CN116850275A

CN116850275A - Mixed cell cancer vaccine containing dendritic cells and B cells activated in vitro and application thereof

Info

Publication number: CN116850275A
Application number: CN202310818760.2A
Authority: CN
Inventors: 刘密
Original assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Current assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Priority date: 2023-06-07
Filing date: 2023-07-05
Publication date: 2023-10-10

Abstract

The invention relates to a mixed cell cancer vaccine containing dendritic cells and B cells activated in vitro and application thereof, wherein the mixed cell vaccine is obtained by simultaneously activating the dendritic cells and the B cells in vitro by antigen delivery particles; wherein the antigen delivery particles are nanoparticles and/or microparticles carrying an antigen component; the antigenic component is derived from one or more of the following: (1) A water-soluble component and/or a water-insoluble component of cells in cancer cells and/or tumor tissue; (2) protein and polypeptide components thereof; (3) an RNA component therein; (4) In vitro synthesized protein and/or polypeptide containing antigen polypeptide epitope; (5) Nucleic acids capable of expressing epitopes of antigenic polypeptides are synthesized in vitro. The invention overcomes the technical prejudice of the prior effect of the B cell on the primary T cell activation, and discovers that the effect of a dendritic cell and B cell mixed cell vaccine system is obviously superior to that of the prior dendritic cell vaccine.

Description

Mixed cell cancer vaccine containing dendritic cells and B cells activated in vitro and application thereof

Technical Field

The present disclosure relates to the field of immunotherapy, and in particular to an in vitro activated mixed cell cancer vaccine containing dendritic cells and B cells and applications thereof.

Background

Cancer vaccines are one of the important approaches to cancer immunotherapy. Cancer vaccines are one of the important approaches in cancer immunotherapy and prophylaxis. Dendritic Cells (DCs) are the most important antigen presenting cells, the most important cells for activation of characteristic specific immune responses. Dendritic cells are derived from bone marrow lymphocytes, can colonise whole body tissues, monitor the surrounding environment, and transmit captured information to an adaptive immune system (T lymphocytes (hereinafter abbreviated as T cells) and B lymphocytes (hereinafter abbreviated as B cells)) at any time, are professional antigen presenting cells expressing Major Histocompatibility Complex (MHC) class I and II molecules, and are core ligaments for innate immunity and adaptive immunity. The dendritic cells internalize and break down antigens obtained from the periphery into short peptide fragments, expressed on the surface of the dendritic cells in the form of peptide fragment-MHC complex, which is also the process of maturation of the dendritic cells, and then the dendritic cells loaded with antigen peptides migrate to secondary lymphoid organs where the T cells are activated. Compared with other antigen presenting cells, the dendritic cell antigen presenting efficiency is extremely high, and can induce few T cell responses, so that the dendritic cell antigen presenting efficiency becomes the most effective endogenous stimulation of T cell and B cell responses.

Clinically, many older cancer patients or cancer patients treated by chemotherapy and the like have poor immune status and small number of immune cells in the body, and if polypeptide or mRNA cancer vaccine is directly injected into the body of the cancer patients, the vaccine effect is poor. Because the reduced dendritic cell content in the patient can result in the vaccine not being taken up and presented by the dendritic cells to the T cells, the cancer cell specific immune response cannot be activated effectively.

Because dendritic cells have the ability to activate specific immunity in vivo, dendritic Cell (DC) cancer vaccines are one type of cancer vaccine, and the process of activating immune responses in vivo is shorter than for non-living cell vaccines that require homing to the lymph nodes and phagocytosis by dendritic cells in the lymph nodes before reactivating T cell immune responses. The development of dendritic cancer vaccines at present mainly comprises the steps of selecting a plurality of polypeptide antigens or a plurality of protein antigens or supernatant fluid in cancer cell lysate to activate dendritic cells in vitro and then reinjecting the dendritic cells in vivo. The first dendritic cell vaccine, profnge, was approved by the U.S. Food and Drug Administration (FDA) in 2010 for the treatment of refractory prostate cancer, but has relatively limited clinical efficacy due to the profnge vaccine. After that, the dendritic cell vaccine successively achieves a certain curative effect in the treatment of breast cancer, bladder cancer, kidney cancer, colon cancer, and rectal cancer, lung cancer, and melanoma, but the curative effect is still not ideal.

The basis of cancer vaccines is the selection of appropriate cancer antigens to activate the recognition of abnormally mutated cancer cells by the human immune system, which are highly heterogeneous and highly mutated, so that cancer cells or cancer tumor tissue itself are the best source of cancer antigens. The more dendritic cells, the more antigen is phagocytized, and the better the therapeutic effect of the vaccine. However, the tumor lysate is directly mixed with dendritic cells for incubation, because the cell membrane is fat-soluble and the components in the supernatant of the cell lysate used by previous researchers are water-soluble and not easily phagocytized by dendritic cells. It has been confirmed by studies that when T cell immune responses are first activated in vivo by antigen presenting cells, dendritic cells are the most important and most efficient antigen presenting cells for the first activation of T cell responses because of the advantage that phagocytic antigens are more likely to cross antigen presentation in cells. The antigen presenting cells such as B cells and macrophages hardly play a role in primary activation of T cells, and play a role in secondary activation or tertiary activation of activated T cells, so that the primary activation of the T cells can be completed by independent dendritic cells. Thus, all of the currently worldwide cancer vaccines prepared based on live antigen presenting cells are based solely on dendritic cell vaccines. Since the effect of the primary activation of B cells and macrophages is far lower than that of dendritic cells, scientists believe that antigens are hardly dependent on B cells and macrophages to activate T cells after phagocytosis by B cells and macrophages, and thus it is not considered necessary to use B cells and macrophages in the primary activation of immune responses. The inventors have also filed a dendritic cell cancer vaccine and its use (application number 202111603486.4), demonstrating that dendritic cells activated by nanoparticles or microparticles carrying the whole cell component of cancer cells can be used as vaccines. Since dendritic cells are the most central antigen presenting cells that activate specific immune responses, prior studies have also considered that dendritic cells alone can be used as a cancer vaccine. All dendritic cell vaccines at home and abroad only use dendritic cell activation as vaccine, but the curative effect of the dendritic cell vaccine is not optimal all the time. Existing research and scientists have considered that mixing antigen presenting cells does not improve the efficacy of dendritic cell live cell vaccines, as current live cell vaccines based on antigen presenting cells are also based on dendritic cells. The inventor discovers that by mixing dendritic cells and B cells in vitro and then incubating the dendritic cells and the B cells with antigen-loaded nano particles/micro particles for a certain time, and then reinjecting the dendritic cells and the B cells together as a vaccine, unexpected better effects can be obtained, and knowledge of traditional cancer vaccines based on the dendritic cells is subverted.

Disclosure of Invention

To solve the above technical problems, the present disclosure provides a mixed antigen presenting cell vaccine activated by antigen delivery particles, and for convenience of description, the present disclosure refers to a vaccine comprising mixed antigen presenting cells of DC and B cells activated by antigens simply as DB vaccine. The DB vaccine can contain DC and B cells activated by the antigen in vitro, and can also contain DC, B cells and macrophages activated by the antigen in vitro.

The research of the disclosure finds that in the process that dendritic cells phagocytose antigen in vitro and are activated by antigen, if a proper amount of B cells are added, and the dendritic cells and the B cells are used as mixed cell cancer vaccine (DB vaccine), the curative effect of the DB cancer vaccine is obviously improved compared with that of the dendritic cell vaccine (DC vaccine). Furthermore, the mixed cell cancer vaccine using DC cells and B cells is better than the mixed cell vaccine using DC cells and macrophages,

the present disclosure mixes antigen delivery particles loaded with an antigen component with Dendritic Cells (DCs) and B cells, then co-incubates them simultaneously in the same system, activates the dendritic cells and B cells simultaneously, and then uses the DC and B cell mix as a cancer vaccine.

Wherein the antigen delivery particles are nanoparticles and/or microparticles. The delivery particles are comprised of (i) a particle preparation scaffold material and (ii) an antigen component; the antigen component (ii) is derived from: (1) A water-soluble component and/or a water-insoluble component of cells in cancer cells and/or tumor tissue; (2) Or a water-soluble component and/or a protein and polypeptide component of a non-water-soluble component of cells in cancer cells and/or tumor tissue; (3) Or the protein and polypeptide components and the RNA component of the water-soluble component and/or the water-insoluble component of the cells in the cancer cells and/or tumor tissue (or only the mRNA component); (4) Or the above three cases, plus in vitro custom-synthesized polypeptide and/or RNA components containing cancer specific and/or cancer associated antigens (or mRNA components alone); (5) Proteins and polypeptides containing epitopes of an antigenic polypeptide and/or nucleic acids (mRNA and DNA) which can express epitopes of an antigenic polypeptide.

When the dendritic cells and the B cells are incubated with the antigen delivery particles, the number ratio of the dendritic cells to the B cells is 50:1-1:100.

When the dendritic cells and the B cells are incubated with the antigen delivery particles, the concentration of the antigen delivery particles in the co-incubation system is 0.002 mug/mL to 80mg/mL; preferably, the concentration of antigen delivery particles in the co-incubation system is from 0.001mg/mL to 30mg/mL, more preferably, the concentration of antigen delivery particles in the co-incubation system is from 0.05mg/mL to 10mg/mL.

The dendritic cells are autologous dendritic cells and/or allogeneic dendritic cells, dendritic cell lines or dendritic cells differentiated from stem cells; the B cells are autologous B cells, allogeneic B cells, B cell lines or B cells differentiated from stem cells.

The activation of the present disclosure is the simultaneous co-incubation of antigen component loaded delivery particles with dendritic cells and B cells in the same system, during which specific molecules may be added.

Macrophages may also be included in the co-incubation system of the present disclosure. The macrophages are autologous macrophages, allogeneic macrophages, macrophage lines or macrophages differentiated from stem cells.

Specific molecules that are added when the antigen delivery particles of the present disclosure are co-incubated with mixed cells include, but are not limited to, one or more of cytokines, chemokines, growth factors, antibodies. The cytokines include, but are not limited to, one or more of interleukins, interferons, tumor necrosis factorsThe method comprises the steps of carrying out a first treatment on the surface of the Such as granulocyte-macrophage colony stimulating factor (GM) CSF), interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 14 (IL-14), interleukin 4 (IL-4), interleukin 15 (IL- - 15 Interleukin 21 (IL-21), interleukin 17 (IL-17), interleukin 12 (IL-12), interleukin 6 (IL-6), interleukin 33 (IL-33), gamma interferon (IFN-gamma), TNF-alpha, etc.; preferably, the co-incubation system comprises granulocyte-macrophage colonies Stimulating factor (GM-CSF), interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 15 (IL-15), and interleukin 12 (IL-12) One or more of the following。

When the antigen component loaded by the delivery particle for activating the dendritic cell and B cell mixed cell cancer vaccine in vitro is a whole cell antigen component, the whole cell antigen component is a whole cell component, or protein and polypeptide components in the whole cell component, or protein and polypeptide components obtained after separation, purification and/or immunogenicity enhancing treatment in the whole cell component are added with RNA components (or mRNA components only), and cancer specific and/or cancer related antigen polypeptides and/or nucleic acids synthesized in vitro; when the antigen component carried by the delivery particle is a specific cancer-specific or cancer-associated antigen component, it is a protein and polypeptide comprising an epitope of an antigen polypeptide and/or a nucleic acid (mRNA and DNA) capable of expressing an epitope of an antigen polypeptide.

When the antigen component loaded by the delivery particles is a whole cell component, the whole cell component contains a water-soluble component and a water-insoluble component dissolved by using a dissolving solution containing a specific dissolving agent; when the antigen component loaded by the delivery particle is whole cell antigen, the whole cell antigen component contains protein and polypeptide components which are subjected to separation and purification treatment and/or immunogenicity enhancing treatment by specific means in cells and/or RNA components (or mRNA components only) in cells.

Methods of isolating and purifying protein and polypeptide components in a treated and/or enhanced immunogenicity treated cell and/or RNA components in a cell (or using only mRNA components) include, but are not limited to, one or more of salting out, heating, enzymatic treatment, immobilization, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, use of RNA isolation kits, use of mRNA isolation kits, oxidation, reduction, mineralization, and the like.

The salting-out is one-time salting-out, repeated salting-out or segmented salting-out. The cations contained in the reagent used for salting-out include, but are not limited to, those contained in the reagent used for salting-out include, but are not limited to, al ³⁺ 、Fe ³⁺ 、Fe ²⁺ 、Mg ²⁺ 、Sn ²⁺ 、Zn ²⁺ 、Ca ²⁺ 、Li ⁺ 、Na ⁺ 、NH ₄ ⁺ 、K ⁺ 、Cu ²⁺ 、Ag ⁺ 、Ba ²⁺ Etc. Anions contained in reagents used for salting out include, but are not limited to, cl ^- 、SO ₄ ^2- 、NO ₃ ^- 、CO ₃ ^2- 、SiO3 ^2- 、S ₂ O ₇ ^2- 、B ₄ O ₇ ^2- 、PO4 ^3- 、COO ^- 、NO ₂ ^- 、S ₂ O ₈ ^2- 、S ^2- 、CrO ₄ ^2- 、MnO ₄ ^- 、P ₂ O ₇ ^4- Etc.

The heating is performed at 40 ℃ or higher.

The enzyme treatment methods include, but are not limited to, the use of one or more of nucleases, dnases, pepsin, chymotrypsin, trypsin, other proteolytic enzymes, protease inhibitors, and the like.

The oxidizing agent for oxidizing antigen components includes, but is not limited to, hypochlorous acid, persulfates, KIO ₃ 、KBrO ₃ Chlorine, dichromate, nitric acid, hydrogen peroxide, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, hydrogen peroxide, sodium percarbonate, sodium perborate, potassium perborate, bromine, iodine, perchlorate, permanganate, dichromate, sodium peroxide, oxygen, chlorine, sodium dichromate, potassium permanganate, nitric acid, clO ₃ ^- 、ClO ₄ ^- 、Na ₂ O ₂ 、K ₂ O ₂ 、MgO ₂ 、CaO ₂ 、BaO ₂ 、H ₂ O ₂ 、NO ₃ ^- 、MnO ₄ ^- 、F ₂ 、Cl ₂ 、O ₂ 、Br ₂ 、I ₂ 、S、Si、HNO ₃ 、MnO ₂ 、FeCl ₃ And the like, and one or more of various types of oxidizing agents.

The mineralization includes, but is not limited to, one or more of the mineralization methods of siliconization, calcification, magnesian, biomineralization, and the like.

The reduction is to reduce the antigen component using a component that can reduce the antigen, and reducing agents for reducing the antigen component include, but are not limited to, dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), and the like.

The chromatography includes, but is not limited to, column chromatography, gas chromatography, high pressure liquid chromatography, adsorption chromatography, partition chromatography, thin layer chromatography, high performance liquid chromatography, ion exchange chromatography, thin film chromatography, affinity chromatography, gel chromatography, etc.

The chromatography includes, but is not limited to, column chromatography, thin layer chromatography, liquid chromatography, gas chromatography, supercritical fluid chromatography, and the like.

The electrophoresis method includes, but is not limited to, SDS electrophoresis, isoelectric focusing electrophoresis, isotachophoresis, immunoelectrophoresis, serum protein electrophoresis, nucleic acid electrophoresis, DNA sequencing electrophoresis, gel electrophoresis, preparative electrophoresis, and the like.

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

R ₁ c, N, S or O, R ₂ ～R ₅ Independently selected from hydrogen, alkyl, carboxyl, substituted or unsubstituted amino, mercapto, guanidino, and the like.

The compound containing the structure of the structural formula 1 comprises, but is not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other guanidine-containing compounds, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, urea salt, urea, carbamyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazole, etc.), nitrosoureas, etc.

The whole cell component preparation process loaded on the delivery particle comprises the following steps: firstly, cracking cancer cells or tumor tissues, then respectively collecting water-soluble components and non-water-soluble components in the cracking liquid, and dissolving the non-water-soluble components by using a dissolving liquid containing a specific dissolving agent and then using the water-soluble components together;

R ₁ c, N, S or O, R ₂ ～R ₅ Independently selected from hydrogen, alkyl, carboxyl, substituted or unsubstituted amino, mercapto, substituted or unsubstituted guanidino, and the like.

The compound containing the structure of the structural formula 1 comprises, but is not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other compounds containing guanidine groups, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamyl urea, acetylurea, urea salt, urea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazole, etc.), nitrosoureas, etc.

Or the whole cell component preparation process loaded on the delivery particle comprises the following steps: the method comprises the steps of lysing cancer cells or tumor tissues by using a lysis solution containing a specific lysis agent, and then lysing whole-cell lysis solution by using the lysis solution containing the specific lysis agent.

The compound containing the structure of the structural formula 1 comprises, but is not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other guanidine-containing compounds, guanidine carbonate, arginine, guanidinoacetic acid, urea salt, urea, guanidino phosphoric acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glimepiride, etc.), thiourea compounds (thiouracil, imidazole, etc.), nitrosoureas, etc.

Alternatively, the whole antigen component loaded in the whole cell component of the delivery particle of the present disclosure may be prepared by a process comprising: firstly, cracking cancer cells or tumor tissues, then respectively collecting water-soluble components and non-water-soluble components in the cracking liquid, and re-dissolving the precipitate part by using a dissolving liquid containing a dissolving agent after all the water-soluble components are treated by salting out, heating, enzyme treatment, oxidation, reduction, mineralization, fixation, chromatography, electrophoresis, chromatography, irradiation, recrystallization, precipitation, extraction, dialysis, and other methods; the water-insoluble part contains the dissolving solution of the dissolving agent and is directly used after being dissolved, or the obtained precipitate component is dissolved again by using the dissolving solution containing the dissolving agent after being treated by salting out, heating, enzyme treatment, oxidation, reduction, mineralization, chromatography, electrophoresis, chromatography, fixation, recrystallization, precipitation, extraction, dialysis, irradiation and other methods; the protein polypeptide component in the water-soluble component is mixed with the water-insoluble component or the antigen component which is secondarily dissolved after the purification treatment of the water-insoluble component forms the whole antigen component in the whole cell component. The RNA component or mRNA component in the water-soluble component and/or the water-insoluble component can be simultaneously separated and extracted in the above treatment steps, and the RNA component or mRNA component can be used as an antigen component alone or in combination with the protein polypeptide component.

Or the preparation method of the whole antigen component loaded on the delivery particle comprises the following steps: after the cancer cells and/or tumor tissues are lysed by using a lysing solution containing a lysing agent, the lysate component is lysed by using a lysing solution containing a lysing agent, and then the obtained lysate component after lysis is subjected to salting out, heating, enzyme treatment, oxidation, mineralization and other methods, and then the obtained precipitate component is re-lysed by using a lysing solution containing a lysing agent. The RNA component or mRNA component of the lysate may be separated and extracted simultaneously in the above-mentioned treatment steps, and used alone as an antigen component or combined with a protein polypeptide component and used as an antigen component.

R ₁ c, N, S or O, R ₂ ～R ₅ Independently selected from hydrogen, alkanesA group, a carboxyl group, a substituted or unsubstituted amino group, a mercapto group, a substituted or unsubstituted guanidino group, or the like.

The antigen component, the non-water soluble component, the whole cell component, or the whole cell antigen component, the precipitated and/or denatured protein and polypeptide component of the present disclosure are loaded into the delivery particles after being solubilized by a solubilization solution containing a lysing agent,

The mixed cell cancer vaccine disclosed by the disclosure is characterized in that: means for loading the antigen component onto the surface of the delivery particle include, but are not limited to, at least one of adsorption, covalent attachment, charge interactions, hydrophobic interactions, one or more steps of solidification, mineralization, and encapsulation; the antigen component is supported within and/or on the surface of the delivered particle, either separately or simultaneously.

