Preparation method for antigen delivery particle and use thereof

Abstract

A preparation method for an antigen delivery particle and use thereof. The antigen delivery particle is used to load an antigen component subjected to fixation treatment with a tissue fixation liquid. The tissue fixation liquid consists of an alcohol and / or a ketone and / or an acid, and an aqueous solution containing an oxidizing agent. The preparation method is simple, rapid, and low-consumption. The solution viscosity is low during centrifugation, eliminating the need for high-speed centrifugation to obtain a precipitate. In addition, freezing of cells or tissue samples is not required, thereby facilitating the storage and transportation of biological samples such as tumor tissues or cancer cells. Meanwhile, oxidizing substances such as hypochlorous acid or hydrogen peroxide in the tissue fixation liquid can oxidize the antigen component in the cells, thereby improving the immunogenicity of the antigen component.

Description

Preparation method and application of antigen delivery particles

[0001] This disclosure claims priority to the Chinese patent application filed with the Patent Office of China on November 28, 2023, with the invention name “A method for preparing antigen delivery particles and their application” and application number 202311600664.7. The entire contents of the above application are incorporated into this disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of immunotherapy, and in particular to a preparation method and application of antigen delivery particles. Background Art

[0003] Cancer vaccines are one of the important methods of immunotherapy for diseases such as cancer. The main factors affecting vaccines include antigens, adjuvants, and delivery formulations. Taking cancer vaccines as an example, the main factors that have the greatest impact on cancer vaccines include tumor antigens, adjuvants, and delivery formulations. Among them, antigens can induce and activate specific immune responses, adjuvants can amplify specific immune responses, and formulations can affect the efficiency of antigen-presenting cells (APCs) in phagocytosis of vaccines and subsequent activation of antigen-specific T cells. Among these three, antigens are the main components that can trigger specific immune responses and are therefore the most critical factors. Cancer cells and / or tumor tissues contain all cancer cell-specific antigens and cancer cell-associated antigens, and are the best raw materials for the preparation of tumor antigen delivery particles or cancer vaccines.

[0004] However, cancer cells or tumor tissue cannot be stored at room temperature for a long time, especially tumor tissue after surgical resection. If stored at room temperature for a long time, the cells in the tumor tissue will undergo autophagy or death, and many antigen proteins will be degraded, thereby reducing the content of antigen components. Therefore, the storage of cancer cells or tumor tissue requires freezing them to below -20 degrees Celsius and using cold chain transportation during transportation. Freezing below -20 degrees Celsius requires low-temperature refrigerators, etc., and cold chain transportation also incurs high storage and transportation costs.

[0005] Summary of the Invention

[0006] Problems to be solved by the invention

[0007] In response to the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a tissue fixative that can directly fix cells or tissues for storage and transportation at room temperature, while enhancing the immunogenicity of antigen components in cells or tissues and improving the efficacy of antigen delivery particles subsequently prepared using antigen components fixed with the tissue fixative.

[0008] Solutions for solving problems

[0009] A method for preparing antigen delivery particles comprises using the antigen delivery particles to load an antigen component, wherein the antigen component is fixed with a tissue fixative, wherein the tissue fixative comprises alcohols and / or ketones and / or acids and an aqueous solution containing an oxidant, wherein the content of the alcohols and / or ketones and / or acids in the tissue fixative is 1 wt%-99 wt%, and the content of the oxidant in the aqueous solution is 0.001 wt%-30 wt%.

[0010] Preferably, the content of alcohols and / or ketones and / or acids in the tissue fixative is 10wt%-95wt%; preferably, the content of oxidant in the aqueous solution is 0.01wt%-25wt%, preferably 0.1wt%-20wt%.

[0011] Preferably, the alcohols and / or ketones and / or acids are alcohols and / or ketones and / or acids that are liquid at 0-50 degrees Celsius, and the oxidizing agent can oxidize the antigen component;

[0012] Preferably, the alcohols and / or ketones and / or acids are selected from one or more of ethanol, methanol, propanol, isopropanol, butanol, isobutanol, acetone, butanone, formic acid, acetic acid, propionic acid, and butyric acid, preferably one or more of ethanol, methanol, acetone, and acetic acid;

[0013] Preferably, the oxidant is selected from hypochlorous acid, hydrogen peroxide, persulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO3, KBrO3, ClO3 - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, NO3 - 、MnO4 - , one or more of F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3, preferably hydrogen peroxide and / or hypochlorous acid.

[0014] Preferably, the method comprises the following steps:

[0015] (1) Fixing cells and / or tissues containing antigen components using a tissue fixative consisting of an alcohol and / or ketone and / or acid and an aqueous solution containing an oxidant;

[0016] (2) After centrifugation at 200g-15000g, the supernatant was removed and the precipitate was collected;

[0017] (3) lysing the collected cells and / or tissues using a lysis solution containing a lysis agent, and dissolving the lysate components after lysis using a lysis solution containing a lysis agent;

[0018] (4) directly using the dissolved lysate component obtained in step (3) as the antigen component; or subjecting the lysate component obtained in step (3) to separation and purification steps such as dilution, salting out, organic solvent precipitation, and heating to obtain a purified component, which is then re-dissolved in a dissolving solution containing a dissolving agent and used as the antigen component;

[0019] Wherein, the dissolving agent is independently selected from one or more of a compound containing a structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

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

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

[0022] Preferably, the fixed time in step (1) is greater than 1 hour;

[0023] Preferably, the organic solvent precipitation is performed using one or more of alcohols, phenols, ketones, acids, and nitriles;

[0024] Preferably, the cells are cells containing antigen components, and the tissues are tissues containing antigen components; more preferably, the cells are cancer cells, and the tissues are tumor tissues.

[0025] Preferably, the method further comprises the steps of:

[0026] (5) The antigen component is further loaded into the interior and / or surface of the nanoparticles and / or microparticles to obtain antigen delivery particles.

[0027] Preferably, the antigen delivery particle has the following components:

[0028] (i) nanoparticle and / or microparticle framework structures formed by particulate materials;

[0029] (ii) lysing a lysate fraction of cells and / or tissue using a lysis solution containing a lytic agent, or isolating and purifying an antigen fraction therefrom after lysis;

[0030] Preferably, the lysate component is a whole-cell lysate component from cells and / or tissues, more preferably, the lysate component is a partial cell and / or tissue lysate component containing an antigen component;

[0031] Preferably, the antigen component is loaded inside and / or on the surface of the skeleton structure;

[0032] Preferably, appropriate methods can be used to enhance the immunogenicity of the antigenic component before or after cell lysis;

[0033] Preferably, the method for enhancing the immunogenicity of the antigen component includes irradiation, oxidation, reduction, modification with a hapten substance, enzyme treatment, denaturation, heating, and mineralization. Preferably, the irradiation includes any commonly used irradiation method;

[0034] Preferably, the irradiation includes one or more of radioactive material irradiation, electron beam irradiation, microwave irradiation, ultraviolet irradiation, X-ray irradiation, α-ray irradiation, β-ray irradiation, and γ-ray irradiation.

[0035] Preferably, the hapten substance includes one or more of 2,4-dinitrofluorobenzene, 2,4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, Ovalbumin, N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other hapten substances containing aldehyde groups, rhamnose, galactose, and aminogalactose.

[0036] Preferably, the immunogenicity of the whole cell lysate fraction and / or the partial cell lysate fraction containing the antigenic component from cells and / or tissues can be enhanced by appropriate methods;

[0037] Preferably, the whole cell lysate component from cells and / or tissues and / or the partial cell lysate component containing the antigen component that enhances immunogenicity are loaded inside and / or on the surface of the skeleton structure;

[0038] Preferably, the antigen component contained in the partial cell lysate component comprises the protein and polypeptide components in the cell and / or tissue lysate and / or the RNA component or mRNA component in the cell lysate;

[0039] Preferably, the antigen component contained in the partial cell lysate component comprises protein and polypeptide components in cell and / or tissue lysate and / or lipid components in cell lysate;

[0040] Preferably, 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 mRNA component in a cell lysate;

[0041] Preferably, the antigen component is separated and purified using an appropriate method from a lysate component dissolved in a lysis solution containing a lytic agent;

[0042] Preferably, the antigen components separated and purified from the lysate components can also be irradiated before or after cell or tumor tissue lysis, or can be irradiated after the separated and purified antigen components are loaded onto nanoparticles and / or microparticles.

[0043] Preferably, the antigen delivery particle is further loaded with at least one component as shown below:

[0044] (iii) immune adjuvants;

[0045] (iv) positively charged substances;

[0046] Preferably, the immune adjuvant comprises at least one of the following: pattern recognition receptor agonists, Toll-like receptor agonists, Bacillus Calmette-Guérin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyantigen A, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhancing reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic Streptococcus preparation, coenzyme QIO, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokine, mRNA, MF59, double-stranded RNA, double-stranded DNA, single-stranded DNA, aluminum adjuvant, manganese adjuvant, calcium adjuvant, STING agonist, endotoxin adjuvant, liposome adjuvant, CAF01, ginseng active ingredient, astragalus active ingredient;

[0047] More preferably, the immune adjuvant comprises at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist;

[0048] Further preferably, the immune adjuvant comprises at least one of Poly(I:C), Poly ICLC, Class A CpG-OND, Class B CpG-OND and Class C CpG-OND;

[0049] Preferably, the positively charged substance is selected from one or more of positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and / or positively charged inorganic substances;

[0050] More preferably, the positively charged substance is selected from one or more of melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

[0051] Preferably, the particle size of the nanoparticles is 1 nm-1000 nm, preferably 50-500 nm, more preferably 100-400 nm; preferably, the particle size of the microparticles is 1 μm-1000 μm, preferably 1-10 μm, more preferably 1-5 μm.

[0052] The present invention also provides an antigen delivery particle prepared according to the method.

[0053] The present invention also provides a use of the antigen delivery particle in at least one of the following (1)-(4):

[0054] (1) Preparation of drugs for preventing and / or treating diseases;

[0055] (2) Used to activate antigen-presenting cells and prepare cellular vaccines based on antigen-presenting cells;

[0056] (3) Assisted activation of antigen-specific T cells and detection of antigen-specific T cell content;

[0057] (4) After assisting in the activation of antigen-specific T cells, the activated antigen-specific T cells are isolated and amplified, and used to prevent or treat diseases.

[0058] Preferably, the antigen delivery particles can be used directly as a vaccine;

[0059] Preferably, after the antigen delivery particles activate dendritic cells and / or B cells, the activated dendritic cells and / or B cells can be used as a cell vaccine;

[0060] More preferably, the antigen delivery particles can be used to detect the content of antigen-specific T cells after assisting in the activation of antigen-specific T cells in vitro, or the activated antigen-specific T cells can be sorted and / or amplified after assisting in the activation of antigen-specific T cells by the antigen delivery particles for the prevention and / or treatment of diseases.

[0061] Preferably, the disease is cancer or tumor;

[0062] More preferably, the disease is a solid tumor or a hematological tumor.

[0063] Effects of the Invention

[0064] The present invention provides a tissue fixative containing alcohols, ketones, and / or acids, as well as an oxidant. It also provides a method for using the tissue fixative to first fix cells and / or tissues, then lyse the cells and / or tissues, and then prepare antigen delivery particles. This method is simple and rapid, does not require freezing of cell or tissue samples, and facilitates the storage and transportation of biological samples such as tumor tissue or cancer cells. After fixation with the tissue fixative, proteins and other substances within the cells and / or tissues lose their activity, thus preventing the degradation of proteins within the cells by proteolytic enzymes and other substances within the cells or tissues due to inactive proteins. Furthermore, oxidizing substances such as hypochlorous acid or hydrogen peroxide in the tissue fixative can oxidize antigen components within the cells, thereby increasing their immunogenicity. Using a mixed fixative containing alcohols, ketones, and / or acids with an aqueous solution containing an oxidant can rapidly inactivate proteases and other substances within the cells, increase the immunogenicity of the antigen components through oxidation, and facilitate the rapid and convenient collection and transportation of samples for preparing antigen delivery particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG1 is a schematic diagram showing the preparation process and application of using a tissue fixative to fix cells or tissues, and then using antigen components treated with the tissue fixative to prepare antigen delivery particles.

[0066] Figure 2 shows the structure of formula 1, wherein R1 is C, S, P, N or O, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine, and any other groups.

[0067] Figures 3 to 7 are the experimental results of tumor growth rate and survival when nanovaccines and / or micron vaccines or antigen presenting cell vaccines or assisted sorting and amplified T cells loaded with separated and purified antigen components are used to prevent or treat cancer in Examples 1-5; wherein, a is the experimental result of tumor growth rate when preventing or treating cancer (n≥8); b is the experimental result of mouse survival when preventing or treating cancer (n≥8), and each data point is the mean ± standard error (mean±SEM); wherein, the significant difference in the tumor growth inhibition experiment in Figure a was analyzed by ANOVA, and the significant difference in Figure b was analyzed by Kaplan-Meier and log-ranktest.

