components, delivery particles containing the same, and applications thereof

[0001] This disclosure claims priority to the Chinese patent application filed with the Patent Office of China on November 28, 2023, with the invention name “A method for extracting an antigen component and delivery particles containing the same and applications thereof” and application number 202311600143.1. The entire contents of the above application are incorporated by reference into this disclosure. Technical Field

[0002] The present invention relates to the field of immunotherapy, and in particular to a method for extracting antigen components and delivery particles containing the same and applications thereof. Background Art

[0003] Cancer vaccines are an important immunotherapy approach for cancer and other diseases. The main factors influencing cancer vaccines include antigens, adjuvants, and delivery formats. Taking cancer vaccines as an example, the factors that have the greatest impact on cancer vaccines include tumor antigens, adjuvants, and delivery formats. Antigens can induce and activate specific immune responses, adjuvants can amplify specific immune responses, and the delivery format can influence the efficiency of vaccine phagocytosis by antigen-presenting cells (APCs) and the subsequent activation of antigen-specific T cells. Of these three factors, antigens are the primary component capable of triggering specific immune responses and are therefore the most critical factor. Cancer cells and / or tumor tissues contain a full range of cancer cell-specific and cancer cell-associated antigens, making them the ideal raw materials for preparing tumor antigen delivery particles or cancer vaccines. However, unpurified whole cell fractions contain other substances such as DNA and carbohydrates, which reduces the content of tumor antigen components such as proteins and peptides. When using high-concentration salt substances to salt out the lysate dissolved with a solvent, the system contains both high-concentration salts and solvents, which will cause the solution to have a very high viscosity. During centrifugation, a relatively high speed is required to centrifuge out the salted-out precipitate, which places higher requirements on the centrifugal speed and centrifugal instrument.

[0004] Summary of the Invention

[0005] Problems to be solved by the invention

[0006] The present invention provides a simple and convenient method for separating and purifying antigen components. This separation and purification method is simple and easy to perform, does not require the use of saline substances for salting out, and does not require the use of high centrifugal speeds to completely precipitate the salted-out substances due to the high viscosity of the saline substances. Furthermore, the antigen components obtained by the method provided by the present invention have strong immunogenicity, achieving unexpected results.

[0007] Solutions for solving problems

[0008] In a first aspect, the present invention provides a method for extracting an antigen component, wherein the method comprises the following steps:

[0009] (1) lysing cells and / or tissues to obtain cell lysate components;

[0010] (2) using water and / or a solution containing the first dissolving solution to dissolve the lysate components after lysis in step (1);

[0011] (3) mixing the solution containing the lysate component obtained in step (2) with an organic solvent to obtain the antigen component;

[0012] Wherein, the organic solvent includes one or more of alcohols, ketones, phenols, nitriles and acids.

[0013] Preferably, after the mixing in step (3), centrifugation is performed, and the lower layer is removed and mixed with a second dissolving solution containing a dissolving agent to obtain the antigen component; or, after the mixing in step (3), centrifugation is performed, the lower layer is removed and mixed with a second dissolving solution containing a dissolving agent and then mixed with the component obtained by separation and purification of the supernatant after the centrifugation to obtain the antigen component.

[0014] Preferably, the organic solvent is selected from one or more of ethanol, methanol, isopropanol, propanol, butanol, butyric acid, propionic acid, acetic acid, formic acid, acetone, phenol and acetonitrile.

[0015] Further preferably, the organic solvent is selected from one or more of ethanol, isopropanol, acetone and phenol.

[0016] Preferably, the volume ratio of the solution containing the lysate components to the organic solvent is 1:0.5-30.

[0017] Further preferably, the volume ratio of the solution containing the lysate components to the organic solvent is 1:0.5-25.

[0018] More preferably, the volume ratio of the solution containing the lysate components to the organic solvent is 1:0.5-20.

[0019] Preferably, the dissolving 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; wherein the structural formula 1 is as follows:

[0020] R1 is C, S, P, N or O, and R2-R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl.

[0021] Further preferably, the dissolving agent is selected from one or more of metformin hydrochloride, guanidine isothiocyanate, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, spermine, spermidine and choline.

[0022] More preferably, the dissolving agent is selected from one or more of urea, urea peroxide, guanidine isothiocyanate, guanidine sulfate, guanidine hydrochloride and spermidine.

[0023] Preferably, the method further comprises treating the cells and / or tissues before and / or after lysis of the cells and / or tissues.

[0024] Further preferably, the treatment comprises enhancing the immunogenicity of the antigenic component and / or fixing the cells and / or tissues.

[0025] Still more preferably, methods for enhancing the immunogenicity of the antigenic component include irradiation, oxidation, reduction, modification with haptenic substances, fixation, enzyme treatment, denaturation, heating, and mineralization.

[0026] Still further preferably, the irradiation includes any commonly used irradiation method; more preferably, the irradiation includes one or more of radioactive material irradiation, electron beam irradiation, microwave irradiation, ultraviolet irradiation, X-ray irradiation, α-ray irradiation, β-ray irradiation, and γ-ray irradiation.

[0027] Still further preferably, the oxidation is oxidation of the antigen component using an oxidant; more preferably, the oxidant includes hypochlorous acid, hydrogen peroxide, persulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO3, KBrO3, ClO3 - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, NO3 - , one or more of MnO4-, F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, and FeCl3.

[0028] Still further preferably, the hapten substance includes one or more of 2,4-dinitrofluorobenzene, 2,4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, Ovalbumin, N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other hapten substances containing aldehyde groups, rhamnose, galactose, and aminogalactose.

[0029] Still more preferably, formaldehyde, paraformaldehyde, glutaraldehyde, other substances containing aldehyde groups, ethanol, methanol, acetone, acetic acid, propionic acid, butyric acid, formic acid, formalin, dichromate, potassium permanganate, chromic acid, picric acid, Zamboni fixative, PLP fixative, FPA fixative, TAF fixative, Rossman fixative, Regaud fixative, PLPD fixative, PAPG fixative, Orth fixative, Muller fixative, McDoWell fixative, neutral formaldehyde The fixation is carried out by one or more of calcium fixative, FAB fixative, Carnoy fixative, Clarke fixative, B-5 ​​fixative, Bouin fixative, FineFIX fixative, AGM fixative, Helly fixative, Zenker fixative, Kolmer fixative, AAF fixative, Hollande fixative, Gendre fixative, aldehyde fixative, mercury fixative, alcohol fixative, oxidant fixative, picrate fixative and diethylene oxide.

[0030] In a second aspect, the present invention provides a delivery particle loaded with the antigen component prepared by the method described in the first aspect.

[0031] Preferably, the delivery particle further has the following components:

[0032] (i) the skeleton structure formed by the particle material;

[0033] (ii) immune adjuvants;

[0034] (iii) Positively charged substances.

[0035] Further preferably, the immune adjuvant is selected from 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 Q10, 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, one or more of ginseng active ingredients and astragalus active ingredients; preferably one or more selected from Toll-like receptor 3 agonists and Toll-like receptor 9 agonists; more preferably one or more selected from Poly(I:C), Poly ICLC, class A CpG-OND, class B CpG-OND and class C CpG-OND.

[0036] Further preferably, the antigen component is loaded inside and / or on the surface of the skeleton structure.

[0037] Further preferably, the positively charged substance is selected from one or more of positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers and positively charged inorganic substances.

[0038] Still further preferably, the positively charged substance is selected from one or more of melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

[0039] Preferably, the delivery particles are nano-delivery particles and / or micro-delivery particles.

[0040] Preferably, the particle size of the nano delivery particles is 1 nm-1000 nm, preferably 50-500 nm, more preferably 100-400 nm.

[0041] Preferably, the particle size of the micron delivery particles is 1 μm-1000 μm, preferably 1-10 μm, more preferably 1-5 μm.

[0042] Preferably, the amount of the protein or polypeptide component in the antigen component loaded per mg of the nano- and / or micro- delivery particles is 0.01-3 mg, preferably 0.02-2 mg.

[0043] Preferably, 1 mg of the nano- and / or micro-delivery particles is loaded with 0.001-2 mg of the immune adjuvant, preferably 0.002-0.8 mg.

[0044] Preferably, the antigen component contained in part of the cell lysate fraction comprises the protein and polypeptide components in the cell and / or tissue lysate and / or the RNA component or mRNA component in the cell lysate.

[0045] Preferably, the antigen component contained in part of the cell lysate fraction comprises the protein and polypeptide components in the cell and / or tissue lysate and / or the lipid component in the cell lysate.

[0046] Preferably, the antigen component contained in the partial cell lysate fraction comprises a lipid component in a cell and / or tissue lysate and / or an RNA component or mRNA component in a cell lysate.

[0047] Preferably, the antigen component is separated and purified using an appropriate method from a lysate component dissolved in a lysis solution containing a lytic agent.

[0048] In a third aspect, the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the antigen component prepared by the method described in the first aspect or the delivery particle described in the second aspect.

[0049] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0050] In a fourth aspect, the present invention provides a use of the antigen component prepared by the method of the first aspect, or the delivery particle according to any one of the second aspect, or the pharmaceutical composition according to the third aspect in at least one of the following (1) to (4):

[0051] (1) Preparation of drugs for preventing or treating diseases;

[0052] (2) Used to activate antigen-presenting cells and prepare cellular vaccines based on antigen-presenting cells;

[0053] (3) Assisted activation of antigen-specific T cells and detection of antigen-specific T cell content;

[0054] (4) After assisting in the activation of antigen-specific T cells, the activated antigen-specific T cells are isolated and amplified, and used to prevent or treat diseases.

[0055] Preferably, the antigen components separated and purified from the lysate components can also be irradiated before or after cell or tumor tissue lysis, or can be irradiated after the separated and purified antigen components are loaded onto nanoparticles or microparticles.

[0056] Preferably, the delivery particles are used directly as vaccines.

[0057] Preferably, after the delivery particles activate dendritic cells and / or B cells, the activated dendritic cells (DC) and / or B cells are used as cell vaccines.

[0058] Preferably, the delivery particles are used to detect the content of cancer antigen-specific T cells after assisting in the activation of cancer cell-specific T cells in vitro, or the delivery particles are used to isolate and / or amplify the activated antigen-specific T cells after assisting in the activation of antigen-specific T cells for the prevention and treatment of diseases.

[0059] Preferably, the disease is cancer or tumor.

[0060] More preferably, the disease is a solid tumor or a hematological tumor.

