Terminal sterilization method for cancer particle vaccine loaded with cancer cell lysate component and use thereof

Abstract

A terminal sterilization method for a cancer particle vaccine loaded with a cancer cell lysate component and use thereof. According to the terminal sterilization method adopting specific irradiation sterilization, irradiation sterilization can be carried out by using rays and the like after a vaccine freeze-dried formulation has been prepared and sealed, so that the operation is more convenient, and the requirements for sites, instruments, and the like are relatively low. The irradiation sterilization treatment can produce a certain influence on components contained in an irradiated substance, but does not reduce the effect of a nano / micron vaccine or particle loaded with the lysate component. The irradiation-sterilized nano / micron vaccine exhibits better efficacy compared to a nano / micron vaccine prepared aseptically throughout the process, whether injected directly into an organism or utilized for in-vitro activation of other immune cells, so that the method has important application prospects in the field of oncotherapy.

Description

Terminal sterilization method for cancer particle vaccine loaded with cancer cell lysate components and its application Technical Field

[0001] The present invention relates to the field of immunotherapy, and in particular to a terminal sterilization method for a nano-vaccine (delivery particle) or micro-vaccine (delivery particle) loaded with all or part of the components of cancer cells and / or tumor tissue lysates and its application. Background Art

[0002] Cancer vaccines are an important approach to cancer immunotherapy. Key factors influencing cancer vaccine efficacy include tumor antigens, adjuvants, and delivery formats. Antigens can induce and activate specific immune responses, adjuvants can amplify these responses, and the delivery format influences the efficiency of vaccine phagocytosis by antigen-presenting cells (APCs) and the subsequent activation of antigen-specific T cells. Of these three components, antigens are the primary component that triggers specific immune responses and are therefore the most critical. Cancer cells and / or tumor tissues contain a full complement of cancer cell-specific and cancer cell-associated antigens, making cancer cell or tumor tissue lysates the optimal raw material for cancer vaccine preparation. The inventors have developed nano- or micro-vaccines loaded with whole-cell lysate fractions or partial lysate fractions of cancer cells / tumor tissues. The resulting nano- or micro-vaccines typically have particle sizes ranging from several hundred nanometers to several microns. Vaccines are typically administered by injection, requiring sterile preparations with stringent sterility requirements, including either terminal sterilization or full sterility throughout the entire production process. Process sterility places higher demands on the company's facilities, equipment, and instruments, resulting in higher costs and stricter management requirements. For terminal sterilization, filtration through a 0.22μm (220nm) filter is generally used. This is feasible for nanovaccines with diameters less than 150nm, but not for nanovaccines with diameters greater than 200nm. Therefore, there is an urgent need to develop feasible terminal sterilization methods, which are crucial for the industrial application of nanovaccines, reducing costs, and improving efficiency.

[0003] Summary of the Invention

[0004] Problems to be solved by the invention

[0005] In order to solve the problem that the site, equipment, management, etc. required for the whole process of aseptic preparation of nano vaccines are higher, and thus the cost, etc. are also higher, the present invention provides a method for terminal sterilization using specific irradiation sterilization, which can be sterilized by irradiation such as radiation after the vaccine freeze-dried preparation is prepared and sealed. The operation is more convenient and the requirements for site, equipment, etc. are also lower. It has been verified that irradiation treatment has a certain effect on the components contained in the irradiated substance, but does not reduce the efficacy of the nano / micro vaccine or particles of the loaded lysate components. Moreover, after irradiation sterilization, the efficacy of the nano / micro vaccine (particles) directly injected into the body or activated other immune cells in vitro is better than the nano / micro vaccine (particles) prepared by the whole process of aseptic preparation, and an unexpected effect has been achieved.

[0006] Solutions for solving problems

[0007] A terminal sterilization method for cancer nanoparticles (vaccines) or micron particles (vaccines) loaded with antigen components from lysates of cancer cells or tumor tissues, wherein the sterilization method is irradiation sterilization for 1 to 48 hours; the irradiation is treatment with one or more of X-rays, gamma rays, alpha rays, beta rays, and 1-100 MeV electron beams at a dose of 12 to 60 kGy;

[0008] The nanoparticles or microparticles comprise: (i) a nanoparticle and / or microparticle skeleton structure formed of a particle preparation material, and (ii) a whole cell lysate component and / or a partial cell lysate component containing an antigen component from cancer cells and / or tumor tissue; wherein the whole cell lysate component and / or the partial cell lysate component containing an antigen component from cancer cells and / or tumor tissue is loaded inside and / or on the surface of the skeleton structure;

[0009] Wherein, all the whole cell components in the lysate component from the cancer cell and / or tumor tissue are dissolved in a dissolving solution containing a dissolving agent; or the whole cell components in the lysate component from the cancer cell and / or tumor tissue include a water-soluble component and a water-insoluble component dissolved in a dissolving solution containing a dissolving agent; the antigen component contained in the partial cell lysate component includes a protein and polypeptide component in the cancer cell and / or tumor tissue lysate and / or an RNA component or mRNA component in the cell lysate, and the partial lysate component containing the antigen component is obtained by separating and purifying from the lysate using an appropriate method;

[0010] The cancer nanoparticles (vaccines) or microparticles (vaccines) loaded with antigen components in the lysate of cancer cells or tumor tissues that have been terminally sterilized by irradiation can be directly injected into the body for use as a vaccine or used as antigen delivery particles for activating other cells in vitro.

[0011] Furthermore, the terminal sterilization method is irradiation sterilization for 1 hour to 48 hours, 12-60 kGy or 1-100 MeV; preferably irradiation sterilization for 4-24 hours; more preferably irradiation for 6-16 hours.

[0012] Furthermore, the irradiation is a treatment using one or more of different irradiation methods such as X-rays, gamma rays, alpha rays, electron beams, beta rays, etc.; preferably, one or more of gamma rays, alpha rays, beta rays, electron beams, and X-rays are used, and more preferably, gamma rays, X-rays, or electron beams are used.

[0013] Furthermore, the particle size of the cancer nanovaccine or nanoparticle (antigen delivery nanoparticle) is 1nm-1000nm; preferably 50-500nm; more preferably 100-400nm; the particle size of the micron vaccine or micron particle (antigen delivery micron particle) is 1μm-1000μm; preferably 1-10μm; more preferably 1-5μm.

[0014] Furthermore, when a mixed vaccine (particle) of nano / micro vaccine (particles) is used, the particle size of the nano vaccine or nanoparticle (antigen delivery nanoparticle) is 100-600 nm, and the particle size of the micro vaccine or micro particle (antigen delivery micro particle) is 1.5-5 μm; preferably, the particle size of the nano vaccine or nano particle (antigen delivery nano particle) is 150-500 nm, and the particle size of the micro vaccine or micro particle (antigen delivery micro particle) is 2.0-3.5 μm.

[0015] Furthermore, the materials (primary materials such as PLGA or PLA) used to prepare the nanovaccine or nanoparticles (antigen delivery nanoparticles) or microvaccine or microparticles (antigen delivery microparticles) are modified by adding an appropriate amount of a substance that improves stability or promotes targeting. Preferably, PEG-modified PLGA or PLA can better achieve long-term circulation and passive targeting effects; the mass ratio of PEG-modified PLGA or PLA to unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.

[0016] Furthermore, the source of cancer cells described in the present invention is any method that can obtain cancer cells, including but not limited to cancer cell lines, cancer cells isolated and extracted from tumor tissues and obtained by in vitro amplification, cancer cells isolated and extracted from blood and obtained by amplification, or cancer cells differentiated and cultured from stem cells.

[0017] Preferably, the cancer cells are from one or more organisms, or from one or more cancer cell lines; the tumor tissue is from one or more organisms; the protein and polypeptide components / mRNA components (or RNA components) in the lysate of the cancer cells or the lysate of the tumor tissue contain antigen components; the protein and polypeptide components / mRNA components (or RNA components) in the water-soluble components of the cancer cells and / or tumor tissue and the protein and polypeptide components / mRNA components (or RNA components) in the water-insoluble components / water-insoluble components of the cancer cells and / or tumor tissue contain antigen components.

[0018] Furthermore, the antigen component may be a protein polypeptide component, a mixture of a protein polypeptide component and a total RNA component, or a mixture of a protein polypeptide component and a total mRNA component.