[0068] FIG8 shows the experimental results of detecting antigen-specific T cells using antigen delivery nanoparticles in Example 6.

[0069] In the above figure, *** indicates that p < 0.005 is significantly different compared with the PBS control group; ** indicates that p < 0.01 is significantly different compared with the PBS control group; ### indicates that p < 0.005 is significantly different compared with the two groups; ## indicates that p < 0.01 is significantly different compared with the two groups; # indicates that p < 0.05 is significantly different compared with the two groups. DETAILED DESCRIPTION

[0070] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following is a detailed description by listing specific embodiments. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0071] The experimental techniques and methods used in this example are conventional unless otherwise specified. For example, in the following examples, where specific conditions are not specified, the experimental methods were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. The materials and reagents used in the examples were all obtained through regular commercial channels unless otherwise specified.

[0072] In the claims and / or description of the present invention, the term "a" or "an" or "the" may mean "one", but may also mean "one or more", "at least one" and "one or more than one".

[0073] As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0074] The term "suffering from a disease" means that the body is experiencing symptoms of a disease.

[0075] The term "treatment" means that after a subject has contracted a disease, the subject is exposed to (e.g., administered) a delivery particle, delivery system, vaccine, antigen-loaded drug, or pharmaceutical composition, thereby alleviating the symptoms of the disease compared to the absence of such exposure, and does not necessarily mean that the symptoms of the disease are completely suppressed.

[0076] The term "prevention" means that before a subject develops a disease, the subject is exposed to (e.g., administered) the delivery particles, delivery systems, vaccines, antigen-loaded drugs, or pharmaceutical compositions of the present disclosure, thereby alleviating symptoms after developing the disease compared to when the subject has not developed the disease. It does not necessarily mean that the disease must be completely suppressed.

[0077] The vaccines of the present disclosure can be prepared using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding and / or lyophilizing processes.

[0078] In the present disclosure, the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the nature of the drug, the convenience of the patient and medical staff, and other relevant factors.

[0079] The terms "individual," "patient," or "subject" as used herein include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0080] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors. The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0081] The term "antigen-presenting cell" or "APC" refers to a type of immune cell that can take up, process, and present antigens to T cells. APCs primarily include monocytes, dendritic cells, B cells, Langerhans cells, and virally infected target cells of tumor cells. "Dendritic cell" and "DC" are used interchangeably herein.

[0082] The terms "hydrogen peroxide," "hydrogen peroxide," or "H2O2" are used interchangeably herein.

[0083] When preparing the antigen components disclosed in the present invention, some embodiments use tumor tissue or cancer cell lines. In actual applications, cancer cells / tumor tissues obtained by several methods, such as tumor tissue, cancer cell lines, cancer cells isolated from tumor tissue and cultured and amplified, and cancer cells obtained by cultured and amplified circulating tumor cells isolated from peripheral blood, can be used to prepare antigen components. In actual applications, any other feasible approach can also be used to obtain cancer cells. In actual applications, other cells or tissues can also be used to prepare antigen components for other diseases, such as using β cells to prepare antigen components for type 1 diabetes, and using pancreatic tissue or pancreatic islet tissue to prepare antigen components for type 1 diabetes.

[0084] Due to space limitations, the cancers described in the examples of this disclosure are solid tumors. In practical applications, the nano- or micro-vaccines described herein can also be used to treat hematologic malignancies and lymphomas. Because the immune microenvironment of hematologic malignancies and lymphomas is less complex than that of solid tumors, the nano- or micro-vaccines described herein are more effective in treating hematologic malignancies and lymphomas.

[0085] The present invention provides a method for preparing antigen delivery particles, which uses antigen delivery particles to load antigen components, wherein the antigen components are fixed with a tissue fixative, wherein the tissue fixative consists of alcohols and / or ketones and / or acids and an aqueous solution containing an oxidant, wherein the content of the alcohols and / or ketones and / or acids in the tissue fixative is 1wt%-99wt%, and the content of the oxidant in the aqueous solution is 0.001wt%-30wt%.

[0086] In certain embodiments, the content of alcohols and / or ketones and / or acids in the tissue fixative is 10 wt % to 95 wt %.

[0087] In certain embodiments, the content of alcohols and / or ketones and / or acids in the tissue fixative is 30 wt % to 75 wt %.

[0088] In certain embodiments, the content of alcohols and / or ketones and / or acids in the tissue fixative is 50 wt % to 70 wt %.

[0089] In some embodiments, the content of alcohols and / or ketones and / or acids in the tissue fixative is 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt% or 95 wt%.

[0090] In certain embodiments, the content of the oxidant in the aqueous solution is 0.01 wt%-25 wt%.

[0091] In certain embodiments, the content of the oxidant in the aqueous solution is 0.1 wt%-20 wt%.

[0092] In certain embodiments, the content of the oxidant in the aqueous solution is 0.5 wt % to 3 wt %.

[0093] In certain embodiments, the amount of oxidant in the aqueous solution is 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt% or 20 wt%. In certain embodiments, the alcohols and / or ketones and / or acids are alcohols and / or ketones and / or acids that are liquid at 0-50 degrees Celsius.

[0094] In certain embodiments, the alcohols and / or ketones and / or acids are selected from one or more of ethanol, methanol, propanol, isopropanol, butanol, isobutanol, acetone, butanone, formic acid, acetic acid, propionic acid, and butyric acid.

[0095] In certain embodiments, the alcohols and / or ketones and / or acids are selected from one or more of ethanol, methanol, acetone, and acetic acid.

[0096] In certain embodiments, the alcohol is ethanol and / or methanol.

[0097] In certain embodiments, the alcohol is ethanol.

[0098] In certain embodiments, the alcohol is methanol.

[0099] In certain embodiments, the ketone is acetone.

[0100] In certain embodiments, the acid is acetic acid.

[0101] In certain embodiments, the oxidizing agent can oxidize the antigenic component.

[0102] In certain embodiments, the oxidant is selected from hypochlorous acid, hydrogen peroxide (hydrogen peroxide, H2O2), persulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO3, KBrO3, ClO3 - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, NO3 - 、MnO4 -, one or more of F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, and FeCl3.

[0103] In certain embodiments, the oxidizing agent is hydrogen peroxide and / or hypochlorous acid.

[0104] In certain embodiments, the oxidizing agent is hydrogen peroxide.

[0105] In certain embodiments, the oxidizing agent is hypochlorous acid.

[0106] In certain embodiments, the method comprises the steps of:

[0107] (1) Fixing cells and / or tissues containing antigen components using a tissue fixative consisting of an alcohol and / or ketone and / or acid and an aqueous solution containing an oxidant;

[0108] (2) After centrifugation at 200g-15000g, the supernatant was removed and the precipitate was collected;

[0109] (3) lysing the collected cells and / or tissues using a lysis solution containing a lysis agent, and dissolving the lysate components after lysis using a lysis solution containing a lysis agent;

[0110] (4) directly using the dissolved lysate component obtained in step (3) as the antigen component; or subjecting the lysate component obtained in step (3) to separation and purification steps such as dilution, salting out, organic solvent precipitation, and heating to obtain a purified component, which is then re-dissolved in a dissolving solution containing a dissolving agent and used as the antigen component;

[0111] wherein the dissolving agent is independently selected from one or more of a compound containing the structure of formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline;

[0112] Wherein, structural formula 1 is as follows:

[0113] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0114] In certain embodiments, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, spermine, spermidine, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

[0115] In certain embodiments, the fixed time in step (1) is greater than 1 hour;

[0116] In certain embodiments, the organic solvent precipitation is performed using one or more of alcohols, phenols, ketones, acids, and nitriles.

[0117] In certain embodiments, the organic solvent precipitation is ethanol precipitation, methanol precipitation, isopropanol precipitation, acetone precipitation, phenol precipitation, acetonitrile precipitation, or acetic acid precipitation.

[0118] In certain embodiments, the cell is a cell containing an antigenic component, and the tissue is a tissue containing an antigenic component.

[0119] In certain embodiments, the cell is a cancer cell and the tissue is a tumor tissue.

[0120] In certain embodiments, the method further comprises the steps of:

[0121] (5) The antigen component is further loaded into the interior and / or surface of the nanoparticles and / or microparticles to obtain antigen delivery particles.

[0122] In certain embodiments, after cells and / or tissues are fixed using the tissue fixative described in the present invention, the fixed cells and / or tissues are used to prepare vaccines and / or antigen delivery particles. The method comprises first fixing the cells and / or tissues using a tissue fixative, then removing the tissue fixative by centrifugation and collecting the fixed cells and / or tissues, then lysing the cells and / or tissues using a lysis solution containing a solvent, and then solubilizing the lysate using a lysis solution containing a solvent, and then directly using the solubilized lysate component as an antigen component, or purifying the lysate component using a dilution method, a heating method, an organic solvent precipitation (such as ethanol precipitation, methanol precipitation, isopropanol precipitation, acetone precipitation, phenol precipitation, acetonitrile precipitation, acetic acid precipitation, etc.), or a salting-out method, and then re-dissolving the obtained component using a lysis solution containing a solvent and using it as an antigen component.

[0123] The antigen components fixed with the fixative are then loaded onto nanoparticles and / or microparticles for use as vaccines or antigen delivery particles. When used as vaccines, they can be used to prevent or treat diseases. When used as antigen delivery particles, they can be used to activate antigen-presenting cells in vitro to prepare antigen-presenting cell vaccines, or to assist in activating antigen-specific T cells and then detecting the content of antigen-specific T cells, or to assist in activating antigen-specific T cells and then sorting and amplifying the antigen-specific T cells for use in preventing or treating diseases.

[0124] In certain embodiments, the other treatment methods include but are not limited to salting out, organic solvent precipitation (such as ethanol precipitation, methanol precipitation, isopropanol precipitation, acetone precipitation, phenol precipitation, acetonitrile precipitation, acetic acid precipitation, etc.), oxidation, reduction, heating, radiation irradiation (irradiation), etc.

[0125] In certain embodiments, the steps for preparing the lysate from the cells and / or tissues are: first, using an aqueous solution containing alcohols and / or ketones and / or acids and an oxidant to fix the cells and / or tissues, then centrifuging to remove the tissue fixative and collecting the precipitated cells and / or tissues, then using a lysis solution containing a dissolving agent to lyse the cells and / or tissues to obtain their lysates, and then using a lysis solution containing a dissolving agent to dissolve the lysate components.

[0126] In certain embodiments, the step of isolating and purifying antigenic components from cells and / or tissues comprises:

[0127] (1) Fixing cells and / or tissues using the fixative for a certain period of time (greater than 1 hour);

[0128] (2) after centrifugation, the supernatant of the tissue fixative was removed and the precipitated cells and / or tissues were collected;

[0129] (3) Lysing cells and / or tissues using a lysis solution containing a lytic agent;

[0130] (4) using a dissolving solution containing a dissolving agent to dissolve the lysate components after lysis in step (1);

[0131] (5) directly using the above-mentioned lysate components as antigen components; or purifying the above-mentioned lysate components through dilution, salting out, organic solvent precipitation (such as ethanol precipitation, methanol precipitation, isopropanol precipitation, acetone precipitation, phenol precipitation, acetonitrile precipitation, acetic acid precipitation, etc.), heating, irradiation, etc., and then re-dissolving the precipitated part with a dissolving solution containing a dissolving agent and using it as an antigen component;

[0132] In certain embodiments, methods by which the antigenic component can be purified include, but are not limited to, salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, immobilization, mineralization, irradiation, and the like.

[0133] In certain embodiments, the dissolving agent is independently selected from one or more of a compound containing a structure of formula 1, deoxycholate, dodecyl sulfate, glycerol, a protein degrading enzyme, a polypeptide, an amino acid, a glycoside, and choline; wherein formula 1 is as follows:

[0134] R1 is C, S, P, N or O, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine.

[0135] In certain embodiments, compounds containing the structure of structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, urea peroxide, guanidine hydrochloride, guanidine isothiocyanate, guanidine sulfate, guanidine sulfonate, guanidine salts, urea salts, urea, spermine, spermidine, other compounds containing guanidine groups, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine sulfamate, guanidine succinic acid, semicarbazide hydrochloride, aminocarbamylurea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0136] In certain embodiments, the urea and guanidine hydrochloride contain the structure of Structural Formula 1. The inventors have discovered that substances having the structure of Structural Formula 1 can be used as dissolving agents in a dissolving solution to dissolve water-insoluble components in cells or tumor tissues. Therefore, in addition to common compounds containing the structure of Structural Formula 1, such as urea and guanidine hydrochloride, other compounds containing this structure also have the ability to act as dissolving agents to dissolve water-insoluble components.

