CN117653720A

CN117653720A - Extraction method of antigen component, delivery particle containing antigen component and application of delivery particle

Info

Publication number: CN117653720A
Application number: CN202311600143.1A
Authority: CN
Inventors: 刘密
Original assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Current assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Priority date: 2023-11-28
Filing date: 2023-11-28
Publication date: 2024-03-08

Abstract

The invention relates to an extraction method of an antigen component, delivery particles comprising the antigen component and application of the antigen component, wherein the method comprises the following steps of: (1) Lysing the cells and/or tissue to obtain a cell lysate component; (2) Dissolving the lysate component after the lysing of step (1) with water and/or a first dissolution liquid comprising a lysing 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 comprises one or more of alcohols, ketones, phenols, nitriles or acids. The preparation method is simple and quick, consumes little, has low solution viscosity during centrifugation, and does not need high-speed centrifugation to obtain the antigen component. Meanwhile, the antigen component obtained by the invention has higher immunogenicity.

Description

Extraction method of antigen component, delivery particle containing antigen component and application of delivery particle

Technical Field

The invention relates to the field of immunotherapy, in particular to an extraction method of an antigen component, a delivery particle containing the antigen component and application of the delivery particle.

Background

Cancer vaccines are one of the important approaches for immunotherapy of diseases such as cancer. Major factors affecting cancer vaccines include antigens, adjuvants, and delivery dosage forms. Taking cancer vaccine as an example, the major factors that affect cancer vaccine the greatest include tumor antigen, adjuvant and delivery formulation. Wherein, the antigen can induce and activate specific immune response, the adjuvant can amplify specific immune response, and the dosage form can influence the phagocytosis of antigen-presenting cells (APCs) on vaccine and the subsequent efficiency of activating antigen-specific T cells. Among the three, the antigen is the main component capable of eliciting a specific immune response and is thus the most critical factor. The cancer cells and/or tumor tissue contains all the cancer cell specific antigens and cancer cell related antigens, and is the best tumor antigen delivery particle or raw material for preparing cancer vaccine. However, the unpurified whole cell fraction contains other substances such as DNA and saccharides, thereby reducing the content of tumor antigen components such as proteins and polypeptides. When the high-concentration salt substance is used for salting out and the lysate containing the solvent is used, the system contains the high-concentration salt and the solvent at the same time, so that the viscosity of the solution is very high, the precipitate which is salted out can be centrifuged out only by using a relatively high rotating speed during centrifugation, and the requirements on the centrifugal rotating speed and a centrifugal instrument are higher.

Disclosure of Invention

Problems to be solved by the invention

The invention aims to provide a simple and convenient method for separating and purifying antigen components. The separation and purification method is simple and easy to implement, does not need to use salt-containing substances for salting out, and does not have the potential trouble that the salt-containing substances are required to be subjected to centrifugal precipitation at a very high centrifugal speed due to high viscosity of the salt-containing substances during salting out. Meanwhile, the antigen component obtained by the method provided by the invention has strong immunogenicity and achieves unexpected effects.

Solution for solving the problem

In a first aspect, the present invention provides a method for extracting an antigen component, wherein the method comprises the steps of:

(1) Lysing the cells and/or tissue to obtain a cell lysate component;

(2) Dissolving the lysate component after the lysing of step (1) with water and/or with a first dissolution solution;

(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 and acids.

Preferably, after said mixing in step (3), centrifuging, and mixing the lower layer with a second dissolution solution containing a dissolution agent to obtain said antigen component; or, after the mixing in the step (3), centrifuging, mixing the lower layer with a second dissolving solution containing a dissolving agent, and then mixing with a component obtained by separating and purifying the supernatant after centrifuging to obtain the antigen component.

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.

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

Preferably, the volume ratio of the lysate component-containing solution to the organic solvent is 1:0.5-30.

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

Still more preferably, the volume ratio of the lysate component-containing solution to the organic solvent is 1:0.5-20.

Preferably, the lytic agent is independently selected from one or more of a compound comprising the 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:

R ₁ c, S, P, N or O, R ₂ -R ₅ Independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino.

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

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

Preferably, the method further comprises treating the cells and/or tissue before and/or after lysis.

Further preferably, the treatment comprises enhancing the immunogenicity of the antigen component and/or immobilizing the cells and/or tissue.

Still more preferably, the method of enhancing the immunogenicity of the antigenic component comprises irradiation, oxidation, reduction, modification with hapten materials, immobilization, enzymatic treatment, denaturation, heating, mineralization.

Still further preferably, the irradiation includes any commonly used irradiation method; more preferably, the irradiation comprises one or more of radioactive substance irradiation, electron beam irradiation, microwave irradiation, ultraviolet irradiation, X-ray irradiation, alpha-ray irradiation, beta-ray irradiation, gamma-ray irradiation.

Still further preferably, the oxidizing is oxidizing the antigenic component with an oxidizing agent; more preferably, the oxidizing agent comprises hypochlorous acid, hydrogen peroxide, persulfates, dichromates, peroxyacetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate Sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO ₃ 、KBrO ₃ 、ClO ₃ ^- 、ClO ₄ ^- 、Na ₂ O ₂ 、K ₂ O ₂ 、MgO ₂ 、CaO ₂ 、BaO ₂ 、NO ₃ ^- 、MnO ₄ ^- 、F ₂ 、Cl ₂ 、O ₂ 、Br ₂ 、I ₂ 、S、Si、HNO ₃ 、MnO ₂ 、FeCl ₃ One or more of the following.

Still further preferably, the hapten substance comprises one or more of 2, 4-dinitrofluorobenzene, 2, 4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, ovabumin, N-iodoacetyl-N' - (5-sulfo-1-naphthyl) ethylene diamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other aldehyde group-containing hapten substances, rhamnose, galactose, and galactosamine.

Still more 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 fixing solution, PLP fixing solution, FPA fixing solution, TAF fixing solution, rosman fixing solution, regaud fixing solution, PLPD fixing solution, PAPG fixing solution, orth fixing solution, muller fixing solution, mcDoWell fixing solution, neutral calcium formaldehyde fixing solution, FAB fixing solution, carnoy fixing solution, clarke fixing solution, B-5 fixing solution, bouin fixing solution, fineFIX fixing solution, a.g. m fixing solution, helly fixing solution, zenker fixing solution, kolmer fixing solution, AAF fixing solution, hollande fixing solution, gendre fixing solution, aldehyde fixing solution, mercury fixing solution, alcohol fixing solution, oxidizing agent fixing solution, picrate fixing solution, and ethylene oxide fixing solution are used.

In a second aspect, the present invention provides a delivery particle loaded with an antigen component prepared by the method of the first aspect.

Preferably, the delivery particles also have the following composition:

(i) A framework structure formed by the particle materials;

(ii) An immunoadjuvant;

(iii) Positively charged species.

Further preferably, the immune adjuvant is selected from one or more of a pattern recognition receptor class agonist, a Toll-like receptor agonist, BCG cell wall skeleton, BCG methanol extraction residue, BCG cell wall dipeptide, mycobacterium phlei, polyoxin a, mineral oil, virus-like particles, immunopotentiating reconstituted influenza virus minibodies, cholera enterotoxin, saponins and derivatives thereof, resiquimod, thymosin, nascent bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly (I: C), immune adjuvant poly ICLC, short coryneform vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukins, interferons, polymyonic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokines, mRNA, MF59, double stranded RNA, double stranded DNA, aluminum adjuvant, manganese adjuvant, calcium adjuvant, STING agonist, endotoxin, astragalus, single stranded RNA, CAF01, and an active ingredient of ginseng; preferably one or more selected from Toll-like receptor 3 agonists and Toll-like receptor 9 agonists; more preferably one or more selected from the group consisting of Poly (I: C), poly ICLC, A class CpG-OND, B class CpG-OND and C class CpG-OND.

Further preferably, the antigenic component is supported within and/or on the surface of the backbone structure.

Further preferably, the positively charged substance is selected from one or more of a positively charged amino acid, a positively charged polypeptide, a positively charged lipid, a positively charged protein, a positively charged polymer and a positively charged mineral.

Even more preferably selected from the group consisting of melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH ₄ HCO ₃ One or more of the following.

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

Preferably, the nano-delivery particles have a particle size of 1nm to 1000nm, preferably 50 to 500nm, more preferably 100 to 400nm.

Preferably, the micrometer delivery particles have a particle size of 1 μm to 1000 μm, preferably 1 to 10 μm, more preferably 1 to 5 μm.

Preferably, 1mg of the nano-and/or micro-delivery particles load the protein or polypeptide component in the antigen component in an amount of 0.01-3mg, preferably 0.02-2mg.

Preferably, 1mg of the nano-and/or micro-delivery particles are loaded with 0.001-2mg of the immunoadjuvant, preferably 0.002-0.8mg.

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

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

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

Preferably, the antigen component is isolated and purified from the lysate component dissolved after lysis with a lysis solution containing a lysing agent using a suitable method.

In a third aspect, the present invention provides a pharmaceutical composition comprising an antigenic component prepared by the method of the first aspect or a delivery particle of the second aspect.

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

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

(1) Preparing a medicament for preventing or treating a disease;

(2) For activating antigen presenting cells, preparing a cell vaccine based on the antigen presenting cells;

(3) Auxiliary activation of antigen-specific T cells and detection of antigen-specific T cell content;

(4) After helper activation of antigen-specific T cells, activated antigen-specific T cells are isolated and expanded for use in preventing or treating a disease.

Preferably, the antigen component obtained by separation and purification in the lysate component can be irradiated before or after the lysis of the cells or tumor tissues, or can be irradiated after the separated and purified antigen component is loaded on the nano-particles or the micro-particles.

Preferably, the delivery particles are used directly as a vaccine.

Preferably, the delivery particles activate dendritic cells and/or B cells, and the activated Dendritic Cells (DCs) and/or B cells are used as a cellular vaccine.

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

Preferably, the disease is cancer or tumor.

Further preferably, the disease is a solid tumor or a hematological tumor.

ADVANTAGEOUS EFFECTS OF INVENTION

The method can enrich antigen components such as protein, polypeptide, RNA and the like by separating and purifying the antigen components in cells (such as cancer cells) and/or tissues (such as tumor tissues) by using an organic solvent precipitation method, and avoids potential toxic and side effects caused by substances such as saccharides and the like. The antigen substances such as protein, polypeptide and the like can be dissolved after being solubilized in the aqueous solution, and the antigen substances such as protein, polypeptide and the like can be separated out after being added with the organic solvents such as ethanol, acetone and the like, so that the aim of separating the antigen substances such as protein, polypeptide and the like is fulfilled, and meanwhile, the immunogenicity of the antigen component obtained by the method is higher. The method is simple and rapid, and does not need to add substances such as high-concentration salts and the like for salting out.