[0019] Furthermore, the nano / micro vaccine (particle) is further loaded with at least one component as shown below:

[0020] (iii) mRNA component or RNA component in the water-soluble component and / or the water-insoluble component;

[0021] (iv) immune adjuvants;

[0022] (v) positively charged substances,

[0023] Preferably, the immune adjuvant comprises at least one of the following: pattern recognition receptor agonists, Toll-like receptor agonists, Bacillus Calmette-Guérin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhancing reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn calf liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic Streptococcus preparation, coenzyme QIO, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokine, mRNA, MF59, double-stranded RNA, double-stranded DNA, single-stranded DNA, aluminum adjuvant, manganese adjuvant, calcium adjuvant, STING agonist, endotoxin adjuvant, liposome adjuvant, CAF01, active ingredients of Panax ginseng, active ingredients of Astragalus membranaceus;

[0024] More preferably, the immune adjuvant comprises at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist;

[0025] Most 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;

[0026] Preferably, the positively charged substance is selected from positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and / or positively charged inorganic substances;

[0027] More preferably, the positively charged substance is selected from any one or more of melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH4HCO3.

[0028] Furthermore, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-10;

[0029] More preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.001-2;

[0030] Most preferably, the mass ratio of the particle skeleton structure, protein and polypeptide components is 1:0.05-1.

[0031] Furthermore, the mass ratio of the particle skeleton structure to the RNA component or mRNA component is 1:0.001-10;

[0032] More preferably, the mass ratio of the particle skeleton structure to the RNA component or mRNA component is 1:0.01-2;

[0033] Most preferably, the mass ratio of the particle skeleton structure to the RNA component or mRNA component is 1:0.05-1.

[0034] Furthermore, before lysing the cancer cells / tumor tissue, the cancer cells / tumor tissue may be irradiated with radiation to inactivate the cancer cells / tumor tissue. The radiation includes but is not limited to γ ​​rays, X rays, β rays, α rays, etc.

[0035] The terminal sterilization method of the present invention is used to prepare a sterilized cancer nano-vaccine (particles) or micro-vaccine (particles) as follows:

[0036] (1) first using a lysis solution containing a lysis agent to lyse cancer cells or tumor tissue, and then using a lysis solution containing a lysis agent to dissolve lysate components;

[0037] (2) purifying or enhancing the immunogenicity of the lysate components dissolved in the lysate solution, and then re-dissolving the lysate components with a lysate solution containing a dissolving agent to separate and purify or enhance the immunogenicity of the components from the lysate solution;

[0038] (3) directly loading the lysate components dissolved in the lysate obtained in step (1) onto the nano / micro vaccine (particles); or loading the components dissolved in the lysate obtained in step (2) onto the nano / micro vaccine (particles) after purification or immunogenicity enhancement.

[0039] Wherein, the dissolving agent is independently selected from one or more of the compound represented by structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0040] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0041] Preferably, the dissolving agent is selected from the group consisting of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, urea peroxide, guanidine hydrochloride, urea peroxide, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine sulfamate, guanidine succinic acid, other guanidine-containing compounds, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzymes, polypeptides, amino acids, glycosides and choline.

[0042] Another preparation process of the cancer nano-vaccine or micro-vaccine sterilized by the terminal sterilization method of the present invention is as follows:

[0043] (1) First, cancer cells or tumor tissues are lysed using ultrapure water or an aqueous solution, and then the lysate is collected and centrifuged. The supernatant is the water-soluble component, and the precipitated water-insoluble component is solubilized using a dissolving solution containing a dissolving agent;

[0044] (2) purifying or enhancing the immunogenicity of the water-soluble component and / or the water-insoluble component, and then performing secondary dissolution using a dissolving solution containing a dissolving agent to separate and purify the component or enhance the immunogenicity from the lysate;

[0045] (3) The water-soluble component and / or the water-insoluble component obtained in step (1) are then directly or simultaneously loaded onto the nano / micro vaccine (particles), or the water-soluble component and / or the water-insoluble component obtained in step (2) are purified or subjected to immunogenicity enhancement treatment and then loaded onto the nano / micro vaccine (particles).

[0046] Wherein, the dissolving agent is independently selected from one or more of the compound represented by structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein structural formula 1 is as follows:

[0047] R1 is C, S, P, N or O, R2 to R5 are independently selected from hydrogen, alkyl, thiol, amino, carboxyl, substituted or unsubstituted guanidinyl;

[0048] Preferably, the dissolving agent is selected from the group consisting of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, urea peroxide, guanidine hydrochloride, urea peroxide, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine sulfamate, guanidine succinic acid, other guanidine-containing compounds, semicarbazide hydrochloride, aminoformyl urea, acetyl urea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracils, imidazoles, etc.), nitrosoureas, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzymes, polypeptides, amino acids, glycosides and choline.

[0049] Furthermore, before the cancer cells or tumor tissues are lysed, the cancer cells or tumor tissues can also be appropriately treated to enhance the immunogenicity of the antigen components in the cancer cells or tumor tissues. The treatment methods for enhancing immunogenicity include but are not limited to one or more of heating (greater than 50°C), incubation with specific substances that stimulate cancer cells, irradiation, fixation, co-action with hapten substances, enzyme treatment, oxidation, reduction, radiation, fixation, mineralization, etc.

[0050] After the cancer cells or tumor tissues are lysed, before the lysate components are loaded onto the nano / micro vaccine (particles), all or part of the lysate components of the cancer cells or tumor tissues can also be appropriately treated to purify the lysate components or enhance the immunogenicity of the antigen components in the cancer cells or tumor tissues. The treatment methods include but are not limited to one or more of heating (greater than 50°C), oxidation, reduction, fixation, mineralization, salting out, dilution of solvents, irradiation, co-action with hapten substances, enzyme treatment, extraction of RNA components, extraction of whole protein components, extraction of mRNA components, ultrafiltration, chromatography, electrophoresis, chromatography, recrystallization, precipitation, dialysis, extraction, etc.

[0051] The enzyme treatment method includes, but is not limited to, using one or more of nuclease, DNA enzyme, pepsin, chymotrypsin, trypsin, other protein digestive enzymes, protease inhibitors, and the like.

[0052] Among them, the oxidizing agent used to oxidize the antigen component includes but is not limited to hypochlorous acid, hydrogen peroxide (hydrogen peroxide), persulfate, KIO3, KBrO3, chlorine, dichromate, nitric acid, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, hydrogen peroxide, bromine, iodine, perchlorate, permanganate, dichromate, sodium peroxide, oxygen, chlorine, sodium dichromate, potassium dichromate, potassium permanganate, nitric acid, ClO3 - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, H2O2, NO3 - 、MnO4 - , F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3, etc. One or more of various oxidants. The oxidation can enhance the immunogenicity of some antigen components.

[0053] 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 such as ginseng, plant root extracts, etc.), interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate with cancer cells or tumor tissues to stimulate cancer cells or tumor tissues is to make the cancer cells produce more antigen components.

[0054] Among them, the mineralization method includes but is not limited to using one or more mineralization methods such as silicification, calcification, magnesiumization, and biomineralization.

[0055] The reduction is to reduce the antigen component using a component that can reduce the antigen. The reducing agent used to reduce the antigen component includes but is not limited to dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP) and other reducing agents.

[0056] The irradiation may be any commonly used irradiation method, including but not limited to one or more of X-ray irradiation, α-ray irradiation, β-ray irradiation, and γ-ray irradiation.

[0057] The chromatography described in the present invention 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, etc.

[0058] The chromatography method of the present invention includes but is not limited to column chromatography, thin layer chromatography, liquid chromatography, gas chromatography, supercritical fluid chromatography, etc.

[0059] The electrophoresis method described in the present invention 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, etc.

[0060] The method of isolating and extracting mRNA components or RNA components from cancer cells and / or tumor tissues described in the present invention includes but is not limited to one or more methods such as using an mRNA separation and extraction reagent, using an RNA separation and extraction kit, using a DNA removal kit, and using enzymes to degrade DNA.

[0061] The co-action with the hapten substance can be performed by using the hapten substance to co-act with cells in cancer cells or tumor tissues for a certain period of time to modify the antigen components in the cancer cells or tumor tissues, and then lysing the cancer cells and / or tumor tissues to obtain their lysates; or first lysing the cancer cells or tumor tissues to obtain their lysates, and then using the hapten substance to modify the antigen components in the cancer cell or tumor tissue lysates.