The mixed cell cancer vaccine disclosed by the disclosure is characterized in that: the delivery particles are internally and/or surface loaded with an immunopotentiating adjuvant.

Cancer cells or tumor tissue components carried by antigen delivery particles of the present disclosure, the cancer cells being cancer cells from one or more organisms, or from one or more cancer cell lines; the tumor tissue is tumor tissue from one or more organisms; the protein and polypeptide component/RNA component (or mRNA component) in the water-soluble component and/or the protein and polypeptide component/RNA component (or mRNA component) in the water-insoluble component in the cancer cell and/or the tumor tissue and/or the water-insoluble component/protein and polypeptide component/RNA component (or mRNA component) in the water-insoluble component and/or the tumor tissue; the cancer specific and/or related antigen polypeptide and/or the nucleic acid capable of expressing the cancer specific and/or related antigen polypeptide are custom synthesized in vitro and then mixed with components from cell/tumor tissue lysate to be co-loaded in nano-or micro-vaccine.

Nucleic acids that can express cancer-specific and/or related antigenic polypeptides include mRNA and DNA.

The protease inhibitor or protease may be added to the cancer cell or tumor tissue lysate fraction prior to isolation and purification of the antigen fraction; alternatively, the antigen may be enhanced in immunogenicity by one or more of irradiation, heating, oxidation, reduction, recrystallization, precipitation, extraction, dialysis, denaturation, etc. prior to isolation and purification of the antigen component.

After separation and purification of the antigen component, a protease inhibitor or protease may be added to the cancer cell or tumor tissue lysate component; alternatively, the antigen component may be isolated and purified and then subjected to one or more of irradiation, heating, oxidation, reduction, immobilization, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, denaturation, etc. to enhance the immunogenicity of the antigen.

The source of cancer cells described in the present disclosure is any method by which cancer cells can be obtained, including, but not limited to, cancer cell lines, cancer cells obtained by in vitro expansion of cancer cells isolated from tumor tissue, cancer cells obtained by expansion of circulating tumor cells isolated from blood, or cancer cells differentiated from stem cells, etc.

The cancer vaccine disclosed by the disclosure, wherein the cancer cells are from one or more organisms or from one or more cancer cell lines; the tumor tissue is tumor tissue from one or more organisms; the protein and polypeptide component/RNA component (or mRNA component) in the water-soluble component and the protein and polypeptide component/RNA component (or mRNA component) in the water-insoluble component/non-water-soluble component of the cancer cell and/or tumor tissue contain an antigen component; the cancer specific and/or related antigen polypeptide and/or the nucleic acid capable of expressing the cancer specific and/or related antigen polypeptide are custom synthesized in vitro and then mixed with components from cell/tumor tissue lysate to be co-loaded in nano-or micro-vaccine.

The cancer cells or tumor tissue may be co-incubated with a specific chemical prior to lysis to stimulate the cancer cells or tumor tissue and then the cancer cells or tumor tissue may be lysed, the specific chemical to stimulate the cancer cells including, but not limited to, small molecule compounds (e.g., doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), plant extracts (e.g., important extracts of ginseng, plant rhizome extracts, etc.), growth factors, cytokines, chemokines, interferons, bacterial secretions, bacterial extracellular vesicles, etc.

The delivery particles of the in vitro activated mixed cell cancer vaccine of the present disclosure are further loaded with at least one component as shown below:

(i) An RNA component or an mRNA component in the water-soluble component or the non-water-soluble component;

(ii) An immunoadjuvant;

(iii) A positively charged substance selected from positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and/or positively charged inorganics;

preferably, the immunoadjuvant comprises at least one of: pattern recognition receptor agonists, toll-like receptor agonists, BCG cell wall scaffold, BCG methanol extraction residue, BCG cell wall dipeptide, mycobacterium, polyoxin a, mineral oil, virus-like particles, immunopotentiating reconstituted influenza virus minibodies, cholera enterotoxin, saponins and derivatives thereof, resiquimod, thymosin, neobovine liver active peptide, imiquimod, polysaccharide, curcumin, immunoadjuvant CpG, immunoadjuvant poly (I: C), immunoadjuvant poly ICLC, short coryneform bacterin, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukins, interferons, polyminosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokines, mRNA, MF59, double-stranded RNA, double-stranded DNA, single-stranded DNA, aluminum adjuvant, manganese adjuvant, calcium adjuvant, STING agonist, endotoxin adjuvant, liposome, CAF01, yellow ginseng, active ingredient;

Preferably, the immunoadjuvant comprises at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist; further, the immunopotentiating adjuvant comprises (1) Poly (I: C) and/or Poly (ICLC); (2) CpG-ODN; wherein the CpG-ODN is at least one of A class CpG-ODN, B class CpG-ODN and C class CpG-ODN; preferably at least two, and at least one of them is a B class CpG-ODN or a C class CpG-ODN.

In the context of the present disclosure, a "CpG" or "CpG-ODN" (CpG oligonucleotide, cpG oligodeoxynucleotide) is a synthetic Oligodeoxynucleotide (ODN) that contains unmethylated cytosine-guanine dinucleotides (CpG). The structural features and immune effects of different types of CpG-ODNs are different, and generally classified into A, B, C.

In some embodiments, cpG-ODNs include, but are not limited to: cpG 1018 (B class), cpG 7909 (B class), cpG 2006 (B class), cpG-BW006 (B class), cpG 2395 (C class), cpG SL01, cpG 1585 (A class), cpG 2216 (A class), cpG SL03, cpG 2395 (C class), cpG M362 (C class), cpG 2336 (A class).

In the present disclosure, the positively charged species include, but are not limited to, at least one of the following: positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, positively charged inorganics. Exemplary, the positively charged species include melittin, RALA polypeptide, KALA polypeptide, R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine, and NH ₄ HCO ₃ Any one or any combination of the above.

Illustratively, positively charged polypeptides include, but are not limited to, arginine-containing polypeptides, histidine-containing polypeptides, and/or histidine-and/or lysine-containing KALA polypeptides, RALA polypeptides, melittin, and the like.

Illustratively, positively charged amino acids include, but are not limited to, arginine, histidine, lysine, and the like.

Illustratively, positively charged high molecular polymers include, but are not limited to, polyarginine, polylysine, polyhistidine, and the like.

Illustratively, positively charged lipids include, but are not limited to DOTAP and the like.

Illustratively, positively charged proteins include, but are not limited to, protamine, histone, and the like.

Exemplary positively charged minerals include, but are not limited to, NH ₄ HCO ₃ Aluminum hydroxide, and the like.

In the delivery particles of the in vitro activated mixed cell vaccine, the particles are used for preparing a framework material, a protein and a polypeptide component according to the mass ratio of 1:0.001-10; preferably, the mass ratio of the components of the particle preparation framework material, protein and polypeptide is 1:0.01-2; most preferably, the mass ratio of the components of the particle preparation framework material, protein and polypeptide is 1:0.05-1.

When RNA or mRNA is loaded in the delivery particles of the in vitro activated mixed cell vaccine described in the present disclosure, the particles produce a mass ratio of scaffold material to RNA/mRNA components of 1:0.001-10; preferably, the mass ratio of the particle preparation framework material to the RN a/mRNA component is 1:0.01-2; most preferably, the particles are prepared with a mass ratio of backbone material to RNA/mRNA component of 1:0.05-1.

In the delivery particles of the in vitro activated mixed cell vaccine of the present disclosure, the mass ratio of the protein and polypeptide component/RNA component (or mRNA component) from the lysate to the cancer specific and/or related antigen polypeptide/nucleic acid component from the artificial synthesis is 1:0.001-10; preferably, the mass ratio of protein and polypeptide components/RNA components (or mRNA components) from the lysate to the cancer specific and/or related antigen polypeptide/nucleic acid components from the artificial synthesis is 1:0.01-2; most preferably, the mass ratio of protein and polypeptide components/mRNA components from the lysate to cancer specific and/or related antigen polypeptide/nucleic acid components from the artificial synthesis is 1:0.05-1.

The delivery particles of the in vitro mixed cell cancer vaccine of the present disclosure further comprise at least one component on the surface and/or inside as shown below:

(a) Cancer cell membrane fraction derived from tumor tissue and/or tumor cells;

(b) An extracellular vesicle membrane fraction derived from extracellular vesicle lysates, the extracellular vesicles being secreted by bacteria or tumor cells;

(c) A bacterial membrane component derived from a bacterial lysate;

(d) An extracellular vesicle membrane fraction derived from extracellular vesicle lysates, the extracellular vesicles being secreted by bacteria or tumor cells or antigen presenting cells;

(e) A membrane fraction of antigen presenting cells or an extracellular vesicle fraction of antigen presenting cells.

When the membrane component is on the surface of the antigen delivery nanoparticle or microparticle, methods of loading the membrane component onto the surface of the nanoparticle or microparticle include, but are not limited to, one or more of ultrasound, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, microfluidic, chemical bond attachment, agitation, dialysis, homogenization, and homogenization.

Preferably, the bacteria include, but are not limited to, at least one of the following: bacillus calmette-guerin, escherichia coli, bifidobacterium longum, bifidobacterium breve, bifidobacterium lactis, lactobacillus acidophilus, lactobacillus formans, lactobacillus reuteri, lactobacillus rhamnosus and the like.

Further, in some embodiments, the antigen component in the cancer cells and/or tumor tissue from which the antigen delivery nanoparticles or microparticles are prepared is selected from one or both of the following: (1) Protein and polypeptide components in the water-soluble component + water-insoluble component; (2) Protein and polypeptide components in the water-soluble component + protein components or polypeptides in the water-insoluble component. In some preferred embodiments, the mass ratio of the protein and polypeptide in the water-soluble component to the protein and polypeptide in the water-insoluble component/water-insoluble component is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Illustratively, the mass ratio of protein and polypeptide components in the water-soluble component to protein and polypeptide components in the water-insoluble component/water-insoluble component is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.

In some embodiments, the immunogenic proteins and/or polypeptides are derived from a portion of the components and extracellular vesicle lysates in cancer cells/tumor tissue. Further, the extracellular vesicle lysate is selected from an extracellular vesicle lysate of a cancer cell and/or an extracellular vesicle lysate of a bacterium. The mass ratio of a part of the components in the cancer cells and/or tumor tissues to the extracellular vesicle lysate components is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Illustratively, the mass ratio is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.

In some embodiments, the immunogenic proteins and/or polypeptides are derived from a portion of the components and bacterial lysates in cancer cells/tumor tissue. Further, the mass ratio of a part of the components in the cancer cells/tumor tissue to the bacterial lysate is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2). Illustratively, the mass ratio is 1:1, 0.5:1, 0.8:1, 1:1.2, 1:1.5, 1:2, 2:1, 3:1, 4:1, 5:1, 1:3, 1:4, 1:5, and the like.

The preparation method of the mixed cell cancer vaccine comprises the following steps of: firstly, cracking cancer cells and/or tumor tissues to obtain a lysate thereof; then separating the water-soluble component and the water-insoluble component in the lysate by using one or more of centrifugation, filtration, dialysis, ultrafiltration and the like to obtain the water-soluble component and the water-insoluble component in the lysate respectively; then the water-soluble components in the obtained lysate are treated by salting out and/or heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation and other methods, and then protein and polypeptide components in the lysate are precipitated; and then dissolving the protein and polypeptide components in the precipitated fraction by using a dissolving solution containing a dissolving agent to obtain the protein and polypeptide components in the water-soluble component. The dissolving agent is selected from one or more of guanidine salt, urea and other compounds containing the structure shown in the structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glucoside and choline.

The cancer vaccine disclosed by the disclosure, wherein the preparation steps of protein and polypeptide components in water insoluble components in cancer cells and/or tumor tissues loaded by antigen delivery particles for activating the cell vaccine are as follows: firstly, cracking cancer cells and/or tumor tissues to obtain a lysate thereof; then separating the water-soluble component and the water-insoluble component in the lysate by using one or more of centrifugation, filtration, dialysis, ultrafiltration and the like to obtain the water-soluble component and the water-insoluble component in the lysate respectively; dissolving the water-insoluble component in the obtained lysate by using a dissolving solution containing a dissolving agent, and then precipitating protein and polypeptide components in the lysate after salting-out and/or heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation and other methods; and then the protein and polypeptide components in the precipitated part are secondarily dissolved by using a dissolving solution, so that the protein and polypeptide components in the water-insoluble components are obtained. The dissolving agent is selected from one or more of guanidine salt, urea and other compounds containing the structure shown in the structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glucoside and choline.

The preparation method of the cancer vaccine disclosed by the disclosure comprises the following steps of: firstly, cracking cancer cells and/or tumor tissues to obtain a lysate thereof; then using a dissolution solution containing a dissolution agent to dissolve all lysate components; then salting out and/or heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation and other methods are carried out on the dissolved lysate components, and protein and polypeptide components in the lysate components are precipitated; and then secondarily dissolving the protein and polypeptide components of the precipitated part by using a dissolving solution containing a dissolving agent to obtain the protein and polypeptide components in all lysates. The dissolving agent is selected from one or more of guanidine salt, urea and other compounds containing the structure shown in the structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glucoside and choline.

The process of separating and purifying the antigen component in the water-soluble component and/or the non-water-soluble component or the antigen component in the whole cell lysate component in the preparation process of the antigen component loaded by the delivery particles of the in vitro activated mixed cell vaccine can comprise salting out; the process of separating and purifying the antigen component in the water-soluble component and/or the non-water-soluble component or the antigen component in the whole cell lysate component may include heating; the process of separating and purifying the antigen component in the water-soluble component and/or the non-water-soluble component or the antigen component in the whole cell lysate component may include an enzymatic treatment (such as enzymolysis); the process of separating and purifying the antigen component in the water-soluble component and/or the non-water-soluble component or the antigen component in the whole cell lysate component may include oxidation; the process of separating and purifying the antigen component in the water-soluble component and/or the non-water-soluble component or the antigen component in the whole cell lysate component may include reduction; the process of separating and purifying the antigen component in the water-soluble component and/or the non-water-soluble component or the antigen component in the whole cell lysate component may include mineralization; the process of separating and purifying the antigen component in the water-soluble component and/or the non-water-soluble component or the antigen component in the whole cell lysate component may include irradiation.

The preparation method of the mixed cell cancer vaccine comprises the following steps of: firstly, cracking cancer cells and/or tumor tissues to obtain a lysate thereof; then using a dissolution solution containing a dissolution agent to dissolve all lysate components; then the lysate component dissolved by the dissolving solution is treated by salting out and/or heating, enzyme treatment (such as enzymolysis), oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation and other methods, and then protein and polypeptide components in the lysate component are precipitated; and then secondarily dissolving the protein and polypeptide components in the precipitate by using a dissolving solution containing a dissolving agent to obtain the protein and polypeptide components in the lysate. The dissolving agent is selected from one or more of a compound containing a structure of a structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycerol, glucoside and choline.

The mixed cell cancer vaccine disclosed by the disclosure, wherein the bacterial lysate and/or extracellular vesicle lysate loaded by the delivery particles of the in-vitro activated mixed cell vaccine is obtained by lysing bacteria and/or extracellular vesicles by a lysate containing a lysing agent; the cleavage agent is selected from one or more of a compound containing a structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, triton, tween, polypeptide, amino acid, glycoside and choline.

The water insoluble antigen, the bacterial lysate or the extracellular vesicle lysate of the present disclosure are dissolved independently of each other in a dissolution solution comprising at least one of the following dissolution agents: comprises a compound of the structure of formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, triton, tween, polypeptide, amino acid, glycoside and choline.

The preparation process of the antigen component loaded by the delivery particles of the in vitro activated mixed cell vaccine can comprise salting out, and the salting out process can use segmented salting out or disposable salting out. The cations contained in the reagent used for salting-out include, but are not limited to, those contained in the reagent used for salting-out include, but are not limited to, al ³⁺ 、Fe ³⁺ 、Fe ²⁺ 、Mg ²⁺ 、Sn ²⁺ 、Zn ²⁺ 、Ca ²⁺ 、Li ⁺ 、Na ⁺ 、NH ₄ ⁺ 、K ⁺ 、Cu ²⁺ 、Ag ⁺ 、Ba ²⁺ Etc. Anions contained in reagents used for salting out include, but are not limited to, cl ^- 、SO ₄ ^2- 、NO ₃ ^- 、CO ₃ ^2- 、SiO3 ^2- 、S ₂ O ₇ ^2- 、B ₄ O ₇ ^2- 、PO4 ^3- 、RCOO ^- 、NO ₂ ^- 、S ₂ O ₈ ^2- 、S ^2- 、CrO ₄ ^2- 、MnO4 ^- 、P ₂ O ₇ ^4- Etc.

The preparation process of the antigen component loaded by the delivery particles of the in vitro activated mixed cell vaccine can comprise heating, wherein the heating temperature is greater than 40 ℃.

The preparation process of the antigen component loaded by the delivery particles of the in vitro activated mixed cell vaccine can comprise enzyme treatment, such as enzymolysis, enzyme inhibition and other enzyme treatment methods. The enzyme treatment methods include, but are not limited to, the use of one or more of nucleases, dnases, pepsin, chymotrypsin, trypsin, other proteolytic enzymes, protease inhibitors, and the like.

The mixed cell cancer vaccine of the present disclosure, wherein the preparation process of the antigen component loaded by the delivery particle of the in vitro activated mixed cell vaccine may include oxidation for a certain time period in conjunction with an oxidizing agent that can oxidize the antigen. The oxidizing agent for oxidizing antigen components includes, but is not limited to, hypochlorous acid, persulfates, KIO ₃ 、KBrO ₃ Chlorine, dichromate, hydrogen peroxide, 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 permanganate, nitric acid, clO ₃ ^- 、ClO ₄ ^- 、Na ₂ O ₂ 、K ₂ O ₂ 、MgO ₂ 、CaO ₂ 、BaO ₂ 、H ₂ O ₂ 、NO ₃ ^- 、MnO ₄ ^- 、F ₂ 、Cl ₂ 、O ₂ 、Br ₂ 、I ₂ 、S、Si、HNO ₃ 、MnO ₂ 、FeCl ₃ And the like, and one or more of various types of oxidizing agents.

The preparation process of the antigen component loaded by the delivery particles of the in vitro activated mixed cell vaccine can comprise reduction, wherein the reduction process is to reduce the antigen component by using a reducing agent, and the reducing agent comprises but is not limited to DTT, TCEP and the like.

The mixed cell cancer vaccine of the present disclosure, wherein the in vitro activated mixed cell vaccine delivery particle loaded antigen component preparation process may include mineralization including, but not limited to, biomineralization, siliconization, magnesization, calcification, and the like.

The preparation process of the antigen component loaded by the delivery particles of the in vitro activated mixed cell vaccine can comprise irradiation, wherein the irradiation method is any common irradiation method, including but not limited to one or more of radioactive substance irradiation, ultraviolet irradiation, X-ray irradiation, gamma-ray irradiation, alpha-ray irradiation, beta-ray irradiation and the like.

The preparation materials of the delivery particles of the in vitro activated mixed cell vaccine are selected from natural high polymer materials, synthetic high polymer materials and/or inorganic materials.

Exemplary organic synthetic polymeric materials include, but are not limited to PLGA, PLA, PGA, PEG, PCL, poloxamer, PVA, PVP, PEI, PTMC, polyanhydrides, PDON, PPDO, PMMA, polyamino acids, synthetic polypeptides, and the like.

Illustratively, natural polymeric materials include, but are not limited to, lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, saccharides, polypeptides, and the like.

Exemplary inorganic materials include, but are not limited to, ferric oxide, carbonates, phosphates, and the like.

The shape of the delivery particles of the in vitro activated mixed cell vaccine of the present disclosure is any shape, including but not limited to spherical, ellipsoidal, barrel, polygonal, rod, tablet, wire, worm, square, triangular, butterfly, disc, vesicle, and the like.