When the method for adding the organic solvent for precipitation is adopted for separation and purification, the steps are simple and convenient, no additional reagent is needed, and high rotating speed is not needed during centrifugation, so that the purification operation and process are simpler, faster and more convenient.

Drawings

FIG. 1 is a schematic representation of a portion of a possible preparation process and application of the invention for purifying an antigen component in a cell or tissue lysate.

FIG. 2 is a structure of formula 1, wherein R ₁ C, S, P, N or O, R ₂ -R ₅ Independently selected from any other group such as hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino, and the like.

FIGS. 3-7 are experimental results of tumor growth rate and survival time in examples 1-5 using nano-and/or micro-vaccine loaded with purified antigen components or Antigen Presenting Cell (APC) vaccine or assisted sorting expanded T cells to prevent or treat cancer; wherein a is the experimental result of the tumor growth rate (n is more than or equal to 8) when preventing or treating cancer; b is the result of a mice survival experiment (n.gtoreq.8) when cancer is prevented or treated, and each data point is the mean.+ -. Standard error (mean.+ -. SEM); wherein, the significant difference of the tumor growth inhibition experiment in the a graph is analyzed by an ANOVA method, and the significant difference in the b graph is analyzed by Kaplan-Meier and log-rank test.

FIG. 8 is the experimental results of detection of tumor antigen specific T cells using antigen delivery nanoparticles in example 6.

In the above figures, p < 0.005 compared to the PBS control group is shown to have significant differences; # # # represents a significant difference between the two groups compared with p < 0.005; # represents that the two groups have significant differences compared with p < 0.01; # represents that the two groups have significant differences in comparison with p < 0.05.

Detailed Description

In order to make the technical scheme and the beneficial effects of the invention more obvious and understandable, the following detailed description is given by way of example. Unless defined otherwise, technical and scientific terms used herein have the same meaning as technical and scientific terms in the technical field to which this application belongs.

The experimental techniques and experimental methods used in this example are conventional techniques unless otherwise specified, and for example, the experimental methods in the following examples are not specified under the specific conditions, and are generally performed under the conventional conditions or under the conditions recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.

In the claims and/or the specification of the present invention, the terms "a" or "an" or "the" may refer to "one" but may also refer to "one or more", "at least one" and "one or more".

As used in the claims and specification, the words "comprise," "have," "include" or "contain" mean including or open-ended, and do not exclude additional, unrecited elements or method steps.

The term "suffering from a disease" means: the body develops symptoms of the disease.

The term "treatment" refers to: after suffering from the disease, the subject is contacted (e.g., dosed) with a delivery particle, delivery system, vaccine, antigen-loaded drug, pharmaceutical composition, thereby alleviating the symptoms of the disease compared to when not contacted, and does not mean that the symptoms of the disease must be completely inhibited.

The term "preventing" refers to: by contacting (e.g., administering) a subject with the delivery particles, delivery systems, vaccines, antigen-loaded drugs, pharmaceutical compositions of the present invention prior to the onset of disease, the symptoms after the onset of disease are reduced compared to when not contacted, and do not mean that complete inhibition of the disease is necessary.

The vaccine of the present invention may be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, milling, encapsulating, entrapping and/or lyophilizing processes.

In the present invention, the route of administration can be varied or adjusted in any suitable manner to meet the nature of the drug, the convenience of the patient and medical personnel, and other related factors.

The term "individual", "patient" or "subject" as used in the context of the present invention includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors. The term "tumor" refers to all neoplastic (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.

In the preparation of the antigen component, part of examples use tumor tissues or cancer cell lines, in practical application, the antigen component can be prepared by using tumor tissues, cancer cell lines, cancer cells obtained by culturing and amplifying cancer cells separated from the tumor tissues, cancer cells obtained by culturing and amplifying circulating tumor cells separated from peripheral blood and the like, and in practical application, any other feasible scheme can be used for obtaining the cancer cells; in practice, other cells or tissues may be used to prepare antigen components in other diseases, such as beta cells to prepare antigen components for type 1 diabetes and pancreatic or islet tissue to prepare antigen components for type 1 diabetes.

The cancer species in the embodiment of the invention is solid tumor, and the nano vaccine or the micro vaccine can be used for treating blood tumor, lymphoma and the like in practical application. Because the immune microenvironment of the hematological tumor and the lymphoma is not complicated by the solid tumor, the nano vaccine or the micro vaccine has better effect in the hematological tumor and the lymphoma.

The invention provides a method for preparing antigen delivery particles, which comprises the steps of firstly lysing cells or tissues, then solubilizing the lysed lysate by using a solubilizing solution containing a solubilizing agent, and then adding an organic solvent to precipitate antigen components such as solubilized proteins, polypeptides and the like in the solubilizing solution, thereby obtaining purified antigen components from the lysate components.

The isolated and purified antigen component is then loaded onto nanoparticles or microparticles for use as a vaccine or antigen delivery particle: can be used as vaccine for preventing or treating diseases; the antigen delivery particles can be used for preparing antigen presenting cell vaccines by activating antigen presenting cells in vitro, or detecting the content of antigen specific T cells after activating antigen specific T cells in an auxiliary way, or can be used for preventing or treating diseases after sorting and amplifying antigen specific T cells after activating antigen specific T cells in an auxiliary way.

(1) Lysing the cells and/or tissue to obtain a cell lysate component;

(2) Dissolving the lysate component after the lysing of step (1) with water and/or a first dissolution liquid comprising a lysing agent;

In certain embodiments, after the mixing in step (3), centrifuging, removing the lower layer and mixing with a second dissolution solution containing a dissolution agent to obtain the antigen component.

In certain embodiments, after the mixing in step (3), centrifuging, mixing the lower layer with a second solution containing a lytic agent, and then mixing with the fraction obtained by separating and purifying the supernatant after centrifuging to obtain the antigen fraction.

In certain embodiments, the first dissolution liquid is pure water or an aqueous solution.

In certain embodiments, the second dissolution solution is pure water or an aqueous solution.

In certain embodiments, a portion of the antigen component contained in the cell lysate component comprises protein and polypeptide components of the cell and/or tissue lysate and/or an RNA component or an mRNA component of the cell lysate.

In certain embodiments, a portion of the antigen component contained in the cell lysate component comprises protein and polypeptide components of the cell and/or tissue lysate and/or lipid components of the cell lysate.

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

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).

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

In certain embodiments, the preparation steps of the lysate in the cancer cells and/or tumor tissue are: the lysate is obtained by lysing cancer cells and/or tumor tissue using a lysing solution containing a lysing agent, and then lysing the lysate components using a lysing solution containing a lysing agent.

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.

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.

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

In certain embodiments, the volume ratio of the lysate component-containing solution to the organic solvent is 1:0.5-30, such as 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, and the like.

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

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

In certain embodiments, the lytic agent is independently selected from one or more of a compound comprising the 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:

In certain embodiments, the lytic agent is selected from one or more of metformin hydrochloride, guanidine isothiocyanate, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, urea, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzymes, polypeptides, amino acids, glycosides, spermine, spermidine, and choline.

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

In certain embodiments, the dissolution agent is selected from urea, metformin, guanidine hydrochloride and the like containing the structure of formula 1. The inventors found that the substance having the structure of formula 1 can be used as a lytic agent in a lysis solution to lyse water-insoluble components in cells or tumor tissues, and thus other compounds containing the structure have the ability to lyse water-insoluble components as lytic agents in addition to common compounds containing a guanidino structure such as urea, guanidine hydrochloride, metformin, and the like.

In certain embodiments, the antigen component of the cancer cell and/or tumor tissue lysate after isolation and purification may be one or both selected from the group consisting of: (1) a protein polypeptide component; (2) A protein polypeptide component and an RNA component (or an mRNA component); (3) lipid component.

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); preferably (0.1-2): (0.1-2).

In certain embodiments, the immunogenic proteins and/or polypeptides may be derived from a portion of the components and extracellular vesicle lysates in cancer cells/tumor tissue. Further, the extracellular vesicle lysate is selected from an extracellular vesicle lysate of a cancer cell and/or an extracellular vesicle lysate of a bacterium. The mass ratio of a part of the components in the cancer cells and/or tumor tissues to the extracellular vesicle lysate components is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Illustratively, 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.

In certain embodiments, the immunogenic proteins and/or polypeptides may be derived from a portion of the components and bacterial lysates in cancer cells/tumor tissue. Further, the mass ratio of a part of the components in the cancer cells/tumor tissue to the bacterial lysate is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Illustratively, 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.

In certain embodiments, the source of cells described herein is any method by which cells can be obtained. By way of example, the source of cancer cells in the present invention is any method by which cancer cells can be obtained, including but not limited to cancer cell lines, cancer cells obtained by in vitro expansion of cancer cells isolated from tumor tissue, cancer cells obtained by expansion of circulating tumor cells isolated from blood, or cancer cells differentiated from stem cells, etc.

In certain embodiments, the cells or tissues may be co-incubated with a specific chemical to stimulate the cells or tissues prior to lysis.

In certain embodiments, the cancer cells or tumor tissue may be co-incubated with a specific chemical to stimulate the cancer cells or tumor tissue prior to lysis.

In certain embodiments, the specific substance that stimulates cancer cells includes, but is not limited to, small molecule compounds (e.g., doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, cytokines, chemokines, plant extracts (e.g., important extracts of ginseng, plant rhizome extracts, etc.), interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of stimulating cancer cells or tumor tissue with a particular substance co-incubation with the cancer cells or tumor tissue is to allow the cancer cells to produce more antigenic components.

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

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

In certain embodiments, the protein and polypeptide components of the lysate component in the cancer cells and/or tumor tissue comprise an antigen component.

In certain embodiments, the methods further comprise treating the cells and/or tissue prior to and/or after lysis.

In certain embodiments, the treatment comprises enhancing the immunogenicity of the antigen component and/or immobilizing the cells and/or tissue.

In certain embodiments, the irradiation comprises any conventional irradiation method.

In certain embodiments, the irradiation comprises one or more of radioactive substance irradiation, electron beam irradiation, microwave irradiation, ultraviolet irradiation, X-ray irradiation, alpha-ray irradiation, beta-ray irradiation, gamma-ray irradiation.

In certain embodiments, the oxidizing is oxidizing the antigenic component using an oxidizing agent.

In certain embodiments, the oxidizing agent comprises hypochlorous acid, hydrogen peroxide, persulfates, dichromates, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO ₃ 、KBrO ₃ 、ClO ₃ ^- 、ClO ₄ ^- 、Na ₂ O ₂ 、K ₂ O ₂ 、MgO ₂ 、CaO ₂ 、BaO ₂ 、NO ₃ ^- 、MnO ₄ ^- 、F ₂ 、Cl ₂ 、O ₂ 、Br ₂ 、I ₂ 、S、Si、HNO ₃ 、MnO ₂ 、FeCl ₃ One or more of the following.

In certain embodiments, the hapten material is a material that increases the immunogenicity of a protein or polypeptide upon interaction with the protein or polypeptide.