[0137] In certain embodiments, the dissolving agent is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, spermine, spermidine, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.

[0138] In certain embodiments, the method further comprises the steps of:

[0139] (6) The separated and purified antigen components are further loaded into the interior and / or surface of nanoparticles and / or microparticles.

[0140] In certain embodiments, the method further comprises the steps of:

[0141] (7) Preparing the nanoparticles and / or microparticles loaded with the separated and purified antigen components into vaccines.

[0142] In certain embodiments, the nanoparticles and / or microparticles loaded with the separated and purified antigen components can be directly used as vaccines.

[0143] In certain embodiments, the nanoparticles and / or microparticles loaded with the separated and purified antigen components can also be used as antigen delivery particles to activate dendritic cells and / or B cells, and the activated dendritic cells and / or B cells can be used as cell vaccines.

[0144] In certain embodiments, the nanoparticles and / or microparticles loaded with separated and purified antigen components can also be used as antigen delivery particles to assist in activating cancer cell-specific T cells (tumor-specific T cells) in vitro and then be used to detect the content of cancer cell-specific T cells, or to assist in activating antigen-specific T cells and then separate and / or amplify the activated antigen-specific T cells for disease prevention and treatment.

[0145] In certain embodiments, the antigen delivery particle has the following components:

[0146] (i) nanoparticle and / or microparticle framework structures formed by particulate materials;

[0147] (ii) a whole cell lysate fraction and / or a partial cell lysate fraction containing an antigen component from cells (e.g., cancer cells) and / or tissues (e.g., tumor tissue);

[0148] In certain embodiments, the whole cell lysate component and / or the partial cell lysate component containing the antigen component is loaded inside and / or on the surface of the scaffold structure.

[0149] In certain embodiments, appropriate methods can be used to enhance the immunogenicity of the antigenic components before or after cell lysis.

[0150] In certain embodiments, the methods for enhancing the immunogenicity of the antigenic component include, but are not limited to, irradiation, oxidation, reduction, modification with haptenic substances, fixation, enzyme treatment, denaturation, heating, mineralization, and the like.

[0151] In certain embodiments, the cancer cells and / or tumor tissues are lysed using a lysis solution containing a dissolving agent, and the lysate obtained by lysis is solubilized using a lysis solution containing a dissolving agent; the antigen components contained in the partial cell components include protein and polypeptide components in the cancer cell and / or tumor tissue lysate and / or RNA components or mRNA components in the cell / tissue lysate.

[0152] In certain embodiments, the antigen component can be appropriately treated to enhance its immunogenicity. The treatment method for enhancing the immunogenicity of the antigen component can be before cell or tumor tissue lysis, after cell or tumor tissue lysis, or after the antigen component is loaded onto nanoparticles or microparticles.

[0153] In certain embodiments, methods for treating antigen components to enhance immunogenicity include, but are not limited to, irradiation with radiation, incubation of cells and / or tissues with specific substances, modification with hapten substances, oxidation, reduction, fixation, mineralization, enzyme treatment, and the like.

[0154] In certain embodiments, the irradiation is performed using one or more of different irradiation methods such as ultraviolet rays, X-rays, microwaves, electron beams, gamma rays, alpha rays, beta rays, radioactive sources, etc. for a period of time.

[0155] In certain embodiments, the precipitation using an organic solvent is but not limited to precipitation using alcohols, ketones, acids, or phenols, such as precipitation using ethanol, methanol, isopropanol, acetone, phenol, acetonitrile, or acetic acid.

[0156] In certain embodiments, the cancer cells and / or tumor tissues can be co-incubated with specific chemical substances to stimulate the cancer cells or tumor tissues before lysis. The specific substances that stimulate cancer cells include but are not limited to small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, cytokines, plant extracts (such as important extracts such as ginseng, plant root extracts, etc.), chemokines, interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate cancer cells and / or tumor tissues to stimulate cancer cells and / or tumor tissues is to cause the cancer cells to produce more antigen components.

[0157] In certain embodiments, the haptenic substance is a substance that can increase the immunogenicity of a protein and / or polypeptide after co-acting with the protein and / or polypeptide.

[0158] In certain embodiments, the hapten substance includes but is not limited to 2,4-dinitrofluorobenzene (DNFB), 2,4-dinitrochlorobenzene (DNCB), trinitrophenol (TNP), dinitrophenol (DNP), albumin, Ovalbumin (OVA), N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide (AED), substituted or unsubstituted benzenesulfonamide, formaldehyde, galactose, rhamnose, aminogalactose, paraformaldehyde, other hapten substances containing aldehyde groups, etc.

[0159] In certain embodiments, the oxidation is oxidation of the antigenic component using an oxidizing agent.

[0160] In certain embodiments, the oxidizing agent includes but is not limited to hypochlorous acid, hydrogen peroxide (H2O2), persulfate, KIO3, KBrO3, chlorine, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO3, KBrO3, ClO3 - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, NO3 - 、MnO4 - , one or more of F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3, etc.

[0161] In certain embodiments, the oxidation can enhance the immunogenicity of a portion of the antigenic component.

[0162] In certain embodiments, the reducing agent includes but is not limited to one or more reducing agents such as dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP).

[0163] In certain embodiments, the enzymatic treatment method includes, but is not limited to, using one or more of nuclease, DNase, pepsin, chymotrypsin, trypsin, other protein digesting enzymes, protease inhibitors, and the like.

[0164] The enzymatic hydrolysis in the present disclosure includes but is not limited to any feasible enzymatic hydrolysis method such as nuclease, pepsin, trypsin, protease inhibitors, chymotrypsin, DNA enzyme, etc.

[0165] In certain embodiments, the irradiation comprises any commonly used irradiation method;

[0166] In certain embodiments, the irradiation includes, but is not limited to, one or more of radioactive material irradiation, ultraviolet irradiation, electron beam irradiation, X-ray irradiation, α-ray irradiation, microwave irradiation, β-ray irradiation, and γ-ray irradiation.

[0167] In certain embodiments, the mineralization includes, but is not limited to, any mineralization or biomineralization method such as silicification, calcification, and magnesiization.

[0168] In certain embodiments, the method for separating and purifying antigen components may use only the dilution method, or may use the dilution method in combination with other methods to separate and purify antigen components.

[0169] In certain embodiments, methods for separating and purifying antigen components that can be used in conjunction with the dilution method include, but are not limited to, salting out, heating, enzyme treatment, oxidation, reduction, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, fixation, mineralization, irradiation, and the like.

[0170] In certain embodiments, the chromatography includes but is not limited to column chromatography, gas chromatography, high pressure liquid chromatography, adsorption chromatography, partition chromatography, thin layer chromatography, high performance liquid chromatography, ion exchange chromatography, thin film chromatography, affinity chromatography, gel chromatography, and the like.

[0171] In certain embodiments, the chromatography includes, but is not limited to, column chromatography, thin layer chromatography, liquid chromatography, gas chromatography, supercritical fluid chromatography, and the like.

[0172] In certain embodiments, the electrophoresis method includes but is not limited to SDS electrophoresis, isoelectric focusing electrophoresis, isotachophoresis, immunoelectrophoresis, serum protein electrophoresis, nucleic acid electrophoresis, DNA sequencing electrophoresis, gel electrophoresis, preparative electrophoresis, and the like.

[0173] In certain embodiments, a hapten substance is used to co-act with cells (e.g., cancer cells) and / or cells in a tissue (e.g., tumor tissue) for a certain period of time to modify the antigenic components in the cells and / or tissue, and then the cells and / or tissue are lysed to obtain a lysate thereof; or the cells (e.g., cancer cells) and / or tissue (e.g., tumor tissue) are first lysed to obtain a lysate thereof, and then the hapten substance is used to modify the antigenic components in the cell and / or tissue lysate.

[0174] In certain embodiments, before lysing cells (such as cancer cells) and / or tissues (such as tumor tissue) or before interacting the hapten substance with cells (such as cancer cells) and / or tissues (such as tumor tissue), the cells (such as cancer cells) and / or tissues (such as tumor tissue) can be irradiated with radiation to inactivate the cells (such as cancer cells) and / or tissues (such as tumor tissue). The radiation includes but is not limited to γ ​​rays, X-rays, electron beams, microwaves, β rays, α rays, etc.

[0175] In certain embodiments, the irradiation used to enhance the immunogenicity of the antigen component is any commonly used irradiation method, including but not limited to one or more of radioactive material irradiation, electron beam, microwave, ultraviolet irradiation, X-ray irradiation, α-ray irradiation, β-ray irradiation, γ-ray irradiation and the like.

[0176] In certain embodiments, the enzymatic treatment method includes, but is not limited to, using one or more of nuclease, DNase, pepsin, chymotrypsin, trypsin, other protein digesting enzymes, protease inhibitors, and the like.

[0177] In certain embodiments, the enzymes used for enzymatic hydrolysis include, but are not limited to, one or more of nuclease, pepsin, trypsin, protease inhibitors, chymotrypsin, DNA enzyme, and the like.

[0178] In certain embodiments, the cells described herein are derived from any method that can obtain cells. Taking cancer cells as an example, the cancer cells described herein are derived from any method that can obtain cancer cells, including but not limited to cancer cell lines, cancer cells isolated and extracted from tumor tissue and expanded in vitro, cancer cells isolated and extracted from blood and expanded from circulating tumor cells, or cancer cells differentiated and cultured from stem cells.

[0179] In certain embodiments, the cells or tissues may be incubated with specific chemicals to stimulate the cells and / or tissues prior to lysis.

[0180] In certain embodiments, the cancer cells and / or tumor tissues may be co-incubated with specific chemicals to stimulate the cancer cells and / or tumor tissues before lysis.

[0181] In certain embodiments, the specific substances that stimulate cancer cells include, but are not limited to, small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, cytokines, chemokines, plant extracts (such as important extracts of ginseng, plant root extracts, etc.), interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate with cancer cells and / or tumor tissues to stimulate cancer cells or tumor tissues is to cause the cancer cells to produce more antigen components.

[0182] In certain embodiments, the antigen component is obtained by purification using a dilution method, or is separated and purified from a lysate component dissolved in a lysis solution containing a dissolving agent using a dilution method in combination with other appropriate methods. The antigen component separated and purified in the lysate component can also be irradiated before or after cell and / or tumor tissue lysis, or can be irradiated after the separated and purified antigen component is loaded onto nanoparticles and / or microparticles.

[0183] In certain embodiments, the cancer cells are from one or more organisms, or from one or more cancer cell lines; the tumor tissues are from one or more organisms; and the protein and polypeptide components in the lysate components of the cancer cells and / or tumor tissues contain antigen components.

[0184] In certain embodiments, the cancer cells and / or tumor tissues can be subjected to treatments including but not limited to inactivation or / and denaturation, fixation, chromatography, organic solvent precipitation (such as ethanol precipitation, methanol precipitation, acetone precipitation), electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, heating, salting out, enzyme treatment, oxidation, reduction, mineralization, irradiation, radiation, ionization, chemical modification, nucleic acid separation and purification, protease endo- or degradation, nuclease treatment, etc. before or after lysis, and then lysed using a lysis solution containing a lytic agent to dissolve the lysate components and then extract and separate the protein and polypeptide components therein; or the cancer cells and / or tumor tissues can be subjected to treatments including but not limited to inactivation or / and denaturation, fixation, heating, salting out, oxidation, reduction, mineralization, enzyme treatment, ionization, irradiation, radiation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, radiation, chemical modification, protease endo- or degradation, nuclease treatment before or after cell lysis, and then directly lysed using a lysis solution containing a lytic agent to dissolve the lysate components and then extract and separate the protein and polypeptide components therein. In some embodiments of the present disclosure, tumor tissue cells are irradiated, inactivated by high temperature, or (and) denatured before lysis. In actual use, irradiation or heat treatment may be performed after cell lysis, or irradiation, heat inactivation, or (and) denaturation treatment may be performed both before and after cell lysis. In actual use, treatment methods including but not limited to radiation irradiation, high pressure, nucleic acid separation and purification, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, irradiation, radiation, heating, salting out, oxidation, reduction, enzyme treatment, mineralization, ionization, chemical modification, nuclease treatment, protease endo- or degradation, collagenase treatment, freeze-drying, etc. may also be used. Those skilled in the art will understand that in actual application, technicians may make appropriate adjustments according to specific circumstances.