In some embodiments, the antigen delivery particles used to activate the mixed cell cancer vaccine are nanoparticles, or microparticles, or a mixture of nanoparticles and microparticles. The particle size of the antigen delivery particles is nano-scale or micro-scale, thus ensuring that the delivery particles can be efficiently phagocytized by antigen presenting cells.

Further, the particle size of the nanoparticle is 1nm to 1000nm, more preferably 30nm to 1000nm, still more preferably 50nm to 600nm; more preferably, the particle size is 50-500nm; more preferably, the particle size is 100-400nm. Illustratively, the nanoparticle has a particle size of 10nm, 50nm, 100nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 400nm, 500nm, and the like.

Further, the micrometer particles have a particle size of 1 μm to 1000 μm, more preferably 1 μm to 100 μm, still more preferably 1 μm to 10 μm, still more preferably 1 μm to 5 μm; illustratively, the microparticles have a particle size of 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.5 μm, 3 μm, 5 μm, 10 μm, and the like.

Further, when a mixed particle of nanoparticles and microparticles is used, the nanoparticle size range is 100nm to 600nm, the microparticle size is 1.5 μm to 5 μm, more preferably, the nanoparticle size is 150nm to 500nm, and the microparticle size is 2.0 μm to 3.5 μm.

The surface of the delivery particle of the in vitro activated mixed cell vaccine can be connected with a target head for actively targeting dendritic cells and B cells, so that the efficiency of phagocytizing the delivery particle by the dendritic cells and the B cells during co-incubation is improved. The targeting molecule comprises at least one of the following: mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, and the like.

The antigen delivery particles may also be bacteria and viruses, where the antigen delivery particles are bacteria and viruses, the bacteria and viruses may express tumor-specific antigens and/or tumor-associated antigens, or the bacteria and viruses may contain DNA and/or mRNA within them that may express tumor-specific antigens and/or tumor-associated antigens.

In the present disclosure, the particle material forming the cancer vaccine is formed of a natural polymer material and/or a synthetic polymer material.

The antigen component is a protein polypeptide and/or an RNA component (or an mRNA component) in cancer cells and/or tumor tissues, or the antigen component is a protein polypeptide and/or a nucleic acid synthesized in vitro. The nucleic acid is mRNA or DNA.

The preparation method of the mixed cell cancer vaccine comprises the following steps:

(1) The cancer cells and/or tumor tissue are first lysed to obtain a lysate thereof.

(2) Then separating the water-soluble component and the water-insoluble component in the lysate by using one or more of centrifugation, filtration, dialysis, ultrafiltration and the like to obtain the water-soluble component and the water-insoluble component respectively, wherein the water-insoluble component is dissolved by using a dissolving solution containing a dissolving agent; or dissolving cancer cells and/or tumor tissue directly with a dissolving solution containing a dissolving agent to obtain a lysate thereof, and dissolving a lysate component with a dissolving solution containing a dissolving agent.

(3) Then the water-soluble components in the lysate are subjected to salting-out and/or heating, enzyme treatment (such as enzymolysis and enzyme inhibition), oxidation, reduction, fixation, recrystallization, precipitation, extraction, dialysis, chromatography, electrophoresis, chromatography, mineralization, radiation, irradiation and other methods to obtain a precipitate, and then the precipitate part is dissolved by using a dissolving solution containing a dissolving agent; the water insoluble component in the lysate is dissolved by using a dissolving solution containing a dissolving agent, and then is subjected to salting out and/or heating, enzyme treatment (such as enzymolysis and enzyme inhibition), oxidation, reduction, chromatography, electrophoresis, chromatography, immobilization, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation and other methods, and then is dissolved by using the dissolving solution containing the dissolving agent for the second time, or the dissolved water insoluble component is directly used without treatment. Or subjecting whole cell lysate component dissolved in dissolving solution containing dissolving agent to salting out and/or heating, enzyme treatment (such as enzymolysis and enzyme inhibition), oxidation, reduction, immobilization, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation, etc., to obtain precipitate, and then secondarily dissolving the precipitate part with dissolving solution containing dissolving agent. The dissolving agent in each step is selected independently. In addition to using the protein and polypeptide components of the lysate, the water-soluble component, the water-insoluble component, or the RNA component or mRNA component of the whole cell lysate may be simultaneously extracted and separated, and then the protein and polypeptide components and the RNA component/mRNA component may be mixed and used as the antigen component.

(4) Loading the lysate component from step (2) separately or simultaneously into the interior and/or surface of a nanoparticle or microparticle to obtain the delivery particles; or the protein polypeptide component and/or the non-water soluble component in the water soluble component obtained in the step (3) or the protein polypeptide component in the water soluble component are respectively or simultaneously loaded into the interior and/or the surface of the nano-particle or the micro-particle to obtain the delivery particle; or the protein polypeptide component and the RNA component (mRNA component) in the water-soluble component obtained in the step (3) and/or the non-water-soluble component or the protein polypeptide component and the RNA component (mRNA component) in the water-soluble component are respectively or simultaneously loaded into the interior and/or the surface of the nano-particle or the micro-particle to obtain the delivery particle; or mixing the component obtained in the step (2) with in-vitro synthesized polypeptide containing the epitope or nucleic acid capable of expressing the epitope, and then respectively or simultaneously loading the mixture into the interior and/or the surface of nano-particles or micro-particles to obtain the delivery particles; or mixing the component obtained in the step (3) with in-vitro synthesized polypeptide containing the epitope or nucleic acid capable of expressing the epitope, and then respectively or simultaneously loading the mixture into the interior and/or the surface of nano-particles or micro-particles to obtain the delivery particles; or the polypeptide containing the antigen epitope or the nucleic acid capable of expressing the antigen epitope are respectively or simultaneously loaded in the nanometer particle or the micrometer particle and/or on the surface to obtain the delivery particle.

(5) After incubating the delivery particles with Dendritic Cells (DCs) and B cells in the same system for a certain period of time simultaneously.

(6) Dendritic Cells (DCs) and B cells were collected and used as mixed cell vaccines.

The present disclosure describes a method of preventing or treating a disease, wherein the method comprises administering to a subject a prophylactically or therapeutically effective amount of a cancer vaccine.

The pharmaceutical composition provided by the present disclosure can exert remarkable disease prevention or treatment effects.

The antigen delivery particles in the present disclosure can activate antigen presenting cells in vitro, and the activated antigen presenting cells can activate immune response of an organism, thereby exerting preventive or therapeutic effects of the vaccine.

Use of a cancer vaccine, or pharmaceutical composition, as described in the present disclosure in at least one of the following (1) - (3):

(1) Preventing or treating a disease, or preparing a medicament for preventing or treating a disease;

(2) Inducing an immune response in a subject, or preparing a medicament for inducing an immune response in a subject;

(3) As or for the preparation of cancer vaccines;

in some embodiments, the disease is cancer or tumor;

in some embodiments, the cancer or tumor is a solid tumor or hematological tumor, including but not limited to squamous cell carcinoma, myeloma, lung cancer, glioma, liver cancer, lymphoma, acute myeloma, gastrointestinal (gastrointestinal) cancer, renal cancer, ovarian cancer, liver cancer, leukemia, colon cancer, rectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, sarcoma, cell tumor, pancreatic cancer, cervical cancer, brain cancer, bladder cancer, breast cancer, and head and neck cancer.

In the present disclosure, the vaccine-loaded antigen component is derived from cells or tissues associated with the disease. Further, the antigen component is derived from a lysate component of cancer cells and/or tumor tissue.

A fourth object of the present disclosure is to provide a method for preparing the above mixed cell cancer vaccine.

The preparation method of the mixed cell cancer vaccine disclosed by the disclosure can comprise the following steps:

s1, using ultrapure water, aqueous solution and the like to lyse cancer cells and/or tumor tissues. The cancer cells are one or more cancer cells or cancer cell lines; the tumor tissue is tumor tissue derived from one body or a plurality of bodies. The lysis method is a common lysis method for cancer cells or tumor tissues, and comprises one or more of repeated freezing and thawing, swelling, ultrasonic treatment, high-pressure treatment, homogenization, extrusion, homogenization, high-speed stirring, chemical substance treatment, enzyme treatment, high-shear force treatment, ultrafiltration treatment, shrinkage and other treatment modes.

S2, after the cancer cells or the tumor tissues are lysed, one or more of centrifugation, filtration, dialysis, ultrafiltration and the like are used for separating the water-soluble components and the water-insoluble components in the lysate, so as to obtain the water-soluble components and the water-insoluble components respectively. And dissolving the water-insoluble component by using a dissolving solution containing a specific dissolving agent to obtain the dissolved water-insoluble component. The resulting water-soluble component and water-insoluble component may be used as they are or after the treatment of S3. The dissolving agent includes, but is not limited to, one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, and the like.

S3, treating water-soluble components in the lysate by salting out and/or heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, mineralization, radiation, irradiation and other methods to obtain a precipitate, and dissolving the precipitate part by using a dissolving solution containing a dissolving agent to obtain the water-soluble component; dissolving water insoluble component in lysate with dissolving solution containing dissolving agent, salting out and/or heating, enzyme treating, oxidizing, reducing, fixing, chromatography, electrophoresis, chromatography, recrystallizing, precipitating, extracting, dialyzing, mineralizing, irradiating, and dissolving with dissolving solution containing dissolving agent for the second time; or the water insoluble components of the lysate are dissolved and used without further treatment. The dissolving agent in each step is selected independently. The dissolving agent includes, but is not limited to, one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, and the like.

S4, loading the water-soluble component and the water-insoluble component obtained in the S2 into the interior and/or the surface of the nano-particle or the micro-particle respectively or simultaneously to obtain the antigen delivery particle; or protein and polypeptide components and/or non-water soluble components or protein and polypeptide components in the water soluble components obtained in the step S3 are respectively or simultaneously loaded into the interior and/or the surface of nano-particles or micro-particles to obtain the antigen delivery particles; or mixing the water-soluble component and the water-insoluble component obtained in the step S2 with the artificially synthesized polypeptide and/or nucleic acid, and then respectively or simultaneously loading the mixture into the interior and/or the surface of nano-particles or micro-particles to obtain the antigen delivery particles; or the protein and polypeptide components in the water-soluble components and/or the non-water-soluble components or the protein and polypeptide components in the water-soluble components and the artificially synthesized polypeptide and/or nucleic acid are respectively or simultaneously loaded into the interior and/or the surface of the nano-particle or the micro-particle to obtain the antigen delivery particle. The antigen delivery nanoparticle or microparticle surface may also be loaded with a membrane component. The artificially synthesized polypeptide is a cancer specific and/or cancer related antigen polypeptide, and can be just equal to or longer than the antigen epitope polypeptide, or simultaneously contains several different antigen epitope polypeptides; the artificially synthesized nucleic acid is mRNA or DNA capable of expressing cancer specific and/or cancer related antigen epitope, and can express only the polypeptide equal to the antigen epitope polypeptide, or express longer than the antigen epitope polypeptide, or express several different antigen epitope polypeptides simultaneously, or express antigen epitope and other substances such as cell factor capable of enhancing immune response simultaneously.

S5, co-incubating antigen delivery particles prepared in the step S4 with dendritic cells and B cells in the same system for a certain time at a certain concentration (0.002 mug/mL-80 mg/mL). The co-incubation system may contain cytokines and the like that enhance activation of the mixed cells.

S6, collecting the co-incubated mixed cells to be used as a cancer vaccine.

The preparation method of the mixed cell cancer vaccine can also comprise the following steps:

s1, lysing cancer cells and/or tumor tissues by using a lysis solution containing a lysis agent. The cancer cells are one or more cancer cells or cancer cell lines; the tumor tissue is tumor tissue derived from one body or a plurality of bodies.

S2, after the cancer cells and/or the tumor tissues are lysed, a lysate component of the cancer cells and/or the tumor tissues is dissolved by using a dissolving solution containing a dissolving agent. The dissolving agent includes, but is not limited to, one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, and the like.

S3, treating lysate components dissolved by a dissolving solution containing a dissolving agent by salting out and/or heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation and other methods to obtain a precipitate, and then secondarily dissolving the precipitate part by using the dissolving solution containing the dissolving agent to obtain the required antigen components. The dissolving agent includes, but is not limited to, one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, and the like.

S4, loading the lysate component obtained in the S2 into the interior and/or the surface of the nano-particle or the micro-particle respectively or simultaneously to obtain the antigen delivery nano-particle or the micro-particle; or the antigen component obtained by S3 is respectively or simultaneously loaded into the interior and/or the surface of the nano-particle or the micro-particle to obtain the antigen delivery nano-particle or the micro-particle; or the component obtained in the step S2 is mixed with the artificially synthesized polypeptide and/or nucleic acid and then is respectively or simultaneously loaded into the interior and/or the surface of the nanoparticle or the microparticle to obtain the antigen delivery nanoparticle or microparticle; or mixing the antigen component obtained in the step S3 with the artificially synthesized polypeptide and/or nucleic acid, and then respectively or simultaneously loading the mixture into the interior and/or the surface of the nanoparticle or the microparticle to obtain the antigen delivery nanoparticle or microparticle. The antigen delivery nanoparticle or microparticle surface may also be loaded with a membrane component. The artificially synthesized polypeptide is a cancer specific and/or cancer related antigen polypeptide, and can be just equal to or longer than the antigen epitope polypeptide, or simultaneously contains several different antigen epitope polypeptides; the artificially synthesized nucleic acid is mRNA or DNA capable of expressing cancer specific and/or cancer related antigen epitope, and can express only the polypeptide equal to the antigen epitope polypeptide, or express longer than the antigen epitope polypeptide, or express several different antigen epitope polypeptides simultaneously, or express antigen epitope and other substances such as cell factor capable of enhancing immune response simultaneously.

S6, collecting the co-incubated mixed cells to be used as a cancer vaccine.

s1, using ultrapure water, aqueous solution and the like to lyse cancer cells and/or tumor tissues. The cancer cells are one or more cancer cells or cancer cell lines; the tumor tissue is tumor tissue derived from one body or a plurality of bodies. The lysis method is a common lysis method for cancer cells or tumor tissues, and comprises one or more of repeated freezing and thawing, swelling, ultrasonic treatment, high-pressure treatment, homogenization, extrusion, homogenization, high-speed stirring, chemical substance treatment, high-shear force treatment, ultrafiltration treatment, shrinkage and other treatment modes.

S2, after the cancer cells or the tumor tissues are lysed, one or more of centrifugation, filtration, dialysis, ultrafiltration and the like are used for separating the water-soluble components and the water-insoluble components in the lysate, so as to obtain the water-soluble components and the water-insoluble components respectively.

S3, treating water-soluble components in the lysate by salting out and/or heating, enzyme treatment, oxidation, reduction, mineralization, radiation, irradiation and other methods to obtain two parts of precipitate and supernatant, then secondarily dissolving the precipitate part by using a dissolving solution containing a dissolving agent, simultaneously separating and extracting RNA components or mRNA components in the supernatant by using a proper method, and then mixing the RNA components or mRNA components in the supernatant with the secondarily dissolved precipitate part of the dissolving solution for later use; dissolving the water insoluble component in the lysate by using a dissolving agent, then carrying out salting-out and/or heating, enzyme treatment, oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation and other methods, and then carrying out secondary dissolution by using a dissolving solution containing the dissolving agent, or simultaneously extracting RNA component or mRNA component in the water insoluble component; or the water insoluble components in the lysate are dissolved and then directly processed without other treatments. The dissolving agent in each step is selected independently. The dissolving agent includes, but is not limited to, one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, and the like.

S4, loading the separated and purified components and/or the non-water-soluble components or the protein and polypeptide components in the water-soluble components obtained in the S3 into the interior and/or the surface of the nano-particles or the micro-particles respectively or simultaneously to obtain the antigen delivery nano-particles or the micro-particles; the separated and purified components and/or non-water soluble components in the water soluble components obtained in the step S3 or protein polypeptide components and RNA components (or mRNA components) in the water soluble components are respectively or simultaneously loaded into the interior and/or the surface of nano-particles or micro-particles to obtain the antigen delivery nano-particles or micro-particles; or the separated and purified components and/or non-water-soluble components or proteins and polypeptide components in the water-soluble components obtained in the step S3 are mixed with the artificially synthesized polypeptides and/or nucleic acids and then respectively or simultaneously loaded into the interior and/or the surface of the nano-particles or the micro-particles to obtain the antigen delivery nano-particles or the micro-particles; or the protein and polypeptide component+RNA component (or mRNA component) in the separated and purified components and/or the non-water-soluble components in the water-soluble components obtained in the step S3 are mixed with the artificially synthesized polypeptide and/or nucleic acid and then respectively or simultaneously loaded into the interior and/or the surface of the nano-particle or the micro-particle to obtain the antigen delivery nano-particle or the micro-particle. The antigen delivery nanoparticle or microparticle surface may also be loaded with a membrane component. The artificially synthesized polypeptide is a cancer specific and/or cancer related antigen polypeptide, and can be just equal to or longer than the antigen epitope polypeptide, or simultaneously contains several different antigen epitope polypeptides; the artificially synthesized nucleic acid is mRNA or DNA capable of expressing cancer specific and/or cancer related antigen epitope, and can express only the polypeptide equal to the antigen epitope polypeptide, or express longer than the antigen epitope polypeptide, or express several different antigen epitope polypeptides simultaneously, or express antigen epitope and other substances such as cell factor capable of enhancing immune response simultaneously.

S6, collecting the co-incubated mixed cells to be used as a cancer vaccine.

S2, directly using a dissolving solution containing a dissolving agent to dissolve lysate components after cancer cells and/or tumor tissues are lysed. The dissolving agent includes, but is not limited to, one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, and the like.

S3, treating lysate components dissolved by the dissolving solution by salting out and/or heating, enzyme treatment (such as enzyme degradation or enzyme inhibitor treatment method), oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation and other methods to obtain two parts of precipitation and supernatant, and then secondarily dissolving the precipitation part by using the dissolving solution containing the dissolving agent; RNA or mRNA components are isolated from the supernatant using a suitable method and then mixed with the secondary solubilised precipitate fraction of the solubilised solution as described above to obtain the desired antigen component. The dissolving agent includes, but is not limited to, one or more of a compound containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, and the like.

S4, respectively or simultaneously loading the antigen components obtained in the S3 into the interior and/or the surface of the nano-particles or the micro-particles to obtain the antigen delivery nano-particles or the micro-particles; or mixing the antigen component obtained in the step S3 with the artificially synthesized polypeptide and/or nucleic acid, and then respectively or simultaneously loading the mixture into the interior and/or the surface of the nanoparticle or the microparticle to obtain the antigen delivery nanoparticle or microparticle. The antigen delivery nanoparticle or particle vaccine surface may also be loaded with a membrane component. The artificially synthesized polypeptide is a cancer specific and/or cancer related antigen polypeptide, and can be just equal to or longer than the antigen epitope polypeptide, or simultaneously contains several different antigen epitope polypeptides; the artificially synthesized nucleic acid is mRNA or DNA capable of expressing cancer specific and/or cancer related antigen epitope, and can express only the polypeptide equal to the antigen epitope polypeptide, or express longer than the antigen epitope polypeptide, or express several different antigen epitope polypeptides simultaneously, or express antigen epitope and other substances such as cell factor capable of enhancing immune response simultaneously.