[0062] The hapten substance is a substance that can increase the immunogenicity of a protein or polypeptide after co-acting with the protein or polypeptide.

[0063] The hapten substances include but are not limited to 2,4-dinitrofluorobenzene (DNFB), 2,4-dinitrochlorobenzene (DNCB), trinitrophenol (TNP), dinitrophenol (DNP), albumin, ovalbumin (OVA), N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide (AED), substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, and other hapten substances containing aldehyde groups.

[0064] The substances that can be used for fixing tissues / cells or treating precipitation include but are not limited to formaldehyde, paraformaldehyde, glutaraldehyde, other hapten substances containing aldehyde groups, ethanol, methanol, acetone, acetic acid, propionic acid, butyric acid, formic acid, formalin, dichromate, chromic acid, potassium permanganate, picric acid, Zamboni fixative, PLP fixative, FPA fixative, TAF fixative, Rossman fixative, Regaud fixative, PLPD fixative, PAPG fixative, Orth fixative, Muller fixative, McDoWell fixative, AAF fixative, Holla fixative, nde fixative, Gendre fixative, aldehyde fixative, mercury fixative, alcohol fixative, oxidant fixative, neutral formaldehyde calcium fixative, FAB fixative, Carnoy fixative, Clarke fixative, B-5 ​​fixative, Bouin fixative, FineFIX fixative, AGM (70% ethanol 80ml + glacial acetic acid 10ml + methanol 10ml) fixative, Helly fixative, Zenker fixative, Kolmer fixative, picrate fixative, other cell or tissue fixatives or fixatives, one or more of diethylene oxide.

[0065] The components obtained by the separation and purification process are protein and polypeptide components and / or RNA components (or mRNA components) in the lysate.

[0066] Preferably, the nano / micro vaccine (particle) may further contain at least one component as shown below:

[0067] (a) cancer cell membrane components derived from tumor tissue and / or tumor cells;

[0068] (b) extracellular vesicle membrane fractions derived from extracellular vesicle lysates, wherein the extracellular vesicles are secreted by bacteria or tumor cells;

[0069] (c) bacterial membrane fractions derived from bacterial lysates;

[0070] (d) membrane fractions derived from antigen-presenting cells;

[0071] Preferably, the bacteria include at least one of the following: BCG, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus gestellii, Lactobacillus reuteri, and Lactobacillus rhamnosus.

[0072] Preferably, the particle material is selected from natural polymer materials, synthetic polymer materials and / or inorganic materials.

[0073] Preferably, the particle material may be a component of bacterial or viral origin, including but not limited to one or more of bacterial cell walls, bacterial proteins, viral proteins, and the like.

[0074] Preferably, the shape of the nano / micro vaccine (particle) is any shape, including but not limited to sphere, ellipsoid, barrel, polygon, rod, sheet, line, worm, square, triangle, butterfly, disc, vesicle, etc.

[0075] The present invention also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the cancer nanovaccine / microvaccine or the cancer vaccine prepared according to the method;

[0076] Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0077] The present invention also provides a method for preventing or treating a disease, wherein the method comprises administering to a subject a preventively or therapeutically effective amount of the cancer vaccine, or the cancer vaccine prepared according to the method, or the pharmaceutical composition.

[0078] The present invention also provides a method for terminal sterilization of the nanoparticles or microparticles, or use of the nanoparticles or microparticles loaded with cancer cell / tumor tissue lysate components prepared according to the method in at least one of the following:

[0079] (1) Preparation of nanoparticles or microparticles for direct injection into the body for the prevention or treatment of diseases;

[0080] (2) preparing antigen delivery particles for in vitro activation of antigen-presenting cells and preparation of antigen-presenting cell vaccines;

[0081] (3) preparing antigen delivery particles for assisting activation of antigen-specific T cells (indirect activation through antigen-presenting cells or direct activation) and detecting the content of antigen-specific T cells;

[0082] (4) After preparing antigen-specific T cells for auxiliary activation (indirect activation through antigen-presenting cells or direct activation), the activated antigen-specific T cells are isolated and amplified and used for the prevention or treatment of diseases;

[0083] Optionally, the disease is cancer or tumor;

[0084] Optionally, the cancer or tumor is a solid tumor or a hematological tumor.

[0085] Effects of the Invention

[0086] The present invention uses a specific ray radiation method to achieve terminal sterilization of nano / micro vaccines (particles) loaded with lysate antigen components, overcoming the problem that filtration sterilization cannot be used for terminal sterilization. After irradiation sterilization, the preparation meets the requirements of the Chinese Pharmacopoeia Sterility Test Method and meets the sterility requirements of injectable preparations. The preparation remains stable and can be stored for a long time, and the sterilization effect is good. Moreover, the nano / micro vaccine (particles) after irradiation sterilization has a better effect, achieving unexpected results. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG1 is a schematic diagram of the process of terminal sterilization of nanoparticles / microparticles loaded with cancer cell / tumor tissue lysate components by irradiation in the present invention.

[0088] 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.

[0089] Figures 3-9 are the experimental results of tumor growth rate and survival when nanovaccines and / or microvaccines are used to prevent or treat cancer in Examples 1-7; wherein, a is the experimental result of tumor growth rate when preventing or treating cancer (n≥8); b is the experimental result of mouse survival when preventing or treating cancer (n≥8), and each data point is the mean ± standard error (mean ± SEM); wherein, the significant difference in the tumor growth inhibition experiment in Figure a was analyzed by ANOVA, and the significant difference in Figure b was analyzed by Kaplan-Meier and log-rank test; *** indicates that there is a significant difference compared with the PBS blank control group at p < 0.005; ** indicates that there is a significant difference compared with the PBS blank control group at p < 0.01; * indicates that there is a significant difference compared with the PBS blank control group at p < 0.05; ### indicates that there is a significant difference between the two groups at p < 0.005; ## indicates that there is a significant difference between the two groups at p < 0.01; # indicates that there is a significant difference between the two groups at p < 0.05. DETAILED DESCRIPTION

[0090] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0091] 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.

[0092] 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".

[0093] 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.

[0094] The term "suffering from a disease" means that the body is experiencing symptoms of a disease.

[0095] 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.

[0096] The term "prevention" means that before a subject develops a disease, the subject is exposed to (e.g., administered) the delivery particles, delivery systems, vaccines, antigen-loaded drugs, or pharmaceutical compositions of the present invention, thereby alleviating the symptoms after developing the disease compared to when the subject has not developed the disease. It does not necessarily mean that the disease must be completely suppressed.

[0097] The vaccines of the present invention can be prepared using any method known to those skilled in the art, for example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding and / or lyophilizing processes.

[0098] In the present invention, the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the properties of the drug, the convenience of patients and medical staff, and other relevant factors.

[0099] The terms "individual," "patient," or "subject" as used in the context of the present invention include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0100] 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.

[0101] When preparing the cancer vaccine described in the present invention, antigen components can be prepared using cancer cells / tumor tissues obtained by various methods, such as tumor tissue, cancer cell lines, cancer cells isolated from tumor tissue and cultured and expanded, and cancer cells obtained by cultured and expanded circulating tumor cells isolated from peripheral blood. In actual applications, any other feasible approach can also be used to obtain cancer cells.

[0102] Due to space limitations, the cancers described in the examples of this invention are solid tumors. In practical applications, the nano- or micro-vaccines of this invention can also be used to treat hematologic malignancies and lymphomas. Because the immune microenvironment of hematologic malignancies and lymphomas is less complex than that of solid tumors, the nano- or micro-vaccines of this invention are more effective in treating hematologic malignancies and lymphomas.