[0185] In certain embodiments, the loaded antigen delivery particle has the following components:

[0186] (i) nanoparticle and / or microparticle framework structures formed by particulate materials;

[0187] (ii) lysing a lysate fraction of cells and / or tissue using a lysis solution containing a lytic agent, or isolating and purifying an antigen fraction therefrom after lysis;

[0188] In certain embodiments, the lysate fraction is a whole cell lysate fraction from cells and / or tissues.

[0189] In certain embodiments, the lysate fraction is a partial cell and / or tissue lysate fraction containing an antigenic component.

[0190] In certain embodiments, the antigen component is loaded inside and / or on the surface of the scaffold structure.

[0191] In certain embodiments, appropriate methods can be used to enhance the immunogenicity of the antigenic components before or after cell lysis.

[0192] In certain embodiments, the method for enhancing the immunogenicity of the antigenic component comprises irradiation, oxidation, reduction, modification with a haptenic substance, enzyme treatment, denaturation, heating, or mineralization.

[0193] In certain embodiments, the irradiating comprises any commonly used irradiation method.

[0194] In certain embodiments, the irradiation comprises one or more of radioactive material irradiation, electron beam irradiation, microwave irradiation, ultraviolet irradiation, X-ray irradiation, α-ray irradiation, β-ray irradiation, and γ-ray irradiation.

[0195] In certain embodiments, the hapten substance comprises one or more of 2,4-dinitrofluorobenzene, 2,4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, Ovalbumin, N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other hapten substances containing aldehyde groups, rhamnose, galactose, and aminogalactose.

[0196] In certain embodiments, the immunogenicity of whole cell lysate fractions and / or partial cell lysate fractions containing antigenic components from cells and / or tissues can be enhanced by appropriate methods;

[0197] In certain embodiments, the whole cell lysate component from cells and / or tissues and / or the partial cell lysate component containing the antigen component that enhances immunogenicity are loaded inside and / or on the surface of the scaffold structure;

[0198] In certain embodiments, the antigen component contained in the partial cell lysate component comprises a protein and polypeptide component in a cell and / or tissue lysate and / or an RNA component or mRNA component in a cell lysate;

[0199] Preferably, the antigen component contained in the partial cell lysate component comprises protein and polypeptide components in cell and / or tissue lysate and / or lipid components in cell lysate;

[0200] Preferably, 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 mRNA component in a cell lysate;

[0201] In certain embodiments, the antigen component is separated and purified using an appropriate method from a lysate component that is lysed using a lysis solution containing a lytic agent;

[0202] In certain embodiments, the separated and purified antigen components in the lysate components can also be irradiated before or after cell or tumor tissue lysis, or can be irradiated after the separated and purified antigen components are loaded onto nanoparticles and / or microparticles.

[0203] In certain embodiments, the nanoparticles and / or microparticles loaded with the separated and purified antigen components are further loaded with at least one component as shown below:

[0204] (iii) immune adjuvants;

[0205] (iv) Positively charged substances.

[0206] In certain embodiments, the immune adjuvant includes but is not limited to at least one of the following: pattern recognition receptor agonists, Toll-like receptor agonists, Bacillus Calmette-Guérin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyantigen A, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhancing reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic Streptococcus preparation, coenzyme QIO, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokine, mRNA, MF59, double-stranded RNA, double-stranded DNA, single-stranded DNA, aluminum adjuvant, manganese adjuvant, calcium adjuvant, STING agonist, endotoxin adjuvant, liposome adjuvant, CAF01, ginseng active ingredients, astragalus active ingredients, etc.

[0207] In certain embodiments, the immune adjuvant comprises one or more of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist.

[0208] In certain embodiments, the immune adjuvant includes one or more of Poly(I:C), Poly ICLC, A-class CpG-OND, B-class CpG-OND, and C-class CpG-OND.

[0209] In certain embodiments, the positively charged substance includes, but is not limited to, one or more of positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and / or positively charged inorganic substances.

[0210] In certain embodiments, the positively charged substance is selected from one or more of melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

[0211] In certain embodiments, the positively charged species is a RALA polypeptide.

[0212] In certain embodiments, the amino acid sequence of the RALA polypeptide is WEARLARALARALARHLARALARALRACEA.

[0213] In the present disclosure, organic polymer substances are used as the preparation materials of nanoparticles or micron particles. In practical applications, any other preparation materials that can load antigens and prepare nano- or micron-sized particles can also be used, including but not limited to inorganic materials, viruses (such as viral proteins), bacteria (such as bacterial walls, bacterial proteins or whole bacteria), and materials of other biological sources (exosomes, extracellular vesicles, bacterial membrane components).

[0214] In certain embodiments, the nanoparticles have a particle size of 1 nm to 1000 nm.

[0215] In certain embodiments, the nanoparticles have a particle size of 50-500 nm.

[0216] In certain embodiments, the nanoparticles have a particle size of 100-400 nm.

[0217] In certain embodiments, the particle size of the microparticles is 1 μm to 1000 μm.

[0218] In certain embodiments, the microparticles have a particle size of 1-10 μm.

[0219] In certain embodiments, the microparticles have a particle size of 1-5 μm.

[0220] In the present disclosure, the nanoparticles or microparticles loaded with isolated and purified antigen components contain 0.001-2000 μg of protein or polypeptide components per 1 mg of particle material. In some embodiments, the nanoparticles or microparticles also contain an immunopotentiating adjuvant, wherein the immunopotentiating adjuvant is loaded at 1-800 μg per 1 mg of particle material. In practical applications, the content of antigen components such as proteins and polypeptides or the content of adjuvants loaded per 1 mg of particle material can be even higher.

[0221] In the present disclosure, the interior and / or surface of the nanoparticles or microparticles loaded with the separated and purified antigen components may further contain membrane components, and the membrane components inside and / or on the surface of the nanoparticles or microparticles are one or more selected from the group consisting of cell membranes of antigen-presenting cells, extracellular vesicles of antigen-presenting cells, cell membranes of cancer cells, extracellular vesicles of cancer cells, cell membranes of bacteria, and extracellular vesicles of bacteria.

[0222] When the membrane component is located on the surface of nanoparticles or microparticles, methods for loading the membrane component onto the surface of nanoparticles and / or microparticles 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.

[0223] Nanoparticles and / or microparticles can also be bacteria and viruses; or the cell walls of bacteria can be used to prepare nanoparticles and / or microparticles, or bacterial proteins or viral proteins can be used to prepare nanoparticles and / or microparticles.

[0224] In some exemplary embodiments of the present disclosure, a solvent volatilization method is used to prepare nanoparticles and / or microparticles loaded with antigen components. In practical applications, any other method for preparing anti-nanoparticles and / or microparticles may also be used, including but not limited to precipitation, dialysis, dispersion, microfluidics, high-pressure homogenization, stirring, spray drying, phase separation, electrostatic spraying, emulsion polymerization, machine stirring shearing, membrane emulsification, etc.

[0225] In certain embodiments, the antigenic components after separation and purification of the cancer cell and / or tumor tissue lysate can be one or two selected from the following: (1) protein polypeptide components; (2) protein polypeptide components and RNA components (or mRNA components); (3) lipid components.

[0226] 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).

[0227] In certain embodiments, when an RNA component or an mRNA component is loaded simultaneously in addition to the protein polypeptide component, the mass ratio of the particle skeleton structure to the RNA component or the mRNA component is 1:0.001-1:10.

[0228] In certain embodiments, when an RNA component or an mRNA component is loaded simultaneously in addition to the protein polypeptide component, the mass ratio of the particle skeleton structure to the RNA component or the mRNA component is 1:0.01-1:2.

[0229] In certain embodiments, when an RNA component or an mRNA component is loaded simultaneously in addition to the protein polypeptide component, the mass ratio of the particle skeleton structure to the RNA component or the mRNA component is 1:0.05-1:1.

[0230] In certain embodiments, the mass ratio of the protein and polypeptide components to the RNA component / mRNA component is 1:0.001-1:10.

[0231] In certain embodiments, the mass ratio of the protein and polypeptide component to the RNA component / mRNA component is 1:0.01-1:2.

[0232] Most preferably, the mass ratio of the protein and polypeptide components to the RNA component / mRNA component is 1:0.05-1:1.

[0233] In certain embodiments, the immunogenic protein and / or polypeptide may be derived from a portion of the components in cancer cells / tumor tissues and extracellular vesicle lysates. Furthermore, the extracellular vesicle lysate is selected from extracellular vesicle lysates of cancer cells and / or extracellular vesicle lysates of bacteria. The mass ratio of the portion of the components in the cancer cells and / or tumor tissues to the extracellular vesicle lysate components is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Exemplarily, the mass ratio is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.

[0234] In certain embodiments, the immunogenic protein and / or polypeptide can be derived from a portion of a component in a cancer cell / tumor tissue and a bacterial lysate. Furthermore, the mass ratio of the portion of the component in the cancer cell / tumor tissue to the bacterial lysate is (0.1-10):(0.1-10); preferably (0.5-2):(0.5-2). Exemplarily, the mass ratio is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.

[0235] In certain embodiments, the particle material may be PEG-modified or unPEG-modified. Preferably, when preparing the backbone structure, the mass ratio of the unPEG-modified particle material to the PEG-modified particle material is 9-200:1. In the preparation of nanoparticles or microparticles, an appropriate amount of PEG-modified PLGA or PLA can be added to a primary material such as PLGA or PLA. This can enhance long-circulation and passive targeting effects after injection into the body. The mass ratio of the PEG-modified PLGA or PLA to the unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.

[0236] In certain embodiments, the mass ratio of the framework material, protein and polypeptide components for particle preparation of the nanoparticles and / or microparticles is 1:0.001-10; preferably, the mass ratio of the framework material, protein and polypeptide components for particle preparation is 1:0.01-2; most preferably, the mass ratio of the framework material, protein and polypeptide components for particle preparation is 1:0.05-1.

[0237] In certain embodiments, the nanoparticles and / or microparticles are prepared from materials selected from natural polymer materials, biological materials, microbial materials, synthetic polymer materials and / or inorganic materials.

[0238] In certain embodiments, the shape of the antigen delivery particles (nanoparticles and / or microparticles) is any shape, including but not limited to spheres, ellipsoids, barrels, polygons, rods, sheets, threads, worms, squares, triangles, butterflies, discs, vesicles, etc. In certain embodiments, the particle size of the nanoparticles is 1 nm to 1000 nm, preferably 50 to 500 nm, and more preferably 100 to 400 nm.

[0239] In certain embodiments, the particle size of the micronized particles is 1 μm-1000 μm, preferably 1-10 μm, and more preferably 1-5 μm.

[0240] In certain embodiments, the surface of the nanoparticles or microparticles may also be loaded with membrane components, which may be derived from one or more of antigen-presenting cells, cancer cells, bacteria, or extracellular vesicles.

[0241] In certain embodiments, the antigen-presenting cells used to prepare the biofilm components loaded on the surface of the nanoparticles or microparticles can be derived from autologous or allogeneic sources, or from cell lines or stem cells. The antigen-presenting cells can be dendritic cells, B cells, macrophages, or any mixture of the above three, or other cells with antigen-presenting function. The antigen-presenting cells can be activated by the antigen-loaded nanoparticles or microparticles.

[0242] In certain embodiments, when the biofilm components carried on the surface of the nanoparticles or microparticles are derived from extracellular vesicles, they can be one or more of the extracellular vesicles of cancer cells, extracellular vesicles of bacteria, or extracellular vesicles of antigen-presenting cells.

[0243] In certain embodiments, any method for preparing nanoparticles or microparticles known to those skilled in the art can be used to prepare the nanoparticles or microparticles described in the present disclosure, including but not limited to solvent evaporation, dialysis, phase separation, spray drying, emulsion polymerization, machine stirring and shearing, membrane emulsification, microfluidics, ultrafiltration, homogenization emulsification, dispersion, precipitation, and the like.

[0244] The present invention also provides an antigen delivery particle prepared according to the method. The antigen delivery particle can be used directly as a vaccine, or used to activate immune cells to prepare a cell vaccine, or used to assist in detecting specific cells after activating immune cells, or used to assist in sorting and amplifying specific cells after activating immune cells.

[0245] The present invention also provides a pharmaceutical composition comprising the antigen delivery particle.

[0246] In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0247] The present invention also provides a use of the antigen delivery particle or the pharmaceutical composition in at least one of the following (1)-(4):

[0248] (1) Preparation of drugs for preventing and / or treating diseases;

[0249] (2) Used to activate antigen-presenting cells and prepare cellular vaccines based on antigen-presenting cells;

[0250] (3) Assisted activation of antigen-specific T cells and detection of antigen-specific T cell content;

[0251] (4) After assisting in the activation of antigen-specific T cells, the activated antigen-specific T cells are isolated and amplified, and used to prevent and / or treat diseases.