In certain embodiments, the hapten material comprises one or more of 2, 4-dinitrofluorobenzene, 2, 4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, ovabumin, N-iodoacetyl-N' - (5-sulfonate 1-naphthyl) ethylene diamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other aldehyde group-containing hapten materials, rhamnose, galactose, and galactosamine.

In certain embodiments, the hapten material is used to interact with cells (e.g., cancer cells) or cells in a tissue (e.g., tumor tissue) for a time period to modify the antigen component of the cells or tissue and then lyse the cells and/or tissue to obtain a lysate thereof; or by first lysing cells (e.g., cancer cells) or tissue (e.g., tumor tissue) to obtain a lysate thereof, and then modifying the antigen component of the cell or tissue lysate with a hapten material.

In certain embodiments, the lysate fraction of cells (e.g., cancer cells) or tissues (e.g., tumor tissues) modified with hapten materials may be loaded directly as an antigen fraction into a nanovaccine or a minivaccine, or may be isolated and purified by appropriate treatment or further immunogenicity enhanced before being loaded as an antigen fraction into a nanovaccine or a minivaccine.

In certain embodiments, prior to lysing or co-acting hapten materials with cells (e.g., cancerous cells) or tissues (e.g., tumor tissue), the cells (e.g., cancerous cells) or tissues (e.g., tumor tissue) can be irradiated with radiation, including but not limited to gamma radiation, X-rays, electron beams, microwaves, beta radiation, alpha radiation, and the like, to inactivate the cells (e.g., cancerous cells) or tissues (e.g., tumor tissue).

In certain embodiments, the reduction is reduction of the antigen component using a reducing agent.

In certain embodiments, the reducing agent includes, but is not limited to, one or more of Dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), and the like.

In certain embodiments, the enzymatic treatment methods include, but are not limited to, use of one or more of nucleases, dnases, pepses, chymotrypsins, trypsin, other protein digestive enzymes, protease inhibitors, and the like. Enzymatic hydrolysis in the present invention includes, but is not limited to, any viable enzymatic hydrolysis method using nucleases, pepsin, trypsin, protease inhibitors, chymotrypsin, dnase, and the like.

In certain embodiments, the enzymatic treatment methods include, but are not limited to, use of one or more of nucleases, dnases, pepses, chymotrypsins, trypsin, other protein digestive enzymes, protease inhibitors, and the like.

In certain embodiments, enzymes for enzymatic hydrolysis include, but are not limited to, one or more of nucleases, pepsin, trypsin, protease inhibitors, chymotrypsin, dnase, and the like.

In certain embodiments, the mineralization includes, but is not limited to, any mineralization or biomineralization method using siliconization, calcification, magnesian, and the like.

In certain embodiments, the cancer cells and/or tumor tissue may be subjected to a treatment including, but not limited to, inactivation or (and) denaturation, immobilization, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, heating, salting-out, enzymatic treatment, oxidation, reduction, mineralization, irradiation, ionization, chemical modification, nucleic acid separation and purification, endoprotease or degradation, nuclease treatment, and the like, prior to or after lysis, and the lysate fraction is lysed by the use of a lysis solution containing a lytic agent prior to extraction and separation of the protein and polypeptide fractions therefrom; the protein and polypeptide components may also be separated by direct use of a lysing solution containing a lysing agent to lyse the lysate components without any inactivation or (and) denaturation, immobilization, 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, or the like before or (and after) cell lysis. In some embodiments of the present invention, tumor tissue cells are subjected to irradiation, high-temperature inactivation or (and) denaturation treatment before being lysed, or irradiation and heating treatment can be performed after cell lysis in the actual use process, or irradiation, heating inactivation or (and) denaturation treatment can be performed before and after cell lysis; in the actual use, treatment methods including but not limited to radiation irradiation, high pressure, nucleic acid separation and purification, immobilization, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, irradiation, radiation, heating, salting out, oxidation, reduction, enzyme treatment, mineralization, ionization, chemical modification, nuclease treatment, protease endo-or degradation, collagenase treatment, lyophilization and the like can also be employed. Those skilled in the art will appreciate that the actual application process can be appropriately adjusted according to the specific circumstances.

In the invention, the fixation can be performed by using a conventional fixation liquid.

In certain embodiments, one or more of formaldehyde, paraformaldehyde, glutaraldehyde, other aldehyde-containing substances, ethanol, methanol, acetone, acetic acid, propionic acid, butyric acid, formic acid, formalin, dichromate, potassium permanganate, chromic acid, picric acid, zamboni fixation, PLP fixation, FPA fixation, TAF fixation, rossman fixation, regaud fixation, PLPD fixation, PAPG fixation, orth fixation, muller fixation, mcDoWell fixation, neutral calcium formaldehyde fixation, FAB fixation, carnoy fixation, clarke fixation, B-5 fixation, bouin fixation, fineFIX fixation, a.g.m fixation, helly fixation, zenker fixation, kolmer fixation, AAF fixation, hollande fixation, gendre fixation, aldehydes fixation, mercury fixation, alcohols fixation, oxidants fixation, picrates, and ethylene oxide fixation are selected.

In the present invention, the A.G.M. fixative solution is a mixture of 70% ethanol (80 mL), glacial acetic acid (10 mL) and methanol (10 mL).

In certain embodiments, the delivery particles further have a composition as shown below:

(i) A framework structure formed by the particle materials;

(ii) An immunoadjuvant;

(iii) Positively charged species.

In certain embodiments, the immunoadjuvant is selected from the group consisting of pattern recognition receptor class agonists, toll-like receptor agonists, BCG cell wall backbones, BCG methanol extraction residues, BCG cell wall dipeptides, mycobacterium, polyoxin a, mineral oil, virus-like particles, immunopotentiating reconstituted influenza virus minibodies, cholera enterotoxin, saponins and derivatives thereof, resiquimod, thymosin, nascent bovine liver active peptide, imiquimod, polysaccharides, curcumin, immunoadjuvant CpG, immunoadjuvant poly (I: C), immunoadjuvant poly ICLC, short rod bacterin, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukins, interferons, polymyonic acid, polyadenylation acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokines, mRNA, MF59, double stranded RNA, double stranded DNA, single stranded DNA, aluminum adjuvants, manganese adjuvants, calcium adjuvants, STING agonists, astragalus, adjuvant, CAF01, and ginseng species active ingredients.

In certain embodiments, the immunoadjuvant is selected from one or more of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist.

In certain embodiments, the immunoadjuvant is selected from one or more of Poly (I: C), poly ICLC, A class CpG-OND, B class CpG-OND, and C class CpG-OND.

In certain embodiments, the antigenic component is supported within and/or on the surface of the framework structure.

In certain embodiments, the positively charged species is selected from one or more of a positively charged amino acid, a positively charged polypeptide, a positively charged lipid, a positively charged protein, a positively charged polymer, and a positively charged mineral.

In certain embodiments, the positively charged substance is selected from one or more of melittin, a RALA polypeptide, a KALA polypeptide, an R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine, and NH4HCO 3.

In certain embodiments, the isolated and purified antigen component of the lysate component may also be irradiated prior to or after lysis of the cells or tumor tissue, or may be irradiated after loading the isolated and purified antigen component into the nanoparticle or microparticle.

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

In certain embodiments, the nano-delivery particles have a particle size of 1nm to 1000nm.

In certain embodiments, the nano-delivery particles have a particle size of 50-500nm.

In certain embodiments, the nano-delivery particles have a particle size of 100-400nm.

In certain embodiments, the micrometer delivery particles have a particle size of 1 μm to 1000 μm.

In certain embodiments, the micrometer delivery particles have a particle size of 1-10 μm.

In certain embodiments, the micrometer delivery particles have a particle size of 1-5 μm.

In certain embodiments, 1mg of the nano-and/or micro-delivery particles load the protein or polypeptide component of the antigen component in an amount of 0.01-3mg.

In certain embodiments, 1mg of the nano-and/or micro-delivery particles load the protein or polypeptide component of the antigen component in an amount of 0.02-2mg.

In certain embodiments, 1mg of the nano-delivery particles load the antigen component with an amount of protein or polypeptide component of 0.01-2mg, e.g., 0.02mg, 0.04mg, 0.08mg, 0.1mg, 0.2mg, 0.4mg, 0.6mg, 0.8mg, 1.0mg, 1.5mg, 2.0mg.

In certain embodiments, 1mg of the micro-delivery particles load the antigen component with an amount of protein or polypeptide component of 0.01-2mg, e.g., 0.02mg, 0.04mg, 0.08mg, 0.1mg, 0.2mg, 0.4mg, 0.6mg, 0.8mg, 1.0mg, 1.5mg, 2.0mg. In practical application, the content of antigen components such as protein polypeptide loaded per 1mg of particle material can be higher.

In certain embodiments, 1mg of the nano-and/or micro-delivery particles are loaded with 0.001-2mg of the immunoadjuvant.

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

In certain embodiments, 1mg of the nano-delivery particles are loaded with 0.001-0.5mg of the immunoadjuvant, e.g., 0.002mg, 0.008mg, 0.02mg, 0.03mg, 0.04mg, 0.05mg, 0.06mg, 0.07mg, 0.08mg, 0.09mg, 0.1mg, 0.15mg, 0.2mg, 0.25mg, 0.3mg, 0.35mg, 0.40mg, 0.45mg, etc.

In certain embodiments, 1mg of the micron delivery particles are loaded with 0.001-0.5mg of the immunoadjuvant, e.g., 0.002mg, 0.004mg, 0.006mg, 0.008mg, 0.02mg, 0.03mg, 0.04mg, 0.05mg, 0.06mg, 0.07mg, 0.08mg, 0.09mg, 0.1mg, 0.15mg, 0.2mg, 0.25mg, 0.3mg, 0.35mg, 0.40mg, 0.45mg, etc. In the present invention, the inside and/or surface of the nanoparticle or microparticle carrying the antigen component isolated and purified may further contain a membrane component, and the membrane component inside and/or surface of the nanoparticle or microparticle is one or more selected from the group consisting of a cell membrane of an antigen presenting cell, an extracellular vesicle of an antigen presenting cell, a cell membrane of a cancer cell, an extracellular vesicle of a cancer cell, a cell membrane of a bacterium, and an extracellular vesicle of a bacterium.

When the membrane component is on the surface of the nanoparticle or microparticle, methods of loading the membrane component onto the surface of the nanoparticle or microparticle include, but are not limited to, one or more of sonication, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bonding, stirring, dialysis, homogenization, and homogenization.

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

In certain embodiments, the nanoparticle and/or microparticle is a nanoparticle or microparticle prepared using the cell wall of a bacterium.

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

In some exemplary embodiments of the present invention, the solvent evaporation method is used to prepare nano-particles or micro-particles loaded with antigen components, and any other method capable of preparing anti-nano-particles or micro-particles may be used in practical applications, including but not limited to precipitation, dialysis, dispersion, microfluidic, high-pressure homogenization, stirring, spray drying, phase separation, electrostatic spraying, emulsion polymerization, machine stirring shear, membrane emulsification, etc.