[0061] Effects of the Invention

[0062] The present invention uses an organic solvent precipitation method to separate and purify antigen components in cells (such as cancer cells) and / or tissues (such as tumor tissues), which can enrich antigen components such as proteins, polypeptides and RNA, and avoid potential toxic side effects caused by substances such as sugars. Since antigen substances such as proteins and polypeptides can be dissolved in aqueous solution after solubilization, and will precipitate after adding organic solvents such as ethanol and acetone, the purpose of separating antigen substances such as proteins and polypeptides is achieved. At the same time, the antigen components obtained by the present invention have high immunogenicity. The method is simple and fast, and does not require the addition of high-concentration salts and other substances for salting out.

[0063] When the separation and purification method of adding organic solvent precipitation is adopted, the steps are simple, no additional reagents are required, and a high rotation speed is not required during centrifugation, thereby making the purification operation and process simpler, faster and more convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG1 is a schematic diagram of some feasible preparation processes and applications for purifying antigen components in cell or tissue lysates in the present invention.

[0065] Figure 2 shows the structure of formula 1, wherein R1 is C, S, P, N or O, and R2-R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine, and any other groups.

[0066] Figures 3 to 7 are the experimental results of tumor growth rate and survival when nanovaccines and / or micron vaccines or antigen presenting cell (APC) vaccines or assisted sorting and amplified T cells loaded with purified antigen components are used to prevent or treat cancer in Examples 1-5; 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 was analyzed by ANOVA, and the significant difference in Figure b was analyzed by Kaplan-Meier and log-rank test.

[0067] FIG8 shows the experimental results of detecting tumor antigen-specific T cells using antigen delivery nanoparticles in Example 6.

[0068] In the above figures, *** indicates that p < 0.005 is significantly different compared with the PBS control group; ### indicates that p < 0.005 is significantly different between the two groups; ## indicates that p < 0.01 is significantly different between the two groups; # indicates that p < 0.05 is significantly different between the two groups. DETAILED DESCRIPTION

[0069] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following is a detailed description by listing specific embodiments. 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.

[0070] 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.

[0071] 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".

[0072] 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.

[0073] The term "suffering from a disease" means that the body is experiencing symptoms of a disease.

[0074] 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.

[0075] 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 invention, thereby alleviating the 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.

[0076] The vaccines of the present invention can be prepared using any method known to those skilled in the art, for example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding and / or lyophilizing processes.

[0077] In the present invention, the route of administration can be changed or adjusted in any applicable manner to meet the needs of the properties of the drug, the convenience of patients and medical personnel, and other relevant factors.

[0078] The terms "individual," "patient," or "subject" as used in the context of the present invention 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).

[0079] 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.

[0080] When preparing the antigen components described in the present invention, some embodiments use tumor tissue or cancer cell lines. In actual applications, cancer cells / tumor tissues obtained by several methods, such as tumor tissue, cancer cell lines, cancer cells isolated from tumor tissue and cultured and amplified, and cancer cells obtained by cultured and amplified circulating tumor cells isolated from peripheral blood, can be used to prepare antigen components. In actual applications, any other feasible approach can also be used to obtain cancer cells. In actual applications, other cells or tissues can also be used to prepare antigen components for other diseases, such as using β cells to prepare antigen components for type 1 diabetes, and using pancreatic tissue or pancreatic islet tissue to prepare antigen components for type 1 diabetes.

[0081] Due to space limitations, the cancers described in the examples of this invention are solid tumors. In practical applications, the nano- or micro-vaccines of this invention 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 of this invention are more effective in treating hematologic malignancies and lymphomas.

[0082] The present invention provides a method for preparing antigen delivery particles, which comprises first lysing cells or tissues, then solubilizing the lysate using a dissolving solution containing a dissolving agent, and then adding an organic solvent to precipitate the solubilized antigen components such as proteins and polypeptides in the dissolving solution, thereby obtaining purified antigen components from the lysate components.

[0083] The separated and purified antigen components are then loaded onto nanoparticles or microparticles for use as vaccines or antigen delivery particles: as vaccines, they can be used to prevent or treat diseases; as antigen delivery particles, they can be used to activate antigen-presenting cells in vitro to prepare antigen-presenting cell vaccines, or to assist in activating antigen-specific T cells and then detecting the content of antigen-specific T cells, or to assist in activating antigen-specific T cells and then sorting and amplifying antigen-specific T cells for prevention or treatment of diseases.

[0084] In a first aspect, the present invention provides a method for extracting an antigen component, wherein the method comprises the following steps:

[0085] (1) lysing cells and / or tissues to obtain cell lysate components;

[0086] (2) dissolving the lysate components after lysis in step (1) using water and / or a first dissolving solution containing a dissolving agent;

[0087] (3) mixing the solution containing the lysate component obtained in step (2) with an organic solvent to obtain the antigen component;

[0088] Wherein, the organic solvent includes one or more of alcohols, ketones, phenols, nitriles and acids.

[0089] In certain embodiments, after the mixing in step (3), centrifugation is performed, and the lower layer is removed and mixed with a second dissolving solution containing a dissolving agent to obtain the antigen component.

[0090] In certain embodiments, after the mixing in step (3), centrifugation is performed, and the lower layer is removed and mixed with a second dissolving solution containing a dissolving agent, and then mixed with the components obtained by separation and purification of the supernatant after the centrifugation to obtain the antigen component.

[0091] In certain embodiments, the first dissolving liquid is pure water or an aqueous solution.

[0092] In certain embodiments, the second dissolving liquid is pure water or an aqueous solution.

[0093] In certain embodiments, the antigen component contained in part of the cell lysate fraction comprises a protein and polypeptide component in a cell and / or tissue lysate and / or an RNA component or mRNA component in a cell lysate.

[0094] In certain embodiments, the antigenic component contained in part of the cell lysate fraction comprises a protein and polypeptide component in a cell and / or tissue lysate and / or a lipid component in a cell lysate.

[0095] In certain embodiments, the antigen component contained in the partial cell lysate fraction comprises a lipid component in a cell and / or tissue lysate and / or an RNA component or mRNA component in a cell lysate.

[0096] In some preferred embodiments, the mass ratio of the protein polypeptide to the RNA component (or mRNA component) is (0.1-100): (0.1-100).

[0097] In some preferred embodiments, the mass ratio of the protein polypeptide to the RNA component (or mRNA component) is (0.1-2): (0.1-2).

[0098] In certain embodiments, the steps for preparing the lysate from the cancer cells and / or tumor tissues are: first using a lysis solution containing a dissolving agent to lyse the cancer cells and / or tumor tissues to obtain their lysates, and then using a lysis solution containing a dissolving agent to dissolve the lysate components.

[0099] In certain embodiments, the separation and purification methods include, but are not limited to, salting out, oxidation, reduction, heating, radiation irradiation (irradiation), and the like.

[0100] In certain embodiments, the organic solvent is selected from one or more of ethanol, methanol, isopropanol, propanol, butanol, butyric acid, propionic acid, acetic acid, formic acid, acetone, phenol and acetonitrile.

[0101] In certain embodiments, the organic solvent is selected from one or more of ethanol, isopropanol, acetone and phenol.

[0102] In certain embodiments, the volume ratio of the solution containing the lysate components to the organic solvent is 1:0.5-30, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:22, 1:24, 1:26, 1:28, 1:30, etc.

[0103] In certain embodiments, the volume ratio of the solution containing the lysate components to the organic solvent is 1:0.5-25.

[0104] In certain embodiments, the volume ratio of the solution containing the lysate components to the organic solvent is 1:0.5-20.

[0105] In certain embodiments, the dissolving agent is independently selected from one or more of a compound containing a structure of Structural Formula 1, deoxycholate, dodecyl sulfate, glycerol, a protein degrading enzyme, a polypeptide, an amino acid, a glycoside, and choline; wherein the Structural Formula 1 is as follows:

[0106] R1 is C, S, P, N or O, and R2-R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl.

[0107] In certain embodiments, the dissolving agent is selected from one or more of metformin hydrochloride, guanidine isothiocyanate, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, spermine, spermidine and choline.

[0108] In certain embodiments, the dissolving agent is selected from one or more of urea, urea peroxide, guanidine isothiocyanate, guanidine sulfate, guanidine hydrochloride, and spermidine.

[0109] In certain embodiments, the dissolving agent is selected from urea, metformin, guanidine hydrochloride, and other compounds containing the structure of Structural Formula 1. The inventors have discovered that a substance having the structure of Structural Formula 1 can be used as a dissolving agent 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 dissolving agents to dissolve water-insoluble components.

[0110] In certain embodiments, the antigenic components after separation and purification of the cancer cell and / or tumor tissue lysate can be one or two selected from the following: (1) protein polypeptide components; (2) protein polypeptide components and RNA components (or mRNA components); (3) lipid components.

[0111] In some preferred embodiments, the mass ratio of the protein polypeptide to the RNA component (or mRNA component) is (0.1-100): (0.1-100).

[0112] In other preferred embodiments, the mass ratio of the protein polypeptide to the RNA component (or mRNA component) is preferably (0.1-2): (0.1-2).

[0113] In certain embodiments, the immunogenic protein and / or polypeptide can be derived from a portion of components in cancer cells / tumor tissues and extracellular vesicle lysates. Furthermore, the extracellular vesicle lysates are selected from extracellular vesicle lysates of cancer cells and / or extracellular vesicle lysates of bacteria.

[0114] In certain embodiments, the mass ratio of a 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).

[0115] In some preferred embodiments, the mass ratio of the portion of the components in the cancer cells and / or tumor tissue to the extracellular vesicle lysate components is (0.5-2):(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.

[0116] In certain embodiments, the immunogenic protein and / or polypeptide may be derived from a portion of a cancer cell / tumor tissue component and a bacterial lysate. Furthermore, the mass ratio of the portion of the cancer cell / tumor tissue component to the bacterial lysate is (0.1-10):(0.1-10).

[0117] In some preferred embodiments, the mass ratio of the portion of the components in the cancer cell / tumor tissue to the bacterial lysate is (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.

[0118] In certain embodiments, the cells described herein are derived from any method that can obtain cells. Taking cancer cells as an example, the cells described herein are derived from any method that can obtain cancer cells, including but not limited to cancer cell lines, cancer cells isolated and extracted from tumor tissue and expanded in vitro, cancer cells isolated and extracted from blood and expanded from circulating tumor cells, or cancer cells differentiated and cultured from stem cells.

[0119] In certain embodiments, the cells or tissues may be incubated with specific chemicals to stimulate the cells or tissues prior to lysis.

[0120] In certain embodiments, the cancer cells or tumor tissues may be co-incubated with specific chemicals to stimulate the cancer cells or tumor tissues before lysis.

[0121] In certain embodiments, 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, chemokines, plant extracts (such as important extracts of ginseng, plant root extracts, etc.), interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate with cancer cells or tumor tissues to stimulate cancer cells or tumor tissues is to enable the cancer cells to produce more antigen components.

[0122] In certain embodiments, the cancer cells are from one or more organisms.