[0103] A method for terminal sterilization of cancer nanoparticles (vaccines) or micron particles (vaccines) loaded with antigen components from lysates of cancer cells or tumor tissues, wherein the sterilization method is irradiation sterilization for 1 to 48 hours; the irradiation is treatment using one or more of different irradiation methods such as X-rays, gamma rays, alpha rays, beta rays, and 1-100 MeV electron beams at a dose of 12 to 60 kGy;

[0104] The nanoparticles or microparticles comprise: (i) a nanoparticle and / or microparticle skeleton structure formed of a particle preparation material, and (ii) a whole cell lysate component and / or a partial cell lysate component containing an antigen component from cancer cells and / or tumor tissue; wherein the whole cell lysate component and / or the partial cell lysate component containing an antigen component from cancer cells and / or tumor tissue is loaded inside and / or on the surface of the skeleton structure;

[0105] Wherein, all the whole cell components in the lysate component from the cancer cell and / or tumor tissue are dissolved in a dissolving solution containing a dissolving agent; or the whole cell components in the lysate component from the cancer cell and / or tumor tissue include a water-soluble component and a water-insoluble component dissolved in a dissolving solution containing a dissolving agent; the antigen component contained in the partial cell lysate component includes a protein and polypeptide component in the cancer cell and / or tumor tissue lysate and / or an RNA component or mRNA component in the cell lysate, and the partial lysate component containing the antigen component is obtained by separating and purifying from the lysate using an appropriate method;

[0106] The cancer nanoparticles (vaccines) or microparticles (vaccines) loaded with antigen components in the lysate of cancer cells or tumor tissues that have been terminally sterilized by irradiation can be directly injected into the body for use as a vaccine or used as antigen delivery particles for activating other cells in vitro.

[0107] Furthermore, the lysate components of the cancer cells and / or tumor tissues loaded by the nano / micro vaccine (particles) can be one or more selected from the following: (1) all lysate components solubilized by a lysate containing a dissolving agent; (2) protein polypeptide components in all lysate components solubilized by a lysate containing a dissolving agent; (3) protein polypeptide components + RNA components (or mRNA components) in all lysate components solubilized by a lysate containing a dissolving agent; (4) water-soluble components + water-insoluble components; (5) protein polypeptide components + water-insoluble components in the water-soluble components; (6) protein polypeptide components in the water-soluble components + protein polypeptide components in the water-insoluble components; (7) protein polypeptide components and RNA components (or mRNA components) in the water-soluble components + protein polypeptide components and RNA components (or mRNA components) in the water-insoluble components. In some preferred embodiments, the mass ratio of the protein and polypeptide in the water-soluble component to the protein and polypeptide in the water-insoluble component / the water-insoluble component is (0.1-10):(0.1-10); preferably (0.5-2):(0.5-2). Exemplarily, the mass ratio of the protein and polypeptide component in the water-soluble component to the protein and polypeptide in the water-insoluble component / the water-insoluble component is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.

[0108] Furthermore, the particle material can 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 25-200:1. In the preparation of the nano / micro vaccine (particle) of the present invention, an appropriate amount of PEG-modified PLGA or PLA can be added to the main material such as PLGA or PLA to better achieve the effects of long circulation and passive targeting; wherein the mass ratio of PEG-modified PLGA or PLA to unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.

[0109] The nano / micro vaccine (particles) of the present invention, wherein, in the vaccine (particles), the mass ratio of the skeleton material, protein and polypeptide components for particle preparation is 1:0.001-10; preferably, the mass ratio of the skeleton material, protein and polypeptide components for particle preparation is 1:0.01-2; most preferably, the mass ratio of the skeleton material, protein and polypeptide components for particle preparation is 1:0.05-1.

[0110] The surface of the nano / micro vaccine (particle) of the present invention 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.

[0111] The antigen-presenting cells used to prepare the biofilm components loaded on the surface of the nano / micro vaccine (particles) of the present invention can be derived from autologous or allogeneic sources, or from cell lines or stem cells. The antigen-presenting cells can be DCs, B cells, macrophages, or any mixture of the three, or other cells with antigen-presenting function. The antigen-presenting cells can be activated by antigen-loaded nanoparticles or microparticles.

[0112] When the biofilm components carried on the surface of the nano / micro vaccine (particle) of the present invention 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.

[0113] Any method for preparing nanoparticles and micron particles known to those skilled in the art can be used to prepare the nano / micro vaccine (particles) described in the present invention, including but not limited to solvent evaporation method, dialysis method, phase separation method, spray drying method, emulsion polymerization method, machine stirring shear method, membrane emulsification method, microfluidics method, ultrafiltration method, homogenization emulsification method, dispersion method, precipitation method, etc.

[0114] In some specific embodiments, the present invention provides the following exemplary particle preparation and irradiation sterilization methods, taking the solvent volatilization method as an example:

[0115] Step 1, using a lysate containing a lytic agent to crack tumor tissue or cancer cells, and then using a lysate containing a lytic agent to dissolve the lysate component; or after using ultrapure water or an aqueous solution to crack cancer cells / tumor tissue, the lysate component is centrifuged to collect the water-soluble component and the non-water-soluble component solubilized by the lysate containing a lytic agent. The above-obtained lysate component can be directly loaded onto the nano / micron vaccine (particle), or it can be loaded onto the nano / micron vaccine (particle) after the treatment (purification or enhancement of antigen component immunogenicity) of the following steps 2 and 3.

[0116] Optionally, before tissue or cell lysis, appropriate treatment can be performed to enhance the immunogenicity of the antigen component. Treatment methods to enhance immunogenicity include but are not limited to the use of radiation, heating, oxidation, reduction, modification with hapten substances, enzyme treatment, mineralization, etc.

[0117] The irradiation is performed using one or more irradiation methods such as X-rays, gamma rays, alpha rays, beta rays, etc. for a period of time.

[0118] The cancer cells or tumor tissues can be co-incubated with specific chemical substances to stimulate the cancer cells or tumor tissues before lysis. The specific substances that stimulate cancer cells include but are not limited to small molecule compounds (such as doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, cytokines, plant extracts (such as important extracts such as ginseng, plant root extracts, etc.), chemokines, interferons, bacterial secretions, bacterial extracellular vesicles, etc. The purpose of using specific substances to co-incubate with cancer cells or tumor tissues to stimulate cancer cells or tumor tissues is to make the cancer cells produce more antigen components.

[0119] The hapten substance is a substance that can increase the immunogenicity of a protein or polypeptide after co-acting with the protein or polypeptide.

[0120] The hapten substances include but are not limited to 2,4-dinitrofluorobenzene (DNFB), 2,4-dinitrochlorobenzene (DNCB), trinitrophenol (TNP), dinitrophenol (DNP), albumin, ovalbumin (OVA), N-iodoacetyl-N'-(5-sulfonic acid 1-naphthyl)ethylenediamide (AED), substituted or unsubstituted benzenesulfonamide, formaldehyde, paraformaldehyde, and other hapten substances containing aldehyde groups.

[0121] Any oxidizing agent that can oxidize the antigen component can be used in the present invention, including but not limited to hypochlorous acid, sulfate, hydrogen peroxide (hydrogen peroxide), KIO3, KBrO3, chlorine, dichromate, nitric acid, hydrogen peroxide, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, bromine, iodine, perchlorate, permanganate, dichromate, sodium peroxide, oxygen, chlorine, sodium dichromate, potassium dichromate, potassium permanganate, nitric acid, ClO3 - 、ClO4 - , Na2O2, K2O2, MgO2, CaO2, BaO2, H2O2, NO3 - 、MnO4 - , F2, Cl2, O2, Br2, I2, S, Si, HNO3, MnO2, FeCl3 and other oxidants.

[0122] The reducing agent of the present invention includes but is not limited to dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) and the like.

[0123] The enzymatic treatment method of the present invention includes but is not limited to the use of one or more of nuclease, DNA enzyme, pepsin, chymotrypsin, trypsin, other protein digestive enzymes, protease inhibitors, etc. The enzymatic hydrolysis of the present invention includes but is not limited to the use of any feasible enzymatic hydrolysis method such as nuclease, pepsin, trypsin, protease inhibitors, chymotrypsin, DNA enzyme, etc.

[0124] The mineralization method described in the present invention includes but is not limited to any mineralization or biomineralization method such as silicification, calcification, and magnesiumization.

[0125] 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 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, homogenization, extrusion, homogenization, high-speed stirring, chemical treatment, high-shear treatment, ultrafiltration, shrinkage, and the like.

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

[0127] 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, 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.

[0128] Step 2: purify the lysate components or enhance the immunogenicity of the lysate components by heating, diluting a dissolving solution containing a dissolving agent, dissolving the lysate components, salting out, oxidation, reduction, co-action with a hapten substance, fixation, irradiation, enzyme treatment, extracting protein and polypeptide components, extracting RNA components, extracting mRNA components, mineralization, etc.

[0129] In step 3, the antigen component (the lysate component that has been purified or treated to enhance immunogenicity) obtained in step 2 is re-dissolved using a dissolving solution containing a dissolving agent.