[0252] In certain embodiments, the antigen delivery particles can be used directly as vaccines.

[0253] In certain embodiments, after the antigen delivery particles activate dendritic cells and / or B cells, the activated dendritic cells and / or B cells can be used as cellular vaccines.

[0254] In certain embodiments, the antigen delivery particles can also be used to detect the content of cancer cell-specific T cells after assisting in the activation of cancer cell-specific T cells (tumor-specific T cells) in vitro, or to sort and / or expand the activated antigen-specific T cells after assisting in the activation of antigen-specific T cells by the antigen delivery particles for the prevention and / or treatment of diseases.

[0255] In certain embodiments, the disease is cancer or tumor;

[0256] In certain embodiments, the disease is a solid tumor or a hematological tumor.

[0257] In some specific embodiments, the present disclosure provides the following exemplary preparation methods, taking the example of fixing tumor tissue or cancer cells with the tissue fixative, lysing the tumor tissue or cancer cells, and loading the lysate components onto nanoparticles or microparticles using a solvent volatilization method:

[0258] Step 1: fix the tumor tissue or cancer cells for a certain period of time (more than 1 hour) using the tissue fixative.

[0259] Step 2: Centrifuge the fixed tissue or cells at 200g-15000g for 3-60 minutes, discard the supernatant of the tissue fixative, and collect the precipitated cells or tissue.

[0260] Step 3: Use a dissolving solution containing a dissolving agent to lyse the tumor tissue or cancer cells, and then use a dissolving solution containing a dissolving agent to dissolve the lysate component; or first add ultrapure water to dissolve the water-soluble component, and then centrifuge, the supernatant part is the water-soluble component, and the precipitate part is dissolved with a dissolving solution containing a dissolving agent as the water-insoluble component, and the water-soluble component and the water-insoluble component are used together as the cell or tissue lysate component.

[0261] In certain embodiments, before tissue or cell lysis, appropriate treatment can be performed to enhance the immunogenicity of the antigen component. Treatment methods for enhancing immunogenicity include but are not limited to the use of radiation irradiation, heating, incubation with certain substances that can enhance intracellular antigen synthesis, oxidation, fixation, reduction, modification with hapten substances, enzyme treatment, mineralization, etc.

[0262] In certain embodiments, the irradiation is performed using one or more of different irradiation methods such as ultraviolet rays, X-rays, gamma rays, electron beams, microwaves, alpha rays, beta rays, radioactive sources, etc. for a period of time.

[0263] In certain embodiments, the cancer cells or tumor tissues can be co-incubated with specific chemical substances to stimulate the cancer cells or tumor tissues before lysis. The specific substances that stimulate cancer cells include but are not limited to small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, cytokines, plant extracts (such as important extracts such as ginseng, plant root extracts, etc.), chemokines, interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate and stimulate cancer cells or tumor tissues is to make the cancer cells produce more antigen components.

[0264] In certain embodiments, the haptenic substance is a substance that can increase the immunogenicity of a protein or polypeptide after co-acting with the protein or polypeptide.

[0265] In certain embodiments, the haptenic substance includes but is not limited to 2,4-dinitrofluorobenzene (DNFB), 2,4-dinitrochlorobenzene (DNCB), trinitrophenol (TNP), dinitrophenol (DNP), albumin, Ovalbumin (OVA), N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide (AED), substituted or unsubstituted benzenesulfonamide, rhamnose, galactose, aminogalactose, formaldehyde, paraformaldehyde, other haptenic substances containing aldehyde groups, etc.

[0266] In certain embodiments, any oxidizing agent that can oxidize an antigen component can be used in the present disclosure, including but not limited to hypochlorous acid, hydrogen peroxide (H2O2), persulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO3, KBrO3, ClO3, - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, NO3 - 、MnO4 - , one or more of F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, and FeCl3.

[0267] In certain embodiments, the reducing agents described herein include, but are not limited to, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), and the like.

[0268] In certain embodiments, the enzymatic treatment methods of the present disclosure include, but are not limited to, the use of one or more of nucleases, DNA enzymes, pepsin, chymotrypsin, trypsin, other protein digestive enzymes, protease inhibitors, etc. Enzymatic hydrolysis of the present disclosure includes, but is not limited to, the use of any feasible enzymatic hydrolysis method such as nucleases, pepsin, trypsin, protease inhibitors, chymotrypsin, DNA enzyme, etc.

[0269] In certain embodiments, the mineralization methods described herein include, but are not limited to, any mineralization or biomineralization methods such as silicification, calcification, and magnesiization.

[0270] In certain embodiments, the cancer cells can be one or more cancer cells or cancer cell lines, cancer cells obtained by culturing cancer cells from tumor tissue, or cancer cells obtained by amplifying circulating tumor cells; the tumor tissue can be tumor tissue from one or more organisms. The lysis method is a commonly used lysis method for cancer cells and / or tumor tissue, including but not limited to one or more of freeze-thaw cycles, swelling, sonication, high-pressure treatment, lysis with a lysate, homogenization, extrusion, homogenization, high-speed stirring, treatment with chemicals, high-shear treatment, ultrafiltration, and shrinkage.

[0271] In certain embodiments, the dissolving agent is selected from one or more of a compound containing a structure of Structural Formula 1, deoxycholate, dodecyl sulfate, glycerol, a protein degrading enzyme, a polypeptide, an amino acid, a glycoside, and choline; wherein Structural Formula 1 is as follows:

[0272] R1 is C, S, P, N or O, and R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidine. Compounds containing structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, urea salts, urea, spermine, spermidine, other compounds containing guanidine groups, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.

[0273] Step 4: adding more than 20% of an organic solvent (such as one or more of ethanol, methanol, acetone, isopropanol, phenol, butyric acid, acetic acid, formic acid, acetonitrile, etc.) to the lysate component dissolved in the dissolving solution containing the dissolving agent to precipitate the protein and polypeptide components, and collecting the precipitate after centrifugation to obtain the antigen component; or adding a salting-out reagent such as ammonium sulfate to salt out the protein and polypeptide components in the lysate component, and collecting the precipitate after centrifugation to obtain the antigen component; or heating at above 50 degrees Celsius to precipitate the protein and polypeptide components, and collecting the precipitate after centrifugation to obtain the antigen component; or adding more than 8 times the volume of pure water or aqueous solution to dilute the content of the dissolving agent, thereby precipitating the protein and polypeptide components, and collecting the precipitate after centrifugation to obtain the antigen component.

[0274] The centrifugal speed is 1000RPM-20000RPM.

[0275] In step 5, the lysate component dissolved in step 3 is directly used as the antigen component for subsequent preparation of antigen delivery particles; or the precipitate component (antigen component) collected in step 4 is re-dissolved in a dissolving solution containing a dissolving agent and used as the antigen component for subsequent preparation of antigen delivery particles.

[0276] Step 6: Add the antigen component obtained in step 5 as the initial aqueous phase to the organic phase to prepare a colostrum sample.

[0277] During preparation, the initial aqueous phase and the organic phase are mixed, specifically, a first predetermined volume of aqueous solution containing a first predetermined concentration of antigen components is added to a second predetermined volume of organic phase containing a second predetermined concentration of raw materials for preparing particles.

[0278] In some embodiments, the aqueous solution may contain at least one of the following i) to ii): i) antigen components in the lysate; ii) antigen components in the lysate and immune enhancing adjuvants. The antigen components in the lysate are separated and purified antigen components dissolved in a dissolving solution containing a solvent such as urea or guanidine hydrochloride during preparation. The first predetermined concentration is the concentration of proteins and polypeptides contained in the aqueous solution, or the concentration of antigen components contained in the aqueous solution. The first predetermined concentration requires that the protein and polypeptide concentration content be greater than 1 ng / mL so that sufficient antigen components can be loaded to activate relevant cells. The concentration of the immune enhancing adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0279] In some embodiments, the organic solvent is selected from dichloromethane or ethyl acetate. In addition, in some embodiments, the second predetermined concentration of the raw material for preparing particles ranges from 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0280] In practice, the second predetermined volume of the organic phase is set based on the ratio of the second predetermined volume of the organic phase to the first predetermined volume of the aqueous phase. In the present disclosure, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably 1:10. During specific implementations, the first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.

[0281] In some embodiments, when the aqueous phase solution is a solution containing antigen components, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL. In some embodiments, when the aqueous phase solution is a solution containing antigen components in a lysate and an immune adjuvant, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of the immune adjuvant is greater than 0.01 ng / mL, preferably 0.001 mg / mL to 20 mg / mL. In some embodiments, in the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane or ethyl acetate; the concentration of the organic phase is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0282] Step 7: subjecting the mixed solution obtained in step 6 to any of the following treatments: i) ultrasonic treatment for more than 2 seconds; ii) stirring for more than 1 minute; iii) homogenization; iv) microfluidic treatment. Preferably, during mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, such as a stirring speed of 50 rpm to 1500 rpm and a stirring time of 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5 W and the time is greater than 0.1 second, such as 2 to 200 seconds; during homogenization, a high-pressure / ultra-high-pressure homogenizer or a high-shear homogenizer is used, and when a high-pressure / ultra-high-pressure homogenizer is used, the pressure is greater than 5 psi, such as 20 psi to 100 psi, and when a high-shear homogenizer is used, the speed is greater than 100 rpm, such as 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, such as 0.1 mL / min to 100 mL / min. Nano- and / or micron-size can be achieved through ultrasound, stirring, homogenization, or microfluidic processing. The size of the prepared nanoparticles or micron particles can be controlled by the length of ultrasound time, stirring speed, or homogenization pressure and time. Too large or too small a particle size will result in a change in particle size.

[0283] Step 8: Add the mixture obtained after the treatment in step 7 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of an emulsifier and perform any of the following treatments: i) ultrasonic treatment for greater than 2 seconds; ii) stirring for greater than 1 minute; iii) homogenization; or iv) microfluidic treatment. In this step, the mixture obtained in step 2 is added to the aqueous emulsifier solution and continues ultrasonication, stirring, homogenization, or mixing to perform nano- or micronization. In the present disclosure, the ultrasonication time is greater than 0.1 seconds, such as 2 to 200 seconds; the stirring speed is greater than 50 rpm, such as 50 rpm to 500 rpm; and the stirring time is greater than 1 minute, such as 60 to 6000 seconds. Preferably, when stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, such as the stirring speed is 50 rpm to 1500 rpm and the stirring time is 0.5 hour to 5 hours; when ultrasonic treatment is performed, the ultrasonic power is 50 W to 500 W and the time is greater than 0.1 second, such as 2 to 200 seconds; when homogenizing, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and the pressure when using a high pressure / ultra-high pressure homogenizer is greater than 20 psi, such as 20 psi to 100 psi, and the speed when using a high shear homogenizer is greater than 1000 rpm, such as 1000 rpm to 5000 rpm; when using microfluidics, the flow rate is greater than 0.01 mL / min, such as 0.1 mL / min-100 mL / min. Ultrasonic or stirring or homogenization or microfluidics is used for nano- or micronization, and the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared nanoparticles or micron particles. Too large or too small will bring about changes in particle size.

[0284] In certain embodiments, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg / mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present disclosure, the range of the ratio of the second predetermined volume to the third predetermined volume is 1:1.1-1:1000, preferably 2:5. In order to control the size of the nanoparticles or micron particles during specific implementation, the ratio of the second predetermined volume to the third predetermined volume can be adjusted. Similarly, the ultrasonic time or stirring time or homogenization time of this step, the volume of the emulsifier aqueous solution and the concentration are based on the values ​​​​all in order to obtain nanoparticles or micron particles of appropriate size.

[0285] Step 9: adding the liquid obtained after the treatment in step 8 to a fourth predetermined volume of an emulsifier aqueous solution with a fourth predetermined concentration, and stirring until a predetermined stirring condition is met.

[0286] In this step, the emulsifier aqueous solution is a PVA solution or other solutions.

[0287] The fourth predetermined concentration is 5 mg / mL. The fourth predetermined concentration is selected to obtain nanoparticles or microparticles of appropriate size. The fourth predetermined volume is selected based on the ratio of the third predetermined volume to the fourth predetermined volume. In the present disclosure, the ratio of the fourth predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. In specific implementations, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted to control the size of the nanoparticles or microparticles.

[0288] In the present disclosure, the predetermined stirring condition of this step is until the organic solvent is completely volatilized, that is, the dichloromethane or ethyl acetate in step 1 is completely volatilized.