[0123] In certain embodiments, the cancer cells are from one or more cancer cell lines.

[0124] In certain embodiments, the protein and polypeptide components in the lysate components of the cancer cells and / or tumor tissues contain antigen components.

[0125] In certain embodiments, the method further comprises treating the cells and / or tissue before and / or after lysis of the cells and / or tissue.

[0126] In certain embodiments, the treatment comprises enhancing the immunogenicity of the antigenic component and / or fixing the cells and / or tissue.

[0127] Still more preferably, methods for enhancing the immunogenicity of the antigenic component include irradiation, oxidation, reduction, modification with haptenic substances, fixation, enzyme treatment, denaturation, heating, and mineralization.

[0128] In certain embodiments, the irradiating comprises any commonly used irradiation method.

[0129] In certain embodiments, the irradiation comprises one or more of radioactive material irradiation, electron beam irradiation, microwave irradiation, ultraviolet irradiation, X-ray irradiation, α-ray irradiation, β-ray irradiation, and γ-ray irradiation.

[0130] In certain embodiments, the oxidation is oxidation of the antigenic component using an oxidizing agent.

[0131] In certain embodiments, the oxidizing agent comprises hypochlorous acid, hydrogen peroxide, persulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO3, KBrO3, ClO3 - 、ClO4 -, Na2O2, K2O2, MgO2, CaO2, BaO2, NO3 - 、MnO4 - , one or more of F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, and FeCl3.

[0132] In certain embodiments, the haptenic substance is a substance that can increase the immunogenicity of a protein or polypeptide after co-acting with the protein or polypeptide.

[0133] In certain embodiments, the hapten substance comprises one or more of 2,4-dinitrofluorobenzene, 2,4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, Ovalbumin, N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other hapten substances containing aldehyde groups, rhamnose, galactose, and aminogalactose.

[0134] In certain embodiments, a hapten substance is used to co-act with cells (e.g., cancer cells) or cells in a tissue (e.g., tumor tissue) for a certain period of time to modify the antigenic components in the cells or tissues, and then the cells and / or tissues are lysed to obtain their lysates; or cells (e.g., cancer cells) or tissues (e.g., tumor tissues) are first lysed to obtain their lysates, and then the hapten substance is used to modify the antigenic components in the cell or tissue lysates.

[0135] In certain embodiments, the lysate components of cells (such as cancer cells) or tissues (such as tumor tissues) modified with hapten substances can be directly loaded into nanovaccines or microvaccines as antigen components, or can be loaded into nanovaccines or microvaccines as antigen components after appropriate treatment, separation and purification, or further enhancement of immunogenicity.

[0136] In certain embodiments, before lysing cells (such as cancer cells) or tissues (such as tumor tissue) or before allowing the hapten substance to act on cells (such as cancer cells) or tissues (such as tumor tissue), the cells (such as cancer cells) or tissues (such as tumor tissue) can be irradiated with radiation to inactivate the cells (such as cancer cells) or tissues (such as tumor tissue). The radiation includes but is not limited to γ ​​rays, X-rays, electron beams, microwaves, β rays, α rays, etc.

[0137] In certain embodiments, the reducing is the reduction of the antigenic component using a reducing agent.

[0138] In certain embodiments, the reducing agent includes but is not limited to one or more reducing agents such as dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP).

[0139] In certain embodiments, the enzymatic treatment method includes but is not limited to the use of one or more of nuclease, DNA enzyme, pepsin, chymotrypsin, trypsin, other protein digestive enzymes, protease inhibitors, etc. Enzymatic hydrolysis in the present invention includes but is not limited to the use of any feasible enzymatic hydrolysis method such as nuclease, pepsin, trypsin, protease inhibitors, chymotrypsin, DNA enzyme, etc.

[0140] In certain embodiments, the enzymatic treatment method includes, but is not limited to, using one or more of nuclease, DNase, pepsin, chymotrypsin, trypsin, other protein digesting enzymes, protease inhibitors, and the like.

[0141] In certain embodiments, the enzymes used for enzymatic hydrolysis include, but are not limited to, one or more of nuclease, pepsin, trypsin, protease inhibitors, chymotrypsin, DNase, and the like.

[0142] In certain embodiments, the mineralization includes, but is not limited to, any mineralization or biomineralization method such as silicification, calcification, and magnesiization.

[0143] In certain embodiments, the cancer cells and / or tumor tissues can be subjected to treatments including but not limited to inactivation or / and denaturation, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, heating, salting out, enzyme treatment, oxidation, reduction, mineralization, irradiation, radiation, ionization, chemical modification, nucleic acid separation and purification, protease endo- or degradation, nuclease treatment, etc. before or after lysis, and then lysed using a lysis solution containing a lytic agent to dissolve the lysate components and then extract and separate the protein and polypeptide components therein; or the cancer cells and / or tumor tissues can be subjected to treatments including but not limited to inactivation or / and denaturation, fixation, heating, salting out, oxidation, reduction, mineralization, enzyme treatment, ionization, irradiation, radiation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, chemical modification, protease endo- or degradation, nuclease treatment before or after cell lysis, and then directly lysed using a lysis solution containing a lytic agent to dissolve the lysate components and then extract and separate the protein and polypeptide components therein. In some embodiments of the present invention, tumor tissue cells are irradiated, inactivated by high temperature, or (and) denatured before lysis. In actual use, irradiation or heat treatment may be performed after cell lysis, or irradiation, heat inactivation, or (and) denaturation treatment may be performed both before and after cell lysis. 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-cleavage or degradation, collagenase treatment, freeze-drying, etc. may also be used. It will be understood by those skilled in the art that in actual application, the technician may make appropriate adjustments according to the specific circumstances.

[0144] In the present invention, the fixation can be performed using conventional fixatives.

[0145] In certain embodiments, the fixation is performed by selecting one or more of formaldehyde, paraformaldehyde, glutaraldehyde, other substances containing aldehyde groups, ethanol, methanol, acetone, acetic acid, propionic acid, butyric acid, formic acid, formalin, dichromate, potassium permanganate, chromic acid, picric acid, Zamboni fixative, PLP fixative, FPA fixative, TAF fixative, Rossman fixative, Regaud fixative, PLPD fixative, PAPG fixative, Orth fixative, Muller fixative, McDoWell fixative, neutral calcium formaldehyde fixative, FAB fixative, Carnoy fixative, Clarke fixative, B-5 ​​fixative, Bouin fixative, FineFIX fixative, AGM fixative, Helly fixative, Zenker fixative, Kolmer fixative, AAF fixative, Hollande fixative, Gendre fixative, aldehyde fixative, mercury fixative, alcohol fixative, oxidant fixative, picrate fixative and diethylene oxide.

[0146] In the present invention, the AGM fixative is a mixture of 70% ethanol (80 mL), glacial acetic acid (10 mL) and methanol (10 mL).

[0147] In a second aspect, the present invention provides a delivery particle loaded with the antigen component prepared by the method described in the first aspect.

[0148] In certain embodiments, the delivery particle further comprises the following components:

[0149] (i) the skeleton structure formed by the particle material;

[0150] (ii) immune adjuvants;

[0151] (iii) Positively charged substances.

[0152] In certain embodiments, the immune adjuvant is selected from 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-enhanced 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 Q10, 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, one or more of ginseng active ingredients and astragalus active ingredients.

[0153] In certain embodiments, the immune adjuvant is selected from one or more of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist.

[0154] In certain embodiments, the immune adjuvant is selected from one or more of Poly(I:C), Poly ICLC, class A CpG-OND, class B CpG-OND, and class C CpG-OND.

[0155] In certain embodiments, the antigen component is loaded inside and / or on the surface of the scaffold structure.

[0156] In certain embodiments, the positively charged substance is selected from one or more of positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers and positively charged inorganic substances.

[0157] In certain embodiments, the positively charged substance is selected from one or more of melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

[0158] In certain embodiments, the separated and purified antigen components in the lysate components can also be irradiated before or after cell or tumor tissue lysis, or can be irradiated after the separated and purified antigen components are loaded onto nanoparticles or microparticles.

[0159] In certain embodiments, the delivery particles are nano-delivery particles and / or micro-delivery particles.

[0160] In certain embodiments, the nanodelivery particles have a particle size of 1 nm to 1000 nm.

[0161] In certain embodiments, the nanodelivery particles have a particle size of 50-500 nm.

[0162] In certain embodiments, the nanodelivery particles have a particle size of 100-400 nm.

[0163] In certain embodiments, the micron delivery particles have a particle size of 1 μm to 1000 μm.

[0164] In certain embodiments, the micron delivery particles have a particle size of 1-10 μm.

[0165] In certain embodiments, the micron delivery particles have a particle size of 1-5 μm.

[0166] In certain embodiments, the amount of the protein or polypeptide component in the antigen component loaded per mg of the nano- and / or micro- delivery particles is 0.01-3 mg.

[0167] In certain embodiments, the amount of the protein or polypeptide component in the antigen component loaded into 1 mg of the nano- and / or micro- delivery particles is 0.02-2 mg.

[0168] In certain embodiments, the amount of the protein or polypeptide component in the antigen component loaded per mg of the nanodelivery particles is 0.01-2 mg, for example, 0.02 mg, 0.04 mg, 0.08 mg, 0.1 mg, 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 1.0 mg, 1.5 mg, or 2.0 mg.

[0169] In certain embodiments, the amount of the protein or polypeptide component in the antigen component loaded per mg of the micron delivery particles is 0.01-2 mg, such as 0.02 mg, 0.04 mg, 0.08 mg, 0.1 mg, 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 1.0 mg, 1.5 mg, or 2.0 mg. In actual applications, the amount of antigen components such as proteins and polypeptides loaded per mg of particle material can be even higher.

[0170] In certain embodiments, 1 mg of the nano- and / or micro-delivery particles is loaded with 0.001-2 mg of the immune adjuvant.

[0171] In certain embodiments, 1 mg of the nano- and / or micro-delivery particles is loaded with 0.002-0.8 mg of the immunoadjuvant.

[0172] In certain embodiments, 1 mg of the nanodelivery particles is loaded with 0.001-0.5 mg of the immune adjuvant, for example, 0.002 mg, 0.008 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.40 mg, 0.45 mg, etc.

[0173] In certain embodiments, 1 mg of the micron delivery particle carries 0.001-0.5 mg of the immune adjuvant, for example, 0.002 mg, 0.004 mg, 0.006 mg, 0.008 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.40 mg, 0.45 mg, etc. In the present invention, the interior and / or surface of the nanoparticles or microparticles carrying the separated and purified antigen components may further contain membrane components, and the membrane components inside and / or on the surface of the nanoparticles or microparticles are one or more selected from the group consisting of cell membranes 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.