In certain embodiments, the particle material is PEG-modified or not PEG-modified, preferably, the mass ratio of the PEG-unmodified particle material to the PEG-modified particle material is 9-200:1 when preparing the backbone structure. The preparation material of the nano particles or the micro particles can be prepared by adding a proper amount of PEG modified PLGA or PLA into main materials such as PLGA or PLA, and the like, so that the long circulation and passive targeting effect can be better achieved after the injection into a body; wherein the mass ratio of PEG modified PLGA or PLA or the like to unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.

In certain embodiments, the nanoparticle or microparticle particles produce a mass ratio of the framework material, protein, and polypeptide components of 1:0.001-10; preferably, the mass ratio of the components of the particle preparation framework material, protein and polypeptide is 1:0.01-2; most preferably, the mass ratio of the components of the particle preparation framework material, protein and polypeptide is 1:0.05-1.

In certain embodiments, the nanoparticle or microparticle preparation material is selected from natural polymeric materials, biological materials, microbial materials, synthetic polymeric materials, and/or inorganic materials.

In the present invention, an organic polymer is used as the preparation material of the nano-particles or micro-particles, and in practical applications, any other preparation material that can support antigen and prepare nano-or micro-sized particles may be used, including but not limited to inorganic materials, viruses (such as viral proteins), bacteria (such as bacterial walls, bacterial proteins or whole bacteria), other biological sources (exosomes, extracellular vesicles, bacterial membrane components) and the like.

In certain embodiments, the antigen delivery particles (nanoparticles or microparticles) are in any shape, including but not limited to spheres, ellipsoids, barrels, polygons, rods, flakes, lines, worms, squares, triangles, butterflies, discs, vesicles, and the like.

In certain embodiments, the nanoparticle or microparticle surface may also be loaded with a membrane component, which may be from one or more of an antigen presenting cell, a cancer cell, a bacterium, or an extracellular vesicle.

In certain embodiments, the antigen presenting cells used to prepare the biofilm component supported on the surface of the nanoparticle or microparticle may be derived from autologous or allogeneic sources, as well as from cell lines or stem cells. The antigen presenting cells may be DC cells, B cells, macrophages or any mixture of the three, or may be other cells having an antigen presenting function. Antigen presenting cells may be activated by antigen-loaded nanoparticles or microparticles.

In certain embodiments, the biofilm component carried by the nanoparticle or microparticle surface may be one or more of an extracellular vesicle of a cancer cell, an extracellular vesicle of a bacterium, or an extracellular vesicle of an antigen presenting cell when derived from an extracellular vesicle.

In certain embodiments, any nanoparticle, microparticle preparation method known to those skilled in the art may be used to prepare the nanoparticles or microparticles described herein, including but not limited to solvent evaporation, dialysis, phase separation, spray drying, emulsion polymerization, machine agitation shear, membrane emulsification, microfluidics, ultrafiltration, homogeneous emulsification, dispersion, precipitation, and the like.

The preparation method of the delivery particle is as follows:

step 1, extracting an antigen component according to the extraction method of the first aspect, specifically, lysing tumor tissue or cancer cells using pure water or a lysing solution containing a lysing agent, and then lysing the lysate component using pure water and/or a first lysing solution containing a lysing agent.

And 2, adding a certain amount of alcohols (such as ethanol, methanol, isopropanol and the like), ketones (such as acetone and the like), phenols (such as phenol and the like) or nitriles (such as acetonitrile and the like) and other organic solvents into pure water or a lysate component dissolved by a first dissolving solution containing a dissolving agent, so as to precipitate antigen components such as protein, polypeptide and the like, and collecting a precipitated part after centrifugation to obtain the antigen component. In the invention, alcohols (such as ethanol, methanol, isopropanol and the like), ketones (such as acetone and the like), phenols (such as phenol and the like) or nitriles (such as acetonitrile and the like) and other organic solvents are used, and in practical application, many organic solvents can precipitate antigen components such as protein polypeptides and the like from aqueous solution, and the purpose can be achieved by using the organic solvents capable of precipitating protein polypeptides, so that other organic solvents capable of precipitating proteins from aqueous solution can also be used.

In certain embodiments, the centrifugation speed is 1000RPM to 20000RPM.

In certain embodiments, the centrifugation speed is 1000RPM to 12000RPM.

In certain embodiments, the centrifugation speed is 1000RPM to 10000RPM.

In certain embodiments, the centrifugation speed is 1000RPM to 8000RPM.

In certain embodiments, the centrifugation speed is from 1000RPM to 5000RPM.

Step 3, secondarily dissolving the precipitate component (antigen component) collected in the step 2 by using a dissolving solution containing a dissolving agent, and directly using the precipitate component; or dissolving the precipitate component (antigen component) collected in the step 2 by using a dissolving solution containing a dissolving agent for the second time, carrying out proper treatment on the supernatant obtained by centrifuging in the step 2 to obtain other components, and mixing the antigen component dissolved by the dissolving solution with the component obtained by separating and purifying the supernatant to obtain the antigen component.

In certain embodiments, the supernatant component obtained in step 2 may be further processed by salting out, heating, enzyme treatment, oxidation, reduction, immobilization, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction of RNA component, extraction of mRNA component, degradation of DNA component, extraction, radiation, mineralization, irradiation, radiation, and other suitable methods, and then separated and purified to obtain other components; the other components separated and purified from the supernatant may be combined with the antigen component secondarily dissolved in the second dissolution liquid containing the dissolution agent and used as the antigen component. For example, an antigen component is obtained by separating an mRNA component or an RNA component from a supernatant by a suitable method and then mixing the mRNA component or the RNA component with a precipitate portion in which a dissolution solution containing a dissolution agent is secondarily dissolved.

And 4, taking the antigen component obtained in the step 3 as an initial water phase, and adding the initial water phase into an organic phase to prepare a initial emulsion sample.

In the preparation, the primary aqueous phase is mixed with the organic phase, specifically by adding a first predetermined volume of an aqueous phase solution containing an antigen component at a first predetermined concentration to a second predetermined volume of an organic phase containing a raw material for preparing particles at a second predetermined concentration.

In certain embodiments, the aqueous phase solution may contain at least one of the following i) -ii): i) An antigen component in the lysate; ii) an antigenic component in the lysate and an immunopotentiating adjuvant. The antigen component in the lysate is separated and purified in the form of a solution containing urea or guanidine hydrochloride. The first predetermined concentration is the concentration of the protein and polypeptide contained in the aqueous solution or the concentration of the antigen component contained in the aqueous solution, and the first predetermined concentration requires a concentration of the protein and polypeptide of greater than 1ng/mL so as to be capable of supporting sufficient antigen component to activate the cells of interest. The concentration of immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01ng/mL.

In certain embodiments, the organic solvent is selected from dichloromethane or ethyl acetate. Additionally, in some embodiments, the second predetermined concentration of the starting material for the preparation of particles ranges from 0.5mg/mL to 5000mg/mL, preferably 100mg/mL.

In practice, the second predetermined volume of the organic phase is set according to the ratio of the same 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. In a specific implementation process, the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as required to adjust the size of the prepared nanoparticles or microparticles.

In certain embodiments, the aqueous solution is a solution comprising the antigenic component in the cell and/or tissue lysate, wherein the concentration of the protein and polypeptide is greater than 1ng/mL, preferably 1mg/mL to 100mg/mL.

In certain embodiments, the aqueous solution is a solution comprising the antigen component of the lysate and the immunoadjuvant, wherein the concentration of the protein and the polypeptide is greater than 1ng/mL, preferably between 1mg/mL and 100mg/mL, and the concentration of the immunoadjuvant is greater than 0.01ng/mL, preferably between 0.001mg/mL and 20mg/mL. In some embodiments, the solvent in the organic phase solution is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, and the like, preferably dichloromethane and ethyl acetate; the concentration of the organic phase is 0.5mg/mL-5000mg/mL, preferably 100mg/mL.

In certain embodiments, when the aqueous solution is a solution comprising the antigen component in the lysate, the concentration of the protein and polypeptide components therein is greater than 0.01ng/mL, preferably 1 μg/mL to 1mg/mL. In some embodiments, when the aqueous solution is a solution comprising the antigen component and the immunoadjuvant, wherein the concentration of the protein and polypeptide component is greater than 1ng/mL, preferably between 1 μg/mL and 1mg/mL, the concentration of the immunoadjuvant is greater than 0.01ng/mL, preferably between 0.001mg/mL and 20mg/mL. In some embodiments, the solvent in the organic phase solution is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, and the like, preferably dichloromethane and ethyl acetate; the concentration of the organic phase is 0.5mg/mL-5000mg/mL, preferably 100mg/mL.

And 5, performing any one of the following treatments on the mixed solution obtained in the step 4: i) Sonication for greater than 2 seconds; ii) stirring for more than 1 minute; iii) Homogenizing; iv) microfluidic processing. Preferably, the stirring speed is greater than 50rpm and the stirring time is greater than 1 minute, such as 50rpm-1500rpm and 0.1-24 hours, when mechanically or magnetically stirring; during ultrasonic treatment, the ultrasonic power is more than 5W, and the time is more than 0.1 seconds, such as 2-200 seconds; the homogenizing treatment is performed by using a high pressure/ultra high pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 5psi, such as 20psi-100psi, and the rotating speed is more than 100rpm, such as 1000rpm-5000rpm; the microfluidic processing flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min. The nano-scale and/or micro-scale is carried out by ultrasonic or stirring or homogenizing treatment or microfluidic treatment, the ultrasonic time or stirring speed or homogenizing treatment pressure and time can control the size of the prepared nano-scale particles or micro-scale particles, and the particle size change can be caused by the excessive or excessive small size.

Step 6, adding the mixture obtained after the treatment in step 5 into a third predetermined volume of aqueous solution containing a third predetermined concentration of emulsifier and performing any one of the following treatments: i) Sonication for greater than 2 seconds; ii) stirring for more than 1 minute; iii) Homogenizing; iv) microfluidic processing. The mixture obtained in the step 5 is added into the aqueous solution of the emulsifier to continue ultrasonic treatment or stirring or homogenization or mixing so as to carry out nanocrystallization or microminiaturization. In the present invention, the ultrasound time is greater than 0.1 seconds, such as 2-200 seconds; the stirring speed is greater than 50rpm, such as 50rpm-500rpm; the stirring time is greater than 1 minute, such as 60-6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm-1500rpm, and the stirring time is 0.5 hours-5 hours; during ultrasonic treatment, the ultrasonic power is 50W-500W, and the time is more than 0.1 seconds, such as 2-200 seconds; the high pressure/ultra-high pressure homogenizer or high shear homogenizer is used for homogenizing, the pressure is more than 20psi, such as 20psi-100psi, and the rotating speed is more than 1000rpm, such as 1000 rpm-5000 rpm; the microfluidic processing flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min. The ultrasonic treatment, stirring or homogenizing treatment or micro-fluidic treatment is performed to carry out nanocrystallization or microminiaturization, the ultrasonic time, stirring speed, homogenizing treatment pressure, homogenizing treatment time and the like can control the size of the prepared nano particles or micro particles, and the particle size change can be brought by too large or too small.