[0289] Step 10, after the mixed solution that meets the predetermined stirring conditions obtained in step 9 is centrifuged at a speed greater than 100 RPM for more than 1 minute, the supernatant is removed, and the remaining precipitate is re-suspended in a fifth predetermined volume of an aqueous solution containing a fifth predetermined concentration of a lyoprotectant or a sixth predetermined volume of PBS (or normal saline); or ultrafiltration centrifugation or dialysis that can remove substances with a specific molecular weight is used to remove free PVA and other substances, and the solution in the system is replaced with a fifth predetermined volume of an aqueous solution containing a fifth predetermined concentration of a lyoprotectant or a sixth predetermined volume of PBS (or normal saline).

[0290] Step 11: freeze-dry the suspension containing the lyoprotectant obtained in step 10, and keep the lyophilized material for later use.

[0291] Optionally, the prepared nanoparticles or microparticles loaded with antigen components are irradiated for a certain period of time using radiation shielding.

[0292] Step 12: The suspension containing nanoparticles / microparticles obtained in step 10 is resuspended in PBS (or normal saline) with a sixth predetermined volume, or the freeze-dried material containing nanoparticles or microparticles and a lyoprotectant obtained in step 11 is resuspended in a sixth predetermined volume of PBS (or normal saline) and then used directly; or the above sample is mixed with the seventh predetermined volume of the antigen component and then used.

[0293] In the present disclosure, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1; preferably, the volume ratio is 1:100 to 100:1; most preferably, the volume ratio is 1:30 to 30:1.

[0294] In step 13, the nanoparticles or microparticles prepared in step 12 are used to prevent or treat diseases such as cancer; or the nanoparticles or microparticles are used as antigen delivery particles to activate antigen-presenting cells (such as dendritic cells or mixed antigen-presenting cells) in vitro to prepare antigen-presenting cell vaccines; or the nanoparticles or microparticles are used to assist in activating antigen-specific T cells to detect the content of antigen-specific T cells; or after using the nanoparticles or microparticles to assist in activating antigen-specific T cells, the antigen-specific T cells are sorted or amplified, and the sorted or amplified antigen-specific T cells are used to prevent or treat diseases.

[0295] Example 1: Nano-vaccine loaded with antigen components for the treatment of pancreatic cancer

[0296] In this example, the antigen component was derived from the KPC mouse pancreatic cancer cell line, the organic polymer material PLGA was used as the nanoparticle skeleton material, Polyinosinic-polycytidylic acid (poly(I:C)), CpG7909 (i.e., CpG2006), and CpG2395 were used as immune adjuvants, and the nanovaccine was prepared by the solvent evaporation method.

[0297] (1) Preparation of antigen components

[0298] Cultured KPC mouse pancreatic cancer cells were collected and then fixed with a mixed tissue fixative (containing 75% ethanol, 25% water; the water contained 20% hydrogen peroxide). After standing for 1 hour, the sample was centrifuged at 3000RPM for 5 minutes. After removing the supernatant, the precipitate was lysed and dissolved using an 8M urea PBS aqueous solution to prepare the antigen component 1 of the nanovaccine 1.

[0299] Cultured KPC mouse pancreatic cancer cells were collected, and then the cells were directly lysed and dissolved using 6 mL of PBS aqueous solution containing 8 M urea to prepare the antigen component 2 of the nanovaccine 2.

[0300] (2) Preparation of nanovaccines

[0301] In this embodiment, nanovaccine 1 was prepared using the double emulsion method, a solvent evaporation method. The vaccine preparation material used, PLGA, has a molecular weight of 10KDa-20KDa, and the immune adjuvants used are poly(I:C), CpG7909, and CpG2395. The preparation method is as described above. During the preparation process, the antigen component 1 and the adjuvant are co-loaded into the nanoparticles using the double emulsion method. Then, 100mg of nanoparticles 1 are centrifuged at 12000g for 30 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 1 is approximately 250nm. Each 1mg of PLGA nanoparticles 1 is loaded with approximately 1.0mg of protein and polypeptide components, and 0.05mg each of poly(I:C), CpG7909, and CpG2395.

[0302] Nanovaccine 2 in this example was prepared using the same methods and materials as Nanovaccine 1. During the preparation process, the antigen component 2 and adjuvant were co-loaded into the nanoparticles using a double emulsion method. 100 mg of nanoparticles 2 were then centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The nanoparticles 2 had an average particle size of approximately 250 nm. Each 1 mg of PLGA nanoparticles 2 was loaded with approximately 1.0 mg of protein and peptide components, with 0.05 mg each of poly(I:C), CpG7909, and CpG2395 loaded.

[0303] (3) Nano-vaccines for cancer treatment

[0304] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare pancreatic cancer tumor-bearing mice. On day 0, 2.0×10 6 KPC cells. 2 mg of nanovaccine (nanovaccine 1 or nanovaccine 2) or 100 μL of PBS was subcutaneously injected into the mice on the 3rd, 6th, 9th, 14th, 19th, and 26th days after tumor inoculation. The tumor growth rate and survival of the mice were monitored. In the experiment, the size of the mouse tumor was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculate, where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0305] (4) Experimental results

[0306] As shown in Figure 3, tumors in mice treated with the PBS control group grew rapidly, leading to their death. Mice treated with both Nanovaccine 1 and Nanovaccine 2 experienced significantly slower tumor growth and prolonged survival, with the majority of mice recovering tumor-free. Furthermore, Nanovaccine 1 was more effective than Nanovaccine 2, demonstrating that using the oxidant-containing mixed tissue fixative described herein to fix cancer cells can enhance the efficacy of subsequently prepared nanovaccines.

[0307] In this embodiment, cultured cancer cell lines were used to prepare antigen components. In practical applications, cancer cells or tumor tissues obtained by methods such as tumor tissue, cancer cells isolated from tumor tissue and cultured and amplified, and circulating tumor cells isolated from blood or peripheral blood can also be used as sources of antigen components.

[0308] In this example, hydrogen peroxide (H2O2) was used to oxidize the tumor tissue. In practical applications, hypochlorous acid, potassium permanganate, or other oxidants may also be used to oxidize cancer cells, tumor tissue, single-cell suspensions of tumor tissue, or antigen components in the aforementioned cell lysate fractions or tissue lysate fractions. Alternatively, reducing agents such as dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) may be used to reduce cancer cells, tumor tissue, single-cell suspensions of tumor tissue, or antigen components in the aforementioned cell lysate fractions or tissue lysate fractions.

[0309] In this embodiment, ethanol and an aqueous solution containing an oxidant are used to fix cancer cells. In actual applications, other fixatives can also be used to fix tumor tissue or cancer cells. Possible fixatives include, but are not limited to, one or more of methanol, acetone, propionic acid, acetic acid, butyric acid, and formic acid.

[0310] Example 2: Micronized vaccines loaded with purified antigen components for the prevention of brain cancer

[0311] (1) Preparation of antigen components

[0312] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 6 GL261 cells, and when the tumors grew to a volume of approximately 1000 mm 3The mice were killed and the tumor tissues were removed. Half of the tumor tissues were fixed for 96 hours in a mixed tissue fixative containing an oxidant (containing 30% methanol, 70% water; the water contained 0.1% hypochlorous acid), and half of the tumor tissues were fixed for 96 hours in a mixed tissue fixative without an oxidant (containing 30% methanol, 70% water; the water did not contain any oxidant). The two samples were then centrifuged at 1000RPM for 60 minutes, and after removing the supernatant, the two precipitates were lysed and dissolved using an 8M urea peroxide PBS aqueous solution, and then the two were mixed. Thereafter, 2 volumes of ethanol were added to the dissolved mixed lysate and allowed to stand for 1 hour. The sample was then centrifuged at 3000RPM for 5 minutes. After removing the supernatant, the precipitate was redissolved using an 8M urea peroxide PBS aqueous solution to obtain antigen component 1.

[0313] (2) Preparation of micronized vaccines

[0314] In this example, micron vaccine 1 (Micronvaccine 1) was prepared using the double emulsion method within the solvent evaporation method. The micron particle preparation material used, PLA, has a molecular weight of 30-50 kDa. The immune adjuvants used were poly(I:C), CpG 1018, and CpG 2395. The positively charged substance used was the RALA polypeptide (N-WEARLARALARALARHLARALARALRACEA-C). The preparation method is as described above. During the preparation process, the antigen component 1, adjuvant, and RALA polypeptide are first loaded into the micron particles. Then, 100 mg of micron particles 1 are centrifuged at 8000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the PLA microparticles 1 was approximately 2.0 μm. Each mg of PLA microparticles 1 was loaded with approximately 20 μg of protein or peptide components, including 0.012 mg of poly(I:C), 0.005 mg each of CpG 1018 and CpG 2395, and 0.1 mg of RALA peptide. The PLGA microparticles 1 loaded with the antigen components and adjuvant were irradiated with gamma rays for 16 hours to obtain the irradiated microparticle vaccine 1.

[0315] (3) Micron vaccines for cancer prevention

[0316] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare glioma-bearing mice. 1 mg of micro-vaccine or 100 μL of PBS was injected subcutaneously on days -35, -28, -21, -14, and -7 before tumor inoculation. On day 0, 1.5×10 6 The methods for monitoring the tumor growth rate and survival of mice were the same as above.

[0317] (4) Experimental results

[0318] As shown in Figure 4, the tumors in the PBS group grew rapidly, leading to early death. Mice treated with Micronvaccine 1 experienced significantly slower tumor growth, significantly prolonged survival, and all mice remained tumor-free. This demonstrates that the oxidant-containing tissue fixative described herein can be used in conjunction with non-oxidant tissue fixatives.

[0319] Example 3 Nano-vaccine for the treatment of liver cancer

[0320] (1) Preparation of antigen components

[0321] Each Balb / c mouse was subcutaneously inoculated with 6.0×10 5 4T1 breast cancer cells, when the tumors grew to a volume of approximately 1000 mm 3 Mice were sacrificed and tumor tissues were removed. Half of the tumor tissues were fixed for 24 hours in a mixed tissue fixative containing an oxidant (95% acetone, 5% water; the water contained 0.5% hydrogen peroxide), and the other half of the tumor tissues were fixed for 24 hours in a mixed tissue fixative containing no oxidant (95% acetone, 5% water; the water contained no oxidant). The two samples were then centrifuged at 10,000 RPM for 5 minutes. After removing the supernatant, the two precipitates were lysed and dissolved in an 8M aqueous solution, and the two were mixed to obtain antigen component 1.

[0322] Each Balb / c mouse was subcutaneously inoculated with 6.0×10 5 4T1 breast cancer cells, when the tumors grew to a volume of approximately 1000 mm 3 The mice were killed and the tumor tissues were removed. The tumor tissues were fixed in a mixed tissue fixative containing an oxidant (containing 95% acetone, 5% water; the water contained 0.5% hydrogen peroxide) for 24 hours, and then the sample was centrifuged at 10000RPM for 5 minutes. After removing the supernatant, the precipitate was lysed and dissolved using an 8M aqueous solution to obtain antigen component 2.

[0323] Each Balb / c mouse was subcutaneously inoculated with 6.0×10 5 4T1 breast cancer cells, when the tumors grew to a volume of approximately 1000 mm 3The mice were killed and the tumor tissues were removed. The tumor tissues were fixed in a mixed tissue fixative (containing 30% methanol and 70% water; the water did not contain any oxidants) without any oxidants for 24 hours. The samples were then centrifuged at 10,000 RPM for 5 minutes. After removing the supernatant, the precipitate was lysed and dissolved using a 6M guanidine hydrochloride aqueous solution in PBS to obtain antigen component 3.

[0324] Each Balb / c mouse was subcutaneously inoculated with 6.0×10 5 4T1 breast cancer cells, when the tumors grew to a volume of approximately 1000 mm 3 The mice were killed and the tumor tissues were removed and fixed in formalin tissue fixative (containing 10% formaldehyde) for 24 hours. The samples were then centrifuged at 10,000 RPM for 5 minutes. After removing the supernatant, the precipitate was lysed and dissolved using a 6M guanidine hydrochloride solution in PBS to obtain antigen component 4.

[0325] (2) Preparation of nanovaccines

[0326] In the present embodiment, nano vaccine 1 (Nanovaccine 1) is prepared by the multiple emulsion method in the solvent evaporation method. The nano vaccine preparation material PLGA molecular weight adopted is 10KDa-20KDa, and the PEG5000-PLGA molecular weight is 15KDa-25KDa. The mass ratio of PLGA and PEG5000-PLGA used is 98.5:1.5. The immune adjuvant adopted is poly (I: C), CpG 2395 and CpG SL03. The preparation method is as described above. During preparation, the cell antigen component 1 and adjuvant are first loaded inside the nanoparticles, and then 100mg of nanoparticles 1 are centrifuged at 12000g for 30 minutes and lyophilized for 48h after being resuspended in 10mL of ultrapure water containing 6% trehalose. The average particle size of the nanoparticles 1 is about 300 nm. Each 1 mg of PLGA nanoparticles 1 is loaded with about 600 μg of protein or polypeptide components, and 0.2 mg each of poly(I:C), CpG 2395, and CpG SL03.