[0174] When the membrane component is located on the surface of nanoparticles or microparticles, methods for loading the membrane component onto the surface of nanoparticles or microparticles include, but are not limited to, one or more of sonication, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bonding, stirring, homogenization, and homogenization.

[0175] In certain embodiments, the nanoparticles and / or microparticles may also be bacteria and viruses.

[0176] In certain embodiments, the nanoparticles and / or microparticles are prepared using bacterial cell walls.

[0177] In certain embodiments, the nanoparticles and / or microparticles are prepared using bacterial proteins or viral proteins.

[0178] In some exemplary embodiments of the present invention, a solvent volatilization method is used to prepare nanoparticles or microparticles loaded with antigen components. In practical applications, any other method for preparing nanoparticles or microparticles can 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.

[0179] In certain embodiments, the immunogenic protein and / or polypeptide can be derived from a portion of a cancer cell / tumor tissue component and an extracellular vesicle lysate. Furthermore, the extracellular vesicle lysate is selected from an extracellular vesicle lysate of a cancer cell and / or a bacterial extracellular vesicle lysate. The mass ratio of the portion of the cancer cell and / or tumor tissue component to the extracellular vesicle lysate component is (0.1-10):(0.1-10).

[0180] In certain embodiments, the mass ratio of the portion of the components in the cancer cells and / or tumor tissue to the extracellular vesicle lysate components is (0.5-2):(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.

[0181] In certain embodiments, the immunogenic protein and / or polypeptide may be derived from a portion of a cancer cell / tumor tissue component and a bacterial lysate. Furthermore, the mass ratio of the portion of the cancer cell / tumor tissue component to the bacterial lysate is (0.1-10):(0.1-10).

[0182] In certain embodiments, the mass ratio of the portion of the components in the cancer cell / tumor tissue to the bacterial lysate is (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.

[0183] In certain embodiments, the particle material may be PEG-modified or unPEG-modified. Preferably, when preparing the backbone structure, the mass ratio of the unPEG-modified particle material to the PEG-modified particle material is 9-200:1. In the preparation of nanoparticles or microparticles, an appropriate amount of PEG-modified PLGA or PLA can be added to a primary material such as PLGA or PLA. This can enhance long-circulation and passive targeting effects after injection into the body; the mass ratio of the PEG-modified PLGA or PLA to the unmodified PLGA or PLA is 0.05% to 20%.

[0184] In certain embodiments, the mass ratio of PEG-modified PLGA or PLA to unmodified PLGA or PLA is 0.1% to 10%.

[0185] In certain embodiments, the mass ratio of the framework material, protein and polypeptide components used to prepare the nanoparticles or microparticles is 1:0.001-10.

[0186] In certain embodiments, the mass ratio of the framework material, protein and polypeptide components in particle preparation is 1:0.01-2.

[0187] In certain embodiments, the mass ratio of the framework material, protein and polypeptide components in particle preparation is 1:0.05-1.

[0188] In certain embodiments, the nanoparticles or microparticles are prepared from materials selected from natural polymer materials, biological materials, microbial materials, synthetic polymer materials and / or inorganic materials.

[0189] In the present invention, organic polymer substances are used as the preparation materials of nanoparticles or micron particles. 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 (such as viral proteins), bacteria (such as bacterial walls, bacterial proteins or whole bacteria), and materials of other biological sources (exosomes, extracellular vesicles, bacterial membrane components).

[0190] In certain embodiments, the shape of the antigen delivery particles (nanoparticles or microparticles) is any shape, including but not limited to spheres, ellipsoids, barrels, polygons, rods, sheets, threads, worms, squares, triangles, butterflies, discs, vesicles, etc.

[0191] In certain embodiments, the surface of the nanoparticles or microparticles 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.

[0192] In certain embodiments, the antigen-presenting cells used to prepare the biofilm components loaded on the surface of the nanoparticles or microparticles can be derived from autologous or allogeneic sources, or from cell lines or stem cells. The antigen-presenting cells can be dendritic cells, B cells, macrophages, or any mixture of the above three, or other cells with antigen-presenting function. The antigen-presenting cells can be activated by the antigen-loaded nanoparticles or microparticles.

[0193] In certain embodiments, when the biofilm components carried on the surface of the nanoparticles or microparticles 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.

[0194] In certain embodiments, any method for preparing nanoparticles or microparticles known to those skilled in the art can be used to prepare the nanoparticles or microparticles described in the present invention, 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.

[0195] The preparation method of the delivery particles of the present invention is as follows:

[0196] Step 1: extract the antigen component according to the extraction method described in the first aspect. Specifically, use pure water or a dissolving solution containing a dissolving agent to lyse the tumor tissue or cancer cells, and then use pure water and / or a first dissolving solution containing a dissolving agent to dissolve the lysate component.

[0197] Step 2: Add a certain amount of organic solvents such as alcohols (such as ethanol, methanol, isopropanol, etc.), ketones (such as acetone, etc.), phenols (such as phenol, etc.) or nitriles (such as acetonitrile, etc.) to the lysate components dissolved in pure water or the first lysate containing a solubilizer, so as to precipitate antigen components such as proteins and polypeptides, and collect the precipitated portion after centrifugation as the antigen components. In the present invention, organic solvents such as alcohols (such as ethanol, methanol, isopropanol, etc.), ketones (such as acetone, etc.), phenols (such as phenol, etc.) or nitriles (such as acetonitrile, etc.) are used. In practical applications, many organic solvents can precipitate antigen components such as proteins and polypeptides from aqueous solutions. The use of organic solvents that can precipitate proteins and polypeptides can achieve the purpose, and thus other organic solvents that can precipitate proteins from aqueous solutions can also be used.

[0198] In certain embodiments, the centrifugation speed is 1000 RPM-20000 RPM.

[0199] In certain embodiments, the centrifugation speed is 1000 RPM-12000 RPM.

[0200] In certain embodiments, the centrifugation speed is 1000 RPM-10000 RPM.

[0201] In certain embodiments, the centrifugation speed is 1000 RPM-8000 RPM.

[0202] In certain embodiments, the centrifugation speed is 1000 RPM-5000 RPM.

[0203] Step 3, using a lytic solution containing a lytic agent to dissolve the precipitated components (antigen components) collected in step 2 for a secondary time, and directly using them; or using a lytic solution containing a lytic agent to dissolve the precipitated components (antigen components) collected in step 2 for a secondary time, and then appropriately treating the supernatant obtained by centrifugation in step 2 to obtain other components, and mixing the antigen components dissolved by the lytic solution with the components obtained by separation and purification in the supernatant to use as the antigen components.

[0204] In certain embodiments, in the present invention, the supernatant components obtained in step 2 can also be separated and purified after salting out, heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction of RNA components, extraction of mRNA components, degradation of DNA components, extraction, radiation, mineralization, irradiation, radiation and other appropriate methods; The other components separated and purified in the supernatant can be combined with the antigen component dissolved twice by the second lysate containing a solubilizer and used as an antigen component. For example, an mRNA component or an RNA component is isolated from the supernatant using an appropriate method, and then the mRNA component or the RNA component is mixed with the precipitate dissolved twice by the lysate containing a solubilizer to obtain the antigen component.

[0205] Step 4: Add the antigen component obtained in step 3 as the initial aqueous phase to the organic phase to prepare a colostrum sample.

[0206] 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.

[0207] In certain embodiments, the aqueous solution may contain at least one of the following i)-ii): i) antigen components in the lysate; ii) antigen components in the lysate and an immunopotentiating adjuvant. The antigen components in the lysate are separated and purified antigen components dissolved in a lysate containing a solvent such as urea or guanidine hydrochloride during preparation. The first predetermined concentration is the concentration of proteins and polypeptides contained in the aqueous solution, or the concentration of antigen components contained in the aqueous solution. The first predetermined concentration requires that the protein and polypeptide concentration content be greater than 1 ng / mL so that sufficient antigen components can be loaded to activate relevant cells. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0208] In certain embodiments, the organic solvent is selected from dichloromethane or ethyl acetate. In addition, in some examples, the second predetermined concentration of the raw material for preparing the particles ranges from 0.5 mg / mL to 5000 mg / mL.

[0209] In certain embodiments, the second predetermined concentration of the raw material for preparing the particles is in the range of 100 mg / mL.

[0210] 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 invention, 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 implementation, 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.

[0211] In certain embodiments, when the aqueous solution is a solution comprising antigenic components in cell and / or tissue lysates, the concentration of proteins and polypeptides therein is greater than 1 ng / mL.

[0212] In certain embodiments, when the aqueous solution is a solution comprising antigenic components in cell and / or tissue lysates, the concentration of proteins and polypeptides therein is 1 mg / mL-100 mg / mL.

[0213] In certain embodiments, when the aqueous phase solution is a solution comprising the antigen components in the lysate and the immune adjuvant, the concentration of the protein and polypeptide is greater than 1 ng / mL, preferably 1 mg / mL-100 mg / mL, and the concentration of the immune adjuvant is greater than 0.01 ng / mL, preferably 0.001 mg / mL-20 mg / mL. In some embodiments, the solvent in the organic phase solution is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane and ethyl acetate; the concentration of the organic phase is 0.5 mg / mL-5000 mg / mL, preferably 100 mg / mL.

[0214] In certain embodiments, when the aqueous phase solution is a solution comprising the antigen component in the lysate, the concentration of the protein and polypeptide components is greater than 0.01 ng / mL, preferably 1 μg / mL-1 mg / mL. In some embodiments, when the aqueous phase solution is a solution comprising the antigen component and the immune adjuvant, the concentration of the protein and polypeptide components is greater than 1 ng / mL, preferably 1 μg / mL-1 mg / mL, and the concentration of the immune adjuvant is greater than 0.01 ng / mL, preferably 0.001 mg / mL-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 and ethyl acetate; the concentration of the organic phase is 0.5 mg / mL-5000 mg / mL, preferably 100 mg / mL.

[0215] 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-1500 rpm and a stirring time of 0.1 hour-24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5 W and the time is greater than 0.1 second, such as 2-200 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-100 psi, and when a high-shear homogenizer is used, the speed is greater than 100 rpm, such as 1000 rpm-5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, such as 0.1 mL / min-100 mL / min. Nano- and / or micron-size can be achieved 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.