S6, collecting the co-incubated mixed cells to be used as a cancer vaccine.

s1, mixing the artificially synthesized polypeptide and/or nucleic acid, and respectively or simultaneously loading the mixture into the interior and/or the surface of nano-particles or micro-particles to obtain the antigen delivery nano-particles or micro-particles. The antigen delivery nanoparticle or particle vaccine surface may also be loaded with a membrane component. The artificially synthesized polypeptide is a cancer specific and/or cancer related antigen polypeptide, and can be just equal to or longer than the antigen epitope polypeptide, or simultaneously contains several different antigen epitope polypeptides; the artificially synthesized nucleic acid is mRNA or DNA capable of expressing cancer specific and/or cancer related antigen epitope, and can express only the polypeptide equal to the antigen epitope polypeptide, or express longer than the antigen epitope polypeptide, or express several different antigen epitope polypeptides simultaneously, or express antigen epitope and other substances such as cell factor capable of enhancing immune response simultaneously. Or constructing a virus or bacterium that can express a polypeptide or mRNA/DNA of the antigen of interest as antigen delivery particles.

S2, incubating antigen delivery particles (prepared nano/micron particles or constructed bacteria and/or viruses) prepared in the S1 with dendritic cells and B cells in the same system for a certain time at a certain concentration. The co-incubation system may contain cytokines and the like that enhance activation of the mixed cells.

S3, collecting the co-incubated mixed cells to be used as a cancer vaccine.

The invention has the beneficial effects that:

the present disclosure uses dendritic cells and B cells to simultaneously incubate and activate in vitro in the same system, and the use of mixed cells as a cancer vaccine after activation achieves better results than the prior use of activated dendritic cells alone as a cancer vaccine. Especially after the organism suffers from the illness, the autoimmune cell number is extremely easy to be reduced, the activity is reduced, the invention can directly supplement the living cells with the immunity, and is helpful for the execution of the immunotherapy and the cure of the illness.

In some exemplary embodiments of the present disclosure, a solvent evaporation method is used to prepare antigen delivery particles, and any other method that can prepare antigen delivery particles may be used in practical applications, including but not limited to precipitation, dialysis, dispersion, microfluidic, high pressure homogenization, stirring, spray drying, phase separation, electrostatic spraying, emulsion polymerization, machine stirring shear, membrane emulsification, and the like.

The foregoing description is only an overview of the technical solutions of the present disclosure, and in order to make the technical means of the present disclosure more clearly understood, it can be implemented according to the content of the specification, and the following description of the preferred embodiments of the present disclosure will be given with reference to the detailed drawings.

Drawings

In order that the present disclosure may be more readily understood, a more particular description of the disclosure will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.

Fig. 1 is a structural formula 1. Wherein R is ₁ C, N, S or O, R ₂ ～R ₅ Independently selected from hydrogen, alkyl, carboxyl, substituted or unsubstituted amino, mercapto, substituted or unsubstituted guanidino, and the like.

FIG. 2 is a schematic illustration of some of the preparation processes and applications of the present disclosure for activating mixed cells and preparing cancer vaccines using delivery particles; wherein a and b are schematic illustrations of the preparation of antigen delivery nanoparticles/microparticles; c schematic of the process of using antigen delivery particles to activate mixed antigen presenting cells for use as a cancer vaccine.

FIGS. 3-18 are experimental results of tumor growth rate and survival when cancer vaccine is used to prevent or treat cancer in examples 1-16, respectively; in FIGS. 3-14, a is the experimental result of tumor growth rate (n.gtoreq.8) in the prevention or treatment of cancer; b is the result of a mice survival experiment (n.gtoreq.8) when cancer is prevented or treated, and each data point is the mean.+ -. Standard error (mean.+ -. SEM); wherein, the significant difference of the tumor growth inhibition experiment in the a graph is analyzed by adopting an ANOVA method, and the significant difference in the b graph is analyzed by adopting Kaplan-Meier and log-rank test; * P < 0.005 compared to PBS blank, with significant differences; * P < 0.01, showing significant differences compared to PBS blank; # # # indicates that the two groups have significant differences compared with p < 0.005; # represents that the two groups have significant differences compared with p < 0.01; # represents that the two groups have significant differences in comparison with p < 0.05.

Detailed Description

The present disclosure is further described in connection with the accompanying drawings and the specific embodiments so that those skilled in the art may better understand the disclosure and be able to practice it, but the examples are not intended to be limiting of the disclosure.

Unless stated to the contrary, the terms used in the present invention have the following meanings.

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

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

The term "treatment" refers to: after suffering from the disease, the subject is exposed (e.g., administered) to a vaccine, pharmaceutical composition, thereby alleviating the symptoms of the disease as compared to when not exposed, and does not mean that the symptoms of the disease must be completely inhibited. The suffering from the disease is: the body develops symptoms of the disease.

The term "preventing" refers to: by contacting (e.g., administering) a subject with a vaccine, or pharmaceutical composition of the present disclosure prior to the onset of a disease, thereby alleviating the symptoms after the onset of the disease as compared to when not contacted, is not meant to necessarily completely inhibit the disease.

The term "pharmaceutically acceptable excipients" or "pharmaceutically acceptable carriers" refers to auxiliary materials widely used in the field of pharmaceutical production. The main purpose of the use of auxiliary substances is to provide a pharmaceutical composition which is safe to use, stable in nature and/or has specific functionalities, and to provide a method so that the active ingredient can be dissolved at a desired rate after administration of the drug to a subject, or so that the active ingredient is effectively absorbed in the subject to whom it is administered. Pharmaceutically acceptable excipients may be inert fillers or may be functional ingredients that provide some function to the pharmaceutical composition (e.g., to stabilize the overall pH of the composition or to prevent degradation of the active ingredients in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, binders, disintegrants, lubricants, anti-adherent agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, reinforcing agents, adsorbents, buffers, chelating agents, preservatives, coloring agents, flavoring agents, sweetening agents, and the like.

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

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

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

Unless defined otherwise or clearly indicated by context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

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

The vaccine system described in the present disclosure is used to activate antigen delivery particles of mixed antigen presenting cells, which are internally loaded with antigen components, and the preparation process and application fields thereof are shown in fig. 2.

In preparing the mixed cell cancer vaccine, an antigen component is prepared or prepared, then the antigen component is loaded to antigen delivery nano/micro particles, and then the antigen delivery nano/micro particles are used for co-incubation with dendritic cells and B cells to prepare the mixed cell cancer vaccine. The nanoparticle or microparticle alone may be used for co-incubation with dendritic cells and B cells, or a mixed particle of nanoparticle and microparticle may be used for co-incubation with dendritic cells and B cells.

In preparing antigen components derived from cancer cells or tumor tissues, the antigen components in the water-soluble components and the antigen components in the water-insoluble components can be collected respectively after the cells or tissues are lysed, and antigen delivery particles loaded with the antigen components are prepared respectively; alternatively, the cells or tissues may be directly lysed by direct use of a lysis solution containing a lysing agent, and the lysate fraction is lysed, and the protein and polypeptide fraction and/or RNA fraction (or mRNA fraction) is then isolated by extraction using a suitable method, and the resulting fraction is reloaded to antigen delivery particles. The cancer cells and/or tumor tissue of the present disclosure may be subjected to treatments including, but not limited to, inactivation or (and) denaturation, immobilization, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, ionization, chemical modification, oxidation, reduction, nucleic acid separation and purification, protease endo-or degradation, nuclease treatment, etc., prior to or (and after) lysis, and then the protein and polypeptide components thereof are extracted and separated; the protein and polypeptide components can also be directly extracted and separated without any inactivation or (and) denaturation, mineralization, ionization, oxidation, immobilization, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, reduction, chemical modification, protease endo-or degradation, and nuclease treatment before or (and) after cell lysis. In some embodiments of the disclosure, the tumor tissue cells are subjected to inactivation or (and) denaturation before lysis, or may be subjected to inactivation or (and) denaturation after cell lysis in the actual use process, or may be subjected to inactivation or (and) denaturation before and after cell lysis; the inactivation or denaturation treatment method before or (and) after the cell lysis in some embodiments of the present disclosure is ultraviolet irradiation and high temperature heating, and treatment methods including, but not limited to, radiation irradiation, high pressure, nucleic acid separation and purification, immobilization, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, oxidation, reduction, ionization, chemical modification, nuclease treatment, protease endo-or degradation, collagenase treatment, lyophilization, and the like may also be employed in the actual use. Those skilled in the art will appreciate that the actual application process can be appropriately adjusted according to the specific circumstances.

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

The antigen presenting cells used to prepare the biofilm component carried on the surface of the antigen delivery nano/microparticles may be derived from autologous or allogenic sources, or from cell lines or stem cells. The antigen presenting cells may be DC cells, B cells, macrophages or any mixture of the three, or may be other cells having an antigen presenting function. Antigen presenting cells may be activated by antigen-loaded nano/micro particles.

When the biofilm component carried on the surface of the antigen delivery nano/micro particles is derived from extracellular vesicles, the biofilm component can be one or more of extracellular vesicles of cancer cells, extracellular vesicles of bacteria or extracellular vesicles of antigen presenting cells.

Any nanoparticle, microparticle preparation method known to those of skill in the art may be used to prepare the nanovaccine or the micropattern described in the present disclosure, including, but not limited to, solvent evaporation, dialysis, microfluidic, membrane emulsification, machine agitation shear, emulsion polymerization, spray drying, phase separation, ultrafiltration, homogeneous emulsification, dispersion, precipitation, and the like.

In some embodiments, the mixed cell cancer vaccine further comprises macrophages in the system when incubated.

In some specific embodiments, the present disclosure provides the following exemplary methods of preparation, taking as an example the activation of mixed cell cancer vaccines after antigen delivery particles are prepared using a multiple emulsion process in a solvent evaporation process:

step 1, lysing cancer cells and/or tumor tissues to obtain a lysate thereof. Any method that can lyse cancer cells or tumor tissue, such as ultrapure water, aqueous solution, ultrasound, tissue homogenization, and the like, may be used. The cancer cells are one or more cancer cells or cancer cell lines; the tumor tissue is tumor tissue derived from one body or a plurality of bodies. The lysis method is a common lysis method for cancer cells or tumor tissues, and comprises one or more of repeated freezing and thawing, swelling, ultrasonic treatment, high-pressure treatment, homogenization, extrusion, homogenization, high-speed stirring, chemical substance treatment, high-shear force treatment, ultrafiltration treatment, shrinkage and other treatment modes.

Alternatively, a lysis solution containing a lysing agent may be used to lyse cancer cells or tumor tissue. The cancer cells are one or more cancer cells or cancer cell lines; the tumor tissue is tumor tissue derived from one body or a plurality of bodies.

The cancer cells or tumor tissue may be co-incubated with a specific chemical prior to lysis to stimulate the cancer cells or tumor tissue and then the cancer cells or tumor tissue may be lysed, the specific chemical including, but not limited to, small molecule compounds (e.g., doxorubicin, paclitaxel, vincristine, retinoic acid, arsenic trioxide, etc.), growth factors, plant extracts (e.g., important extracts of ginseng, plant rhizome extracts, etc.), cytokines, chemokines, interferons, bacterial secretions, bacterial extracellular vesicles, etc.

And 2, after the cancer cells or the tumor tissues are lysed by using ultrapure water or an aqueous solution, separating the water-soluble components and the water-insoluble components in the lysate by using one or more of centrifugation, filtration, dialysis, ultrafiltration and the like to obtain the water-soluble components and the water-insoluble components respectively. The water-insoluble component is dissolved by using a dissolving solution containing a dissolving agent to obtain a dissolved water-insoluble component. Directly using water-soluble component and/or non-water-soluble component as antigen component to load on nano-particle/micrometer particle; or mixing water-soluble component and/or non-water-soluble component with artificially synthesized polypeptide and/or nucleic acid (mRNA or DNA) to obtain antigen component loaded on nanoparticle/micrometer particles; or the water-soluble component and the water-insoluble component obtained are treated in the step 3 and then used as antigen components to be loaded on nano particles/micro particles; or mixing the water-soluble component and the water-insoluble component with synthetic polypeptide and/or nucleic acid (mRNA or DNA) after the treatment of step 3, and using the mixture as antigen component to load on nano particles/micro particles. The dissolving agent includes, but is not limited to, one or more of compounds containing structural formula 1 (such as urea, guanidine hydrochloride, and the like containing guanidine groups and urea structures), deoxycholate, dodecyl sulfate, glycerol, protein degrading enzymes, polypeptides, amino acids, glycosides, choline, and the like.

Or after lysing cancer cells or tumor tissue using a lysing solution containing a lysing agent, directly lysing the lysate component using a lysing solution containing a lysing agent. Directly using the dissolved lysate component as an antigen component to be loaded on nano particles/micro particles; or mixing the dissolved lysate component with artificially synthesized polypeptide and/or nucleic acid to be used as antigen component and loaded on nano-particle/micro-particle; or the dissolved lysate component is used as an antigen component to be loaded on nano particles/micro particles after being subjected to the treatment of the step 3; or the lysate component after being dissolved is processed in the step 3 and then is mixed with the artificially synthesized polypeptide and/or nucleic acid to be used as an antigen component to be loaded on nano particles/micro particles. The dissolving agent includes, but is not limited to, one or more of compounds containing structural formula 1 (such as urea, guanidine hydrochloride, and the like containing guanidine groups and urea structures), deoxycholate, dodecyl sulfate, glycerol, protein degrading enzymes, polypeptides, amino acids, glycosides, choline, and the like.

Step 3, the water-soluble components in the lysate are subjected to salting out, heating, enzyme treatment (enzymolysis, enzyme inhibition and other treatment methods), oxidation, reduction, fixation, mineralization, irradiation and other methods to obtain a precipitate, and then the precipitate part is dissolved by using a dissolving solution containing a dissolving agent; dissolving water insoluble component in lysate with dissolving solution containing dissolving agent, salting out, heating, enzyme treatment (enzymolysis, enzyme inhibition, etc.), oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, irradiation, etc., and dissolving with dissolving solution containing dissolving agent for the second time; or the water insoluble components of the lysate are used without treatment. The dissolving agent in each step is selected independently. The dissolving agent includes, but is not limited to, one or more of a compound containing structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, etc., such as urea and guanidine hydrochloride.

Or salting out, heating, enzyme treating (enzymolysis, enzyme inhibition, etc.), oxidizing, reducing, fixing, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation, irradiation, etc. water-soluble components in the lysate are treated to obtain two parts of precipitate and supernatant, then the precipitate part is dissolved by using a dissolving solution containing a dissolving agent, RNA or mRNA components in the supernatant are separated and extracted by using a proper method, and then the RNA or mRNA components in the supernatant and the precipitate part dissolved by the dissolving solution containing the dissolving agent are mixed for standby; dissolving water insoluble components in the lysate by using a dissolving agent, then performing salting-out and/or heating, enzyme treatment (enzymolysis, enzyme inhibition and other treatment methods), oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, radiation, recrystallization, precipitation, extraction, dialysis, mineralization, irradiation and other methods, and then performing secondary dissolution by using a dissolving solution containing the dissolving agent; or the water insoluble components of the lysate are used without treatment. The dissolving agent in each step is selected independently. The dissolving agent includes, but is not limited to, one or more of a compound containing structure 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, etc., such as urea and guanidine hydrochloride.

Or directly lysing cancer cells/tumor tissues by using a lysis solution containing a lytic agent, then dissolving lysate components by using the lysis solution, then performing salting-out, heating, enzyme treatment (enzymolysis, enzyme inhibition and other treatment methods), oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, radiation, mineralization, irradiation and other methods to obtain precipitation, and then secondarily dissolving the precipitation part by using the lysis solution containing the lytic agent to obtain antigen components. The dissolving agent includes, but is not limited to, one or more of a compound containing structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, etc., such as urea and guanidine hydrochloride.

Or directly lysing cancer cells/tumor tissues by using a lysis solution containing a lysis agent, dissolving the whole cell lysate component by using the lysis solution, and then performing salting-out and/or heating, enzyme treatment (enzymolysis, enzyme inhibition and other treatment methods), oxidation, reduction, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, radiation, mineralization, irradiation and other methods to obtain two parts of precipitation and supernatant, and secondarily dissolving the precipitation part by using the lysis solution containing the lysis agent; the RNA or mRNA component is separated from the supernatant by a suitable method, and then mixed with a precipitation fraction in which a dissolution solution containing a dissolution agent is secondarily dissolved to obtain an antigen component. The dissolving agent includes, but is not limited to, one or more of a compound containing structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside, choline, etc., such as urea and guanidine hydrochloride.

In the case of separating and purifying an antigen component by salting out, the salting-out process may use fractional salting-out or one-time salting-out. The cations contained in the reagent used for salting-out include, but are not limited to, those contained in the reagent used for salting-out include, but are not limited to, al ³⁺ 、Fe ³⁺ 、Fe ²⁺ 、Mg ²⁺ 、Sn ²⁺ 、Zn ²⁺ 、Ca ²⁺ 、Li ⁺ 、Na ⁺ 、NH ₄ ⁺ 、K ⁺ 、Cu ²⁺ 、Ag ⁺ 、Ba ²⁺ Etc. Anions contained in reagents used for salting out include, but are not limited to, cl ^- 、SO ₄ ^2- 、NO ₃ ^- 、CO ₃ ^2- 、SiO3 ^2- 、S ₂ O ₇ ^2- 、B ₄ O ₇ ^2- 、PO4 ^3- 、RCOO ^- 、NO ₂ ^- 、S ₂ O ₈ ^2- 、S ^2- 、CrO ₄ ^2- 、MnO4 ^- 、P ₂ O ₇ ^4- Etc.

When heating is used to separate and purify the antigen component, the heating temperature is greater than 40 ℃.

In some embodiments of the present disclosure, hypochlorous acid is used as the oxidizing agent to oxidize the antigenic component, and any other oxidizing agent that can oxidize the antigenic component may be used in practice, including but not limited to persulfates, KIO ₃ 、KBrO ₃ Hydrogen peroxide (hydrogen peroxide), chlorine, dichromate, nitric acid, hydrogen peroxide, peroxyacetic 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 permanganate, nitric acid, clO ₃ ^- 、ClO ₄ ^- 、Na ₂ O ₂ 、K ₂ O ₂ 、MgO ₂ 、CaO ₂ 、BaO ₂ 、H ₂ O ₂ 、NO ₃ ^- 、MnO ₄ ^- 、F ₂ 、Cl ₂ 、O ₂ 、Br ₂ 、I ₂ 、S、Si、HNO ₃ 、MnO ₂ 、FeCl ₃ And the like. Oxidation may enhance the immunogenicity of a portion of the antigen component.

In the present disclosure, the enzyme treatment method in some embodiments uses nuclease, pepsin, trypsin, protease inhibitor, etc., and any other feasible enzymolysis method such as chymotrypsin, dnase, etc. may be used in practical use.

For the sake of brevity, the examples of the present disclosure do not exemplify the use of mineralization to isolate and purify or enhance the immunogenicity of the antigen component, and any mineralization method such as siliconization, calcification, magnesian may be used in practical applications.

For the sake of brevity, the methods of isolating and purifying or enhancing the immunogenicity of the antigen component using reduction are not exemplified in the examples of the present disclosure, and in practice, reduction may be used to isolate and purify the antigen component or enhance the immunogenicity of the antigen component. Reducing agents that may be used include, but are not limited to, DTT, TCEP, and the like.

The antigen component may be prepared by irradiation, which may be any conventional irradiation method including, but not limited to, one or more of radioactive substance irradiation, ultraviolet irradiation, X-ray irradiation, gamma-ray irradiation, alpha-ray irradiation, beta-ray irradiation, and the like.