In certain embodiments, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) 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 to the second predetermined volume. In the present invention, the ratio of the second predetermined volume to the third predetermined volume ranges from 1:1.1 to 1:1000, preferably may be 2:5. In order to control the size of the nano-or micro-particles during the implementation process, the ratio of the second predetermined volume to the third predetermined volume may be adjusted. Similarly, the ultrasonic time or stirring time or homogenizing time, the volume of the aqueous solution of the emulsifier and the concentration of the aqueous solution of the emulsifier are taken as values according to the step, so that the nano-particles or the micro-particles with proper size can be obtained.

And 7, adding the liquid obtained after the treatment in the step 6 into a fourth preset volume of a fourth preset concentration emulsifier water solution, and stirring until a preset stirring condition is met.

In this step, the aqueous emulsifier solution is a PVA solution or other solution.

The fourth preset concentration is 5mg/mL, and the nano particles or the micro particles with proper size are obtained through the selection of the fourth preset concentration. The fourth predetermined volume is selected based on a 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. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted in order to control the size of the nanoparticles or microparticles in the implementation process.

In the present invention, the predetermined stirring condition of this step is until the evaporation of the organic solvent is completed, i.e., the evaporation of methylene chloride or ethyl acetate in step 4 is completed.

Step 8, centrifuging the mixed solution which meets the preset stirring condition and is obtained in the step 7 at a rotating speed of more than 100RPM for more than 1 minute, removing supernatant, and re-suspending the rest precipitate in a fifth preset volume of fifth preset concentration aqueous solution containing a lyoprotectant or a sixth preset volume of PBS (or physiological saline); the solution in the system is replaced with a fifth predetermined volume of aqueous solution or a sixth predetermined volume of PBS (or physiological saline) containing a fifth predetermined concentration of lyoprotectant while removing substances such as free PVA by ultrafiltration centrifugation or dialysis capable of removing substances of a specific molecular weight.

And 9, performing freeze drying treatment on the suspension containing the freeze-drying protective agent obtained in the step 8, and then, reserving the freeze-dried substance.

In certain embodiments, the prepared antigen component-loaded nanoparticle or microparticle is irradiated with an anti-radiation for a certain period of time.

Step 10, re-suspending the nanoparticle/micrometer particle-containing suspension obtained in step 8 in PBS (or physiological saline) in a sixth predetermined volume, or re-suspending the freeze-dried lyophilized substance containing nanoparticles or microparticles and a lyoprotectant obtained in step 9 in PBS (or physiological saline) in a sixth predetermined volume, and then directly using; or mixing the sample with a seventh predetermined volume of the antigen component.

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 from 1:30 to 30:1.

Step 11, the nano particles or the micro particles prepared in the step 10 are used for preventing or treating diseases such as cancers and the like; or using the nanoparticle microparticles 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 using nanoparticles or microparticles to assist in activating antigen-specific T cells to detect the content of antigen-specific T cells; or after the antigen-specific T cells are activated with the aid of nano-particles or micro-particles, sorting or amplifying the antigen-specific T cells, and using the sorted or amplified antigen-specific T cells for preventing or treating diseases.

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

(1) Preparing a medicament for preventing or treating a disease;

In certain embodiments, the delivery particles are used directly as a vaccine.

In certain embodiments, the delivery particles activate dendritic cells and/or B cells prior to use of the activated dendritic cells and/or B cells as a cellular vaccine. In the present invention, the cellular immunity of the T cell pathway is the desired activation of cancer vaccines and the like, which recognize the 1-order sequence fragment of the polypeptide, not the 3-order spatial conformational structure recognized by humoral immunity, so that protein denaturation does not adversely affect the immunogenicity of the antigen component of the protein against cellular immunity, and even enhances the immunogenicity of the protein to some extent.

In certain embodiments, the delivery particles are used to detect the level of cancer antigen-specific T cells after in vitro assisted activation of cancer cell-specific T cells, or to isolate and/or expand activated antigen-specific T cells after assisted activation of antigen-specific T cells by the delivery particles for the prevention and treatment of disease.

In certain embodiments, the disease is cancer or tumor.

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

The present invention will be further illustrated by the following examples, wherein "%" represents mass percent unless otherwise specified. The materials and reagents used in the examples which follow, unless otherwise indicated, are those commonly used in the art and may be obtained commercially or synthesized by known methods. The experimental methods, which are not specified in the following examples, are generally carried out according to conventional experimental conditions or conditions recommended by the manufacturer of the relevant reagent (kit).

Example 1 sorting expanded T cells after activation of antigen component-loaded nanoparticles for treatment of breast cancer

(1) Preparation of antigenic components

Back of each Balb/c mouse was inoculated subcutaneously with 6.0X10 s ⁵ Breast cancer cells of 4T1 mice grew to a volume of about 1000mm in the tumor ³ Mice were sacrificed and tumor tissues were harvested, then diced into ultrapure water and single cell suspensions were prepared using a cell sieve, and then the tumor tissues were lysed by repeated freeze thawing 5 times, and the tumor tissue lysate fraction was centrifuged at 5000g for 10 minutes. Collecting supernatant to obtain water-soluble component in lysate; the precipitation part is solubilized by 8M urea aqueous solution, and the water insoluble component in the lysate component is obtained. Adding 10 times volume of water solution containing 50% ethanol and 0.5% phenol into water soluble component and non-water soluble component, standing for 6 hrPrecipitation. And then respectively centrifuging the precipitate obtained in the water-soluble component and the precipitate obtained in the water-insoluble component at 3500rpm for 5 minutes, respectively dissolving the precipitate in the water-soluble component and the precipitate in the water-insoluble component by using 8M urea aqueous solution, and mixing according to a mass ratio of 1:2 to obtain the antigen component 1.

Back of each Balb/c mouse was inoculated subcutaneously with 6.0X10 s ⁵ Breast cancer cells of 4T1 mice grew to a volume of about 1000mm in the tumor ³ Mice were sacrificed and tumor tissues were harvested, then diced into ultrapure water and single cell suspensions were prepared using a cell sieve, and then the tumor tissues were lysed by repeated freeze thawing 5 times, and the tumor tissue lysate fraction was centrifuged at 5000g for 10 minutes. Collecting supernatant to obtain water-soluble component in lysate; the precipitation part is solubilized by 8M urea aqueous solution, and the water insoluble component in the lysate component is obtained. And mixing the water-soluble component and the water-insoluble component according to the mass ratio of 1:2 to obtain the antigen component 2.

(2) Preparation of antigen delivery nanoparticles

In this example, nanoparticle 1 (Nanoparticle 1) was prepared by the multiple emulsion method in the solvent evaporation method. The molecular weight of the PLGA used for preparing the nano particles is 20kDa-40kDa. The immunoadjuvants used were poly (I: C), cpG 1018, and CpG 7909. Preparation method As described above, the preparation was carried out by loading the cell antigen component 1 and the adjuvant into the nanoparticle, and then centrifuging 100mg of the nanoparticle 1 at 12000g for 30 minutes, and freeze-drying for 48 hours after resuspension using 10mL of ultra-pure water containing 6% trehalose. The average particle size of the nanoparticle 1 is about 230nm, and about 200 mug of protein or polypeptide components are loaded per 1mg of PLGA nanoparticle 1, and 0.02mg of poly (I: C), cpG 1018 and CpG 7909 are loaded. Then gamma-rays were used for irradiation for 16 hours.

In this example, nanoparticle 2 (Nanoparticle 2) was prepared in the same manner as in example 1. The preparation method comprises the steps of loading cell antigen component 2 and an adjuvant into the nanoparticle, centrifuging 100mg of the nanoparticle 2 at 12000g for 30 minutes, and freeze-drying for 48 hours after resuspension by using 10mL of ultrapure water containing 6% trehalose. The average particle size of the nanoparticle 2 is about 230nm, and about 200 μg of protein or polypeptide component is loaded per 1mg of PLGA nanoparticle 2, and 0.001mg of each of poly (I: C), cpG 1018 and CpG 7909 is loaded. Then gamma-rays were used for irradiation for 16 hours.

(3) Sorting expansion of antigen-specific T cells

Female Balb/c mice were selected for 6-8 weeks, and each mouse was inoculated subcutaneously with 6.0X10 s on day 0 ⁵ Each mouse was intraperitoneally injected with 150 μg of mouse PD-1 antibody on day 6, day 8, day 10, day 12, day 14, day 16, day 18, day 20, respectively, 4T1 cells. Mice were sacrificed on day 21, peripheral blood was collected from the mice, peripheral Blood Mononuclear Cells (PBMCs) were then isolated from the peripheral blood, then 500 ten thousand PBMC cells, 1mg nanoparticles (nanoparticle 1, or nanoparticle 2) were co-incubated in 2ml of rpm 1640 complete medium for 36 hours, and then the incubated CD3 was sorted using flow cytometry ⁺ CD134 ⁺ T cells are antigen-specific T cells that recognize cancer cell antigens. 5 ten thousand CD3 s obtained by the separation ⁺ CD134 ⁺ T cells with IL-2 (20 ng/mL), IL-7 (10 ng/mL), IL-15 (10 ng/mL), and the alpha CD3 antibody (10 ng/mL) and the alpha CD28 antibody (10 ng/mL) in 10mL DMEM complete medium (37 ℃,5% CO) ₂ ) Co-incubation was performed for 21 days to expand cancer cell-specific T cells (cell viability about 85%). Wherein, the cancer cell specific T cells amplified by using the nanoparticle 1 (nano vaccine 1) in an assisted sorting way are T cells 1 (T cells 1); the amplified cancer cell-specific T cells were sorted as T cells 2 (T cells 2) using nanoparticle 2 (nanovaccine 2) aid.

(4) Sorting expanded cancer cell antigen-specific T cells for treatment of cancer

Female Balb/c of 6-8 weeks was selected as model mice to prepare melanoma-bearing mice, and 6.0X10 s was inoculated subcutaneously on the lower right back of each mouse on day 0 ⁵ Breast cancer cells from 4T1 mice. Each mouse was given cyclophosphamide (dose 100 mg/kg) intraperitoneally on day 6 after tumor inoculation, and 100 tens of thousands of amplified antigen-specific T cells (T cell 1 or T cell 2) or 100 μl of PBS were intravenously injected on day 7, day 14, day 21 and day 28, respectively, in mice; t cell (T cell 1 or T cell 2) treatment groups were injected intravenously with IL-2 (10000U) every two days from day 7 to day 27.The method for monitoring the growth speed and the survival time of the tumor of the mouse adopts a conventional method.