[0216] 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 5 is added to the aqueous emulsifier solution and continues to be ultrasonicated, stirred, homogenized, or mixed to achieve nano- or micron-scaling. In the present invention, the ultrasonication time is greater than 0.1 seconds, such as 2-200 seconds; the stirring speed is greater than 50 rpm, such as 50 rpm-500 rpm; and the stirring time is greater than 1 minute, such as 60-6000 seconds. Preferably, when stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, such as the stirring speed is 50rpm-1500rpm, and the stirring time is 0.5 hour-5 hours; when ultrasonic treatment is performed, the ultrasonic power is 50W-500W, and the time is greater than 0.1 second, such as 2-200 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 20psi, such as 20psi-100psi, and the speed when using a high shear homogenizer is greater than 1000rpm, such as 1000rpm-5000rpm; when using microfluidics, the flow rate is greater than 0.01mL / min, such as 0.1mL / min-100mL / min. Ultrasonication or stirring or homogenization or microfluidics is used for nano- or micronization, and the length of ultrasonication 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.

[0217] In certain embodiments, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20mg / mL. The third predetermined volume is adjusted according to its ratio with the second predetermined volume. In the present invention, the range of the ratio of the second predetermined volume to the third predetermined volume is 1:1.1-1:1000, preferably 2:5. In order to control the size of nanoparticles or micron particles during specific implementation, the ratio of the second predetermined volume to the third predetermined volume can be adjusted. Similarly, the ultrasonic time or stirring time or homogenization time of this step, the volume of the emulsifier aqueous solution and the concentration are based on, all in order to obtain nanoparticles or micron particles of suitable size.

[0218] 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.

[0219] In this step, the emulsifier aqueous solution is a PVA solution or other solutions.

[0220] The fourth predetermined concentration is 5 mg / mL. By selecting the fourth predetermined concentration, nanoparticles or microparticles of appropriate size are obtained. The selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, 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 a specific implementation, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted to control the size of the nanoparticles or microparticles.

[0221] In the present invention, the predetermined stirring condition of this step is until the organic solvent is completely volatilized, that is, the dichloromethane or ethyl acetate in step 4 is completely volatilized.

[0222] 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).

[0223] Step 9: freeze-dry the suspension containing the lyoprotectant obtained in step 8, and keep the lyophilized material for later use.

[0224] In certain embodiments, the prepared nanoparticles or microparticles loaded with antigen components are irradiated with radiation for a certain period of time.

[0225] Step 10: The suspension containing nanoparticles / microparticles obtained in step 8 is resuspended in PBS (or normal saline) with a sixth predetermined volume, or the freeze-dried material containing nanoparticles or microparticles and lyoprotectant obtained in step 9 is resuspended in a sixth predetermined volume of PBS (or normal saline) and then used directly; or the above sample is mixed with the seventh predetermined volume of the antigen component and then used.

[0226] In the present invention, 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.

[0227] In step 11, the nanoparticles or microparticles prepared in step 10 are used to prevent or treat diseases such as cancer; or the nanoparticles or microparticles are used as antigen delivery particles to activate antigen-presenting cells (such as dendritic cells or mixed antigen-presenting cells) in vitro to prepare antigen-presenting cell vaccines; or the nanoparticles or microparticles are used to assist in activating antigen-specific T cells to detect the content of antigen-specific T cells; or after using the nanoparticles or microparticles to assist in activating antigen-specific T cells, the antigen-specific T cells are sorted or amplified, and the sorted or amplified antigen-specific T cells are used to prevent or treat diseases.

[0228] In a third aspect, the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the antigen component prepared by the method described in the first aspect or the delivery particle described in the second aspect.

[0229] In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0230] In a fourth aspect, the present invention provides a use of the antigen component prepared by the method of the first aspect, or the delivery particle according to any one of the second aspect, or the pharmaceutical composition according to the third aspect in at least one of the following (1) to (4):

[0231] (1) Preparation of drugs for preventing or treating diseases;

[0232] (2) Used to activate antigen-presenting cells and prepare cellular vaccines based on antigen-presenting cells;

[0233] (3) Assisted activation of antigen-specific T cells and detection of antigen-specific T cell content;

[0234] (4) After assisting in the activation of antigen-specific T cells, the activated antigen-specific T cells are isolated and amplified, and used to prevent or treat diseases.

[0235] In certain embodiments, the separated and purified antigen components in the lysate components can also be irradiated before or after cell or tumor tissue lysis, or can be irradiated after the separated and purified antigen components are loaded onto nanoparticles or microparticles.

[0236] In certain embodiments, the delivery particles are used directly as vaccines.

[0237] In certain embodiments, after the delivery particles activate dendritic cells and / or B cells, the activated dendritic cells and / or B cells are used as cellular vaccines. In the present invention, cancer vaccines and other targeted therapies are designed to activate cellular immunity via the T cell pathway, which recognizes primary sequence fragments of polypeptides rather than the tertiary conformational structures recognized by humoral immunity. Therefore, protein denaturation has no negative impact on the immunogenicity of the protein antigen component against cellular immunity and may even enhance the protein's immunogenicity to some extent.

[0238] In certain embodiments, the delivery particles are used to detect the content of cancer antigen-specific T cells after assisting in the activation of cancer cell-specific T cells in vitro, or the delivery particles are used to isolate and / or expand the activated antigen-specific T cells after assisting in the activation of antigen-specific T cells for the prevention and treatment of diseases.

[0239] In certain embodiments, the disease is cancer or a tumor.

[0240] In certain embodiments, the disease is a solid tumor or a hematological tumor.

[0241] The present invention is further illustrated below by means of specific examples. Unless otherwise specified herein, "%" represents mass percentage. The materials and reagents used in the following examples, unless otherwise specified, are commonly used in the art and can be obtained from commercial sources or synthesized by known methods. Experimental methods in the following examples where no conditions are specified were generally performed according to conventional experimental conditions or the conditions recommended by the manufacturers of the relevant reagents (kits).

[0242] Example 1: T cells activated by antigen-loaded nanoparticles and sorted and expanded for the treatment of breast cancer

[0243] (1) Preparation of antigen components

[0244] Each Balb / c mouse was subcutaneously inoculated with 6.0×10 5 4T1 mouse breast cancer cells, when the tumor grows to a volume of about 1000mm 3Mice were sacrificed and tumor tissue was removed. The tissue was then cut into pieces and added to ultrapure water. A single-cell suspension was prepared using a cell strainer. The tumor tissue was then lysed by repeated freeze-thawing five times. The tumor tissue lysate fraction was centrifuged at 5000 g for 10 minutes. The supernatant was collected to obtain the water-soluble fraction of the lysate. The precipitate was solubilized with an 8M aqueous urea solution to obtain the water-insoluble fraction of the lysate. Ten volumes of an aqueous solution containing 50% ethanol and 0.5% phenol were added to each of the water-soluble and water-insoluble fractions, respectively. The mixture was allowed to stand for 6 hours to precipitate. The precipitate from the water-soluble fraction and the precipitate from the water-insoluble fraction were then centrifuged at 3500 rpm for 5 minutes. The precipitate from the water-soluble fraction and the precipitate from the water-insoluble fraction were each dissolved with an 8M aqueous urea solution and mixed in a 1:2 mass ratio to obtain antigen fraction 1.

[0245] Each Balb / c mouse was subcutaneously inoculated with 6.0×10 5 4T1 mouse breast cancer cells, when the tumor grows to a volume of about 1000mm 3 Mice were sacrificed and tumor tissue was removed. The cells were then cut into pieces and placed in ultrapure water. A single-cell suspension was prepared using a cell strainer. The tumor tissue was then lysed by repeated freeze-thawing five times. The tumor tissue lysate fraction was centrifuged at 5000 g for 10 minutes. The supernatant was collected to obtain the water-soluble fraction of the lysate. The precipitate was solubilized with 8M urea aqueous solution to obtain the water-insoluble fraction of the lysate. The water-soluble and water-insoluble fractions were mixed in a mass ratio of 1:2 to obtain antigen fraction 2.

[0246] (2) Preparation of antigen delivery nanoparticles

[0247] In this example, nanoparticles 1 were prepared using the double emulsion method within the solvent evaporation method. The PLGA material used for nanoparticle preparation had a molecular weight of 20-40 kDa. The immunoadjuvants employed were poly(I:C), CpG 1018, and CpG 7909. The preparation method was as described above. The nanoparticles were first loaded with the cell antigen component 1 and the adjuvant. Then, 100 mg of nanoparticles 1 were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 6% trehalose, and freeze-dried for 48 hours. The nanoparticles 1 had an average particle size of approximately 230 nm. Each 1 mg of PLGA nanoparticles 1 was loaded with approximately 200 μg of protein or polypeptide components, including 0.02 mg each of poly(I:C), CpG 1018, and CpG 7909. The nanoparticles were then irradiated with gamma rays for 16 hours.

[0248] Nanoparticles 2 in this example were prepared using the same method as in Example 1. The nanoparticles were first loaded with the cell antigen component 2 and an adjuvant. 100 mg of nanoparticles 2 were then centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 6% trehalose, and freeze-dried for 48 hours. The nanoparticles 2 had an average particle size of approximately 230 nm. Each mg of PLGA nanoparticles 2 was loaded with approximately 200 μg of protein or polypeptide components, including 0.001 mg each of poly(I:C), CpG 1018, and CpG 7909. The nanoparticles were then irradiated with gamma rays for 16 hours.

[0249] (3) Sorting and expansion of antigen-specific T cells

[0250] Female Balb / c mice aged 6-8 weeks were selected and inoculated subcutaneously with 6.0×10 5 4T1 cells were added to each mouse, and 150 μg of mouse PD-1 antibody was injected intraperitoneally on days 6, 8, 10, 12, 14, 16, 18, and 20. Mice were sacrificed on day 21, and peripheral blood was collected. Peripheral blood mononuclear cells (PBMCs) were then isolated from the peripheral blood. 5 million PBMCs and 1 mg of nanoparticles (nanoparticle 1 or nanoparticle 2) were co-incubated in 2 mL of RPMI1640 complete medium for 36 hours. The CD3 + CD134 + T cells are antigen-specific T cells that can recognize cancer cell antigens. + CD134 + T cells were co-incubated with IL-2 (20 ng / mL), IL-7 (10 ng / mL), IL-15 (10 ng / mL), and αCD3 and αCD28 antibodies (10 ng / mL) in 10 mL of complete DMEM medium (37°C, 5% CO2) for 21 days to expand cancer cell-specific T cells (cell viability was approximately 85%). Among them, the cancer cell-specific T cells sorted and expanded with the assistance of nanoparticle 1 (nanovaccine 1) are T cells 1 (T cells 1); the cancer cell-specific T cells sorted and expanded with the assistance of nanoparticle 2 (nanovaccine 2) are T cells 2 (T cells 2).

[0251] (4) Sorting and expanding cancer cell antigen-specific T cells for cancer treatment

[0252] Female Balb / c mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 6.0×105 4T1 mouse breast cancer cells were injected intraperitoneally with cyclophosphamide (100 mg / kg) on ​​day 6 after tumor inoculation. One million sorted and expanded antigen-specific T cells (T cell 1 or T cell 2) or 100 μL of PBS were injected intravenously on days 7, 14, 21, and 28. The T cell (T cell 1 or T cell 2)-treated group received intravenous IL-2 (10,000 U) every two days from day 7 to day 27. Tumor growth and survival were monitored using conventional methods.