[0327] Nano vaccine 2 (Nanovaccine 2) is prepared by the multiple emulsion method in the solvent evaporation method in the present embodiment. The nano vaccine preparation material PLGA molecular weight adopted is 10KDa-20KDa, and the PEG5000-PLGA molecular weight is 15KDa-25KDa. The mass ratio of PLGA and PEG5000-PLGA used is 98.5:1.5. The immune adjuvant adopted is poly (I: C), CpG 2395 and CpG SL03. The preparation method is as described above. During preparation, the cell antigen component 2 and adjuvant are first loaded inside the nanoparticles, and then 100mg of nanoparticles 2 are centrifuged at 12000g for 30 minutes, and 10mL of ultrapure water containing 6% trehalose is resuspended and freeze-dried for 48h. The average particle size of the nanoparticles 2 is about 300 nm. Each 1 mg of PLGA nanoparticles 2 is loaded with about 600 μg of protein or polypeptide components, and 0.2 mg each of poly(I:C), CpG 2395, and CpG SL03.

[0328] In the present embodiment, nano vaccine 3 (Nanovaccine 3) is prepared by the multiple emulsion method in the solvent evaporation method. The nano vaccine preparation material PLGA molecular weight adopted is 10KDa-20KDa, and the PEG5000-PLGA molecular weight is 15KDa-25KDa. The mass ratio of PLGA and PEG5000-PLGA used is 98.5:1.5. The immune adjuvant adopted is poly (I: C), CpG 2395 and CpG SL03. The preparation method is as described above. During preparation, the cell antigen component 3 and adjuvant are first loaded inside the nanoparticles, and then 100mg of nanoparticles 3 are centrifuged at 12000g for 30 minutes, and 10mL of ultrapure water containing 6% trehalose is resuspended and freeze-dried for 48h. The average particle size of the nanoparticles 3 is about 300 nm. Each 1 mg of PLGA nanoparticles 3 is loaded with about 600 μg of protein or polypeptide components, and 0.2 mg each of poly(I:C), CpG 2395, and CpG SL03.

[0329] In the present embodiment, nano vaccine 4 (Nanovaccine 4) is prepared by the multiple emulsion method in the solvent evaporation method. The nano vaccine preparation material PLGA molecular weight adopted is 10KDa-20KDa, and the PEG5000-PLGA molecular weight is 15KDa-25KDa. The mass ratio of PLGA and PEG5000-PLGA used is 98.5:1.5. The immune adjuvant adopted is poly (I: C), CpG 2395 and CpG SL03. The preparation method is as described above. During the preparation, the cell antigen component 4 and adjuvant are first loaded inside the nanoparticles, and then 100mg of nanoparticles 4 are centrifuged at 12000g for 30 minutes, and 10mL of ultrapure water containing 6% trehalose is resuspended and freeze-dried for 48h. The average particle size of the nanoparticles 4 is about 300 nm. Each 1 mg of PLGA nanoparticles 4 is loaded with about 600 μg of protein or polypeptide components, and 0.2 mg each of poly(I:C), CpG 2395, and CpG SL03.

[0330] (3) Nano-vaccines for cancer treatment

[0331] Female Balb / c mice aged 6-8 weeks were selected as model mice for breast cancer tumor-bearing mice. On day 0, 6.0×10 5 4T breast cancer cells were inoculated. Mice were subcutaneously injected with 100 μL of 1 mg of nanovaccine (Nanovaccine 1, Nanovaccine 2, Nanovaccine 3, or Nanovaccine 4) or 100 μL of PBS on days 3, 6, 9, 14, 19, and 25 after tumor inoculation. Tumor growth and survival were monitored as described above.

[0332] (4) Experimental results

[0333] As shown in Figure 5, the tumor volume of mice in the PBS group grew rapidly and the mice died soon after. The survival time of mice using nanovaccine 1 (Nanovaccine 1), nanovaccine 2 (Nanovaccine 2), nanovaccine 3 (Nanovaccine 3), and nanovaccine 4 (Nanovaccine 4) was significantly prolonged, and the vast majority of mice were tumor-free and recovered. Moreover, nanovaccine 1 (Nanovaccine 1) and nanovaccine 2 (Nanovaccine 2) performed better than nanovaccine 3 (Nanovaccine 3) and nanovaccine 4 (Nanovaccine 4), indicating that antigen delivery particles prepared using antigen components treated with tissue fixatives containing oxidants as described in the present disclosure are more effective than antigen delivery particles prepared using antigen components treated with tissue fixatives containing no oxidants or other commonly used tissue fixatives.

[0334] Example 4: T cells activated by antigen-loaded nanoparticles and sorted and expanded for the treatment of breast cancer

[0335] (1) Preparation of antigen components

[0336] Each C57BL / 6 mouse was subcutaneously inoculated with 1.0×10 6 E0771 mouse breast cancer cells were grown to a volume of approximately 1000 mm 3 Mice were sacrificed and tumor tissue was removed. One-fifth of the tumor tissue was fixed for 48 hours in a mixed tissue fixative containing an oxidant (50% acetone, 50% water, with 3% hydrogen peroxide in the water). Four-fifths of the tumor tissue was fixed for 48 hours in a mixed tissue fixative containing no oxidant (50% acetone, 50% water, with no oxidant in the water). The cells were then centrifuged at 3500 RPM for 15 minutes. The supernatant was discarded, and the precipitate was lysed and dissolved in an 8M urea solution to prepare antigen component 1 of nanoparticle 1.

[0337] (2) Preparation of antigen delivery nanoparticles

[0338] In this example, nanoparticles 1 were prepared using the double emulsion method within the solvent evaporation method. The PLGA material used for nanoparticle preparation had a molecular weight of 20-40 kDa. The immunoadjuvants employed were poly(I:C), CpG 1018, and CpG 7909. The preparation method was as described above. The nanoparticles were first loaded with the cell antigen component 1 and the adjuvant. Then, 100 mg of nanoparticles 1 were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 6% trehalose, and freeze-dried for 48 hours. The nanoparticles 1 had an average particle size of approximately 230 nm. Each 1 mg of PLGA nanoparticles 1 was loaded with approximately 200 μg of protein or polypeptide components, including 0.02 mg each of poly(I:C), CpG 1018, and CpG 7909. The nanoparticles were then irradiated with gamma rays for 16 hours.

[0339] (3) Sorting and expansion of antigen-specific T cells

[0340] Female C57BL / 6 mice aged 6-8 weeks were selected and each mouse was subcutaneously inoculated with 1.0×10 6E0771 cells were injected intraperitoneally with 150 μg of mouse PD-1 antibody on days 6, 8, 10, 12, 14, 16, 18, and 20. Mice were sacrificed on day 21, and peripheral blood was collected. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood. 5 million PBMCs and 1 mg of nanoparticles 1 were co-incubated in 2 mL of RPMI1640 complete medium for 36 hours. CD3 + CD134 + T cells are antigen-specific T cells that can recognize cancer cell antigens. + CD137 + T cells were co-incubated with IL-2 (20 ng / mL), IL-7 (10 ng / mL), IL-15 (10 ng / mL), and αCD3 and αCD28 antibodies (10 ng / mL) in 10 mL of complete DMEM medium (37°C, 5% CO2) for 21 days to expand cancer cell-specific T cells (cell viability was approximately 85%). Nanoparticle 1 (nanovaccin 1) assisted the sorting and expansion of cancer cell-specific T cells as T cells 1 (T cells 1).

[0341] (4) Sorting and expanding cancer cell antigen-specific T cells for cancer treatment

[0342] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 2.0×10 6 E0771 mouse breast cancer cells were inoculated. On day 5 after tumor inoculation, each mouse in each group received an intravenous injection of cyclophosphamide (100 mg / kg). On days 6, 14, and 22, 3 million sorted and expanded tumor antigen-specific T cells (T cells 1) or 100 μL of PBS were injected intravenously. The T cell 1 group and the IL-2 control group received intravenous IL-2 (10,000 U per mouse) every two days from day 6 to day 24. Tumor growth and survival were monitored using the same methods as above.

[0343] (5) Experimental results

[0344] As shown in Figure 6, the tumors in the PBS group grew rapidly and the mice died soon after. The survival of the mice treated with T cells 1 was significantly prolonged, and all mice recovered without tumors.

[0345] Example 5 Antigen-presenting cell vaccine activated by nanoparticles loaded with antigen components for the treatment of colon cancer

[0346] (1) Preparation of antigen components

[0347] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 MC38 colon cancer cells, when the tumor grows to a volume of about 1000mm 3 Mice were killed and tumor tissues were removed. The tumor tissues were fixed for 2 hours using a mixed tissue fixative containing an oxidant (containing 10% acetic acid, 90% water; the water contained 1% hypochlorous acid) and then centrifuged at 2000 RPM for 5 minutes. The supernatant was discarded and the fixed tumor tissue pellet was lysed and dissolved using an appropriate amount of 8M urea and 0.1M arginine aqueous solution. Three volumes of ethanol were then added to the sample and the mixture was allowed to stand for 3 hours to precipitate the dissolved lysate components. The mixture was then centrifuged at 3000 RPM for 5 minutes. The supernatant was discarded and the fixed tumor tissue pellet was redissolved using an appropriate amount of 8M urea aqueous solution to obtain antigen component 1 for preparing antigen delivery particles 1.

[0348] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 MC38 colon cancer cells, when the tumor grows to a volume of about 1000mm 3 The mice were killed and the tumor tissues were removed. The tumor tissues were then lysed using an appropriate amount of an aqueous solution of 8M urea and 0.1M arginine, and the tumor tissue lysate components were dissolved using an aqueous solution of 8M urea and 0.1M arginine. Three volumes of ethanol were then added to the above sample and the sample was allowed to stand for 3 hours to precipitate the dissolved lysate components. The sample was then centrifuged at 3000 RPM for 5 minutes. After discarding the supernatant, the fixed tumor tissue precipitate was redissolved using an appropriate amount of an 8M urea aqueous solution to obtain antigen component 2 for preparing antigen delivery particles 2.

[0349] (2) Preparation of antigen delivery nanoparticles loaded with antigen components

[0350] In this example, nanoparticles 1 were prepared using the double emulsion method within the solvent evaporation method. The PLGA material used for nanoparticle preparation had a molecular weight of 10 kDa to 20 kDa. The immunoadjuvants employed were poly(I:C), CpG SL01, and CpG SL03. The preparation method was as described above. The nanoparticles were first loaded with the cell antigen component 1 and the adjuvant. Then, 100 mg of nanoparticles 1 were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 6% trehalose, and freeze-dried for 48 hours. The nanoparticles 1 had an average particle size of approximately 280 nm. Each 1 mg of PLGA nanoparticles 1 was loaded with approximately 2.0 mg of the protein or peptide component, and 0.02 mg each of poly(I:C), CpG SL01, and CpG SL03. Electron beam irradiation was then performed for 2 hours.

[0351] Nanoparticle 2 in this example was prepared using the same methods and materials as Nanoparticle 1. The nanoparticles were first loaded with the cell antigen component 2 and an adjuvant. Then, 100 mg of nanoparticle 2 was centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 6% trehalose, and freeze-dried for 48 hours. The nanoparticles had an average particle size of approximately 230 nm. Each 1 mg of PLGA nanoparticle 2 was loaded with approximately 2.0 mg of protein or peptide components, including 0.02 mg each of poly(I:C), CpG SL01, and CpG SL03. Electron beam irradiation was then performed for 2 hours.

[0352] (3) In vitro activation of antigen-presenting cells (dendritic cells + B cells) using antigen delivery particles

[0353] This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare antigen-presenting cells. First, 6-8 week-old C57BL / 6 mice were sacrificed by cervical dislocation. The tibia and femur of the hind legs were surgically removed and placed in PBS. The surrounding muscle tissue was removed using scissors and forceps. The ends of the bone were cut with scissors. PBS solution was then drawn with a syringe. A needle was inserted into the bone marrow cavity at each end of the bone, and the bone marrow was repeatedly flushed into a culture dish. The bone marrow solution was collected and centrifuged at 400g for 3 minutes. After that, 1mL of red blood cell lysate was added to lyse the red blood cells. Lysis was terminated by adding 3mL of RPMI 1640 (10% FBS) medium, centrifuged at 400g for 3 minutes, and the supernatant was discarded. The cells were cultured in 10mm culture dishes using complete RPMI 1640 (10% FBS) medium supplemented with recombinant mouse GM-CSF (20ng / mL) and incubated at 37°C, 5% CO2 for 7 days. On the 3rd day, the culture flask was gently shaken and the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng / mL) was added. On the 6th day, half of the medium was replaced. On the 7th day, a small amount of suspended and semi-adherent cells were collected and detected by flow cytometry. + CD80 + CD11c + When the ratio of BMDC in cells is between 15-20%, the induced cultured BMDC can be used for the next experiment.