Step 4, loading the lysate component obtained in the step 2 on nano particles/micro particles as an antigen component; mixing the lysate component obtained in the step 2 with artificially synthesized polypeptide and/or nucleic acid to be used as an antigen component for loading on nano particles/micro particles; or the protein polypeptide component and/or the non-water-soluble component in the water-soluble component obtained in the step 3 or the protein polypeptide component in the water-soluble component are respectively or simultaneously loaded into the interior and/or the surface of the nano-particle or the micro-particle to obtain the nano-particle or the micro-particle; or mixing the protein polypeptide component and/or the non-water-soluble component in the water-soluble component obtained in the step 3 or the protein polypeptide component in the water-soluble component with the artificially synthesized polypeptide and/or nucleic acid to be used as an antigen component to be loaded into and/or on the interior and/or the surface of nano-particles or micro-particles so as to obtain the nano-particles or the micro-particles; or the protein polypeptide component and the RNA component (or mRNA component) in the water-soluble component obtained in the step 3 and/or the non-water-soluble component or the protein polypeptide component and the RNA component (or mRNA component) in the water-soluble component are respectively or simultaneously loaded into the interior and/or the surface of the nano-particle or the micro-particle to obtain the nano-particle or the micro-particle; or mixing the protein polypeptide component and RNA component (or mRNA component) in the water-soluble component obtained in the step 3 and/or the non-water-soluble component or the protein polypeptide component and RNA component (or mRNA component) in the water-soluble component with the artificially synthesized polypeptide and/or nucleic acid, and then loading the mixture as an antigen component into and/or on the interior of nano-particles or micro-particles to obtain the nano-particles or micro-particles; or loading the antigen component obtained in the step 3 into the interior and/or the surface of the nano-particle or the micro-particle to obtain the nano-particle or the micro-particle; or respectively or simultaneously mixing the antigen component obtained in the step 3 with the artificially synthesized polypeptide and/or nucleic acid to be used as the antigen component to be loaded into and/or on the surfaces of the nano-particles or the micro-particles so as to obtain the nano-particles or the micro-particles.

The following describes how antigen-loaded nano/micro particles are prepared by taking the preparation of the multiple emulsion method in the solvent evaporation method as an example. Any other preparation method that can load antigen to nanoparticles or microparticles may be used in practical applications.

In the case of preparation by the multiple emulsion method, the primary aqueous phase and the organic phase are mixed first, specifically, a first predetermined volume of aqueous phase solution containing an antigen component of a first predetermined concentration is added to a second predetermined volume of organic phase containing a raw material for preparing particles of a second predetermined concentration.

In some embodiments, the aqueous phase solution may contain at least one of the following i) to iii): i) Each component and/or antigenic component in the lysate, ii) each component and/or antigenic component in the lysate, and an immunopotentiating adjuvant. Each component in the lysate contains an antigen component in a water-soluble component and/or an original water-insoluble antigen component dissolved in a dissolving solution, or contains an antigen component in the lysate, or contains an antigen component such as an artificially synthesized protein polypeptide and nucleic acid. The first predetermined concentration is the concentration of protein and polypeptide contained in the aqueous solution, or the concentration of water-soluble antigen and/or the concentration of primary water-insoluble antigen contained in the aqueous solution, the first predetermined concentration requiring a concentration of protein polypeptide of greater than 1ng/mL so as to be capable of supporting sufficient antigen components to activate the cells of interest. The concentration of immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01ng/mL.

In some embodiments, the organic solvent is selected from dichloromethane. Additionally, in some embodiments, the second predetermined concentration of the starting material for the preparation of particles is in the range of 0.5mg/mL to 5000mg/mL, selected to be 100mg/mL.

In practice, the second predetermined volume of the organic phase is set according to the ratio of it to the first predetermined volume of the aqueous phase, in the present disclosure the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably from 1:2 to 1:10. The ratio of the first predetermined volume, the second predetermined volume and the first predetermined volume to the second predetermined volume can be adjusted as needed in the implementation process to adjust the size of the prepared nano-particles or micro-particles.

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

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

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

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

In some embodiments, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20mg/mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present disclosure, the second predetermined volume and the third predetermined volume are set in the range of 1:1.1-1:1000, preferably may be 2:5. In order to control the size of the nano-or micro-particles during the implementation process, the ratio of the second predetermined volume to the third predetermined volume may be adjusted. Similarly, the ultrasonic time or stirring time or homogenizing time, the volume of the aqueous solution of the emulsifier and the concentration of the aqueous solution of the emulsifier are taken as values according to the step, so that the nano-particles or the micro-particles with proper size can be obtained.

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

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

The fourth predetermined concentration is 5mg/mL, and the fourth predetermined concentration is selected based on obtaining nano-particles or micro-particles with proper size. The fourth predetermined volume is selected based on a ratio of the third predetermined volume to the fourth predetermined volume. In the present disclosure, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted in order to control the size of the nanoparticles or microparticles in the implementation process.

In the present disclosure, the predetermined stirring condition of this step is until the evaporation of the organic solvent is completed, that is, the evaporation of the methylene chloride in step 4 is completed.

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

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

Step 10, re-suspending the nanoparticle/microparticle-containing suspension obtained in step 8 in a sixth predetermined volume in PBS (or physiological saline) or re-suspending the lyophilized nanoparticle/microparticle obtained in step 9 with a sixth predetermined volume of PBS (or physiological saline) and then directly using; or the sample is mixed with a seventh predetermined volume of the antigen component.

In the present disclosure, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferential volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1.

In some embodiments, the nano/microparticles are loaded with 0.005 to 2000 μg of protein or polypeptide component per 1mg of particle material. In some embodiments, or the nano/microparticles further carry an immunopotentiating adjuvant, wherein 1-400 μg immunopotentiating adjuvant is carried per 1mg of particle material. In some embodiments, the nano/microparticles further carry an electropositive molecule, wherein 1-800 μg of electropositive molecule is carried per 1mg of particle material.

In some embodiments, the interior and/or surface of the nano/micro particle further comprises a membrane component, the membrane component of the interior and/or surface of the nano/micro particle being one or more mixed membranes selected from the group consisting of a cell membrane of an antigen presenting cell, an extracellular vesicle of an antigen presenting cell, a cell membrane of a cancer cell, an extracellular vesicle of a cancer cell, a cell membrane of a bacterium, and an extracellular vesicle of a bacterium.

Methods of loading the membrane component onto the nano/micro particle surface when the membrane component is on the nano/micro particle surface include, but are not limited to, one or more of sonication, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bonding, stirring, dialysis, homogenization, and homogenization.

In the exemplary embodiments of the present disclosure, a solvent evaporation method is used to prepare antigen delivery particles, and any other method that can prepare antigen delivery particles may be used in practical applications, including but not limited to precipitation, dialysis, dispersion, microfluidic, high pressure homogenization, stirring, spray drying, phase separation, electrostatic spraying, emulsion polymerization, machine stirring shear, membrane emulsification, and the like.

Step 11, co-incubating antigen delivery particles (nanoparticles and/or microparticles) prepared in the previous step with Dendritic Cells (DC) and B cells in the same system at the same time, and activating antigen presenting cells in the system. Macrophages may be present in the co-incubation system in addition to dendritic cells and B cells. The co-incubation system may also contain substances that may assist in the activation of antigen presenting cells, such as cytokines, growth factors, antibodies, and the like. When co-incubating, the concentration of antigen delivery particles in the co-incubating system is (0.002 μg/mL-80 mg/mL); preferably, the concentration of antigen delivery particles in the co-incubation system is from 0.02mg/mL to 30mg/mL, more preferably, the concentration of antigen delivery particles in the co-incubation system is from 0.05 to 10mg/mL. The ratio of DC to B cells in the system is 50:1 to 1:100, preferably 20:1 to 1:20, in cell number at co-incubation.

Step 12, collecting the activated mixed cells obtained in step 11, and using the mixed cells as a cancer vaccine.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

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

Example 1 vaccine for treatment of melanoma

(1) Preparation of antigenic components

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. Preparing single cell suspension of tumor tissue, adding proper amount of ultrapure water and repeatedly freezing and thawing for 5 times to lyse cells in the tumor tissue. After the pyrolysis, centrifuging the pyrolysis at a rotation speed of 5000g for 5 minutes, and taking supernatant fluid to obtain a water-soluble component which is soluble in pure water; adding 8M urea aqueous solution into the obtained precipitation part to dissolve the precipitation part, so as to convert the insoluble component insoluble in pure water into soluble component in the 8M urea aqueous solution. Adding saturated ammonium sulfate aqueous solution dropwise into water-soluble components in the lysate, centrifuging the obtained sample at 3000g for 5 minutes after complete precipitation, dissolving the precipitate in 8M urea aqueous solution for later use, heating supernatant at 40 ℃ for 10 minutes, centrifuging the obtained sample at 3000g for 5 minutes, discarding the supernatant, and dissolving the precipitate in 8M urea aqueous solution; the salted-out precipitate dissolved with 8M urea and the heated precipitate were then combined and used as a part of the water-soluble components. The non-water soluble fraction of the above 8M aqueous urea solution-dissolved lysate and the fraction obtained by salting out and heat-precipitating the 8M aqueous urea-dissolved water-soluble fraction are mixed with four tumor-specific antigens B16-M20 (Tubb 3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn 4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) and two tumor-associated antigens (IO 102: DTLLKALLEIASCL) EKALQVF and IO103: FMTYWHLLNAFTVTVPKDL) according to a mass ratio of 1:1:0.01:0.01:0.01:0.01:0.01:0.01, to obtain antigen component 1 for preparing nanoparticle 1.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. Preparing single cell suspension of tumor tissue, adding proper amount of ultrapure water and repeatedly freezing and thawing for 5 times to lyse cells in the tumor tissue. After the pyrolysis, centrifuging the pyrolysis at a rotation speed of 5000g for 5 minutes, and taking supernatant fluid to obtain a water-soluble component which is soluble in pure water; adding 8M urea aqueous solution into the obtained precipitation part to dissolve the precipitation part, so as to convert insoluble antigen insoluble in pure water into soluble antigen in 8M urea aqueous solution. Adding saturated ammonium carbonate aqueous solution dropwise into water-soluble components in the lysate, centrifuging the obtained sample at 3000g for 5 minutes after complete precipitation, dissolving the precipitate in 8M urea aqueous solution for later use, heating supernatant at 40 ℃ for 10 minutes, centrifuging the obtained sample at 3000g for 5 minutes, discarding the supernatant, and dissolving the precipitate in 8M urea aqueous solution; the salted-out precipitate dissolved with 8M urea and the heated precipitate were then combined and used as a part of the water-soluble components. The non-water soluble component in the lysate dissolved by the 8M urea aqueous solution and the component obtained by salting out and heating and precipitating the 8M urea-dissolved water soluble component are mixed with four tumor specific antigens B16-M20 (Tubb 3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn 4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) and two tumor related antigens (IO 102: DTLLKALLEIASCLEKALQVF and IO103: FMTYWHLLNAFTVTVPKDL) according to the mass ratio of 1:1:0.01:0.01:0.01:0.01:0.01:0.01:0.01 to obtain the antigen component 2 for preparing the nano particles 2.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. Preparing single cell suspension of tumor tissue, adding proper amount of ultrapure water and repeatedly freezing and thawing for 5 times to lyse cells in the tumor tissue. After the pyrolysis, the pyrolysis is centrifuged at 5000g for 5 minutes and the supernatant is taken to obtain the water-soluble pure waterA component (C); adding 8M urea aqueous solution into the obtained precipitation part to dissolve the precipitation part, so as to convert insoluble antigen insoluble in pure water into soluble antigen in 8M urea aqueous solution. The non-water soluble components and water soluble components in the lysate dissolved in the 8M urea aqueous solution are mixed with four tumor specific antigens B16-M20 (Tubb 3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn 4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) and two tumor associated antigens (IO 102: DTLLKALLEIASCLEKALQVF and IO103: FMTYWHLLNAFTVTVPKDL) according to the mass ratio of 1:1:0.01:0.01:0.01:0.01:0.01:0.01 to prepare the antigen component 3 for delivering the nanoparticle 3.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. Preparing single cell suspension of tumor tissue, adding proper amount of ultrapure water and repeatedly freezing and thawing for 5 times to lyse cells in the tumor tissue. After the pyrolysis, centrifuging the pyrolysis at a rotation speed of 5000g for 5 minutes, and taking supernatant fluid to obtain a water-soluble component which is soluble in pure water; adding 8M urea aqueous solution into the obtained precipitation part to dissolve the precipitation part, so as to convert the insoluble component insoluble in pure water into soluble component in the 8M urea aqueous solution. Adding saturated ammonium sulfate aqueous solution dropwise into water-soluble components in the lysate, centrifuging the obtained sample at 3000g for 5 minutes after complete precipitation, solubilizing the precipitate with aqueous solution for later use, heating supernatant at 40 ℃ for 10 minutes, centrifuging the obtained sample at 3000g for 5 minutes, discarding supernatant, and solubilizing the precipitate with 5% PEG5000 aqueous solution; the salted-out precipitate solubilized using 5% peg5000 aqueous solution and the heated precipitate were then combined and used as a part of the water-soluble components. Salting out and precipitating the above 8M urea aqueous solution-dissolved lysate and 5% PEG5000 aqueous solution-dissolved water-soluble fraction with four tumor specific antigens B16-M20 (Tubb 3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn 4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) and two tumor associated antigens (IO 102: DTLLKALLEIASCLEKALQVF and IO103: FMTYWHLLNAF) Tvtvtvpkdl) in a mass ratio of 1:1:0.01:0.01:0.01:0.01:0.01:0.01 to prepare antigen component 4 for delivering nanoparticle 4.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. Preparing single cell suspension of tumor tissue, adding proper amount of ultrapure water and repeatedly freezing and thawing for 5 times to lyse cells in the tumor tissue. After the pyrolysis, centrifuging the pyrolysis at a rotation speed of 5000g for 5 minutes, and taking supernatant fluid to obtain a water-soluble component which is soluble in pure water; and adding 5% PEG5000 aqueous solution into the obtained precipitation part to solubilize the precipitation part, thus obtaining the component of the water-insoluble component soluble in the 5% PEG5000 aqueous solution. Adding saturated ammonium sulfate aqueous solution dropwise into water-soluble components in the lysate, centrifuging the obtained sample at 3000g for 5 min after complete precipitation, solubilizing the precipitate with 5% PEG5000 aqueous solution for later use, heating supernatant at 40 ℃ for 10 min, centrifuging the obtained sample at 3000g for 5 min, discarding supernatant, and solubilizing the precipitate with 5% PEG5000 aqueous solution; the salted-out precipitate solubilized using 5% peg5000 aqueous solution and the heated precipitate were then combined and used as a part of the water-soluble components. Salting out and heating the water-insoluble component in the lysate dissolved in the 5% PEG5000 aqueous solution and the water-soluble component dissolved in the 5% PEG5000 aqueous solution, and precipitating the obtained component, mixing the component with four tumor-specific antigens B16-M20 (Tubb 3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn 4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) and two cancer-related antigens (IO 102: DTLLKALLEIASCLEKALQVF and IO103: FMTYWHLLNAFTVTVPKDL) according to a mass ratio of 1:1:0.01:0.01:0.01:0.01:0.01:0.01:0.01, and obtaining the antigen component 5 for preparing the delivery nanoparticle 5.

(2) Preparation of antigen delivery particles

The antigen delivery nanoparticle 1 in this example was prepared by the multiple emulsion method in the solvent evaporation method. The antigen delivery nanoparticle preparation material PLGA has a molecular weight of 10kDa-20kDa, and the immunological adjuvant used is Polyinosinic-polycytidylic acid (poly (I: C)). Preparation method As previously described, in the preparation process, the cell antigen component 1 and the adjuvant are firstly loaded in the nanoparticle by adopting a multiple emulsion method, 100mg of the nanoparticle is centrifuged at 18000g for 50 minutes, and 10mL of ultra-pure water containing 4% trehalose is used for re-suspension, and then the nanoparticle is freeze-dried for 48 hours. The average particle diameter of the nanoparticle 1 is about 100nm, and about 15 mug protein/polypeptide components and 0.2mg poly (I: C) are loaded per 1mg PLGA nanoparticle.

The antigen delivery nanoparticle 2 preparation method and preparation steps are the same as the antigen delivery nanoparticle 1. The molecular weight of the nanoparticle preparation material PLGA is 10kDa-20kDa, and the immunological adjuvant is poly (I: C). Preparation method As previously described, cell antigen component 2 and adjuvant were loaded inside the nanoparticle, and then 100mg of the nanoparticle was centrifuged at 18000g for 50 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose and then lyophilized for 48 hours. The average particle diameter of the nanoparticle 2 is about 100nm, and about 15 mug protein/polypeptide components and 0.2mg poly (I: C) are loaded per 1mg PLGA nanoparticle.

The antigen delivery nanoparticle 3 preparation method and preparation steps are the same as the antigen delivery nanoparticle 1. The molecular weight of PLGA material prepared from the nanoparticle 3 is 10kDa-20kDa, and the immunoadjuvant is poly (I: C). Preparation method As previously described, cell antigen component 3 and adjuvant were loaded inside the nanoparticle, and then 100mg of the nanoparticle was centrifuged at 18000g for 50 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose and then lyophilized for 48 hours. The average particle diameter of the nanoparticle 3 is about 100nm, and about 15 mug of protein/polypeptide component and 0.2mg of poly (I: C) are loaded per 1mg of PLGA nanoparticle.

The antigen delivery nanoparticle 4 preparation method and preparation steps are the same as the antigen delivery nanoparticle 1. The molecular weight of the nanoparticle preparation material PLGA is 10kDa-20kDa, and the immunological adjuvant is poly (I: C). Preparation method As previously described, cellular antigen component 4 and adjuvant were loaded inside the nanoparticle, and then 100mg of the nanoparticle was centrifuged at 18000g for 50 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose and then lyophilized for 48 hours. The average particle diameter of the nanoparticle 4 is about 100nm, 15 mug of protein/polypeptide components are loaded per 1mg of PLGA nanoparticle, and 0.2mg of poly (I: C) is loaded per 1mg of PLGA nanoparticle.

The antigen delivery nanoparticle 5 preparation method and preparation steps are the same as the antigen delivery nanoparticle 1. The molecular weight of the nanoparticle preparation material PLGA is 10kDa-20kDa, and the immunological adjuvant is poly (I: C). Preparation method As previously described, cell antigen component 5 and adjuvant were loaded inside the nanoparticle, and then 100mg of the nanoparticle was centrifuged at 18000g for 50 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose and then lyophilized for 48 hours. The average particle size of the nanoparticle 5 is about 100nm, and about 15 mug of protein or polypeptide component and 0.2mg of poly (I: C) are loaded per 1mg of PLGA nanoparticle.

(3) Preparation of bone marrow-derived dendritic cells (BMDCs) and preparation of B cells

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

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

(4) Preparation of cell vaccine

BMDC (500 ten thousand) and B cells (500 ten thousand) After mixing in a quantitative ratio of 1:1, 750. Mu.g of nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3, or nanoparticle 4, or nanoparticle 5) were co-incubated in 15mL of RPM 1640 complete medium for 96 hours (37 ℃,5% CO ₂ ). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove free nanoparticles in the system, and then the mixed cells activated by antigen delivery particles were used as cancer vaccines. Wherein, the mixed cells activated by the nanoparticle 1 are the mixed cell cancer vaccine 1; the mixed cells activated by the nano particles 2 are used as the mixed cell cancer vaccine 2; the mixed cells activated by the nano particles 3 are used as the mixed cell cancer vaccine 3; the mixed cells activated by the nanoparticles 4 are mixed cell cancer vaccine 4; the mixed cells activated using the nanoparticles 5 are mixed cell cancer vaccine 5.

Alternatively nanoparticle 1 (750. Mu.g) was co-incubated with BMDC (1000 ten thousand) in 15mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove free nano-sized particles in the system, and then the mixed cells activated by the antigen delivery particles were used as cancer vaccine 6.

Or antigen delivery nanoparticle 1 (750 μg) was co-incubated with BMDC (500 tens of thousands) in 15mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the Simultaneously nanoparticle 1 (750. Mu.g) was co-incubated with B cells (500 ten thousand) in 15mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the activated DC and B cells were centrifuged at 400g for 4 min to remove free antigen delivery particles from the system, and then the DC and B cells, which had been activated individually, were mixed in a number ratio of 1:1, and the mixed cells were used as cancer vaccine 7.