[0253] (5) Experimental results

[0254] As shown in Figure 3, tumors in mice in the PBS group rapidly expanded and the mice died soon after. Mice treated with both T cells 1 (T cells 1) and T cells 2 (T cells 2) had significantly prolonged survival, with the vast majority of mice recovering tumor-free. Furthermore, T cells 1 were more effective than T cells 2, demonstrating that purifying antigen components using the methods described herein can effectively enhance the therapeutic efficacy of T cells sorted and expanded with the assistance of antigen delivery particles.

[0255] Example 2: Nano-vaccine loaded with antigen components for the treatment of pancreatic cancer

[0256] In this example, the antigen component was derived from the Pan02 mouse pancreatic cancer cell line, the organic polymer material PLGA was used as the nanoparticle skeleton material, Polyinosinic-polycytidylic acid (poly(I:C)), CpG7909 (i.e., CpG2006), and CpG2395 were used as immune adjuvants, and the nanovaccine was prepared by the solvent evaporation method.

[0257] (1) Preparation of antigen components

[0258] Collect 2×10 8 Pan 02 mouse pancreatic cancer cells were cultured and irradiated with gamma rays for 2 hours. The cancer cells were then lysed by adding 2 mL of an 8M urea-PBS aqueous solution. The cell lysate fraction was then dissolved in 6 mL of an 8M urea-PBS aqueous solution. 12 mL of ethanol was then added to the lysate fraction. After standing for 1 hour, the sample was centrifuged at 3000 RPM for 5 minutes. The supernatant was removed, and the precipitate was redissolved in an 8M urea-PBS aqueous solution to prepare antigen component 1 of nanovaccine 1.

[0259] Collect 2×10 8Pan 02 mouse pancreatic cancer cells were cultured and irradiated with gamma rays for 2 hours. The cells were then lysed by adding 2 mL of an 8M urea-PBS aqueous solution. The cell lysate fraction was then dissolved in 6 mL of an 8M urea-PBS aqueous solution. A 40% ammonium sulfate aqueous solution was then added dropwise to the lysate fraction to prepare a 16 mL sample. The sample was allowed to stand for 1 hour to obtain a salted-out sample. The sample was centrifuged at 16,000 RPM for 60 minutes, the supernatant removed, and the precipitate redissolved in an 8M urea-PBS aqueous solution to prepare antigen component 2 of nanovaccine 2.

[0260] Cultured Pan 02 mouse pancreatic cancer cells were harvested and irradiated with gamma rays for 2 hours. 2 mL of 8 M urea in PBS was then added to lyse the cancer cells. The cell lysate was then dissolved in 6 mL of 8 M urea in PBS to prepare antigen component 3 of nanovaccine 3.

[0261] (2) Preparation of nanovaccines

[0262] In this embodiment, nanovaccine 1 was prepared using the double emulsion method, a solvent evaporation method. The vaccine preparation material used, PLGA, has a molecular weight of 10KDa-20KDa, and the immune adjuvants used are poly(I:C), CpG7909, and CpG2395. The preparation method is as described above. During the preparation process, the antigen component 1 and adjuvant are co-loaded into the nanoparticles using the double emulsion method. Then, 100mg of nanoparticles 1 are centrifuged at 12000g for 30 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 250nm. Each 1mg of PLGA nanoparticles 1 is loaded with approximately 1.0mg of protein and polypeptide components, and 0.02mg each of poly(I:C), CpG7909, and CpG2395.

[0263] Nanovaccine 2 in this example was prepared using the same methods and materials as Nanovaccine 1. First, the antigen component 2 and adjuvant were co-loaded into the nanoparticles using a double emulsion method. Then, 100 mg of nanoparticles 2 were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The nanoparticles 2 had an average particle size of approximately 250 nm. Each 1 mg of PLGA nanoparticles 2 was loaded with approximately 1.0 mg of protein and peptide components, with 0.02 mg each of poly(I:C), CpG7909, and CpG2395 loaded.

[0264] Nanovaccine 3 in this example was prepared using the same methods and materials as Nanovaccine 1. First, the antigen component 3 and adjuvant were co-loaded into the nanoparticles using a double emulsion method. Then, 100 mg of nanoparticles 3 were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The nanoparticles 3 had an average particle size of approximately 250 nm. Each mg of PLGA nanoparticles 3 was loaded with approximately 1.0 mg of protein and peptide components, including 0.02 mg each of poly(I:C), CpG7909, and CpG2395.

[0265] (3) Nano-vaccines for cancer treatment

[0266] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare pancreatic cancer tumor-bearing mice. On day 0, 1.5×10 6 Pan 02 cells. 2 mg of nanovaccine (nanovaccine 1, nanovaccine 2, or nanovaccine 3) or 100 μL of PBS was subcutaneously injected into the mice on days 3, 6, 9, 14, 19, and 26 after tumor inoculation. The tumor growth rate and survival of the mice were monitored. During the experiment, the size of the mouse tumor was recorded every 3 days starting from day 3. 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. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0267] (4) Experimental results

[0268] As shown in Figure 4, the tumor volume of mice in the PBS control group (PBS control) grew rapidly and the mice died soon. The tumor growth rate of mice using nano vaccine 1 (Nanovaccine 1), nano vaccine 2 (Nanovaccine 2), and nano vaccine 3 (Nanovaccine 3) was significantly slowed, the survival period was significantly prolonged, and most mice recovered without tumors. Moreover, the effect of nano vaccine 1 was comparable to that of nano vaccine 2, and the effects of nano vaccine 1 and nano vaccine 2 were better than nano vaccine 3, indicating that the antigen components in the cell lysate obtained by separation and purification using the alcohol precipitation method were comparable to the antigen components in the cell lysate obtained by separation and purification using the salting-out method, and were better than the cell lysate components that were not separated, purified, and re-dissolved.

[0269] In this embodiment, the cancer cells are irradiated with gamma rays before using a lysis solution containing a lytic agent to lyse the cancer cells and dissolve the cancer cell lysate components. In actual applications, one or more of different irradiation methods such as electron beams, X-rays, alpha rays, beta rays, ultraviolet rays, microwaves, and radioactive sources can also be used for a period of time; or the cancer cells can be lysed and the cancer cell lysate components can be dissolved directly using a lysis solution containing a lytic agent without irradiation treatment.

[0270] In this embodiment, cultured cancer cell lines were used to prepare antigen components. In practical applications, cancer cells or tumor tissues obtained by methods such as tumor tissue, cancer cells isolated from tumor tissue and cultured and amplified, and circulating tumor cells isolated from blood or peripheral blood can also be used as sources of antigen components.

[0271] Example 3: Micro-vaccine loaded with antigen components for the prevention of brain cancer

[0272] (1) Preparation of antigen components

[0273] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 6 GL261 cells, and when the tumors grew to a volume of approximately 1000 mm 3 Mice were sacrificed and tumor tissues were removed. Single-cell suspensions of tumor tissue were prepared, and the cells were then cultured in RPMI1640 (containing 10% FBS) complete medium for 7 days (37°C, 5% CO2). After the culture was completed, cancer cells were collected and centrifuged at 400g for 5 minutes to remove the medium. The precipitated cancer cells were lysed using an appropriate amount of Trizol reagent (containing guanidine isothiocyanate and phenol), and chloroform was added to produce a second (organic) phase. DNA and protein were extracted into the organic phase, while RNA remained in the aqueous supernatant. First, the upper aqueous phase was aspirated, half the volume of isopropanol was added, and the mixture was allowed to stand at room temperature for 15 minutes, followed by centrifugation at 12,000 g for 10 minutes. The supernatant was discarded and the precipitate was collected. The precipitate was then washed with an appropriate amount of 75% ethanol. After standing for 10 minutes, the mixture was centrifuged at 12,000 g for 5 minutes. The supernatant was discarded and the precipitated RNA was collected. The precipitated RNA was dissolved in 6M guanidine hydrochloride. The middle and lower organic phases were collected, and 2 volumes of isopropanol were added to precipitate the protein and DNA therein. The supernatant was discarded and the precipitate was centrifuged at 5,000 g for 10 minutes. The precipitate was collected and dissolved in 6M guanidine hydrochloride. The RNA dissolved in 6M guanidine hydrochloride was combined with the protein and DNA dissolved in 6M guanidine hydrochloride to obtain antigen component 1.

[0274] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 6 GL261 cells, and when the tumors grew to a volume of approximately 1000 mm 3The mice were killed and the tumor tissue was removed. A single cell suspension of the tumor tissue was prepared, and the cells were then cultured in RPMI1640 (containing 10% FBS) complete medium for 7 days (37°C, 5% CO2). After the culture was completed, the cancer cells were collected and centrifuged at 400g for 5 minutes to remove the medium. The precipitated cancer cells were lysed using an appropriate amount of 6M guanidine hydrochloride aqueous solution, and the lysate components were then dissolved using a 6M guanidine hydrochloride aqueous solution to obtain antigen component 2.

[0275] (2) Preparation of micronized vaccines

[0276] In this example, micron vaccine 1 (Micronvaccine 1) was prepared using the solvent evaporation method, a double emulsion method. The micron particle preparation material used, PLA, has a molecular weight of 30-50 kDa. The immune adjuvants used were poly(I:C), CpG 1018, and CpG 2395. The positively charged substance used was the RALA polypeptide (N-WEARLARALARALARHLARALARALRACEA-C). The preparation method is as described above. During the preparation process, the antigen component 1 and adjuvant are first loaded into the micron particles. Then, 100 mg of micron particles 1 are centrifuged at 8000 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 micronized particles 1 is about 2.0 μm. Each 1 mg of PLA micronized particles 1 is loaded with about 20 μg of protein or polypeptide components, 0.012 mg of poly(I:C), 0.005 mg each of CpG 1018 and CpG2395, and 0.1 mg of RALA polypeptide.

[0277] In this example, micron vaccine 2 (Micronvaccine 2) was prepared using the double emulsion method within the solvent evaporation method. The PLA used as the microparticle preparation material has a molecular weight of 30-50 kDa. The immunoadjuvants used were poly(I:C), CpG 1018, and CpG 2395. The positively charged substance used was the RALA polypeptide. The preparation method is as described above. During the preparation process, the antigen component 2 and adjuvant were first loaded into the microparticles. Then, 100 mg of microparticles 2 were centrifuged at 8000 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 microparticles 2 was approximately 2.0 μm. Each 1 mg of PLA microparticles 2 loaded approximately 20 μg of protein or polypeptide component, 0.012 mg of poly(I:C), 0.005 mg each of CpG 1018 and CpG 2395, and 0.1 mg of RALA polypeptide.