[0354] B cells were derived from mouse spleen cells. The mice were first sacrificed, and then a single cell suspension of mouse spleen cells was prepared. Then, CD19 was isolated using magnetic bead sorting. + of B cells.

[0355] BMDCs (10 million) and B cells (10 million) were mixed in a 1:1 ratio and incubated with 2 mg of nanoparticles (nanoparticle 1 or nanoparticle 2) in 10 mL of RPMI1640 complete medium containing 20 ng / mL interleukin 15 (IL-15) for 36 hours (37°C, 5% CO2). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove free nanoparticles in the system, and then the mixed cells activated by the antigen delivery particles were used as cancer vaccines. Among them, the mixed cells activated by nanoparticle 1 are mixed cell cancer vaccine 1 (Cell vaccine 1); the mixed cells activated by nanoparticle 2 are mixed cell cancer vaccine 2 (Cell vaccine 2).

[0356] (4) DC vaccine for cancer treatment

[0357] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 6 MC38 colon cancer cells were inoculated in mice. 500,000 cells of the vaccine (cell vaccine 1 or cell vaccine 2) or 100 μL of PBS were injected subcutaneously on days 3, 6, 9, 14, 19, and 25 after tumor inoculation. Tumor growth and survival were monitored as described above.

[0358] (4) Experimental results

[0359] As shown in Figure 7, the tumors in the PBS group grew rapidly and the mice died soon after. Most of the mice treated with Cell Vaccine 1 and Cell Vaccine 2 recovered without tumors, and Cell Vaccine 1 was more effective than Cell Vaccine 2.

[0360] In this embodiment, acetic acid and an aqueous solution containing an oxidant are used to fix the tumor tissue. In actual applications, other fixatives can also be used to fix tumor tissue or cancer cells. Possible fixatives include, but are not limited to, one or more of acetone, propionic acid, butyric acid, formic acid, and y.

[0361] Example 6: Nanoparticles used to detect cancer cell-specific T cells (tumor antigen-specific T cells) in immune cells

[0362] (1) Preparation of antigen components

[0363] Human lung cancer cell lines A549, H1299, PC9, H1437, H226, HCC1588, H2170, and H520 were cultured separately. A549, H1299, PC9, and H1437 cells belong to the lung adenocarcinoma subtype, while H226, HCC1588, H2170, and H520 cells belong to the lung squamous cell carcinoma subtype. The eight cell types were collected separately and then mixed at a cell number ratio of 1:1:1:1:1:1:1:1:1. Half of the mixed cancer cells were fixed for 2 hours using a mixed tissue fixative (70% ethanol, 30% water, containing 0.5% hypochlorous acid) containing an oxidant; the other half was fixed for 2 hours using a mixed tissue fixative (70% ethanol, 30% water, containing no oxidant). The cells were then centrifuged at 5000 RPM for 5 minutes. The supernatant was discarded, and the precipitate was lysed and dissolved in an 8.5 M urea solution to prepare the antigen component 1 of nanoparticle 1 (NP).

[0364] (2) Preparation of Nanoparticles Loaded with Antigen Components

[0365] In this embodiment, nanoparticle 1 (NP1) was prepared using a double emulsion method within a solvent evaporation method. The PLGA material used for nanoparticle preparation has a molecular weight of 10KDa-30KDa. The preparation method is as described above. During the preparation process, the antigen component 1 is first loaded into the nanoparticles using the double emulsion method. Then, 100mg of the nanoparticles are centrifuged at 15000g for 30 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 1 is approximately 200nm, and each 1mg of PLGA nanoparticles is loaded with approximately 500μg of protein and polypeptide components.

[0366] (3) Detection of cancer cell-specific T cells (tumor-specific T cells)

[0367] Patient A, a non-small cell lung cancer patient, experienced significant tumor reduction after immunotherapy with a PD-1 antibody. 8 mL of peripheral blood was drawn from patient A before and three weeks after immunotherapy. Peripheral blood mononuclear cells (PBMCs) were isolated from this 8 mL of blood using gradient centrifugation.

[0368] Nanoparticles 1 (1.5 mg) were co-incubated with PBMC (3 million) in 3 mL of AIM V serum-free medium for 24 hours (37°C, 5% CO2); or the control group was co-incubated with PBMC (3 million) in 3 mL of AIM V serum-free medium for 24 hours (37°C, 5% CO2). After that, the sample was centrifuged at 400g for 5 minutes, the supernatant was discarded, the cell pellet was collected, and the cells were resuspended in PBS. Live and dead cell dyes and Fc block were first used to incubate the cells to mark live cells and avoid non-specific antibody adsorption, and then CD3 antibody and IFN-γ antibody were used for staining. The stained cells were then analyzed by flow cytometry to analyze CD3 + IFN-γ + T cells in all CD3 + The proportion of T cells is cancer cell-specific T cells.

[0369] (4) Experimental results

[0370] As shown in Figure 8, when PBMCs were incubated alone, almost no activated cancer cell-killing T cells were detected before and after treatment. However, when cells were co-incubated with Nanoparticle 1, a certain amount of cancer cell-specific T cells were detected before and after treatment, and the amount after treatment was significantly higher than before treatment.

[0371] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A method for preparing antigen delivery particles, characterized in that: Antigen delivery particles are used to load antigen components, wherein the antigen components are fixed with a tissue fixative, the tissue fixative consists of alcohols and / or ketones and / or acids and an aqueous solution containing an oxidant, the content of alcohols and / or ketones and / or acids in the tissue fixative is 1wt%-99wt%, and the content of the oxidant in the aqueous solution is 0.001wt%-30wt%.

2. The method according to claim 1, characterized in that The content of alcohols and / or ketones and / or acids in the tissue fixative is 10wt%-95wt%; preferably, the content of oxidant in the aqueous solution is 0.01wt%-25wt%, preferably 0.1wt%-20wt%.

3. The method according to claim 1, characterized in that The alcohols and / or ketones and / or acids are alcohols and / or ketones and / or acids that are liquid at 0-50 degrees Celsius, and the oxidant can oxidize the antigen component; Preferably, the alcohols and / or ketones and / or acids are selected from one or more of ethanol, methanol, propanol, isopropanol, butanol, isobutanol, acetone, butanone, formic acid, acetic acid, propionic acid, butyric acid, preferably one or more of ethanol, methanol, acetone, acetic acid; Preferably, the oxidant is selected from hypochlorous acid, hydrogen peroxide, persulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, KIO3, KBrO3, ClO3 - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, NO3 - 、MnO4 - , one or more of F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3, preferably hydrogen peroxide and / or hypochlorous acid.

4. The method according to claim 1, characterized in that The method comprises the following steps: (1) Fixing cells and / or tissues containing antigen components using a tissue fixative consisting of alcohols and / or ketones and / or acids and an aqueous solution containing an oxidant; (2) after centrifugation at 200 g to 15000 g, remove the supernatant and collect the precipitate; (3) lysing the collected cells and / or tissues using a lysing solution containing a lysing agent, and dissolving the lysate components after lysing using a lysing solution containing a lysing agent; (4) directly using the dissolved lysate component obtained in step (3) as an antigen component; Alternatively, the purified component obtained by subjecting the lysate component obtained in step (3) to separation and purification steps such as dilution, salting out, organic solvent precipitation, heating, etc. is dissolved again in a dissolving solution containing a dissolving agent and then used as an antigen component; Wherein, the dissolving agent is independently selected from one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; Wherein, the structural formula 1 is as follows: R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl; Preferably, the dissolving agent is selected from metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, guanidine isothiocyanate, One or more of metformin, urea, urea peroxide, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, spermine, spermidine, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; Preferably, the fixing time in step (1) is greater than 1 hour; Preferably, the organic solvent precipitation is performed using one or more of alcohols, phenols, ketones, acids, and nitriles; Preferably, the cell is a cell containing an antigen component, and the tissue is a tissue containing an antigen component; more preferably, the cell is a cancer cell, and the tissue is a tumor tissue.

5. The method according to claim 4, characterized in that The method further comprises the steps of: (5) The antigen component is further loaded into the interior and / or surface of the nanoparticles and / or microparticles to obtain antigen delivery particles.

6. The method according to any one of claims 1 to 5, characterized in that The antigen delivery particle has the following components: (i) nanoparticle and / or microparticle framework structure formed by particle materials; (ii) lysing a lysate component of cells and / or tissues using a lysing solution containing a lytic agent, or isolating and purifying an antigen component therefrom after lysis; Preferably, the lysate component is a whole cell lysate component from cells and / or tissues, and more preferably, the lysate component is a partial cell and / or tissue lysate component containing an antigen component; Preferably, the antigen component is loaded inside and / or on the surface of the backbone structure; Preferably, an appropriate method can be used to enhance the immunogenicity of the antigen component before or after cell lysis; Preferably, the method for enhancing the immunogenicity of the antigen component comprises irradiation, oxidation, reduction, modification with hapten substances, enzyme treatment, denaturation, heating, and mineralization.

7. The method according to claim 6, characterized in that The irradiation includes any commonly used irradiation method; Preferably, the irradiation includes one or more of radioactive material irradiation, electron beam irradiation, microwave irradiation, ultraviolet irradiation, X-ray irradiation, α-ray irradiation, β-ray irradiation, and γ-ray irradiation.

8. The method according to claim 6, characterized in that The hapten substance includes one or more of 2,4-dinitrofluorobenzene, 2,4-dinitrochlorobenzene, trinitrophenol, dinitrophenol, albumin, Ovalbumin, N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide, substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, other hapten substances containing aldehyde groups, rhamnose, galactose, and aminogalactose.

9. The method according to claim 6, characterized in that The immunogenicity of whole cell lysate fractions and / or partial cell lysate fractions containing antigenic components from cells and / or tissues can be enhanced by appropriate methods; Preferably, the whole cell lysate component from cells and / or tissues and / or the partial cell lysate component containing the antigen component that enhances immunogenicity are loaded inside and / or on the surface of the skeleton structure; Preferably, the antigen component contained in the partial cell lysate component comprises protein and polypeptide components in cell and / or tissue lysate and / or RNA component or mRNA component in cell lysate; Preferably, the antigen component contained in the partial cell lysate component comprises protein and polypeptide components in cell and / or tissue lysate and / or lipid components in cell lysate; Preferably, 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; Preferably, the antigen component is separated and purified using an appropriate method from a lysate component dissolved after lysing using a lysing solution containing a lytic agent; Preferably, the antigen components separated and purified in the lysate components can also be irradiated before or after cell or tumor tissue lysis, or can be irradiated after the antigen components separated and purified are loaded onto nanoparticles and / or microparticles.

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

11. The method according to any one of claims 1 to 10, characterized in that The particle size of the nanoparticles is 1 nm-1000 nm, preferably 50-500 nm, more preferably 100-400 nm; preferably, the particle size of the micron particles is 1 μm-1000 μm, preferably 1-10 μm, more preferably 1-5 μm.

12. An antigen delivery particle prepared according to the method according to any one of claims 1 to 11.

13. Use of the antigen delivery particle according to claim 12 in at least one of the following (1)-(4): (1) Preparation of drugs for preventing and / or treating diseases; (2) Used to activate antigen-presenting cells and prepare cell vaccines based on antigen-presenting cells; (3) Assist in activating antigen-specific T cells and detect the content of antigen-specific T cells; (4) After assisting in the activation of antigen-specific T cells, the activated antigen-specific T cells are isolated and amplified, and used to prevent and / or treat diseases.

14. The use according to claim 13, characterized in that The antigen delivery particles can be used directly as vaccines; Preferably, after the antigen delivery particles activate dendritic cells and / or B cells, the activated dendritic cells and / or B cells can be used as cell vaccines; More preferably, the antigen delivery particles can also be used to detect the content of antigen-specific T cells after assisting in activating antigen-specific T cells in vitro, or the activated antigen-specific T cells can be sorted and / or amplified after assisting in activating antigen-specific T cells by the antigen delivery particles for the prevention and / or treatment of diseases.

15. The use according to any one of claims 13-14, characterized in that The disease is cancer or a tumor; preferably, the disease is a solid tumor or a blood tumor.

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