[0130] The dissolving agent in the dissolving solution used for the secondary dissolution of the precipitated components is selected from one or more of the compound represented by Structural Formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline; wherein Structural Formula 1 is as follows:

[0131] 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, 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.

[0132] Step 4: The antigen component solubilized by the dissolving solution containing the dissolving agent obtained in step 3 is added to the organic phase as the initial aqueous phase to prepare a colostrum sample.

[0133] 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.

[0134] 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.

[0135] In some embodiments, the organic solvent is dichloromethane. In addition, in some embodiments, the second predetermined concentration of the raw material for preparing the particles ranges from 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0136] In practice, the second predetermined volume of the organic phase is set based on the ratio of the second predetermined volume of the organic phase to the first predetermined volume of the aqueous phase. In the present invention, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably 1:10. During implementation, the first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.

[0137] In some embodiments, when the aqueous phase solution is a solution comprising antigen components in cell and / or tissue lysates, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL. In some embodiments, when the aqueous phase solution is a solution comprising antigen components in lysates and immune adjuvants, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of immune adjuvants is greater than 0.01 ng / mL, preferably 0.001 mg / mL to 20 mg / mL. In some embodiments, in the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0138] In some embodiments, when the aqueous phase solution is a solution containing the antigen component in the lysate, the concentration of the protein and polypeptide components therein is greater than 0.01 ng / mL, preferably 1 μg / mL to 1 mg / mL. In some embodiments, when the aqueous phase solution is a solution containing the antigen component and the immune adjuvant, the concentration of the protein and polypeptide components therein is greater than 1 ng / mL, preferably 1 μg / mL to 1 mg / mL, and the concentration of the immune adjuvant is greater than 0.01 ng / mL, preferably 0.001 mg / mL to 20 mg / mL. In some embodiments, in the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0139] Step 5: subjecting the mixed solution obtained in step 4 to any of the following treatments: i) ultrasonic treatment for more than 2 seconds; ii) stirring for more than 1 minute; iii) homogenization; iv) microfluidic treatment. Preferably, during mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, such as a stirring speed of 50 rpm to 1500 rpm and a stirring time of 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5 W and the time is greater than 0.1 second, such as 2 to 200 seconds; during homogenization, a high-pressure / ultra-high-pressure homogenizer or a high-shear homogenizer is used, and when a high-pressure / ultra-high-pressure homogenizer is used, the pressure is greater than 5 psi, such as 20 psi to 100 psi, and when a high-shear homogenizer is used, the speed is greater than 100 rpm, such as 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, such as 0.1 mL / min to 100 mL / min. Nano- and / or micron-size can be achieved through ultrasound, stirring, homogenization, or microfluidic processing. The size of the prepared nanoparticles or micron particles can be controlled by the length of ultrasound time, stirring speed, or homogenization pressure and time. Too large or too small a particle size will result in a change in particle size.

[0140] Step 6: Add the mixture obtained after the treatment in step 5 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of an emulsifier and perform any of the following treatments: i) ultrasonic treatment for greater than 2 seconds; ii) stirring for greater than 1 minute; iii) homogenization; or iv) microfluidic treatment. In this step, the mixture obtained in step 2 is added to the aqueous emulsifier solution and continues ultrasonication, stirring, homogenization, or mixing to achieve nano- or micronization. In the present invention, 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.

[0141] 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 invention, the range of the ratio of the second predetermined volume to the third predetermined volume is 1:1.1-1:1000, preferably 2:5. In order to control the size of 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.

[0142] Step 7: adding the liquid obtained after the treatment in step 6 to a fourth predetermined volume of an emulsifier aqueous solution with a fourth predetermined concentration, and stirring until a predetermined stirring condition is met.

[0143] In this step, the emulsifier aqueous solution is a PVA solution or other solutions.

[0144] The fourth predetermined concentration is 5 mg / mL. By selecting the fourth predetermined concentration, nanoparticles or microparticles of appropriate size are obtained. The selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the fourth predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. In a specific implementation, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted to control the size of the nanoparticles or microparticles.

[0145] In the present invention, the predetermined stirring condition of this step is until the organic solvent is completely volatilized, that is, the dichloromethane in step 1 is completely volatilized.

[0146] Step 8: After the mixed solution obtained in step 7 and meeting the predetermined stirring conditions 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).

[0147] Step 9: The suspension containing the lyophilization protective agent obtained in step 8 is divided into 20 ml transparent sample bottles of SKJYLEAN (Su Ke) and freeze-dried, and the lyophilized material is set aside.

[0148] Step 10: The freeze-dried nanoparticles or microparticles packed in freeze-dried sample bottles are irradiated with radiation such as gamma rays, alpha rays, or beta rays for a certain period of time to perform terminal sterilization.

[0149] Using irradiated and sterilized nano / microparticles as vaccines to prevent or treat diseases; or using nanoparticles and microparticles as antigen delivery particles to activate antigen-presenting cells (such as dendritic cells or mixed antigen-presenting cells) in vitro to prepare antigen-presenting cell vaccines; or using nanoparticles or microparticles to assist in the activation of antigen-specific T cells to detect the content of antigen-specific T cells; or using nanoparticles or microparticles to assist in the activation of antigen-specific T cells, sorting or amplifying the antigen-specific T cells, and using the sorted or amplified antigen-specific T cells to prevent or treat diseases.

[0150] Example 1 Nano-vaccine for the treatment of pancreatic cancer

[0151] In this example, the antigen component was derived from the Pan02 mouse pancreatic cancer cell line, and the organic polymer material PLGA was used as the nanoparticle skeleton material, and Polyinosinic-polycytidylic acid (poly(I:C)), CpG7909, and CpG2395 were used as immune adjuvants to prepare the nanovaccine using the solvent evaporation method.

[0152] (1) Preparation of antigen components

[0153] Collect 1.5×10 7 Pan 02 mouse pancreatic cancer cells were then resuspended in 1 mL of PBS and exposed to ultraviolet light for 30 minutes. An appropriate amount of ultrapure water was then added and the cells were repeatedly frozen and thawed 5 times (with ultrasound) to lyse the cancer cells. The lysate was then centrifuged at 5000 g for 5 minutes and the supernatant was taken as a water-soluble component soluble in pure water; an 8M urea aqueous solution was added to the resulting precipitate to dissolve the precipitate, thereby converting the water-insoluble component insoluble in pure water into a component soluble in 8M urea aqueous solution. The water-soluble component and the water-insoluble component were mixed in a mass ratio of 1:2 and used as antigen component 1 for preparing nanovaccine 1.

[0154] (2) Preparation of nanovaccines

[0155] In this example, nanovaccine 1 was prepared using the double emulsion method, a solvent evaporation method. The vaccine preparation material used, PLGA, has a molecular weight of 10-20 kDa, and the immunoadjuvants used are poly(I:C), CpG7909, and CpG2395. All of the above substances were filtered through a 0.22 μm filter membrane before use, and the entire preparation process was performed in a Class B sterile cleanroom. The preparation method is as described above. First, the antigen component 1 and the adjuvant were co-loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 1 was approximately 250 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 1.5 mg of protein and peptide components, and 0.05 mg each of poly(I:C), CpG7909, and CpG2395.

[0156] In this example, nanovaccine 1 was prepared using the double emulsion method, a solvent evaporation method. The vaccine preparation material used, PLGA, has a molecular weight of 10 kDa to 20 kDa, and the immunoadjuvants used are poly(I:C), CpG7909, and CpG2395. All of the above substances were filtered through a 0.22 μm filter membrane before use, and the preparation process was performed in a Class C cleanroom. The preparation method is as described above. First, the antigen component 1 and the adjuvant were co-loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 1 was approximately 250 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 1.5 mg of protein and peptide components, and 0.05 mg each of poly(I:C), CpG7909, and CpG2395. After freeze-drying, the nanoparticles were sterilized by irradiation with gamma rays for 24 hours (24 kGy). The sterilized preparation met the Chinese Pharmacopoeia's sterility test requirements, meeting the sterility requirements for injectable preparations, and remained stable after sterilization.