[0278] (3) Micron vaccines for cancer prevention

[0279] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare glioma-bearing mice. 6 mg of micron vaccine 1 or 6 mg of micron vaccine 2 or 100 μL PBS were injected subcutaneously on days -35, -28, -21, -14, and -7 before tumor inoculation. On day 0, 1.5×10 6 The methods for monitoring the tumor growth rate and survival of mice were the same as above.

[0280] (4) Experimental results

[0281] As shown in Figure 5, tumors in mice in the PBS group grew rapidly and the mice died soon after. Mice treated with Micronvaccine 1 and Micronvaccine 2 experienced significantly slower tumor growth, significantly prolonged survival, and the majority of mice remained tumor-free. Micronvaccine 1 was more effective than Micronvaccine 2, demonstrating that loading the micron vaccine with antigen components purified by precipitation with phenolic and alcoholic organic solvents is more effective than directly loading the micron vaccine with cancer cell lysates.

[0282] Example 4 Nano-vaccine for the treatment of liver cancer

[0283] (1) Preparation of antigen components

[0284] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 Hepa 1-6 liver cancer cells, when the tumor grows to a volume of about 1000mm 3 Mice were killed and tumor tissue was removed. The tumor tissue was cut into pieces and fixed in a 75% ethanol aqueous solution containing 1% hydrogen peroxide (hydrogen peroxide) for 2 hours. The pieces were then centrifuged at 1000g for 10 minutes. The supernatant of the tissue fixative was discarded and the tumor tissue was lysed with an appropriate amount of 4M guanidine isothiocyanate and 0.5% phenol aqueous solution. The tumor tissue lysate component was dissolved with a 4M guanidine isothiocyanate and 0.5% phenol aqueous solution, and then precipitated with 1 volume of isopropanol. The mixture was allowed to stand for 3 hours to allow the precipitate to precipitate. The mixture was then centrifuged at 3500RPM for 10 minutes, and the precipitate was redissolved with an 8M urea aqueous solution; this was used as the antigen component 1 for preparing nanovaccine 1.

[0285] (2) Preparation of nanovaccines

[0286] In the present embodiment, nano vaccine 1 (Nanovaccine 1) is prepared by the multiple emulsion method in the solvent evaporation method. The nano vaccine preparation material PLGA molecular weight adopted is 10KDa-20KDa, and the PEG2000-PLGA molecular weight is 15KDa-25KDa. The mass ratio of PLGA and PEG5000-PLGA used is 98.4:1.6. The immune adjuvant adopted is poly (I: C), CpG 2395 and CpG SL03. The preparation method is as described above. During the preparation, the cell antigen component 1 and adjuvant are first loaded inside the nanoparticles, and then 100mg of nanoparticles 1 are centrifuged at 12000g for 30 minutes and lyophilized for 48h after being resuspended in 10mL of ultrapure water containing 4% trehalose. The nanoparticles 1 have an average particle size of approximately 300 nm. Each 1 mg of PLGA nanoparticles 1 is loaded with approximately 500 μg of protein or peptide components, and 0.1 mg each of poly(I:C), CpG 2395, and CpG SL03. The PLGA nanovaccine 1 loaded with the antigen component and adjuvant is then irradiated with gamma rays for 12 hours to obtain the irradiated nanovaccine 1.

[0287] (3) Nano-vaccines for cancer treatment

[0288] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare liver cancer-bearing mice. On day 0, 1.5×10 6 Hepa 1-6 liver cancer cells were inoculated. Mice were subcutaneously injected with 100 μL of 1 mg of nanovaccine 1 or 100 μL of PBS on days 3, 6, 9, 14, 19, and 25 after tumor inoculation. Tumor growth and survival were monitored as described above.

[0289] (4) Experimental results

[0290] As shown in Figure 6, the tumors in the PBS group grew rapidly and the mice died soon after. The survival of the mice using Nanovaccine 1 was significantly prolonged, and the majority of the mice recovered without tumors.

[0291] In this embodiment, hydrogen peroxide (hydrogen peroxide) was used in the tissue fixative to oxidize the tumor tissue. In practical applications, hypochlorous acid, potassium permanganate, or other oxidants may also be used to oxidize cancer cells, tumor tissue, single-cell suspensions of tumor tissue, or antigen components in the aforementioned cell lysate fractions or tissue lysate fractions. Alternatively, in practical applications, reducing agents such as dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) may be used to reduce cancer cells, tumor tissue, single-cell suspensions of tumor tissue, or antigen components in the aforementioned cell lysate fractions or tissue lysate fractions.

[0292] Example 5 Antigen-presenting cell vaccine activated by nanoparticles loaded with antigen components for the treatment of colon cancer

[0293] (1) Preparation of antigen components

[0294] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 MC38 colon cancer cells, when the tumor grows to a volume of about 1000mm 3 The mice were sacrificed and the tumor tissue was removed. The tumor tissue was cut and fixed with a 50% ethanol aqueous solution (containing 0.1% hypochlorous acid) for 2 hours, then centrifuged at 2000 RPM for 5 minutes. After discarding the supernatant, the fixed tumor tissue pellet was lysed with an appropriate amount of 4M guanidine sulfate and 0.1M spermidine aqueous solution. The tumor tissue lysate component was dissolved with a 4M guanidine sulfate and 0.1M spermidine aqueous solution, and then 20 volumes of isopropanol were added and allowed to stand for 3 hours to precipitate. The pellet was then centrifuged at 3500 RPM for 10 minutes, and the pellet was redissolved with a 4M guanidine sulfate and 0.1M spermidine aqueous solution to obtain antigen component 1.

[0295] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 MC38 colon cancer cells, when the tumor grows to a volume of about 1000mm 3 The mice were killed and the tumor tissues were removed. The tumor tissues were cut into pieces and fixed with a 50% ethanol aqueous solution (containing 0.1% hypochlorous acid) for 2 hours, and then centrifuged at 2000RPM for 5 minutes. After discarding the supernatant, the fixed tumor tissue pellet was lysed with an appropriate amount of 4M guanidine sulfate and 0.1M spermidine aqueous solution, and the lysate component of the tumor tissue was dissolved with a 4M guanidine sulfate and 0.1M spermidine aqueous solution, which was the antigen component 2.

[0296] (2) Preparation of antigen delivery nanoparticles loaded with antigen components

[0297] In this example, nanoparticles 1 were prepared using the double emulsion method within the solvent evaporation method. The PLGA material used for nanoparticle preparation had a molecular weight of 10 kDa to 20 kDa. The immunoadjuvants employed were poly(I:C), CpG SL01, and CpG SL03. The preparation method was as described above. During preparation, the nanoparticles were first loaded with the cell antigen component 1 and the adjuvant. Then, 100 mg of nanoparticles 1 were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 6% trehalose, and freeze-dried for 48 hours, followed by electron beam irradiation for 2 hours. The nanoparticles 1 had an average particle size of approximately 280 nm. Each 1 mg of PLGA nanoparticles 1 was loaded with approximately 2.0 mg of the protein or polypeptide component, and 0.02 mg each of poly(I:C), CpG SL01, and CpG SL03.

[0298] Nanoparticle 2 in this example was prepared using the same methods and materials as Nanoparticle 1. The nanoparticles were first loaded with the cell antigen component 2 and adjuvant. 100 mg of nanoparticle 2 was then centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 6% trehalose, and freeze-dried for 48 hours before electron beam irradiation for 2 hours. The average particle size of the nanoparticles was approximately 230 nm. Each 1 mg of PLGA nanoparticle 2 loaded approximately 2.0 mg of protein or peptide component, with 0.02 mg each of poly(I:C), CpG SL01, and CpG SL03 loaded.

[0299] (3) In vitro activation of antigen-presenting cells (dendritic cells + B cells) using antigen delivery particles

[0300] This example illustrates how to prepare DCs from mouse bone marrow cells. First, 6-8 week-old C57BL / 6 mice were sacrificed by cervical dislocation. The tibia and femur of the hind legs were surgically removed and placed in PBS. The surrounding muscle tissue was removed using scissors and forceps. The ends of the bone were cut with scissors. PBS solution was then drawn with a syringe. A needle was inserted into the bone marrow cavity at each end of the bone, and the bone marrow was repeatedly flushed into a culture dish. The bone marrow solution was collected and centrifuged at 400g for 3 minutes. After that, 1 mL of red blood cell lysate was added to lyse the red blood cells. Lysis was terminated by adding 3 mL of RPMI 1640 (10% FBS) medium, centrifuged at 400g for 3 minutes, and the supernatant discarded. The cells were cultured in 10 mm culture dishes using complete RPMI 1640 (10% FBS) medium supplemented with recombinant mouse GM-CSF (20 ng / mL) and incubated at 37°C, 5% CO₂ for 7 days. On the 3rd day, the culture flask was gently shaken and the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng / mL) was added. On the 6th day, half of the medium was replaced. On the 7th day, a small amount of suspended and semi-adherent cells were collected and detected by flow cytometry. + CD80 + CD11c + When the ratio of BMDC in cells is between 15-20%, the induced cultured BMDC can be used for the next experiment.

[0301] B cells were derived from mouse spleen cells. The mice were first sacrificed, and then a single cell suspension of mouse spleen cells was prepared. Then, CD19 was isolated using magnetic bead sorting. + of B cells.

[0302] BMDCs (10 million) and B cells (10 million) were mixed in a 1:1 ratio and incubated with 2 mg of nanoparticles (nanoparticle 1 or nanoparticle 2) in 10 mL of RPMI1640 complete culture medium containing 20 ng / mL interleukin 15 (IL-15) for 24 hours (37°C, 5% CO2). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove free nanoparticles in the system, and then the mixed cells activated by the antigen delivery particles were used as cancer vaccines. Among them, the mixed cells activated by nanoparticle 1 are mixed cell cancer vaccine 1 (Cell vaccine 1); the mixed cells activated by nanoparticle 2 are mixed cell cancer vaccine 2 (Cell vaccine 2).

[0303] (4) DC vaccine for cancer treatment

[0304] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 6 MC38 colon cancer cells were inoculated. Mice were subcutaneously injected with 1 million cells (cell vaccine 1 or cell vaccine 2) or 100 μL of PBS on days 3, 6, 9, 14, 19, and 25 after tumor inoculation. Tumor growth and survival were monitored as described above.

[0305] (4) Experimental results

[0306] As shown in Figure 7, the tumors in the PBS group grew rapidly and the mice died soon after. The survival of mice treated with Cell Vaccine 1 and Cell Vaccine 2 was significantly prolonged, with most mice recovering without tumors. Cell Vaccine 1 was more effective than Cell Vaccine 2.