(5) Experimental results

As shown in fig. 3, the PBS group mice rapidly grew in tumor volume and the mice died quickly. The survival time of the mice treated by the T cells 1 (T cells 1) and the T cells2 (T cells 2) is obviously prolonged, most of the mice are healed without tumor, and the effect of the T cells 1 is better than that of the T cells2, which indicates that the treatment effect of the antigen delivery particles assisted in sorting and amplifying the T cells can be effectively improved by purifying the antigen component by the method.

Example 2 nanovaccines loaded with antigen component for the treatment of pancreatic cancer

In the embodiment, the antigen component is derived from Pan02 mouse pancreatic cancer cell line, organic polymer material PLGA is used as nanoparticle skeleton material, polyinosinic-polycytidylic acid (poly (I: C)), cpG7909 (CpG 2006) and CpG2395 are used as immunological adjuvants, and a solvent volatilization method is used for preparing the nanometer vaccine.

(1) Preparation of antigenic components

Collection of 2X 10 ⁸ The cultured Pan02 mice pancreatic cancer cells were then irradiated with gamma rays for 2 hours, then 2mL of 8M urea in PBS aqueous solution was added to lyse the cancer cells, and then the cell lysate fraction was dissolved using 6mL of 8M urea in PBS aqueous solution. Then adding 12mL of ethanol into the lysate component-containing solution, standing for 1 hour, centrifuging the sample at 3000RPM for 5 minutes, removing the supernatant, and secondarily dissolving the precipitate part by using 8M urea PBS aqueous solution to obtain the antigen component 1 for preparing the nano vaccine 1.

Collection of 2X 10 ⁸ The cultured Pan02 mice pancreatic cancer cells were then irradiated with gamma rays for 2 hours, then 2mL of 8M urea in PBS aqueous solution was added to lyse the cancer cells, and then the cell lysate fraction was dissolved using 6mL of 8M urea in PBS aqueous solution. Then, a 40% ammonium sulfate aqueous solution was added dropwise to the lysate to prepare 16mL of a sample, the sample was left standing for 1 hour to obtain a salted-out sample, the sample was centrifuged at 16000RPM for 60 minutes, the supernatant was removed, and the precipitated fraction was twice using 8M urea in PBS Dissolving to obtain antigen component 2 for preparing nanometer vaccine 2.

Cultured Pan 02 mouse pancreatic cancer cells were collected, then irradiated with gamma rays for 2 hours, then lysed by adding 2mL of 8M urea in PBS, and then cell lysate fractions were lysed using 6mL of 8M urea in PBS. Namely the antigen component 3 for preparing the nano vaccine 3.

(2) Preparation of nanovaccine

In this example, the Nanovaccine 1 (Nanovaccine 1) was prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of the adopted vaccine preparation material PLGA is 10kDa-20kDa, and the adopted immunoadjuvants are poly (I: C), cpG7909 and CpG2395. Preparation method As described above, in the preparation process, the antigen component 1 and the adjuvant were co-supported in the nanoparticle by the multiple emulsion method, and then 100mg of the nanoparticle 1 was centrifuged at 12000g for 30 minutes, resuspended in 10mL of ultra-pure water containing 4% trehalose, and lyophilized for 48 hours. The average particle size of the nanoparticle 1 is about 250nm, and each 1mg of PLGA nanoparticle 1 is loaded with about 1.0mg of protein and polypeptide components, and 0.02mg of poly (I: C), cpG7909 and CpG2395.

The preparation method and the preparation material of the nano vaccine 2 (nanovaccinee 2) in the embodiment are the same as those of the nano vaccine 1. In the preparation process, the antigen component 2 and the adjuvant are loaded in the nano particles by a multiple emulsion method, 100mg of the nano particles 2 are centrifuged at 12000g for 30 minutes, and 10mL of ultra-pure water containing 4% trehalose is used for resuspension and then freeze drying is carried out for 48 hours. The average particle size of the nanoparticle 2 is about 250nm, and each 1mg of PLGA nanoparticle 2 is loaded with about 1.0mg of protein and polypeptide components, and 0.02mg of poly (I: C), cpG7909 and CpG2395.

The preparation method and the preparation material of the nano vaccine 3 (nanovaccinee 3) in this embodiment are the same as those of the nano vaccine 1. In the preparation process, the antigen component 3 and the adjuvant are loaded in the nano particles together by adopting a multiple emulsion method, 100mg of the nano particles 3 are centrifuged at 12000g for 30 minutes, and 10mL of ultra-pure water containing 4% trehalose is used for resuspension and then freeze drying is carried out for 48 hours. The average particle size of the nanoparticle 3 is about 250nm, and each 1mg of PLGA nanoparticle 3 is loaded with about 1.0mg of protein and polypeptide components, and 0.02mg of poly (I: C), cpG7909 and CpG 2395.

(3) Nanometer vaccine for treating cancer

Preparation of pancreatic cancer tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 ⁶ Pan 02 cells. 2mg of nanovaccine (nanovaccine 1, or nanovaccine 2, or nanovaccine 3) or 100 μl of PBS was injected subcutaneously in mice on day 3, 6, 9, 14, 19 and 26, respectively, after tumor inoculation. The mice were monitored for tumor growth rate and mice survival. In the experiment, the size of the tumor volume of the mice was recorded every 3 days starting on day 3. Tumor volume was calculated using the formula v=0.52×a×b ² Calculation, where v is tumor volume, a is tumor length, and b is tumor width. For ethical reasons in animal experiments, the tumor volume of the mice exceeds 2000mm in the life cycle test of the mice ³ I.e. the mice were regarded as dead and euthanized.

(4) Experimental results

As shown in fig. 4, the tumor volume of the PBS control group (PBS control) mice rapidly increased and the mice died rapidly. The mice using the nano vaccine 1 (nanovaccinee 1), the nano vaccine 2 (nanovaccinee 2) and the nano vaccine 3 (nanovaccinee 3) have obviously slowed tumor growth speed, obviously prolonged survival time and recovery of most of the mice without tumor. Moreover, the effect of the nano vaccine 1 is equivalent to that of the nano vaccine 2, and the effects of the nano vaccine 1 and the nano vaccine 2 are better than those of the nano vaccine 3, which shows that the effect of the antigen component in the cell lysate obtained by separating and purifying by using an alcohol precipitation method is equivalent to that of the antigen component in the cell lysate obtained by separating and purifying by using a salting-out method, and is better than that of the cell lysate component which is not separated and purified and secondarily dissolved.

In this embodiment, the cancer cells are irradiated with gamma rays before the cancer cells are lysed and the components of the lysate of the cancer cells are lysed by using a lysing solution containing a lysing agent, and in practical applications, one or more of various irradiation methods such as electron beam, X-ray, α -ray, β -ray, ultraviolet ray, microwave, radiation source, etc. may be used for a period of time; alternatively, the lysis solution containing the lytic agent may be used to lyse cancer cells and lyse the cancer cell lysate component without performing the irradiation treatment.

In this example, the antigen component is prepared using a cultured cancer cell line, and in practical applications, cancer cells obtained by culturing and amplifying cancer cells isolated from tumor tissue, circulating tumor cells isolated from blood or periphery, or tumor tissue may be used as the source of the antigen component.

EXAMPLE 3 antigen component-loaded micrometer vaccine for preventing brain cancer

(1) Preparation of antigenic components

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁶ GL261 cells, growing to a volume of about 1000mm in each tumor ³ Mice were sacrificed and tumor tissue was harvested. A single cell suspension of tumor tissue was prepared and then the cells were cultured in RPMI1640 (containing 10% FBS) complete medium for 7 days (37 ℃,5% CO) ₂ ). After the completion of the culture, 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 Trizol reagent (containing guanidine isothiocyanate and phenol) and chloroform was added to produce a second (organic) phase into which the DNA and proteins were extracted, while the RNA remained in the aqueous supernatant. Firstly sucking the upper water phase, adding half volume of isopropanol, standing at room temperature for 15 minutes, centrifuging at 12000g for 10 minutes, discarding the supernatant, collecting the precipitate, then adding a proper amount of 75% ethanol to wash the precipitate, standing for 10 minutes, centrifuging at 12000g for 5 minutes, discarding the supernatant, collecting the precipitate RNA, and dissolving the precipitate RNA by using 6M guanidine hydrochloride; the middle and lower organic phases were collected, protein and DNA therein were precipitated by adding 2 volumes of isopropanol, the supernatant was discarded, and the precipitate was centrifuged at 5000g for 10 minutes, and then the precipitate was collected and then dissolved using 6M guanidine hydrochloride. Combining RNA dissolved by 6M guanidine hydrochloride and protein dissolved by 6M guanidine hydrochloride with DNA to obtain the antigen component 1.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁶ GL261 cells, growing to a volume of about 1000mm in each tumor ³ Mice were sacrificed and tumor tissue was harvested. Single cell suspensions of tumor tissue were prepared and then the cells were fully cultured in RPMI1640 (containing 10% FBS)Culture in medium for 7 days (37 ℃,5% CO) ₂ ). After the completion of the culture, the cancer cells were collected and centrifuged at 400g for 5 minutes to remove the medium. And (3) lysing the precipitated cancer cells by using a proper amount of 6M guanidine hydrochloride aqueous solution, and then dissolving a lysate component by using the 6M guanidine hydrochloride aqueous solution to obtain an antigen component 2.

(2) Preparation of micrometer vaccine

In this example, micrometer vaccine 1 (Micronvaccine 1) was prepared by the multiple emulsion method of the solvent evaporation method. The molecular weight of PLA is 30-50 kDa, the immunological adjuvant is poly (I: C), cpG 1018 and CpG2395, and the positively charged substance is RALA polypeptide (N-WEARLARALARALARHLARALARALRACEA-C). Preparation method As previously described, antigen component 1 and adjuvant were loaded inside microparticles during the preparation, and 100mg of microparticles 1 were centrifuged at 8000g for 20 minutes, resuspended in 10mL of ultra-pure water containing 4% trehalose, and lyophilized for 48h. The average particle diameter of the micron particles 1 is about 2.0 mu m, about 20 mu g of protein or polypeptide component is loaded per 1mg of PLA micron particles 1, 0.012mg of poly (I: C) is loaded, 0.005mg of CpG 1018 and CpG2395 are respectively loaded, and 0.1mg of RALA polypeptide is loaded.

In this example, micrometer vaccine 2 (Micronvaccine 2) was prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of PLA is 30-50 kDa, the immunological adjuvant is poly (I: C), cpG 1018 and CpG2395, and the positively charged substance is RALA polypeptide. Preparation method As previously described, antigen component 2 and adjuvant were loaded inside microparticles during the preparation, and 100mg of microparticles 2 were centrifuged at 8000g for 20 minutes, resuspended in 10mL of ultra-pure water containing 4% trehalose, and lyophilized for 48h. The average particle diameter of the micron particles 2 is about 2.0 mu m, 20 mu g of protein or polypeptide component is loaded per 1mg of PLA micron particles 2, poly (I: C) is loaded by 0.012mg, cpG 1018 and CpG2395 are respectively loaded by 0.005mg, and RALA polypeptide is loaded by 0.1mg.