[0157] In this example, nanovaccine 1 was prepared using the double emulsion method, a solvent evaporation method. The vaccine preparation material used, PLGA, has a molecular weight of 10 kDa to 20 kDa, and the immunoadjuvants used are poly(I:C), CpG7909, and CpG2395. All of the above substances were filtered through a 0.22 μm filter membrane before use, and the preparation process was performed in a Class C cleanroom. The preparation method is as described above. First, the antigen component 1 and the adjuvant were co-loaded into the nanoparticles using the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 1 was approximately 250 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 1.5 mg of protein and peptide components, and 0.05 mg each of poly(I:C), CpG7909, and CpG2395. After freeze-drying, the nanoparticles were sterilized by irradiation with gamma rays for 48 hours (48 kGy). The sterilized preparation met the Chinese Pharmacopoeia's sterility test requirements, meeting the sterility requirements for injectable preparations and remaining stable.

[0158] (3) Nano-vaccines for cancer treatment

[0159] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare pancreatic cancer tumor-bearing mice. On day 0, 1.5×10 6 Pan 02 cells. On days 3, 6, 9, 14, 20, and 27 after tumor inoculation, mice were subcutaneously injected with 2 mg of nanovaccine 1 (irradiated and non-irradiated) or 100 μL of PBS. Tumor growth rate and survival were monitored. Tumor volume was recorded every three days starting on day 3. 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.

[0160] (4) Experimental results

[0161] As shown in Figure 3, tumors in mice in the PBS group rapidly expanded, leading to early death. Mice treated with Nanovaccine 1 experienced significantly slower tumor growth and prolonged survival, with the majority of mice achieving tumor-free recovery. Furthermore, irradiated Nanovaccine 1 was slightly more effective than non-irradiated Nanovaccine 1.

[0162] Example 2 Micronized vaccine for preventing brain cancer

[0163] (1) Preparation of antigen components

[0164] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 6 GL261 cells, and when the tumors grew to a volume of approximately 1000 mm 3 The mice were killed and the tumor tissues were removed. An appropriate amount of ultrapure water was added to resuspend the sample and the sample was repeatedly frozen and thawed five times to lyse the tumor tissue. The tumor tissue lysate was then heated at 90°C for 5 minutes. The heated sample was then centrifuged at 5000 g for 5 minutes. The supernatant was discarded and all the precipitates were collected. The precipitates were dissolved in 8M urea aqueous solution, which was the antigen component 1.

[0165] (2) Preparation of micronized vaccines

[0166] In this example, micron vaccine 1 (Micronvaccine 1) was prepared using the double emulsion method within the solvent evaporation method. The PLA used as the micron particle preparation material has a molecular weight of 30 kDa to 50 kDa, and the immunoadjuvants used are poly(I:C), CpG 1018, and CpG 2395. All of the above materials were filtered through a 0.22 μm filter membrane before use, and the entire preparation process was performed in a Class B sterile cleanroom. The preparation method is as described above. During the preparation process, the antigen component 1 and adjuvant were first loaded into the micron particles. Then, 100 mg of the micron particles were centrifuged at 8000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of micron vaccine 1 was approximately 2.0 μm. Each 1 mg of PLA microparticles was loaded with approximately 20 μg of protein or polypeptide component, and each 1 mg of PLA microparticles was loaded with 0.01 mg each of poly(I:C), CpG 1018, and CpG 2395. After freeze drying, a portion of the freeze-dried micron vaccine was sterilized by irradiation with gamma rays for 16 hours (16 kGy); after freeze drying, a portion of the freeze-dried micron vaccine was sterilized by irradiation with ultraviolet rays at 280-315 nm for 16 hours;

[0167] (3) Micron vaccines for cancer prevention

[0168] Tumor-bearing mice were prepared by selecting female C57BL / 6 mice aged 6-8 weeks. 4 mg of micronized vaccine 1 (irradiated or non-irradiated) or 100 μL of PBS were injected subcutaneously on days -35, -28, -21, -14, and -7 before tumor inoculation. On day 0, 1.5×10 6 The methods for monitoring the tumor growth rate and survival of mice were the same as above.

[0169] (9) Experimental results

[0170] As shown in Figure 4, tumors in mice in the PBS group grew rapidly, leading to early death. Mice treated with Micronvaccine 1 experienced significantly slower tumor growth, significantly prolonged survival, and the majority of mice remained tumor-free. Micronvaccine 1 irradiated with gamma rays was more effective than both unirradiated and ultraviolet-irradiated versions, demonstrating that irradiation sterilization does not reduce or affect the efficacy of micron vaccines loaded with tumor tissue lysate components, and that appropriate irradiation methods can enhance vaccine efficacy.

[0171] Example 3 Nano-vaccine for the treatment of liver cancer

[0172] (1) Preparation of antigen components

[0173] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 Hepa 1-6 liver cancer cells, when the tumor grows to a volume of about 1000mm 3 The mice were killed and the tumor tissue was removed and irradiated under X-rays for 20 minutes, and then a single-cell suspension of the tumor tissue was prepared. An appropriate amount of 6M guanidine hydrochloride aqueous solution was used to lyse the single-cell suspension of the tumor tissue, and then the lysate component was dissolved using a 6M guanidine hydrochloride aqueous solution. Then, a 40% ammonium sulfate aqueous solution was added dropwise to salt out the protein and polypeptide components in the lysate component dissolved by the 6M guanidine hydrochloride aqueous solution. After precipitation, the protein and polypeptide components were centrifuged at 14000RPM for 30 minutes, and then the precipitated protein and polypeptide components were collected. The protein and polypeptide components precipitated were secondary dissolved using 6M guanidine hydrochloride, which was the antigen component 1 for preparing nanovaccine 1.

[0174] (2) Preparation of nanovaccines

[0175] In this example, nanovaccine 1 was prepared using the double emulsion method within the solvent evaporation process. The PLGA materials used for nanovaccine preparation had a molecular weight of 20-30 kDa, and the PEG5000-PLGA had a molecular weight of 25-35 kDa. The mass ratio of PLGA to PEG5000-PLGA was 99:1. The immunoadjuvants used were poly(I:C), CpG 2395, and CpG SL03. The preparation method was as described above. During preparation, the cell antigen component 1 and the adjuvant were first loaded into the nanoparticles. Then, 100 mg of the nanoparticles were centrifuged at 12,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours. The average particle size of the nanoparticles 1 was approximately 300 nm. Each 1 mg of PLA nanoparticles was loaded with approximately 500 μg of the protein or polypeptide component, and 0.1 mg each of poly(I:C), CpG 2395, and CpG SL03. After freeze-drying, half of the nanovaccine 1 was irradiated with an electron beam (25 MeV) for 1 hour for irradiation sterilization; the other half was used directly without any irradiation treatment.

[0176] (3) Nano-vaccines for cancer treatment

[0177] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. On day 0, 1.5×10 6 Hepatocellular carcinoma cells were grown in mice. Mice were subcutaneously injected with 100 μL of 0.4 mg of nanovaccine 1 (irradiated or non-irradiated) 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.

[0178] (4) Experimental results

[0179] As shown in Figure 5, tumors in the PBS group rapidly expanded, leading to early death. Mice treated with Nanovaccine 1 significantly prolonged their survival, with the vast majority of mice recovering tumor-free. The irradiated Nanovaccine was even more effective.

[0180] Example 4 Nano-vaccine for the treatment of esophageal cancer

[0181] (1) Preparation of antigen components

[0182] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 AKR esophageal cancer cells, when the tumor grows to a volume of approximately 1000 mm 3Mice were sacrificed and tumor tissue was removed. A single-cell suspension of tumor tissue was prepared. 60 μM hypochlorous acid was then added and incubated for 1 hour to oxidize the tumor tissue suspension. The oxidized single-cell suspension was then lysed by adding 8 M urea in PBS, and the lysate was solubilized using 8 M urea in PBS. Ten volumes of ultrapure water were then added to the 8 M urea in PBS solution to dilute the urea concentration. The mixture was allowed to stand for 1 hour. After protein and peptide components were analyzed, the mixture was centrifuged at 3000 RPM for 10 minutes, and the supernatant and precipitate were collected. The precipitate was then redissolved using 8 M urea in PBS. 30% ammonium sulfate was added dropwise to the supernatant to salt out the protein and peptide components. The mixture was then centrifuged at 15000 RPM for 25 minutes. The supernatant was discarded, and the salted-out precipitate was redissolved in 8 M urea in PBS. The two redissolved precipitates were then combined to form antigen component 1.