(5) Vaccine for treating cancer

Preparation of melanoma tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 ⁵ B16F10 cells. 100 ten thousand corresponding mixed cell vaccines (vaccine 1, or Vaccine 2, or vaccine 3, or vaccine 4, or vaccine 5, or vaccine 6, or vaccine 7), or 100 μl PBS. The mice were monitored for tumor growth rate and mice survival. In the experiment, the size of the tumor volume of the mice was recorded every 3 days starting on day 3. Tumor volume was calculated using the formula v=0.52×a×b ² Calculation, where v is tumor volume, a is tumor length, and b is tumor width. For ethical reasons in animal experiments, the tumor volume of the mice exceeds 2000mm in the life cycle test of the mice ³ I.e. the mice were regarded as dead and euthanized.

(4) Experimental results

As shown in fig. 3, the PBS group mice in fig. 3 quickly grew in tumor volume and the mice died quickly. Mice using Vaccine 1 (Vaccine 1), vaccine 2 (Vaccine 2), vaccine 3 (Vaccine 3), vaccine 4 (Vaccine 4), vaccine 5 (Vaccine 5), vaccine 6 (Vaccine 6) and Vaccine 7 (Vaccine 7) all had significantly slower tumor growth rates and significantly longer survival periods, with some mice recovering from tumors. Wherein, the effect of the vaccine 1 is better than that of the vaccine 2, the vaccine 3, the vaccine 4 and the vaccine 5, which shows that the effect of separating and purifying protein and polypeptide components in water-soluble components by ammonium sulfate salt analysis is better than that of ammonium carbonate, and the effect of separating and purifying a part of components in water-soluble components is better than that of directly using the water-soluble components. Furthermore, the use of urea is better in dissolving the antigen component than the use of PEG. Moreover, vaccine 1 is significantly better than vaccine 6, demonstrating that using activated mixed cell cancer vaccine is better than using only activated DCs as vaccine; the effect of the vaccine 1 is obviously better than that of the vaccine 7, which shows that the effect of the mixed cell cancer vaccine in which DC and B cells are activated by antigen delivery particles simultaneously in the same system is better than that of the cancer vaccine obtained by respectively activating DC by antigen delivery particles and B cells by antigen delivery particles and then mixing the DC and B cells.

In this example, BMDC derived from bone marrow culture was used, and in practical application, DC may be obtained by extracting DC from peripheral blood or any other method for obtaining DC. In this example, B cells in spleen cells are used, and in practical applications, B cells in peripheral blood or B cells prepared by any other method may be used.

Example 2 vaccine for treatment of melanoma

(1) Preparation of antigenic components

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. A single cell suspension of tumor tissue was prepared, and then an appropriate amount of ultrapure water was added and freeze thawing was repeated 5 times with ultrasound to lyse the cells. After the pyrolysis, centrifuging the pyrolysis at a rotation speed of 5000g for 5 minutes, and taking supernatant fluid to obtain a water-soluble component which is soluble in pure water; the non-water-soluble component insoluble in pure water can be converted into soluble in 6M guanidine sulfate aqueous solution by adding 6M guanidine sulfate aqueous solution to the obtained precipitation part to dissolve the precipitation part. Adding saturated sodium chloride aqueous solution dropwise into water-soluble components in the lysate, centrifuging the obtained sample at 3000g for 5 minutes after complete precipitation, dissolving the precipitate in 6M guanidine sulfate aqueous solution for later use, heating supernatant at 95 ℃ for 10 minutes, centrifuging the obtained sample at 3000g for 5 minutes, discarding the supernatant, and dissolving the precipitate in 6M guanidine sulfate aqueous solution; the salted-out precipitate dissolved using 6M guanidine sulfate and the heated precipitate were then combined and used as a part of the water-soluble components. The non-water-soluble component in the lysate dissolved in the 6M guanidine sulfate aqueous solution and the component obtained by salting out and heating and precipitating the water-soluble component dissolved in the 6M guanidine sulfate aqueous solution are used together, namely the antigen component 1 for preparing the cancer delivery nanoparticle 1.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. A single cell suspension of tumor tissue was prepared, and then an appropriate amount of ultrapure water was added and freeze thawing was repeated 5 times with ultrasound to lyse the cells. After the pyrolysis, centrifuging the pyrolysis at a rotation speed of 5000g for 5 minutes, and taking supernatant fluid to obtain a water-soluble component which is soluble in pure water; adding 6M guanidine sulfate aqueous solution into the obtained precipitation part to dissolve the precipitation part, so as to convert insoluble water insoluble antigen into water soluble 6M guanidine sulfate aqueous solution. The water-insoluble component and the water-soluble component in the above lysate dissolved in 6M guanidine sulfate aqueous solution are used together,i.e. the antigen component 2 for the preparation of cancer delivery nanoparticles 2.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. A single cell suspension of tumor tissue was prepared, and then an appropriate amount of ultrapure water was added and freeze thawing was repeated 5 times with ultrasound to lyse the cells. After the pyrolysis, centrifuging the pyrolysis at a rotation speed of 5000g for 5 minutes, and taking supernatant fluid to obtain a water-soluble component which is soluble in pure water; the non-water-soluble component insoluble in pure water can be converted into soluble in 6M guanidine sulfate aqueous solution by adding 6M guanidine sulfate aqueous solution to the obtained precipitation part to dissolve the precipitation part. Adding saturated sodium chloride aqueous solution dropwise into water-soluble components in the lysate, centrifuging the obtained sample at 3000g for 5 minutes after complete precipitation, solubilizing the precipitate with 2% Triton X100 aqueous solution for later use, heating supernatant at 95deg.C for 10 minutes, centrifuging the obtained sample at 3000g for 5 minutes, discarding supernatant, and solubilizing the precipitate with 2% Triton X100 aqueous solution; the salted-out precipitate solubilized with 2% Triton X100 aqueous solution and the heated precipitate were then combined and used as a part of the water-soluble components. The non-water-soluble component in the lysate dissolved in the above 6M guanidine sulfate aqueous solution and the component obtained by salting out and precipitating after heating in the water-soluble component dissolved in 2% Triton X100 aqueous solution are used together, namely, antigen component 3 for preparing cancer delivery nanoparticle 3.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. A single cell suspension of tumor tissue was prepared, and then an appropriate amount of ultrapure water was added and freeze thawing was repeated 5 times with ultrasound to lyse the cells. After the pyrolysis, centrifuging the pyrolysis at a rotation speed of 5000g for 5 minutes, and taking supernatant fluid to obtain a water-soluble component which is soluble in pure water; and adding 2% of Triton X100 aqueous solution to the obtained precipitation part to solubilize the precipitation part to obtain the component of which the water-insoluble component is soluble in 2% of Triton X100 aqueous solution. Adding saturated sodium chloride aqueous solution dropwise into water-soluble components in the lysate, centrifuging the obtained sample at 3000g for 5 min after precipitation is completed, and precipitatingSolubilizing with 2% Triton X100 aqueous solution, heating supernatant at 95deg.C for 10 min, centrifuging the obtained sample at 3000g for 5 min, discarding supernatant, and solubilizing precipitate with 2% Triton X100 aqueous solution; the salted-out precipitate solubilized with 20% Triton X100 aqueous solution and the heated precipitate were then combined and used as a part of the water-soluble components. The non-water-soluble component in the lysate of the above 2% Triton X100 aqueous solution and the component obtained by salting out and precipitating after heating in the 2% Triton X100 aqueous solution-dissolved water-soluble component are used together, namely, the antigen component 4 for preparing the cancer antigen delivery nanoparticle 4.

(2) Preparation of antigen delivery particles

The antigen delivery nanoparticle 1 in this example was prepared by the multiple emulsion method in the solvent evaporation method. In preparation, part of the water-soluble components in the tumor tissue lysate are dissolved by using 6M guanidine sulfate aqueous solution after salting out and purification by sodium chloride and heating separation and purification. At the time of preparation, nanoparticles carrying a water-soluble fraction component (component dissolved with 6M aqueous guanidine sulfate solution after salting out with sodium chloride and heating separation purification) in a cancer cell lysate and nanoparticles carrying a water-insoluble antigen (dissolved with 6M aqueous guanidine sulfate solution) in a cancer cell whole-cell antigen were prepared separately and then used together as antigen delivery nanoparticle 1 at the time of use. The molecular weight of the PLGA material prepared by the antigen delivery nano particles is 20kDa-40kDa, and the immunological adjuvant is poly ICLC. Preparation method As previously described, in the preparation process, the cell antigen component 1 and the adjuvant are firstly loaded in the nanoparticle by a multiple emulsion method, 100mg of the nanoparticle is centrifuged at 15000g for 20 minutes, and 10mL of ultra-pure water containing 4% trehalose is used for re-suspension, and then freeze-drying is performed for 48 hours. The average particle size of the nanoparticle 1 is about 250nm, and about 15 mug protein or polypeptide components are loaded per 1mg of PLGA nanoparticle, and 0.01mg of poly ICLC is loaded.

The antigen delivery nanoparticle 2 preparation method and preparation steps are the same as the antigen delivery nanoparticle 1. At the time of preparation, all water-soluble components in tumor tissue lysate were used without any treatment. At the time of preparation, nanoparticles carrying water-soluble components in cancer cell lysate and nanoparticles carrying water-insoluble antigens (6M guanidine sulfate aqueous solution is dissolved) in cancer cell whole-cell antigens were prepared separately, and then used together as antigen delivery nanoparticles 2 at the time of use. The molecular weight of the nanoparticle preparation material PLGA is 20kDa-40kDa, and the immunological adjuvant is poly ICLC. Preparation method As previously described, in the preparation process, the cell antigen component and the adjuvant are firstly loaded in the nanoparticle by adopting a multiple emulsion method, 100mg of the nanoparticle is centrifuged at 15000g for 20 minutes, and 10mL of ultra-pure water containing 4% trehalose is used for re-suspension, and then the nanoparticle is freeze-dried for 48 hours. The average particle size of the nanoparticle 2 is about 250nm, and about 15 mug protein or polypeptide components are loaded per 1mg of PLGA nanoparticle, and 0.01mg of poly ICLC is loaded.

The antigen delivery nanoparticle 3 preparation method and preparation steps are the same as the antigen delivery nanoparticle 1. In preparation, part of the water-soluble components in the tumor tissue lysate are dissolved by using 2% Triton X100 aqueous solution after salting out and purification by sodium chloride and heating separation and purification. Nanoparticles carrying water-soluble partial components (components dissolved in 2% Triton X100 aqueous solution after salting out and heating separation purification) in cancer cell lysate and nanoparticles carrying water-insoluble antigens (dissolved in 6M guanidine sulfate aqueous solution) in cancer cell whole-cell antigens are prepared separately and then used together as antigen delivery nanoparticles 3 at the time of use. The molecular weight of the PLGA material for preparing the nano particles is 20kDa-40kDa, and the immune adjuvant is polyICLC. Preparation method As previously described, cell antigen component 3 and adjuvant were loaded inside the nanoparticle, and then 100mg of the nanoparticle was centrifuged at 15000g for 20 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose and lyophilized for 48 hours. The average particle size of the nanoparticle 3 is about 250nm, and about 15 mug of protein or polypeptide component and 0.01mg of polyICLC0 are loaded per 1mg of PLGA nanoparticle.

The antigen delivery nanoparticle 4 is prepared by the same method and steps as the nanoparticle 1. In preparation, part of the water-soluble components in the tumor tissue lysate are dissolved by using 2% Triton X100 aqueous solution after salting out and purification by sodium chloride and heating separation and purification. In the preparation, nanoparticles carrying water-soluble partial components (components dissolved in 2% Triton X100 aqueous solution after salting out and heating separation purification) in cancer cell lysate and nanoparticles carrying water-insoluble antigens (20% Triton X100 aqueous solution) in cancer cell whole-cell antigens are prepared separately and then used together as nanoparticles 4 in the use. The molecular weight of the PLGA material for preparing the nano particles is 20kDa-40kDa, and the immune adjuvant is polyICLC. Preparation method As described above, the inside of the nanoparticle was loaded with the cell antigen component and the adjuvant, and 100mg of the nanoparticle was centrifuged at 10000g for 20 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose and then lyophilized for 48 hours. The average particle size of the nanoparticle 4 is 250nm, and about 15 mug of protein or polypeptide component and 0.01mg of polyICLC are loaded per 1mg of PLGA nanoparticle.

BMDC and mouse B cells were prepared as in example 1.

(4) Preparation of cell vaccine

BMDC (500 ten thousand) and B cells (5000 ten thousand) were mixed at a number ratio of 1:10, and then incubated with antigen delivery nanoparticle 1 (250. Mu.g water-soluble component-loaded nanoparticle+250. Mu.g water-insoluble component-loaded nanoparticle), antigen delivery nanoparticle 2 (250. Mu.g water-soluble component-loaded nanoparticle+250. Mu.g water-insoluble component-loaded nanoparticle), antigen delivery nanoparticle 3 (250. Mu.g water-soluble component-loaded nanoparticle+250. Mu.g water-insoluble component-loaded nanoparticle), or antigen delivery nanoparticle 4 (250. Mu.g water-soluble component-loaded nanoparticle+250. Mu.g water-insoluble component-loaded nanoparticle) in 15mL RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove free antigen delivery particles in the system, and then the mixed cells activated by the antigen delivery particles were used as cancer vaccines. Wherein the mixed cells activated using the antigen delivery particles 1 are mixed cell cancer vaccine 1; the mixed cells activated using antigen delivery particles 2 are mixed cell cancer vaccine 2; the mixed cells activated using antigen delivery particles 3 are mixed cell cancer vaccine 3; delivery of particles 4 using antigen The activated mixed cells are mixed cell cancer vaccine 4.

Or antigen delivery nanoparticle 1 (250. Mu.g of water-soluble component-loaded nanoparticle+250. Mu.g of water-insoluble component-loaded nanoparticle) was co-incubated with BMDC (5500 ten thousand) in 15mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the DC cells were centrifuged at 400g for 4 minutes to remove free antigen delivery particles in the system, and then the DC cells activated by the antigen delivery particles were used as cancer vaccine 5.

Or antigen delivery nanoparticle 1 (250. Mu.g of water-soluble component-loaded nanoparticle+250. Mu.g of water-insoluble component-loaded nanoparticle) was co-incubated with BMDC (500 ten thousand) in 15mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the Simultaneously antigen delivery nanoparticle 1 (250. Mu.g of water-soluble component-loaded nanoparticle+250. Mu.g of water-insoluble component-loaded nanoparticle) was co-incubated with B cells (5000 ten thousand) in 15mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the activated DC and B cells were centrifuged at 400g for 4 min to remove free antigen delivery particles from the system, and then the DC and B cells, which had been activated individually, were mixed in a number ratio of 1:10, and the mixed cells were used as cancer vaccine 6.

(5) Vaccine for treating cancer

Preparation of melanoma tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 ⁵ The mice were subcutaneously vaccinated with 55 ten thousand corresponding mixed cell cancer vaccines (vaccine 1, or vaccine 2, or vaccine 3, or vaccine 4, or vaccine 5, or vaccine 6) or 100 μl PBS on day 3, day 6, day 9, day 14, and day 20, respectively. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.

(6) Experimental results

As shown in fig. 4, the PBS group mice in fig. 4 quickly grew in tumor volume and died quickly. Mice treated with Vaccine 1 (Vaccine 1), vaccine 2 (Vaccine 2), vaccine 3 (Vaccine 3), vaccine 4 (Vaccine 4), vaccine 5 (Vaccine 5), vaccine 6 (Vaccine 6) had significantly prolonged survival, with some mice healed tumor-free. Wherein, the effect of vaccine 1 is better than that of vaccine 2, vaccine 3 and vaccine 4, which shows that the effect of separating and purifying a part of the water-soluble components is better than that of directly using the water-soluble components. Furthermore, the use of guanidine sulfate to dissolve the water-soluble components resulted in better precipitation by salting out and heat treatment than the use of Triton. Moreover, vaccine 1 is significantly better than vaccine 5, demonstrating that using activated mixed cell cancer vaccine is better than using only activated DCs as vaccine; the effect of the vaccine 1 is obviously better than that of the vaccine 6, which shows that the effect of the mixed cell cancer vaccine in which DC and B cells are activated by antigen delivery particles simultaneously in the same system is better than that of the cancer vaccine obtained by respectively activating DC by antigen delivery particles and B cells by antigen delivery particles and then mixing the DC and B cells.

Example 3 vaccine for treatment of melanoma

In this example, the cancer cells isolated from the tumor tissue of B16F10 melanoma were first lysed using ultrapure water, and after culturing the cancer cells isolated from the portion, an antigen component derived from the lysate component after the cancer cells isolated from the tumor tissue were prepared, and then loaded on microparticles.

(1) Lysis of tumor tissue and collection of fractions

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing up to about 1000mm in tumors ³ Mice were sacrificed and tumor tissue was harvested. A single cell suspension of tumor tissue was prepared, and then cancer cells were isolated from the tumor tissue and cultured in RPMI1640 (10% fbs-containing) complete medium for 7 days (37 ℃,5% co) ₂ ). After the completion of the culture, the cancer cells were collected and centrifuged at 400g for 5 minutes to remove the medium. The precipitated cancer cells were resuspended in an appropriate amount of ultrapure water and then repeatedly freeze-thawed 5 times to lyse the cancer cells. Centrifuging the lysate at 5000g rotation speed for 5 min, and collecting supernatant to obtain water soluble component soluble in pure water; adding 6M guanidine hydrochloride water solution into the obtained precipitation part to dissolve the precipitation part, so as to convert insoluble water insoluble antigen into 6M guanidine hydrochloride water solution.

And respectively dropwise adding a saturated magnesium sulfate aqueous solution into the water-soluble component or the water-insoluble component in the lysate, centrifuging the obtained sample at 3000g for 5 minutes after the precipitation is completed, respectively dissolving the precipitate in a 6M guanidine hydrochloride aqueous solution, and using the precipitate as a part of antigen components in the separated and purified water-soluble component or a part of antigen components in the separated and purified water-insoluble component. Antigen component in separated and purified water-soluble fraction dissolved in 6M guanidine hydrochloride aqueous solution, antigen component in separated and purified water-insoluble fraction dissolved in 6M guanidine hydrochloride aqueous solution, four tumor-specific antigens B16-M20 (Tubb 3), B16-M24 (Dag 1), B16-M46 (Actn 4) and TRP2:180-188, and two tumor-associated antigens (WT 1 _126-134 RMFPNAPYL and WT1 _35-52 WAPVLDFAPPGASAYGSL) and mixing according to the mass ratio of 1:5:0.001:0.001:0.001:0.001:0.0011:0.0011, namely the antigen component 1.

Or four tumor specific antigens B16-M20 (Tubb 3), B16-M24 (Dag 1), B16-M46 (Actn 4) and TRP2:180-188 (SVYDFFVWL) and two tumor associated antigens (WT 1) _126-134 RMFPNAPYL and WT1 _35-52 WAPVLDFAPPGASAYGSL) are mixed according to the mass ratio of 1:1:1:1:1 to obtain the antigen component 2.

(2) Preparation of microparticles

In this example, microparticles 1 (Micronpartcle 1) were prepared by the multiple emulsion method of the solvent evaporation method. The molecular weight of the PLGA used for preparing the microparticles is 38KDa-54KDa, the immunoadjuvant used is poly (I: C) and CpG ODN7909, and the antigen component 1 and the adjuvant are loaded in the microparticles. Preparation method As described above, cell antigen component 1 and adjuvant were loaded inside microparticles, and 100mg microparticles were centrifuged at 10000g for 20 minutes, resuspended in 10mL of ultra-pure water containing 4% trehalose, and lyophilized for 48 hours. The average particle diameter of the microparticles 1 is about 1.2. Mu.m, and about 10. Mu.g of protein and polypeptide components are loaded per 1mg of PLGA microparticles, and 0.001mg of poly (I: C) and CpG ODN are loaded.