(3) Micron vaccine for preventing cancer

Female C57BL/6 of 6-8 weeks is selected as model mice to prepare brain glioma tumor-bearing mice, and the mice are inoculated with tumor on day-35, day-28, day-21, day-14 and day-7, respectivelyMice were subcutaneously injected with 6mg of micrometer vaccine 1 or 6mg of micrometer vaccine 2 or 100 μl of PBS. Day 0, 1.5X10 s were subcutaneously inoculated on the lower right back of each mouse ⁶ And GL261. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.

(4) Experimental results

As shown in fig. 5, the PBS group mice rapidly grew in tumor volume and the mice died quickly. Mice using micrometer vaccine 1 (Micronvaccine 1) and micrometer vaccine 2 (Micronvaccine 2) had significantly slower tumor growth rates, significantly longer survival periods, and most mice had no tumor. The effect of the micrometer vaccine 1 is better than that of the micrometer vaccine 2, which shows that the effect of loading the antigen component obtained by adding phenols and alcohol organic solvents for precipitation and purification to the micrometer vaccine is better than that of directly loading the cancer cell lysate to the micrometer vaccine.

EXAMPLE 4 nanometer vaccine for treatment of liver cancer

(1) Preparation of antigenic components

The back of each C57BL/6 mouse was inoculated subcutaneously with 1.5X10 ⁶ The Hepa 1-6 liver cancer cells grow to a volume of about 1000mm in the tumor ³ Mice were sacrificed and tumor tissues were harvested, the tumor tissues were diced and fixed in 75% ethanol aqueous solution containing 1% hydrogen peroxide (hydrogen peroxide) for 2 hours, then centrifuged at 1000g for 10 minutes, the tissue fixative supernatant was discarded, the tumor tissues were lysed using an appropriate amount of 4M guanidine isothiocyanate and 0.5% phenol aqueous solution, and the tumor tissue lysate fraction was dissolved using 4M guanidine isothiocyanate and 0.5% phenol aqueous solution, then 1 volume of isopropanol was added for precipitation, and standing for 3 hours for precipitation. Then centrifuging at 3500RPM for 10 minutes, and secondarily dissolving the precipitate part by using 8M urea aqueous solution; as antigen component 1 for the preparation of nanovaccine 1.

(2) Preparation of nanovaccine

In this example, the Nanovaccine 1 (Nanovaccine 1) was prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of the adopted nanometer vaccine preparation material PLGA is 10kDa-20kDa, the molecular weight of PEG2000-PLGA is 15kDa-25kDa, and the mass ratio of PLGA to PEG5000-PLGA is 98.4:1.6. The immunoadjuvants used were poly (I: C), cpG 2395 and CpG SL03. Preparation method As described above, the preparation was carried out by loading the cell antigen component 1 and the adjuvant into the nanoparticle, and then centrifuging 100mg of the nanoparticle 1 at 12000g for 30 minutes, and freeze-drying for 48 hours after resuspension using 10mL of ultra-pure water containing 4% trehalose. The average particle size of the nanoparticle 1 is about 300nm, and about 500 mug of protein or polypeptide components are loaded per 1mg of PLGA nanoparticle 1, and 0.1mg of poly (I: C), cpG 2395 and CpG SL03 are loaded. And then irradiating the PLGA nano vaccine 1 loaded with the antigen component and the adjuvant for 12 hours by using gamma rays to obtain the irradiated nano vaccine 1.

(3) Nanometer vaccine for treating cancer

Female C57BL/6 of 6-8 weeks is selected as model mouse to prepare liver cancer tumor-bearing mice, and 1.5X10 s is inoculated subcutaneously on the lower right back of each mouse on day 0 ⁶ And (3) liver cancer cells of the Hepa 1-6. 100 μl of 1mg nanovaccine 1 or 100 μl of PBS was injected subcutaneously in mice on day 3, 6, 9, 14, 19 and 25, respectively, after tumor inoculation. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.

(4) Experimental results

As shown in fig. 6, the PBS group mice rapidly grew in tumor volume and the mice died quickly. The survival time of the mice using the nano vaccine 1 (nanovaccinee 1) is obviously prolonged, and most of the mice are healed without tumor.

In this embodiment, hydrogen peroxide (hydrogen peroxide) is used in the tissue fixing solution to oxidize tumor tissue, and hypochlorous acid, potassium permanganate or other oxidizing agents may be used in practical application to oxidize cancer cells, tumor tissue, single cell suspension of tumor tissue, or antigen component in the cell lysate component or lysate component of tissue. Alternatively, in practice, a reducing agent such as Dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP) or the like may be used to reduce cancer cells, tumor tissues, single cell suspensions of tumor tissues, or antigen components in the above cell lysate components or lysate components of tissues.

EXAMPLE 5 nanoparticle-activated antigen presenting cell vaccine loaded with antigen component for the treatment of colon cancer

(1) Preparation of antigenic components

Back subcutaneous inoculation of 1.5X10 s per C57BL/6 mice ⁶ MC38 colon cancer cells grow to a volume of about 1000mm in a tumor ³ Mice were sacrificed and tumor tissues were harvested, the tumor tissues were diced and fixed with 50% aqueous ethanol (0.1% hypochlorous acid) for 2 hours, then centrifuged at 2000RPM for 5 minutes, the supernatant was discarded, the fixed tumor tissue pellet was lysed with an appropriate amount of aqueous solution of 4M guanidine sulfate and 0.1M spermidine, and the lysate fraction of the tumor tissue was dissolved with an aqueous solution of 4M guanidine sulfate and 0.1M spermidine, then 20 volumes of isopropanol were added, and left to stand for 3 hours for precipitation. And then centrifuging at 3500RPM for 10 minutes, and secondarily dissolving the precipitate part by using a water solution of 4M guanidine sulfate and 0.1M spermidine to obtain the antigen component 1.

Back subcutaneous inoculation of 1.5X10 s per C57BL/6 mice ⁶ MC38 colon cancer cells grow to a volume of about 1000mm in a tumor ³ Mice were sacrificed and tumor tissues were harvested, the tumor tissues were diced and fixed with 50% aqueous ethanol (containing 0.1% hypochlorous acid) for 2 hours, then centrifuged at 2000RPM for 5 minutes, the supernatant was discarded, the fixed tumor tissue pellet was lysed with an appropriate amount of aqueous solution of 4M guanidine sulfate and 0.1M spermidine, and the lysate fraction of the tumor tissue was solubilized with an aqueous solution of 4M guanidine sulfate and 0.1M spermidine, i.e., antigen fraction 2.

(2) Preparation of antigen component loaded antigen delivery nanoparticles

In this example, nanoparticle 1 (Nanoparticle 1) was prepared by the multiple emulsion method in the solvent evaporation method. The molecular weight of the PLGA used for preparing the nano particles is 10kDa-20kDa. The immunoadjuvants used were poly (I: C), cpG SL01 and CpG SL03. Preparation method As described above, the preparation was carried out by loading the cell antigen component 1 and the adjuvant into the nanoparticle, centrifuging 100mg of the nanoparticle 1 at 12000g for 30 minutes, re-suspending with 10mL of ultra-pure water containing 6% trehalose, freeze-drying for 48 hours, and irradiating with electron beam for 2 hours. The average particle size of the nanoparticle 1 is about 280nm, and each 1mg of PLGA nanoparticle 1 is loaded with about 2.0mg of protein or polypeptide component, and 0.02mg of poly (I: C), cpG SL01 and CpG SL03.

Nanoparticle 2 (Nanoparticle 2) was prepared and materials were the same as Nanoparticle 1 in this example. The preparation method comprises the steps of loading cell antigen component 2 and an adjuvant into the nano particles, centrifuging 100mg of the nano particles 2 at 12000g for 30 minutes, re-suspending the nano particles by using 10mL of ultrapure water containing 6% trehalose, freeze-drying the nano particles for 48 hours, and irradiating the nano particles by using an electron beam for 2 hours. The average particle size of the nanoparticle 2 is about 230nm, and each 1mg of PLGA nanoparticle 2 is loaded with about 2.0mg of protein or polypeptide component, and 0.02mg of poly (I: C), cpG SL01 and CpG SL 03.

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

This example illustrates how DCs can be prepared by taking the preparation of dendritic cells from mouse bone marrow cells as an example. Firstly, taking C57BL/6 mice with 6-8 weeks of age, killing cervical dislocation, taking out tibia and femur of the rear leg after operation, putting the tibia and femur into PBS, and removing muscle tissues around the bone by scissors and forceps. Cutting off two ends of bone with scissors, extracting PBS solution with syringe, inserting needle into bone marrow cavity from two ends of bone, and repeatedly flushing bone marrow into culture dish. Bone marrow solution was collected, centrifuged at 400g for 3min, and 1mL of erythrocyte lysate split red was added. Lysis was terminated by adding 3mL of RPMI 1640 (10% FBS) medium, centrifuging 400g for 3min, and discarding the supernatant. Cells were placed in 10mm dishes and cultured using RPMI 1640 (10% FBS) complete medium with the addition of recombinant mouse GM-CSF (20 ng/mL) at 37℃with 5% CO ₂ Culturing for 7 days. The flask was gently shaken on day 3 and the same volume of RPMI1640 (10% FBS) medium containing GM-CSF (20 ng/mL) was supplemented. On day 6, the medium was subjected to half-volume liquid exchange. On day 7, small amounts of suspended and semi-adherent cells were collected and assayed by flow as CD86 ⁺ CD80 ⁺ Cell in CD11c ⁺ The ratio of cells is 15-20%, and the induced BMDC can be used for the next experiment.

B cells derived from mouse spleen cells were used. The preparation method comprises killing mice, preparing single cell suspension of spleen cells, and separating by magnetic bead separation method to obtain CD19 ⁺ B cells of (a).

BMDC (1000 ten thousand) and B cells (1000 ten thousand) were mixed in a quantitative ratioAfter 1:1 mixing, 2mg of nanoparticles (nanoparticle 1, or nanoparticle 2) were co-incubated in 10mL of RPM I1640 complete medium containing 20ng/mL interleukin 15 (IL-15) for 24 hours (37 ℃,5% CO) ₂ ). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove free nanoparticles in the system, and then the mixed cells activated by antigen delivery particles were used as cancer vaccines. Wherein the mixed cells activated by using the nanoparticle 1 are the mixed Cell cancer vaccine 1 (Cell vaccinee 1); the mixed cells activated using nanoparticle 2 were mixed Cell cancer vaccine 2 (Cell vaccinee 2).