[0307] In this embodiment, before using a lytic solution containing a dissolving agent to lyse the tumor tissue and dissolve the tumor tissue lysate components, the tumor tissue is first fixed using a 50% ethanol aqueous solution containing an oxidant. In actual applications, other fixatives can also be used to fix tumor tissue or cancer cells. Useful fixatives include, but are not limited to, one or more of acetone, acetic acid, propionic acid, butyric acid, formic acid, aldehyde fixatives, mercury fixatives, alcohol fixatives, oxidant fixatives, picrate fixatives, other cell or tissue fixatives or fixatives, and diethylene oxide.

[0308] Example 6: Nanoparticles used to detect cancer cell-specific T cells (tumor antigen-specific T cells) in immune cells

[0309] (1) Preparation of antigen components

[0310] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 were cultured separately. A549, H1299, PC9, and H1437 cells belong to the lung adenocarcinoma subtype, while H226, HCC1588, H2170, and H520 cells belong to the lung squamous cell carcinoma subtype. The eight cell lines were collected separately, and then the A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 cells were mixed at a cell number ratio of 1:1:1:1:1:1:1:1. The cells were then resuspended and lysed in 3 mL of an 8M urea peroxide aqueous solution, and the lysate components were dissolved in 5 mL of an 8M urea peroxide aqueous solution. Then, 20 mL of acetone was added to the dissolved lysate component, and after standing for 4 hours, the mixture was centrifuged at 5000 RPM for 5 minutes. After discarding the supernatant, the precipitate was redissolved using 8 M urea peroxide aqueous solution to obtain antigen component 1.

[0311] (2) Preparation of Nanoparticles Loaded with Antigen Components

[0312] In this embodiment, nanoparticle 1 (NP1) was prepared using a double emulsion method within a solvent evaporation method. The PLGA material used for nanoparticle preparation has a molecular weight of 10KDa-30KDa. The preparation method is as described above. During the preparation process, the antigen component 1 is first loaded into the nanoparticles using the double emulsion method. Then, 100mg of the nanoparticles are centrifuged at 15000g for 30 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 200nm, and each 1mg of PLGA nanoparticles is loaded with approximately 500μg of protein and polypeptide components.

[0313] (3) Detection of cancer cell-specific T cells (tumor-specific T cells)

[0314] Patient A, a non-small cell lung cancer patient, experienced significant tumor reduction after immunotherapy with a PD-1 antibody. 8 mL of peripheral blood was drawn from patient A before and three weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from this 8 mL of blood using gradient centrifugation.

[0315] Nanoparticles 1 (1.5 mg) were co-incubated with PBMC (3 million) in 3 mL of AIM V serum-free medium for 24 hours (37°C, 5% CO2); or the control group was co-incubated with PBMC (3 million) in 3 mL of AIM V serum-free medium for 24 hours (37°C, 5% CO2). After that, the sample was centrifuged at 400g for 5 minutes, the supernatant was discarded, the cell pellet was collected, and the cells were resuspended in PBS. Live and dead cell dyes and Fc block were first used to incubate the cells to mark live cells and avoid non-specific antibody adsorption, and then CD3 antibody and IFN-γ antibody were used for staining. The stained cells were then analyzed by flow cytometry to analyze CD3 + IFN-γ + T cells in all CD3 + The proportion of T cells is cancer cell-specific T cells.

[0316] (4) Experimental results

[0317] As shown in Figure 8, when PBMCs were incubated alone, almost no activated cancer cell-killing T cells were detected before and after treatment. However, when cells were co-incubated with Nanoparticle 1, a certain amount of cancer cell-specific T cells were detected before and after treatment, and the amount after treatment was significantly higher than before treatment.

[0318] 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 the present invention. 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 the present invention.

Claims

1. A method for extracting antigen components, characterized in that: The method comprises the following steps: (1) lysing cells and / or tissues to obtain cell lysate components; (2) dissolving the lysate components after lysing in step (1) using water and / or a first dissolving solution containing a dissolving agent; (3) mixing the solution containing the lysate component obtained in step (2) with an organic solvent to obtain the antigen component; Wherein, the organic solvent includes one or more of alcohols, ketones, phenols, nitriles and acids.

2. The method according to claim 1, characterized in that After the mixing in step (3), centrifugation is performed, and the lower layer is taken out and mixed with a second dissolving solution containing a dissolving agent to obtain the antigen component; or, after the mixing in step (3), centrifugation is performed, the lower layer is taken out and mixed with the second dissolving solution containing a dissolving agent, and then mixed with the component obtained by separating and purifying the supernatant after the centrifugation to obtain the antigen component.

3. The method according to claim 1 or 2, characterized in that: The organic solvent is selected from one or more of ethanol, methanol, isopropanol, propanol, butanol, butyric acid, propionic acid, acetic acid, formic acid, acetone, phenol and acetonitrile, preferably one or more of ethanol, isopropanol, acetone and phenol; Preferably, the volume ratio of the solution containing the lysate components to the organic solvent is 1:0.5-30, preferably 1:0.5-25, and more preferably 1:0.5-20.

4. The method according to claim 1, characterized in that The dissolving 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; wherein the structural formula 1 is as follows: R1 is C, S, P, N or O, R2-R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl; Preferably, the dissolving agent is selected from one or more of metformin hydrochloride, guanidine isothiocyanate, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, spermine, spermidine and choline; more preferably, one or more of urea, urea peroxide, guanidine isothiocyanate, guanidine sulfate, guanidine hydrochloride and spermidine.

5. The method according to claim 1, characterized in that The method further comprises treating the cells and / or tissues before and / or after lysis of the cells and / or tissues, Preferably, the treatment comprises enhancing the immunogenicity of the antigenic component and / or fixing the cells and / or tissues; Preferably, methods for enhancing the immunogenicity of the antigenic component include irradiation, oxidation, reduction, modification with haptenic substances, fixation, enzyme treatment, denaturation, heating, mineralization; Preferably, the irradiation includes any commonly used irradiation method; more preferably, the irradiation includes one or more of radioactive material irradiation, electron beam irradiation, microwave irradiation, ultraviolet irradiation, X-ray irradiation, α-ray irradiation, β-ray irradiation, and γ-ray irradiation; Preferably, the oxidation is oxidation of the antigen component using an oxidant; more preferably, the oxidant comprises hypochlorous acid, hydrogen peroxide, persulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO3, KBrO3, ClO3 - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, NO3 - 、MnO4 - , one or more of F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3; Preferably, the hapten substance comprises one or more of 2,4-dinitrofluorobenzene, 2,4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, Ovalbumin, N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other hapten substances containing aldehyde groups, rhamnose, galactose, and aminogalactose; Preferably, one or more of formaldehyde, paraformaldehyde, glutaraldehyde, other substances containing aldehyde groups, ethanol, methanol, acetone, acetic acid, propionic acid, butyric acid, formic acid, formalin, dichromate, potassium permanganate, chromic acid, picric acid, Zamboni fixative, PLP fixative, FPA fixative, TAF fixative, Rossman fixative, Regaud fixative, PLPD fixative, PAPG fixative, Orth fixative, Muller fixative, McDoWell fixative, neutral calcium formaldehyde fixative, FAB fixative, Carnoy fixative, Clarke fixative, B-5 ​​fixative, Bouin fixative, FineFIX fixative, AGM fixative, Helly fixative, Zenker fixative, Kolmer fixative, AAF fixative, Hollande fixative, Gendre fixative, aldehyde fixative, mercury fixative, alcohol fixative, oxidant fixative, picrate fixative and diethylene oxide are selected for the fixation.

6. A delivery particle loaded with an antigen component prepared by the method according to any one of claims 1 to 5.

7. The delivery particle according to claim 6, characterized in that The delivery particle also has the following components: (i) the skeleton structure formed by the particle material; (ii) immune adjuvants; (iii) positively charged substances; Preferably, the immunoadjuvant is selected from the group consisting of pattern recognition receptor agonists, Toll-like receptor agonists, Bacillus Calmette-Guérin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG cell wall acyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhancing reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn calf liver active peptide, imiquimod, polysaccharides, curcumin, immunoadjuvant CpG, immunoadjuvant poly(I:C), immunoadjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, 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, one or more of ginseng effective ingredients and astragalus effective ingredients; preferably one or more selected from Toll-like receptor 3 agonists and Toll-like receptor 9 agonists; more preferably one or more selected from Poly (I: C), Poly ICLC, A-type CpG-OND, B-type CpG-OND and C-type CpG-OND; Preferably, the antigen component is loaded inside and / or on the surface of the backbone structure; Preferably, the positively charged substance is selected from one or more of positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and / or positively charged inorganic substances; preferably selected from one or more of bee venom peptide, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

8. The delivery particle according to claim 6 or 7, characterized in that The delivery particles are nano-delivery particles and / or micro-delivery particles; The particle size of the nano delivery particles is 1 nm-1000 nm, preferably 50-500 nm, more preferably 100-400 nm; Preferably, the particle size of the micron delivery particles is 1 μm-1000 μm, preferably 1-10 μm, more preferably 1-5 μm; Preferably, the amount of the protein or polypeptide component in the antigen component loaded by 1 mg of the nano and / or micro delivery particles is 0.01-3 mg, preferably 0.02-2 mg; Preferably, 1 mg of the nano and / or micro delivery particles is loaded with 0.001-2 mg of the immune adjuvant, preferably 0.002-0.8 mg.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antigen component prepared by the method according to any one of claims 1 to 5 or the delivery particle according to any one of claims 6 to 8; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

10. Use of an antigen component prepared by the method according to any one of claims 1 to 5, or a delivery particle according to any one of claims 6 to 8, or a pharmaceutical composition according to claim 9 in at least one of the following (1) to (4): (1) Preparation of drugs for preventing or treating diseases; (2) Used to activate antigen-presenting cells and prepare cell vaccines based on antigen-presenting cells; (3) Assist in activating antigen-specific T cells and detect the content of antigen-specific T cells; (4) After assisting in the activation of antigen-specific T cells, the activated antigen-specific T cells are isolated and amplified, and used to prevent or treat diseases.

11. The use according to claim 10, characterized in that The delivery particles are used directly as vaccines; Preferably, after the delivery particles activate dendritic cells and / or B cells, the activated dendritic cells and / or B cells are used as cell vaccines; Preferably, the delivery particles are used to detect the content of cancer antigen-specific T cells after assisting in activating cancer cell-specific T cells in vitro, or the delivery particles are used to separate and / or amplify the activated antigen-specific T cells after assisting in activating antigen-specific T cells for disease prevention and treatment.

12. The use according to claim 10 or 11, characterized in that The disease is cancer or a tumor; preferably, the disease is a solid tumor or a blood tumor.

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