The method and procedure for the preparation of microparticles 2 (Micronpartcle 2) in this example are the same as for microparticles 1. In preparation, 4 melanoma cancer specific antigen polypeptides B16-M are loaded in equal amounts20 B16-M24, B16-M46 and TRP2:180-188 and two cancer-associated antigens (WT 1) _126-134 RMFPNAPYL and WT1 _35-52 WAPVLDFAPPGASAYGSL). The molecular weight of the PLGA material for preparing the microparticles is 38KDa-54KDa, the immunoadjuvant is poly (I: C) and CpG ODN7909, and the polypeptide and the adjuvant are simultaneously loaded in the microparticles. Preparation method As described previously, cell antigen component 2 and adjuvant were loaded inside the nanoparticle, and then 100mg of microparticles were centrifuged at 10000g for 20 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose and lyophilized for 48 hours. The average particle diameter of the microparticles 2 was about 1.2. Mu.m, and about 10. Mu.g of the polypeptide component was loaded per 1mg of PLGA microparticles, and 0.001mg of each of poly (I: C) and CpG ODN was loaded.

(3) Preparation of bone marrow-derived dendritic cells (BMDC), B cells, and bone marrow-derived macrophages (BMDM)

BMDC and B cells were prepared as described above.

This example illustrates how macrophages are prepared from mouse bone marrow cells. The BMDM preparation method is as follows: dislocation was sacrificed after C57 mice were anesthetized, the mice were sterilized with 75% ethanol, then a small opening was cut on the backs of the mice with scissors, and the skin was directly torn by hand to the joints of the mice 'legs, removing the joints of the mice' feet and the skin. The hind limbs are detached along the root of the thigh of the mouse by scissors, muscle tissues are removed, the hind limbs are placed in a culture dish containing 75% ethanol for soaking for 5min, and a new culture dish containing 75% ethanol is replaced and moved into an ultra clean bench. The ethanol soaked leg bones are moved into cold PBS for soaking, the ethanol on the surfaces of the tibia and the femur is washed off, and the process can be repeated for 3 times. The washed femur and tibia are separated, two ends of the femur and tibia are cut off respectively by scissors, the bone marrow is blown out of the femur and tibia by sucking cold induction culture medium by a 1mL syringe, and the flushing is repeated for 3 times until no obvious red color is seen in the femur. Repeatedly blowing the culture medium containing bone marrow cells with a 5mL pipetting gun to disperse cell clusters, sieving the cells with a 70 μm cell filter, transferring into a 15mL centrifuge tube, centrifuging at 1500rpm/min for 5min, discarding the supernatant, adding erythrocyte lysate, re-suspending for 5min, centrifuging at 1500rpm/min for 5min, discarding the supernatant, re-suspending with cold prepared bone marrow macrophage induction medium (DMEM high sugar medium containing 15% L929 medium), and plating. Cells were cultured overnight to remove other foreign cells such as fibroblasts, etc. that were relatively fast in attachment. The non-adherent cells were collected and seeded into dishes or cell culture plates according to the experimental design. Macrophage colony-stimulating factor (M-CSF) stimulates differentiation of bone marrow cells into mononuclear macrophages at a concentration of 40 ng/mL. The cells were cultured for 8 days, and the morphological changes of the macrophages were observed under a microscope. After 8 days, cells were collected by digestion, incubated with anti-mouse F4/80 antibody and anti-mouse CD11b antibody at 4deg.C for 30min in the absence of light, and the proportion of macrophages induced to be successful was identified by flow cytometry.

(4) Preparation of cell vaccine

BMDC (500 ten thousand) and B cells (500 ten thousand) were mixed in a quantitative ratio of 1:1 and then co-incubated with 100mg of microparticles 1 or microparticles 2 in 5mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove free antigen delivery particles from the system, and the activated mixed cells were used as cancer vaccines. Wherein, the mixed cells activated by the microparticles 1 are the mixed cell cancer vaccine 1; the mixed cells activated with microparticles 2 are mixed cell cancer vaccine 2.

100mg of microparticles 1 were co-incubated with BMDC (1000 ten thousand) in 5mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove particles from the system, and the activated DCs were then used as cancer vaccine 3.

BMDC (500 ten thousand) and BMDM (500 ten thousand) were mixed in a quantitative ratio of 1:1 and then co-incubated with 100mg microparticles in 5mL RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, activated DCs and macrophages were centrifuged at 400g for 4 min to remove free particles from the system, and the activated mixed cells were used as cancer vaccine 4.

(5) Vaccine for treating cancer

Preparation of melanoma-bearing mice by selecting 6-8 week female C57BL/6 as model mice, and administering to each mouse on day 0Subcutaneous inoculation below 1.5X10 ⁵ B16F10 cells. On day 3, 6, 9, 14 and 20 mice were subcutaneously vaccinated with 100 ten thousand corresponding mixed cell cancer vaccines (vaccine 1, or vaccine 2, or vaccine 3, or vaccine 4, or vaccine 5) or 100 μl PBS, respectively. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.

(6) Experimental results

As shown in fig. 5, the PBS group mice in fig. 5 quickly grew in tumor volume and died quickly. Mice using Vaccine 1 (Vaccine 1), vaccine 2 (Vaccine 2), vaccine 3 (Vaccine 3) and Vaccine 4 (Vaccine 4) all had significantly slower tumor growth rates and significantly longer survival, with some mice recovering from tumor-free. Wherein, the effect of the vaccine 1 is better than that of the vaccine 2, the vaccine 3 and the vaccine 4. Demonstrating that using activated mixed cell cancer vaccines is better than using activated DCs alone as vaccines; and the vaccine prepared by using the DC and B cell mixed antigen presenting cells has obviously better effect than the vaccine prepared by using DC and macrophages as mixed cell vaccines. Furthermore, the simultaneous loading of tumor tissue lysate fraction and cancer specific and related antigenic polypeptides is better than the use of only the antigenic polypeptide fraction.

Example 4 vaccine for treatment of melanoma

(1) Preparation of antigenic components

The cultured B16F10 cells were collected, and after discarding the supernatant, a proper amount of ultrapure water was added to the cancer cell pellet for resuspension and repeated freeze thawing 5 times with sonication to lyse the cells. Then adding 0.02mg/mL nuclease to the lysate for 10 min, and then heating at 95℃for 5 min to inactivate the nuclease. Centrifuging the lysate at 5000g for 5 min and collecting supernatant to obtain water soluble component soluble in pure water; adding 10% sodium deoxycholate aqueous solution into the obtained precipitation part to dissolve the precipitation part, so as to convert insoluble water-insoluble antigen into soluble antigen in 10% sodium deoxycholate aqueous solution.

Dissolving water soluble component in the lysate, water insoluble component in 10% sodium deoxycholate aqueous solution, and cancer related antigen WT1 _126-134 Cancer-associated antigen WT1 _35-52 And mRNA encoding four cancer specific antigens (B16-M20, B16-M24, B16-M46 and TRP 2:180-188) are mixed according to the mass ratio of 10:1:22:22:22, namely the antigen component 1 for preparing the nanoparticle 1.

Or directly binding cancer-associated antigen WT1 _126-134 Cancer-associated antigen WT1 _35-52 And mRNA encoding four cancer-specific antigens (B16-M20, B16-M24, B16-M46 and TRP 2:180-188) simultaneously were mixed in a mass ratio of 22:22:22 as antigen component 2 for the preparation of nanoparticle 2.

(2) Preparation of nanoparticles

In this example, nanoparticle 1 (Nanoparticle 1) was prepared by the multiple emulsion method in the solvent evaporation method. The molecular weight of the PLA nanoparticle preparation material is 20-40 kDa, and the immunological adjuvants used are poly (I: C), cpG SL01 (B class) and CpG SL03 (C class). Preparation method As described above, cell antigen component 1 and adjuvant were loaded inside the nanoparticle, and then 100mg of the nanoparticle was centrifuged at 12000g for 20 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose, and lyophilized for 48 hours. The average particle size of the nanoparticle 1 was about 500nm, and about 50. Mu.g of protein/polypeptide component, 20. Mu.g of mRNA, and 0.2mg of each of poly (I: C), cpG SL01 (B class) and CpG SL03 (C class) were loaded per 1mg of PLA nanoparticle.

The method and procedure for preparing Nanoparticle 2 (Nanoparticle 2) in this example were the same as that of Nanoparticle 1. The average particle size of the nanoparticle 2 is about 500nm, and about 50. Mu.g of polypeptide component, 20. Mu.g of mRNA, and 0.2mg of each of poly (I: C), cpG SL01 (B class) and CpG SL03 (C class) are loaded per 1mg of PLA nanoparticle.

(3) Preparation of bone marrow derived dendritic cells (BMDC) and B cells

BMDC and B cells were prepared as in example 1.

(4) Activation of antigen presenting cells

BMDC (500 ten thousand) and B cells (1000 ten thousand) were mixed in a quantitative ratio of 1:2 and then co-incubated with 10mg of nanoparticle 1 or nanoparticle 2 in 5mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove particles from the system, and the activated mixture was then removedHeterocytes are used as cancer vaccines. Wherein, the mixed cells activated by the nanoparticle 1 are the mixed cell cancer vaccine 1; the mixed cells activated using the nanoparticles 2 are mixed cell cancer vaccine 2.

BMDC (500 ten thousand) and B cells (1000 ten thousand) were mixed in a quantitative ratio of 1:2 and co-incubated with 250mg of nanoparticle 1 in 5mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 3.

BMDC (500 ten thousand) and B cells (500 ten thousand) were mixed in a quantitative ratio of 1:2 and co-incubated with 10ng of nanoparticle 1 in 5mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 4.

(5) Vaccine for treating cancer

Preparation of melanoma tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 ⁵ B16F10 cells. On day 3, 6, 9, 14 and 20 before the mice were vaccinated with tumor, 100 μl of 200 ten thousand cancer vaccines (vaccine 1, or vaccine 2, or vaccine 3, or vaccine 4) or 100 μl of PBS were subcutaneously vaccinated on the mice, respectively. The mice were monitored for tumor growth rate and mice survival. In the experiment, the size of the tumor volume of the mice was recorded every 3 days starting on day 3.

(6) Experimental results

As shown in FIG. 6, the PBS group mice in FIG. 6 have tumor volumes that rapidly grow and mice die quickly, and the mice treated with the vaccine are effective. Furthermore, vaccine 1 (Vaccine 1) is better than Vaccine 2 (Vaccine 2), vaccine 3 (Vaccine 3) and Vaccine 4 (Vaccine 4). This illustrates: the use of whole cell component antigens plus synthetic polypeptides and mRNAs is better than the use of synthetic polypeptides and mRNAs alone, and the use of nanoparticles at appropriate concentrations to deactivate mixed antigen presenting cells is better than the use of nanoparticles at too low or too high concentrations.

Example 5 vaccine for treatment of lung cancer

(1) Preparation of antigenic components

After collecting the cultured cancer cells, the supernatant was discarded, and then an appropriate amount of ultrapure water was added to the cell pellet to resuspend the cancer cells, followed by repeated freeze thawing for 5 times to lyse the cancer cells. Centrifuging the lysate at 5000g for 5 min, and collecting supernatant as water soluble component soluble in pure water; adding 0.1% octyl glucoside aqueous solution into the obtained precipitation part to dissolve the precipitation part, so as to convert the insoluble antigen insoluble in pure water into the soluble antigen in 0.1% octyl glucoside aqueous solution. And mixing the water-insoluble component and the water-soluble component in the lysate dissolved in the 0.1% octyl glucoside aqueous solution according to the mass ratio of 10:1 to obtain the antigen component for preparing the nano particles.

(2) Preparation of nanoparticles

In this example, nanoparticle 1 (Nanoparticle 1) was prepared by the multiple emulsion method in the solvent evaporation method. In the preparation process, the adopted nanoparticle preparation material PLGA has the molecular weight of 7KDa-17KDa, and the immunological adjuvants used are poly (I: C), cpG ODN 1018 (B class) and CpG ODN M362 (C class). Preparation method As described above, the cell antigen component and the adjuvant were loaded inside the nanoparticle, and then 100mg of the nanoparticle was centrifuged at 12000g for 20 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose, followed by lyophilization for 48 hours. The average particle size of the nanoparticle is about 280nm, and about 200 mug protein and polypeptide components are loaded per 1mg of PLGA nanoparticle, and 0.02mg of poly (I: C), cpG ODN 1018 (B class) and CpG ODN M362 (C class) are loaded.

The Nanoparticle 2 (Nanoparticle 2) is prepared from the same material and preparation method as the Nanoparticle 1, wherein the average particle diameter of the Nanoparticle 2 is about 280nm, and 1mg of PLGA Nanoparticle is loaded with 0.02mg of poly (I: C), cpG ODN 1018 (B class) and CpG ODN M362 (C class) respectively, and no antigen component is loaded.

(3) Preparation and activation of antigen presenting cells

Bone marrow derived dendritic cells (BMDCs) and B cells were used as antigen presenting cells. Preparation of BMDC and B cells was as in example 1. BMDC and B cells were mixed in different quantitative ratios and used as mixed antigen presenting cells.

Nanoparticle 1 (500. Mu.g) or nanoparticle 2+ equivalent amounts of the free lysate were co-incubated with 2000 ten thousand mixed antigen presenting cells (1000 ten thousand BMDC+1000 ten thousand B cells) in 10mL of RPM 1640 complete medium for 12 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained the cytokine IL-15 (20 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove particles from the system, and the activated mixed cells were used as cancer vaccines. Wherein, the mixed antigen presenting cell activated by the nanoparticle 1 is used as the vaccine 1; mixed antigen presenting cells activated using nanoparticle 2 are vaccine 2.

Nanoparticle 1 (500. Mu.g) was co-incubated with 2000 ten thousand mixed antigen presenting cells (50 ten thousand BMDC+1950 ten thousand B cells) in 10mL of RPM 1640 complete medium for 12 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained the cytokine IL-15 (20 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 3.

Nanoparticle 1 (500. Mu.g) was co-incubated with 2000 ten thousand mixed antigen presenting cells (1900 ten thousand BMDC+100 ten thousand B cells) in 10mL of RPM 1640 complete medium for 12 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained the cytokine IL-15 (20 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 4.

Nanoparticle 1 (500. Mu.g) was co-incubated with 2000 ten thousand mixed antigen presenting cells (1000 ten thousand BMDC+1000 ten thousand B cells) in 10mL of RPM 1640 complete medium for 12 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the Flt3L (100 ng/mL) was included in the incubation system. After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 5.

Nanoparticle 1 (500. Mu.g) was co-incubated with 2000 ten thousand mixed antigen presenting cells (1000 ten thousand BMDC+1000 ten thousand B cells) in 10mL of RPM 1640 complete medium for 12 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained 2-BP (50. Mu.M).After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 6.

Nanoparticle 1 (500. Mu.g) was co-incubated with 2000 ten thousand mixed antigen presenting cells (1000 ten thousand BMDC+1000 ten thousand B cells) in 10mL of RPM 1640 complete medium for 12 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system does not contain other cytokines or substances assisting activation. After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 7.

(7) Vaccine for the treatment of cancer

Preparation of lung cancer tumor-bearing mice by selecting 6-8 week female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 ⁶ And LLC lung cancer cells. On day 3, 6, 9, 14 and 20 mice were subcutaneously vaccinated with 100 μl of 200 μl of mixed cell vaccine (vaccine 1, or vaccine 2, or vaccine 3, or vaccine 4, or vaccine 5, or vaccine 6 or vaccine 7) or 100 μl of PBS, respectively. The mice were monitored for tumor growth rate and mice survival. In the experiment, the size of the tumor volume of the mice was recorded every 3 days starting on day 3.

(8) Experimental results

As shown in fig. 7, the PBS control mice had very fast tumor growth and very short survival. The tumor growth rate of the mice in the vaccine group is obviously slowed down or the tumor disappears. Among them, vaccine 1 is significantly better than other vaccines, which suggests that co-incubation of DC and B cells after mixing them in appropriate proportions is better and that nanoparticle activation with cancer antigen loading is better than particle with adjuvant + free lysate. Moreover, the addition of IL-15 during co-incubation of the nanoparticle with the mixed antigen presenting cells can significantly enhance the activation effect.

In this example, interleukin-15 (IL-15) is used as the substance for activating the helper antigen presenting cells, and other substances for activating the helper antigen presenting cells, such as GM-CSF, IL-7, IL-15, vincristine, IL-21, IL-12, IL-2, IL-1, M-CSF, TNF- α, IL-6, PGE2, or a combination of various substances, may be used in practical applications.

Example 6 vaccine for treatment of colorectal cancer

In this embodiment, the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells are loaded on the surface of the nanoparticle, and in practical application, the extracellular vesicles of bacteria and the extracellular vesicle components of cancer cells can be loaded inside the nano vaccine according to the requirement; or simultaneously loaded on the surface of the nano vaccine and the inside of the nano vaccine. In the present example, bacterial extracellular vesicles and cancer cell extracellular vesicles are used, and in practical applications, bacterial outer membrane components or cancer cell membrane components may be used; alternatively, any mixed membrane fraction of a membrane fraction of an antigen-presenting cell (cell membrane or extracellular vesicle membrane) loaded with a cancer cell antigen and a membrane fraction derived from a cancer cell and/or a membrane fraction derived from a bacterium may be used.

(1) Preparation of antigenic components

After collecting cultured MC38 mouse colon cancer cells, the supernatant was discarded and the cells were resuspended in a suitable amount of ultrapure water and then lysed by repeated freeze thawing 5 times. Centrifuging the lysate at 5000g for 5 min, and collecting supernatant as water soluble component soluble in pure water; adding 6M guanidine hydrochloride water solution into the obtained precipitation part to dissolve the precipitation part, so as to convert insoluble water insoluble antigen into 6M guanidine hydrochloride water solution. Dissolving the above 6M guanidine hydrochloride aqueous solution in water insoluble component, water soluble component in lysate, and cancer related antigen WT1 _235-243 IO102 (CYTWNQMNL DTLLKALLEIASCLEKALQVF) and cancer-associated antigen WT1 _294-312 IO103 (FRGIQDVRRVSGVAPTLVR-FMTYWHLLNAFTVTVPKDL) is mixed according to the mass ratio of 1:1:1:0.1 to obtain the antigen component 1 for preparing the nano particles. Wherein the cancer-associated antigen WT1 _235-243 Sequence of-IO 102 is WT1 _294-312 And IO103 two cancer related antigen sequences, WT1 _294-312 IO103 by WT1 _294-312 And IO103, and does not contain other redundant amino acid sequences.

(2) Preparation of nanoparticles

In the embodiment, the PLGA molecular weight of the Nanoparticle 1 (Nanoparticle 1) preparation material is 7kDa-17kDa, and the immunological adjuvants adopted are poly (I: C), cpG ODN 7909 (B class) and CpG ODN 2395 (C class). Preparation method As described above, antigen component 1 and adjuvant were loaded into the nanoparticle by multiple emulsion method, and 100mg of the nanoparticle was centrifuged at 12000g for 20 minutes, resuspended in 10mL of ultra-pure water containing 4% trehalose, and lyophilized for 48 hours. The average particle size of the nanoparticle 1 is about 380nm, and about 950 mug of protein or polypeptide components of cancer cells are loaded per 1mg of PLGA nanoparticle, and 0.05mg of each of poly (I: C), cpG ODN 7909 (B class) and CpG ODN 2395 (C class) are loaded.