[0183] (2) Preparation of nanovaccines

[0184] In this example, nanovaccine 1 was prepared using the double emulsion method within the solvent evaporation method. The PLGA material used for nanovaccine preparation has a molecular weight of 20-40 kDa. The immune adjuvants employed were poly(I:C), CpG 1018, and CpG 7909. The preparation method is as described above. During preparation, the cell antigen component 1 and the adjuvant are first loaded into the nanoparticles. Then, 100 mg of the nanoparticles are 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 average particle size of the nanoparticles 1 is approximately 260 nm. Each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of the protein or polypeptide component, and 0.005 mg each of poly(I:C), CpG 1018, and CpG 7909. After freeze-drying, one-third of the nano-vaccine 1 was used directly without irradiation sterilization; the other two-thirds of the vaccine was irradiated with beta rays for 12 hours (12 KGy) or 36 hours (36 KGy) for irradiation sterilization before use.

[0185] (3) Nano-vaccines for cancer treatment

[0186] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare esophageal cancer-bearing mice. On day 0, 1.5×10 6 AKR esophageal cancer cells were inoculated. Mice were subcutaneously injected with 100 μL of 2 mg of nanovaccine 1 or 100 μL of PBS on days 3, 6, 9, 14, 19, and 25 after tumor inoculation. Tumor growth and survival were monitored as described above.

[0187] (4) Experimental results

[0188] As shown in Figure 6, the tumors in the PBS group grew rapidly and the mice died soon after. The survival of the mice using Nanovaccine 1 was significantly prolonged, and the majority of the mice recovered without tumors.

[0189] Example 5: Nanoparticle-activated antigen-presenting cell vaccine for the treatment of colon cancer

[0190] (1) Preparation of antigen components

[0191] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5×10 6 MC38 colon cancer cells, when the tumor grows to a volume of approximately 1000 mm 3 Mice were sacrificed and tumor tissues were removed. The tumor tissues were fixed in a 75% ethanol aqueous solution (containing 2% hydrogen peroxide) for 2 hours, then centrifuged at 1500 RPM for 5 minutes. The supernatant was discarded, and an appropriate amount of 8M urea peroxide aqueous solution was added to the precipitate to lyse the tumor tissue cells. The tumor tissue lysate fraction was dissolved in the 8M urea peroxide aqueous solution. The 8M urea peroxide aqueous solution containing the lysate fraction was then diluted with 40 volumes of ultrapure water. The solution was allowed to stand for 6 hours to allow precipitation to form. The solution was then centrifuged at 3500 RPM for 10 minutes, and the precipitate was redissolved in 8M urea peroxide aqueous solution to obtain antigen component 1 for preparing antigen delivery particle 1.

[0192] (2) Preparation of antigen delivery nanoparticles loaded with antigen components

[0193] In this example, nanoparticle 1 was prepared using the double emulsion method within the solvent evaporation method. The nanovaccine material used, PLGA, had a molecular weight of 10 kDa to 20 kDa. The immune adjuvants used 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 the nanoparticles 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 average particle size of the nanoparticles 1 was approximately 280 nm. Each 1 mg of PLGA nanoparticles loaded approximately 2.0 mg of the protein or peptide component, with 0.02 mg each of poly(I:C), CpG 1018, and CpG 7909 loaded. Half of the nanoparticles 1 were then irradiated with gamma rays for 18 hours (18 kGy); the other half was used directly without irradiation.

[0194] (3) In vitro activation of antigen-presenting cells (dendritic cells + B cells) using antigen delivery particles 1 (irradiated or non-irradiated)

[0195] This example illustrates how to prepare DCs using the method of preparing dendritic cells from mouse bone marrow cells. First, 6-8 week-old C57BL / 6 mice were sacrificed by cervical dislocation. The tibia and femur of the hind legs were surgically removed and placed in PBS. The surrounding muscle tissue was removed using scissors and forceps. The ends of the bone were cut with scissors. PBS solution was then drawn with a syringe. A needle was inserted into the bone marrow cavity at each end of the bone, and the bone marrow was repeatedly flushed into a culture dish. The bone marrow solution was collected and centrifuged at 400g for 3 minutes. After that, 1mL of erythrocyte lysis buffer was added to lyse the erythrocytes. Lysis was terminated by adding 3mL of RPMI 1640 (10% FBS) medium, centrifuged at 400g for 3 minutes, and the supernatant 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.

[0196] 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.

[0197] BMDC (10 million) and B cells (10 million) were mixed in a quantitative ratio of 1:1, and then incubated with 0.5 mg of nanoparticles 1 (irradiated or unirradiated) in 10 mL of RPMI1640 complete culture medium containing 20 ng / mL interleukin 15 (IL-15) for 24 hours (37°C, 5% CO2). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove the free nanoparticles in the system, and then the mixed cells activated by the antigen delivery particles were used as a mixed cell vaccine (Cell vaccine). Among them, the mixed cells activated by irradiated antigen delivery particles were used as mixed cell vaccine 1 (Cell vaccine 1); the mixed cells activated by antigen delivery particles 1 that had not been irradiated were used as mixed cell vaccine 2 (Cell vaccine 2).

[0198] (4) DC vaccine for cancer treatment

[0199] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. On day 0, 1.5×10 6 MC38 colon cancer cells were inoculated. Mice were subcutaneously injected with 1 million cells 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.

[0200] (5) Experimental results

[0201] As shown in Figure 7, tumors in the PBS group rapidly expanded, leading to early death. Mice treated with Cell Vaccine 1 and Cell Vaccine 2 significantly prolonged their survival, with the majority of mice recovering tumor-free. Furthermore, Cell Vaccine 1 was more effective than Cell Vaccine 2.

[0202] Example 6 Antigen-loaded nanoparticles assist in the sorting and expansion of T cells for the treatment of breast cancer

[0203] (1) Preparation of antigen components

[0204] Each C57BL / 6 mouse was subcutaneously inoculated with 1.0×10 6 E0771 mouse 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 to prepare a single-cell suspension of the tumor tissue. 4 mL of 3% hydrogen peroxide (hydrogen peroxide) was then added for oxidation for 1 hour. 16 mL of ultrapure water was then added to lyse the single-cell suspension. The protein and polypeptide components were precipitated by heating at 95°C for 5 minutes. The protein and polypeptide components were then precipitated by centrifugation at 5000 RPM for 5 minutes. The supernatant was discarded and the precipitate was collected. An appropriate amount of 8 M urea was added to dissolve the precipitated component, which was the antigen component 1 for preparing nanoparticle 1.

[0205] (2) Preparation of antigen delivery nanoparticles

[0206] In this example, nanoparticle 1 was prepared using the double emulsion method within the solvent evaporation method. The PLGA nanovaccine material used had a molecular weight of 20-40 kDa. The immune adjuvants used were poly(I:C), CpG 1018, 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 the nanoparticles 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 average particle size of the nanoparticles 1 was approximately 280 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 600 μg of protein or peptide component, with 0.02 mg each of poly(I:C), CpG 1018, and CpG 7909 loaded. Half of the nanoparticles 1 were then terminally sterilized by X-ray irradiation for 30 hours (30 kGy); the other half were used without irradiation.

[0207] (3) Sorting and expansion of antigen-specific T cells

[0208] Female C57BL / 6 mice aged 6-8 weeks were selected and inoculated subcutaneously with 1.0×10 6 E0771 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 2 mg of nanoparticles 1 (irradiated or non-irradiated) were co-incubated in 5 mL of RPMI1640 complete medium for 36 hours. CD3 + CD134 + T cells are antigen-specific T cells (tumor-specific) that can recognize cancer cell antigens. + CD95 + 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 75%). The cancer cell-specific T cells sorted and expanded using irradiated nanoparticles 1 (nanovaccine 1) were designated T cells 1 (T cells 1); the cancer cell-specific T cells sorted and expanded using unirradiated nanoparticles 1 (nanovaccine 1) were designated T cells 2 (T cells 2).

[0209] (4) Sorting and expanding cancer cell antigen-specific T cells for cancer treatment

[0210] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. On day 0, 1.0×10 6 E0771 mouse breast cancer cells were injected intravenously on days 7, 10, 15, 20, and 25 after tumor inoculation. Mice were injected intravenously with 1 million sorted and expanded antigen-specific T cells 1 or 2 (concomitantly with 10,000 units of IL-2 every two days from day 7 to day 27). Alternatively, mice were injected intravenously with 10,000 units of IL-2 or 100 μL of PBS every two days from day 7 to day 27. Tumor growth and survival were monitored as described above.