(4) DC vaccine for treating cancer

Preparation of melanoma tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 ⁶ Individual MC38 colon cancer cells. 100 ten thousand cell vaccines (cell vaccine 1 or cell vaccine 2) or 100 μl of PBS were injected subcutaneously in mice on day 3, 6, 9, 14, 19 and 25, respectively, after tumor inoculation of the mice. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.

(4) Experimental results

As shown in fig. 7, the PBS group mice rapidly grew in tumor volume and the mice died quickly. The survival time of the mice using the Cell vaccine 1 (Cell vaccinee 1) and the Cell vaccine 2 (Cell vaccinee 2) is obviously prolonged, most of the mice are healed without tumor, and the effect of the Cell vaccine 1 is better than that of the Cell vaccine 2.

In this embodiment, before lysing tumor tissue and lysing tumor tissue lysate components using a lysing solution containing a lysing agent, tumor tissue is fixed using a 50% aqueous ethanol solution containing an oxidizing agent, and other fixatives may be used to fix tumor tissue or cancer cells in practice. The fixative solution that may be used includes, but is not limited to, one or more of acetone, acetic acid, propionic acid, butyric acid, formic acid, aldehyde fixative solution, mercury fixative solution, alcohol fixative solution, oxidant fixative solution, picrate fixative solution, fixatives or fixatives for other cells or tissues, ethylene oxide, and the like.

Example 6 nanoparticles for detection of cancer cell-specific T cells (tumor antigen-specific T cells) in immune cells

(1) Preparation of antigenic components

The human lung cancer cell lines A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells and H520 cells were cultured, respectively, wherein the A549 cells, H1299 cells, PC9 cells and H1437 cells belong to lung cancer subtype lung adenocarcinoma cell lines, and the H226 cells, HCC1588 cells, H2170 cells and H520 cells belong to lung cancer subtype lung squamous carcinoma cell lines. After the 8 cells were collected, the a549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed according to a cell number ratio of 1:1:1:1:1:1:1:1, 3mL of an aqueous 8M urea peroxide solution was added to resuspend the cells and lyse cancer cells, and 5mL of an aqueous 8M urea peroxide solution was added to lyse the lysate fraction. Then adding 20mL of acetone into the redissolved lysate component, standing for 4 hours, centrifuging for 5 minutes at 5000RPM, discarding the supernatant, and then secondarily dissolving the precipitate by using 8M urea peroxide aqueous solution to obtain the antigen component 1.

(2) Preparation of antigen component-loaded nanoparticles

In this example, nanoparticle 1 (NP 1) was prepared by the multiple emulsion method of the solvent evaporation method. The PLGA molecular weight of the adopted nanoparticle preparation material is 10kDa-30kDa, the preparation method is as described above, in the preparation process, firstly, antigen component 1 is loaded in the nanoparticle by adopting a multiple emulsion method, then 100mg of the nanoparticle is centrifuged at 15000g for 30 minutes, and 10mL of ultra-pure water containing 4% trehalose is used for resuspension and then freeze drying is carried out for 48 hours. The average particle size of the nanoparticle 1 is about 200nm, and about 500 mug of protein and polypeptide components are loaded per 1mg of PLGA nanoparticle.

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

The tumor volume of the patient A with the non-small cell lung cancer is obviously reduced after the patient A is subjected to PD-1 antibody immunotherapy. 8mL of peripheral blood of the non-small cell lung cancer patient A was withdrawn 3 weeks before and after the immunotherapy, respectively. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from 8mL of peripheral blood of a non-small cell lung cancer patient using gradient centrifugation.

Nanoparticle 1 (1.5 mg) was co-incubated with PBMC (300 ten thousand) in 3mL AIM V serum-free medium for 24 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the Or control group incubated PBMC (300 ten thousand) alone in 3mL AIM V serum-free medium for 24 hr (37deg.C, 5% CO) ₂ ). Afterwards, the samples were centrifuged at 400g for 5 min, the supernatant was discarded, the cell pellet was collected, and the cells were then resuspended in PBS. Living and dead cell dyes and Fc blocks were first used to incubate with cells, respectively, to label living cells and avoid non-specific antibody adsorption, followed by staining with CD3 antibodies and IFN-gamma antibodies. The stained cells were then analyzed by flow cytometry for CD3 ⁺ IFN-γ ⁺ T cells of (c) at all CD3 ⁺ The proportion of T cells is cancer cell specific T cells.

(4) Experimental results

As shown in FIG. 8, incubation of PBMC alone hardly detected any activated and killing cancer cell-specific T cells before (pre-therapy) and after (post-therapy). The cells incubated with Nanoparticle 1 (Nanoparticle 1) can detect a certain amount of cancer cell-specific T cells both before and after treatment, and the amount after treatment is significantly higher than before treatment.

It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the invention. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments 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 an antigen component, comprising the steps of:

(1) Lysing the cells and/or tissue to obtain a cell lysate component;

2. The method of claim 1, wherein after said mixing in step (3), centrifuging, removing the lower layer and mixing with a second dissolution solution containing a dissolution agent to obtain said antigen component; or, after the mixing in the step (3), centrifuging, mixing the lower layer with a second dissolving solution containing a dissolving agent, and then mixing with a component obtained by separating and purifying the supernatant after centrifuging to obtain the antigen component.

3. The method according to claim 1 or 2, wherein 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 lysate component-containing solution to the organic solvent is from 1:0.5 to 30, preferably from 1:0.5 to 25, more preferably from 1:0.5 to 20.

4. The method of claim 1, wherein the lytic agent is independently selected from one or more of a compound comprising the 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:

R ₁ c, S, P, N or O, R ₂ -R ₅ Independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidino;

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

5. The method of claim 1, further comprising treating the cells and/or tissue prior to and/or after lysis,

preferably, the treatment comprises enhancing the immunogenicity of the antigen component and/or immobilizing the cells and/or tissue;

preferably, the method of enhancing the immunogenicity of the antigenic component comprises irradiation, oxidation, reduction, modification with hapten substances, immobilization, enzymatic treatment, denaturation, heating, mineralization;

preferably, the irradiation comprises any commonly used irradiation method; more preferably, the irradiation comprises one or more of radioactive substance irradiation, electron beam irradiation, microwave irradiation, ultraviolet irradiation, X-ray irradiation, alpha-ray irradiation, beta-ray irradiation, gamma-ray irradiation;

preferably, the oxidation is oxidation of the antigenic component using an oxidizing agent; more preferably, the oxidizing agent comprises hypochlorous acid, hydrogen peroxide, persulfates, dichromates, peroxyacetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO ₃ 、KBrO ₃ 、ClO ₃ ^- 、ClO ₄ ^- 、Na ₂ O ₂ 、K ₂ O ₂ 、MgO ₂ 、CaO ₂ 、BaO ₂ 、NO ₃ ^- 、MnO ₄ ^- 、F ₂ 、Cl ₂ 、O ₂ 、Br ₂ 、I ₂ 、S、Si、HNO ₃ 、MnO ₂ 、FeCl ₃ One or more of the following;

Preferably, the hapten substance comprises one or more of 2, 4-dinitrofluorobenzene, 2, 4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, ovlbumin, N-iodoacetyl-N' - (5-sulfonic acid 1-naphthyl) ethylene diamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other hapten substances containing aldehyde groups, rhamnose, galactose and galactosamine;

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 fixing solution, PLP fixing solution, FPA fixing solution, TAF fixing solution, rosman fixing solution, regaud fixing solution, PLPD fixing solution, PAPG fixing solution, orth fixing solution, muller fixing solution, mcDoWell fixing solution, neutral calcium formaldehyde fixing solution, FAB fixing solution, carnoy fixing solution, clarke fixing solution, B-5 fixing solution, bouin fixing solution, fineFIX fixing solution, a.g.m fixing solution, helly fixing solution, zenker fixing solution, kolmer fixing solution, AAF fixing solution, hollande fixing solution, gendre fixing solution, aldehyde fixing solution, mercury fixing solution, alcohol fixing solution, oxidizing agent fixing solution, picrate fixing solution, and ethylene oxide fixing solution are used.

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

7. The delivery particle of claim 6, further having a composition as shown below:

(i) A framework structure formed by the particle materials;

(ii) An immunoadjuvant;

(iii) A positively charged species;

preferably, the immune adjuvant is selected from one or more of pattern recognition receptor class agonists, toll-like receptor agonists, BCG cell wall backbones, BCG methanol extraction residues, BCG cell wall dipeptides, mycobacterium phlei, polyoxin a, mineral oil, virus-like particles, immunopotentiating reconstituted influenza virus minibodies, cholera enterotoxin, saponins and derivatives thereof, resquimod, thymosin, neonatal bovine liver active peptides, imiquimod, polysaccharides, curcumin, immune adjuvant CpG, immune adjuvant poly (I: C), immune adjuvant poly ICLC, short coryneform bacterin, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukins, interferons, polymyonic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokines, mRNA, MF59, double-stranded RNA, double-stranded DNA, single-stranded DNA, aluminum adjuvant, manganese adjuvant, calcium adjuvant, STING agonist, endotoxin, plastid, ginseng adjuvant, CAF01, and an active ingredient; preferably one or more selected from Toll-like receptor 3 agonists and Toll-like receptor 9 agonists; more preferably one or more selected from the group consisting of Poly (I: C), poly ICLC, A class CpG-OND, B class CpG-OND and C class CpG-OND;

Preferably, the antigenic component is supported within and/or on the surface of the scaffold structure;

preferably, the positively charged substance is selected from one or more of a positively charged amino acid, a positively charged polypeptide, a positively charged lipid, a positively charged protein, a positively charged polymer, and/or a positively charged mineral; preferably selected from melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH ₄ HCO ₃ One or more of the following.

8. The delivery particle of claim 6 or 7, wherein the delivery particle is a nano-delivery particle and/or a micro-delivery particle;

the particle size of the nano-delivery particles is 1nm-1000nm, preferably 50-500nm, more preferably 100-400nm;

preferably, the micrometer delivery particles have a particle size of 1 μm to 1000 μm, preferably 1 to 10 μm, more preferably 1 to 5 μm;

preferably, 1mg of the nano-and/or micro-delivery particles load the antigen component with an amount of protein or polypeptide component of 0.01-3mg, preferably 0.02-2mg;

9. A pharmaceutical composition comprising an antigenic component prepared by the method of any one of claims 1-5 or the delivery particle of any one of claims 6-8;

10. Use of an antigenic component prepared by the method of any one of claims 1-5 or the delivery particle of any one of claims 6-8 or the pharmaceutical composition of claim 9 in at least one of the following (1) - (4):

(1) Preparing a medicament for preventing or treating a disease;

11. The use according to claim 10, wherein the delivery particles are used directly as a vaccine;

preferably, after the delivery particles activate the dendritic cells and/or B cells, the activated dendritic cells and/or B cells are used as a cellular vaccine;

12. The use according to claim 10 or 11, wherein the disease is cancer or a tumour; preferably, the disease is a solid tumor or hematological tumor.