[0211] (5) Experimental results

[0212] As shown in Figure 8, the tumors in the PBS group grew rapidly, leading to early death. Mice treated with either T cells 1 (T cells 1) or T cells 2 (T cells 2) had significantly longer survival, with the vast majority of mice recovering tumor-free. Furthermore, T cells 1 performed better than T cells 2.

[0213] Example 7 Nano-vaccine for the treatment of lung cancer

[0214] (1) Preparation of antigen components

[0215] Each C57BL / 6 mouse was subcutaneously inoculated with 2.0×10 6 LLC lung cancer cells, and when the tumor grows to a volume of approximately 1000 mm 3 Mice were sacrificed and tumor tissues were removed. The tumor tissues were fixed with 75% ethanol for 24 hours and then centrifuged at 1000 g for 5 minutes. The supernatant was discarded and the precipitate was lysed with an 8M urea-PBS aqueous solution. The lysate fraction was then dissolved with an 8M urea-PBS aqueous solution. Two volumes of 75% ethanol were then added to the lysate fraction dissolved with the 8M urea-PBS aqueous solution to precipitate the antigen component, etc. The mixture was allowed to stand for 6 hours. After the antigen component, etc. precipitated, the mixture was centrifuged at 3000 RPM for 10 minutes. The precipitate was collected and redissolved with an 8M urea-PBS aqueous solution to obtain antigen component 1.

[0216] (2) Preparation of nanovaccines

[0217] In this example, nanovaccine 1 was prepared using the double emulsion method within the solvent evaporation method. The PLGA nanoparticle used had a molecular weight of 20-40 kDa. The immunoadjuvants used were poly(I:C), CpG SL01, and CpG SL03. The preparation method was as described above. The cell antigen component 1 and the adjuvant were first loaded into the nanoparticles. Then, 100 mg of the nanoparticles 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 average particle size of the nanoparticles 1 was approximately 260 nm. Each 1 mg of PLGA nanoparticles was loaded with approximately 100 μg of protein or peptide component, and 0.05 mg each of poly(I:C), CpG SL01, and CpG SL03. After freeze-drying, half of the nanovaccine 1 was not sterilized by irradiation; the other half was terminally sterilized by alpha irradiation for 48 hours (48 kGy).

[0218] (3) Nano-vaccines for cancer treatment

[0219] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare lung cancer tumor-bearing mice. On day 0, 2.0×10 6 LLC lung cancer cells were inoculated. Mice were subcutaneously injected with 100 μL of 0.5 mg of nanovaccine 1 (irradiated or non-irradiated) 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.

[0220] (4) Experimental results

[0221] As shown in Figure 9, the tumors in the PBS group grew rapidly and the mice died soon after. The survival of mice treated with Nanovaccine 1 was significantly prolonged, and the therapeutic effect of irradiated Nanovaccine 1 was further enhanced compared to unirradiated Nanovaccine 1, with the vast majority of mice recovering without tumors.

[0222] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A terminal sterilization method for a cancer particle vaccine or antigen delivery particle loaded with an antigen component, characterized in that, The terminal sterilization includes: irradiating the encapsulated cancer particle vaccine or antigen delivery particles, where the irradiation is to treat with X-rays, γ-rays, α-rays, β-rays or 1-100 MeV electron beam at a dose of 12-60 kGy for 1-48 hours; the cancer particle vaccine contains antigen delivery particles, and the antigen delivery particles are nanoparticles or microparticles; The nanoparticles or microparticles have: (i) a nanoparticle and / or microparticle framework structure formed by the particle preparation material, (ii) whole cell components and / or partial cell lysate components containing antigen components from cancer cells and / or tumor tissue lysate components; wherein, the whole cell components and / or partial cell lysate components containing antigen components from cancer cells and / or tumor tissue are loaded inside and / or on the surface of the framework structure.

2. The terminal sterilization method according to claim 1, wherein, The specific steps of the terminal sterilization are as follows: S1. Prepare antigen delivery particles and resuspend them in a buffer solution containing a lyoprotectant to obtain a suspension; S2. Encapsulate the suspension, lyophilize the encapsulated product, and then perform irradiation treatment.

3. The terminal sterilization method according to claim 1, characterized in that, The particle preparation material is selected from one or more of natural polymer materials, synthetic polymer materials, inorganic materials, and biomaterials derived from bacteria or viruses.

4. The terminal sterilization method according to claim 3, wherein: The particle preparation material is poly(ethylene glycol)-modified poly(lactic-co-glycolic acid) and / or poly(ethylene glycol)-modified polylactic acid.

5. The terminal sterilization method according to claim 1, characterized in that: The whole cell components in the cancer cell and / or tumor tissue lysate components are obtained by completely dissolving cancer cells and / or tumor tissue with a lysing solution containing a solubilizer, or contain water-soluble components and water-insoluble components dissolved with a lysing solution containing a solubilizer; the antigen components contained in the partial cell lysate components include protein and polypeptide components in cancer cell and / or tumor tissue lysates and / or RNA components or mRNA components in cell lysates.

6. The terminal sterilization method according to claim 5, characterized in that, The preparation method of the antigen delivery particles is as follows: (1) First, lyse cancer cells or tumor tissue with a lysing solution containing a solubilizer, and then dissolve the lysate components with a lysing solution containing a solubilizer; (2) After purifying or enhancing the immunogenicity of the lysate components dissolved in the lysing solution, dissolve the components separated and purified or enhanced in immunogenicity from the lysate with a lysing solution containing a solubilizer again; (3) Directly load the lysate components dissolved in the lysing solution obtained in step (1) onto the antigen delivery particles; Or load the components obtained by purifying or enhancing the immunogenicity of the lysate components dissolved in the lysing solution obtained in step (2) onto the antigen delivery particles again; Wherein, the solubilizer is independently selected from one or more of the compounds shown in Structural Formula 1, deoxycholate, dodecyl sulfate, glycerol, proteolytic enzymes, polypeptides, amino acids, glycosides, and choline; wherein, Structural Formula 1 is as follows: R1 is C, S, P, N or O, and R2-R5 are independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidyl.

7. The terminal sterilization method according to claim 5, characterized in that, The preparation method of the antigen delivery particles is as follows: (1) First, lyse cancer cells or tumor tissues using ultrapure water or an aqueous solution, then collect the lysate and centrifuge it. The supernatant part is the water-soluble component, and the precipitated water-insoluble component is solubilized using a solubilizing solution containing a solubilizer; (2) After purifying or enhancing the immunogenicity of the water-soluble component and / or the water-insoluble component, use a solubilizing solution containing a solubilizer to redissolve the component separated, purified or enhanced in immunogenicity from the lysate; (3) Then directly load the water-soluble component and / or the water-insoluble component obtained in step (1) separately or simultaneously onto antigen delivery particles, or load the component obtained by purifying or enhancing the immunogenicity of the water-soluble component and / or the water-insoluble component obtained in step (2) onto antigen delivery particles after purification or enhancement of immunogenicity; Wherein, the solubilizer is independently selected from one or more of a compound represented by structural formula 1, deoxycholate, dodecyl sulfate, glycerol, proteolytic enzyme, polypeptide, amino acid, glycoside, and choline; wherein, structural formula 1 is as follows: R1 is C, S, P, N or O, and R2-R5 are independently selected from hydrogen, alkyl, mercapto, amino, carboxyl, substituted or unsubstituted guanidyl.

8. Use of the terminal sterilization method according to any one of claims 1-7 or the cancer particle vaccine or antigen delivery particle prepared by this terminal sterilization method in the preparation of a drug for preventing or treating tumors. The use is to inject the cancer particle vaccine containing antigen delivery particles into the body, or to activate immune cells in vitro using antigen delivery particles and then reinfuse the immune cells into the body.

9. The application according to claim 8, wherein: The use at least includes one of the following:

10. The application according to claim 9, characterized in that, (1) Preparation of antigen delivery particles for directly injecting into the body to prevent or treat diseases; (2) Preparation of antigen delivery particles for activating antigen-presenting cells in vitro or preparing an antigen-presenting cell vaccine; (3) Preparation of antigen delivery particles for assisting in activating antigen-specific T cells to detect the content of antigen-specific T cells; (4) Preparation of antigen delivery particles for assisting in activating antigen-specific T cells, then separating and amplifying the activated antigen-specific T cells and using them to prevent or treat diseases; Wherein, the disease is cancer or tumor; Wherein, the cancer or tumor is a solid tumor or a hematological tumor. ​

Images