The Nanoparticle 2 (Nanoparticle 2) is prepared from the same material and method as the Nanoparticle 1, and the average particle size of the Nanoparticle 2 is about 380nm, and each 1mg of PLGA Nanoparticle is loaded with about 950 μg of protein or polypeptide component, only the antigen component 1 is loaded without any adjuvant.

(3) Preparation of bacterial extracellular vesicles (OMVs) and cancer extracellular vesicles

The bifidobacterium longum is centrifuged at 5000g for 30 minutes, the sediment is removed, the supernatant is collected, the supernatant is filtered by a 1 mu m filter membrane, the supernatant is treated by 20W ultrasonic waves for 5 minutes at 4 ℃, and then the sediment is centrifuged at 16000g for 90 minutes, and the sediment is resuspended in PBS to obtain the collected bacterial outer vesicle membrane component.

Alternatively, cancer cells were centrifuged at 5000g for 30 minutes, then the pellet was discarded, the supernatant was collected, filtered with a 1 μm filter membrane, sonicated at 4℃for 5 minutes with 20W, and then centrifuged at 16000g for 90 minutes, and the pellet was resuspended in PBS to obtain the outer vesicle membrane fraction of the collected cancer cells.

And mixing the bacterial outer vesicle membrane component and the cancer cell outer vesicle membrane component according to the mass ratio of 1:1 for later use.

(4) Preparation of nanoparticle internally loaded with antigen component and simultaneously surface-loaded with bacterial and cancer extracellular vesicle membrane component

Mixing 30mg of nano particles (nano particles 1 or nano particles 2) prepared in the step (2) with 5mg of mixed membrane components of cancer cells and bacterial outer vesicles prepared in the step (3), standing for 1 minute, repeatedly co-extruding through a filter membrane with the pore diameter of 0.45 mu m after using low-power 7.5W ultrasound for 1 minute at the temperature of 4 ℃, collecting the obtained filtrate, and freeze-drying to obtain the nano particles. Wherein, the Nanoparticle 3 (Nanoparticle 3) is prepared by using the Nanoparticle 1 to co-act with the mixed membrane component of the cancer cells and the bacterial outer vesicles, and the particle size is 400nm; about 950. Mu.g of protein or polypeptide component, 0.05mg each of poly (I: C), cpG ODN 7909 (class B) and CpG ODN 2395 (class C) were loaded per 1mg PLGA nanoparticle, and 20. Mu.g of membrane component were loaded. The Nanoparticle 4 (Nanoparticle 4) is prepared by using the Nanoparticle 2 to co-act with the mixed membrane component of the cancer cells and the bacterial outer vesicles, and the particle size is 400nm; about 950. Mu.g of protein or polypeptide component was loaded per 1mg of PLGA nanoparticles, without any adjuvant, with 20. Mu.g of membrane component.

(5) Preparation and activation of antigen presenting cells

Bone marrow-derived dendritic cells (BMDCs), bone marrow-derived macrophages (BMDMs), and B cells were used as antigen presenting cells. BMDC, BMDM and B cells were prepared as described above.

Nanoparticle 3 (20 mg) or nanoparticle 4 (20 mg) were co-incubated with 2000 ten thousand mixed antigen presenting cells (DC, B cells and macrophages in a 5:1:4 quantitative ratio) in 10mL of RPM 1640 complete medium for 12 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contains cytokines IL-1 beta (10 ng/mL), IL-6 (10 ng/mL) and PGE ₂ (10 ng/mL) and TNF- α (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove particles from the system, and the activated mixed cells were used as cancer vaccines. Wherein, the mixed antigen presenting cell activated by the nanoparticle 3 is used as the vaccine 1; the mixed antigen presenting cells activated using the nanoparticles 4 are vaccine 2.

(6) Vaccine for the treatment of cancer

Preparation of colon cancer tumor-bearing mice by selecting 6-8 week female C57BL/6 as model mice, and inoculating 1.0X10 s under armpit of each mouse on day 0 ⁶ Individual MC38 colon cancer cells. On day 3, 6, 9, 14 and 20, mice were subcutaneously vaccinated with 100 μl of 200 ten thousand mixed cell vaccines (vaccine 1, or vaccine 2) or 100 μ, respectivelyPBS of L. The mice were monitored for tumor growth rate and mice survival. In the experiment, the size of the tumor volume of the mice was recorded every 3 days starting on day 3.

(7) Experimental results

The results showed that the tumor growth rate was fast and the survival time was short in PBS control mice, as shown in fig. 8. The tumor growth rate of the mice treated by the nano vaccine is obviously slowed down, and part of the tumors of the mice disappear and heal. Furthermore, vaccine 1 is better than vaccine 2 and vaccine 3.

In this embodiment, the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells are loaded on the surface of the nanoparticle, and in practical application, the extracellular vesicles of bacteria and the extracellular vesicle components of cancer cells can be loaded inside the nanoparticle according to the requirement; or both on the nanoparticle surface and within the nanoparticle. The bacterial extracellular vesicles used in the present example and the cancer cell extracellular vesicles may be used as an outer membrane component of the bacterium or a membrane component of the cancer cell in practical applications; alternatively, any mixed membrane fraction of a membrane fraction of an antigen-presenting cell (cell membrane or extracellular vesicle membrane) loaded with a cancer cell antigen and a membrane fraction derived from a cancer cell and/or a membrane fraction derived from a bacterium may be used.

In this embodiment, the membrane component is loaded onto the nanoparticle surface by ultrasonic, co-incubation and co-extrusion methods, and in practical applications, any method that can load the membrane component onto the nanoparticle or microparticle surface by co-action may be used, including but not limited to one or more of ultrasonic, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bonding, stirring, dialysis, homogenization and homogenization.

In this embodiment, the membrane component of the bacteria and cancer cells is located on the surface of the nanoparticle or microparticle, and in practical application, the membrane component of the bacteria or cancer cells may be supported inside the nanoparticle or microparticle.

In summary, the vaccine of the present disclosure has excellent therapeutic effects on cancer.

Example 7 vaccine for treatment of melanoma

(1) Lysis of tumor tissue and collection of fractions

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, growing to a volume of about 1000mm in a tumor ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is prepared into single cell suspension, then CD 45-cancer cells are separated out and cultured for 3 days, the cultured cancer cells are collected by centrifugation, and then a proper amount of ultrapure water is added and the cells are repeatedly frozen and thawed for 5 times to lyse the cells. Centrifuging the lysate at 5000g for 5 min and collecting supernatant to obtain water soluble component soluble in pure water; the water insoluble component insoluble in pure water can be converted into soluble in 0.3M semicarbazide hydrochloride aqueous solution by adding 0.3M semicarbazide hydrochloride aqueous solution to the obtained precipitation part to dissolve the precipitation part. Adding saturated sodium chloride aqueous solution dropwise into water-soluble components in the lysate, centrifuging the obtained sample at 3000g for 5 minutes after complete precipitation, dissolving the precipitate in 0.3M semicarbazide hydrochloride aqueous solution for later use, heating supernatant at 100 ℃ for 5 minutes, centrifuging the obtained sample at 3000g for 5 minutes, discarding the supernatant, and dissolving the precipitate in 0.3M semicarbazide hydrochloride aqueous solution; the salted-out precipitate dissolved using 0.3M semicarbazide hydrochloride and the heated precipitate dissolved using 0.3M semicarbazide hydrochloride are then combined and used as a part of the water-soluble components. The non-water-soluble component in the lysate dissolved by the 0.3M semicarbazide hydrochloride aqueous solution and the component obtained by salting out, heating and precipitating the water-soluble component dissolved by the 0.3M semicarbazide hydrochloride aqueous solution are the antigen component 1 for preparing the cancer nano-particles 1.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, in which the tumor growth to volume was about 1000mm each ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is prepared into single cell suspension, then CD 45-cancer cells are separated out and cultured for 3 days, the cultured cancer cells are collected by centrifugation, and then a proper amount of ultrapure water is added and the cells are repeatedly frozen and thawed for 5 times to lyse the cells. Centrifuging the lysate at 5000g for 5 min and collecting supernatant to obtain water soluble component soluble in pure water; adding 0.3M semicarbazide hydrochloride aqueous solution to the obtained precipitation part for dissolvingThe water insoluble component insoluble in pure water can be converted into a water soluble component in 0.3M semicarbazide hydrochloride aqueous solution by the precipitation section. A saturated aqueous sodium chloride solution was added dropwise to the water-soluble component in the lysate, and after completion of precipitation, the obtained sample was centrifuged at 3000g for 5 minutes, and the precipitate was dissolved in a 0.3M aqueous semicarbazide hydrochloride solution to be used as a part of the water-soluble component. The components obtained by salting out and precipitating the water-insoluble component in the lysate dissolved by the 0.3M semicarbazide hydrochloride aqueous solution and the water-soluble component dissolved by the 0.3M semicarbazide hydrochloride aqueous solution are the antigen component 2 for preparing the cancer nano-particles 2.

Subcutaneous inoculation of the back of each C57BL/6 mouse with 1.5X10 ⁵ B16F10 cells, in which the tumor growth to volume was about 1000mm each ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is prepared into single cell suspension, then CD 45-cancer cells are separated out and cultured for 3 days, the cultured cancer cells are collected by centrifugation, and then a proper amount of ultrapure water is added and the cells are repeatedly frozen and thawed for 5 times to lyse the cells. Centrifuging the lysate at 5000g for 5 min and collecting supernatant to obtain water soluble component soluble in pure water; the water insoluble component insoluble in pure water can be converted into soluble in 0.3M semicarbazide hydrochloride aqueous solution by adding 0.3M semicarbazide hydrochloride aqueous solution to the obtained precipitation part to dissolve the precipitation part. The water-soluble component in the lysate is heated at 100 ℃ for 5 minutes, the obtained sample is centrifuged at 3000g for 5 minutes, the supernatant is removed, and the precipitate is dissolved in 0.3M semicarbazide hydrochloride aqueous solution, namely, a part of the water-soluble component. The components obtained by heating and precipitating the water-insoluble component in the lysate dissolved by the 0.3M semicarbazide hydrochloride aqueous solution and the water-soluble component dissolved by the 0.3M semicarbazide hydrochloride aqueous solution are the antigen component 3 for preparing the cancer nano-particles 3.

(2) Preparation of nanoparticles

In this example, nanoparticle 1 (Nanoparticle 1) was prepared by the multiple emulsion method in the solvent evaporation method. Part of the water-soluble components in the lysate are dissolved by using 0.3M semicarbazide hydrochloride aqueous solution after salting-out purification and heating separation purification. Nanoparticles supporting a part of components in the water-soluble components in the lysate (components dissolved in 0.3M semicarbazide hydrochloride aqueous solution after salting out and heating separation and purification) and nanoparticles supporting the non-water-soluble components in the lysate (dissolved in 0.3M semicarbazide hydrochloride aqueous solution) are prepared separately and then used together as nanoparticle 1 when used. The molecular weight of PLGA used for preparing antigen delivery nano particles is 20kDa-40kDa, and the immunological adjuvant used is poly (I: C). Preparation method As described above, antigen component 1 and adjuvant were loaded inside the nanoparticle, and then 300mg of the nanoparticle was centrifuged at 10000g for 20 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose, followed by lyophilization for 48 hours. The average particle diameter of the nanoparticle 1 is about 300nm, and about 550 mug protein or polypeptide component is loaded per 1mg PLGA nanoparticle, and 0.01mg poly (I: C) is loaded.

In this example, nanoparticle 2 (Nanoparticle 2) was prepared by the multiple emulsion method in the solvent evaporation method. Part of the water-soluble components in the lysate are dissolved by using 0.3M semicarbazide hydrochloride aqueous solution after salting-out and purification. In the preparation, nanoparticles carrying a part of the water-soluble component in the lysate (component dissolved in 0.3M semicarbazide hydrochloride aqueous solution after salting-out separation and purification) and nanoparticles carrying the water-insoluble component in the lysate (0.3M semicarbazide hydrochloride aqueous solution dissolution) are prepared separately and then used together as nanoparticle 2 in use. The molecular weight of the nanoparticle preparation material PLGA is 20kDa-40kDa, and the immunological adjuvant is poly (I: C). Preparation method As described above, antigen component 2 and adjuvant were loaded inside the nanoparticle, and then 300mg of the nanoparticle was centrifuged at 10000g for 20 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose, followed by lyophilization for 48 hours. The average particle diameter of the nanoparticle 2 is about 300nm, and about 550 mug protein or polypeptide component is loaded per 1mg PLGA nanoparticle, and 0.01mg poly (I: C) is loaded.

In this example, nanoparticle 3 (Nanoparticle 3) was prepared by the multiple emulsion method in the solvent evaporation method. Part of the water-soluble components in the lysate are dissolved by using 0.3M semicarbazide hydrochloride aqueous solution after heating, separating and purifying. In the preparation, nanoparticles supporting a part of the water-soluble component in the lysate (component dissolved in 0.3M aqueous semicarbazide hydrochloride solution after separation and purification by heating) and nanoparticles supporting the water-insoluble component in the lysate (dissolved in 0.3M aqueous semicarbazide hydrochloride solution) were prepared separately and then used together as nanoparticles 3 in the use. The molecular weight of the nanoparticle preparation material PLGA is 20kDa-40kDa, and the immunological adjuvant is poly (I: C). Preparation method As described above, antigen component 3 and adjuvant were loaded inside the nanoparticle, and then 300mg of the nanoparticle was centrifuged at 10000g for 20 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose, followed by lyophilization for 48 hours. The average particle diameter of the nanoparticle 3 is about 300nm, about 550 mug of protein or polypeptide component is loaded per 1mg of PLGA nanoparticle, and 0.01mg of poly (I: C) is loaded per 1mg of PLGA nanoparticle.

(3) Preparation and activation of antigen presenting cells

Peripheral blood derived DC and B cells were used as antigen presenting cells. Mouse peripheral blood was collected after sacrifice of mice, and mouse Peripheral Blood Mononuclear Cells (PBMCs) were prepared. Isolation of CD11c from PBMC Using flow cytometry ⁺ DC cells and CD19 of (C) ⁺ The collected DCs and B cells were then used as antigen presenting cells.

Nanoparticle 1 (50. Mu.g of nanoparticle with a part of the water-soluble component+25. Mu.g of nanoparticle with a non-water-soluble component) or nanoparticle 2 (50. Mu.g of nanoparticle with a part of the water-soluble component+25. Mu.g of nanoparticle with a non-water-soluble component) or nanoparticle 3 (50. Mu.g of nanoparticle with a part of the water-soluble component+25. Mu.g of nanoparticle with a non-water-soluble component) were co-incubated with 250 ten thousand mixed antigen presenting cells (DC and B cell number ratio 1:4) in 25mL of RPM complete medium of RPM 1640 for 12 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (50 ng/mL) and GM-CSF (5 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove particles from the system, and the activated mixed cells were used as cancer vaccines. Wherein, the mixed antigen presenting cell activated by the nanoparticle 1 is used as the vaccine 1; mixed antigen presenting cells activated with nanoparticle 2 are vaccine 2; mixed antigen presenting cells activated using nanoparticle 3 are vaccine 3.

(4) Vaccine for treating cancer

Preparation of melanoma tumor-bearing mice by selecting 6-8 weeks female C57BL/6 as model mice, and subcutaneously inoculating 1.5X10 lower right back of each mouse on day 0 ⁵ B16F10 cells. On day 3, 6, 9, 14 and 20, 20 ten thousand vaccine cells (vaccine 1, or vaccine 2, or vaccine 3) or 100 μl PBS were injected subcutaneously in mice, respectively. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.

(5) Experimental results

As shown in FIG. 9, the PBS group mice showed a rapid tumor volume and the mice died rapidly. Mice using Vaccine 1 (Vaccine 1), vaccine 2 (Vaccine 2) and Vaccine 3 (Vaccine 3) all had significantly slower tumor growth rates and some mice had healed without tumors. The effect of the vaccine 1 is better than that of the vaccine 2 and the vaccine 3, which means that the effect of separating and purifying by using salting out and heating at the same time is obviously better than that of separating and purifying by using only salting out or separating and purifying by using only heating.

In this example, protein and polypeptide components in the water-soluble components are separated and purified by salting out, and in practical application, protein and polypeptide components in the water-insoluble components may be purified by salting out and/or heating or other suitable methods, or protein and polypeptide components in all lysates may be purified by salting out and/or heating after lysis and lysis of cancer cells/tumor tissue with a lytic agent.

In this embodiment, protein and polypeptide components in the water-soluble component are separated and purified by salting out, and in practical application, mRNA components can be purified simultaneously after protein and polypeptide components in the water-soluble component are purified, and mixed for use; in practical application, protein and polypeptide components in the water-insoluble components can be purified, and mRNA components can be purified at the same time and used after being mixed; alternatively, the protein and polypeptide components of all lysates may be purified by using methods such as salting out and/or heating after lysis and lysis of cancer cells with a lytic agent, and the mRNA components may be purified simultaneously and mixed for use.

Example 8 vaccine for the treatment of breast cancer

(1) Preparation of antigenic components

Cultured 4T1 breast cancer cell lines were collected and centrifuged at 350g for 5 minutes, the supernatant was discarded and washed twice with PBS, then the cells were resuspended in ultrapure water containing 0.1% protease inhibitor, and repeatedly freeze-thawed 5 times to lyse the cancer cells, and then the lysate fraction was dissolved using a 0.1M aqueous solution of metformin sulfate. Adding 3% hydrogen peroxide into the lysate component and allowing the mixture to act for 10 minutes, then dropwise adding a saturated ammonium sulfate aqueous solution into the sample, centrifuging the sample at 10000g for 25 minutes, discarding the supernatant, and then secondarily dissolving the precipitate into a 0.1M metformin sulfate aqueous solution to obtain the antigen component 1 for preparing antigen delivery microparticles.

(2) Preparation of microparticles

In this example, microparticle 1 (Micronpartcle 1) was prepared by the multiple emulsion method. The PLGA molecular weight of the material prepared by the microparticles 1 is 24-38 kDa, and the immunological adjuvants used are CpG ODN 7909, cpG ODN 2216 and Poly ICLC. Poly ICLC is a Toll-like receptor 3 agonist, whereas classes of CpG are Toll-like receptor 9 agonists, with Toll-like receptor 3 and Toll-like receptor 9 both located in endocytosomal membrane structures within the cell. Firstly, the antigen component 1 and an immune adjuvant are loaded in the microparticles together, and then are centrifuged at 10000g for 15 minutes, and are re-suspended by 10mL of ultrapure water containing 4% trehalose and then are freeze-dried for 48 hours; the particles were resuspended in 7mL PBS before use and then 3mL of antigen component (protein concentration 50 mg/mL) was added and allowed to react at room temperature for 10min to give microparticles 1 loaded with antigen component both internally and externally. The average particle diameter of the microparticles is about 2.50 μm; about 450 μg of protein or polypeptide component was loaded per 1mg of PLGA microparticles, 0.03mg each of loaded CpG 7909 (class B), cpG2216 (class A) and Poly ICLC.

The preparation material and the preparation method of the micron particles 2 (micron particles 2) are the same, and the loaded immunoadjuvants are CpG2336 (A class), cpG2216 (A class) and Poly ICLC. The particle size of the microparticles 2 is about 2.50 μm, about 450 μg of protein or polypeptide component is loaded per 1mg of PLGA microparticles, and each of CpG2336 (A class), cpG2216 (A class) and Poly ICLC immunoadjuvant loaded per 1mg of PLGA microparticles is 0.03mg.

The preparation material and the preparation method of the micron particles 3 (micron particles 3) are the same, and the loaded immunoadjuvants are CpG 7909 (B class) and CpG 2216 (A class). The particle size of the micrometer vaccine 3 is about 2.50 mu m per 1mgPLGA microparticle, about 450 mu g of protein or polypeptide component is loaded per 1mg of PLGA microparticle, and 0.045mg of CpG 7909 (B class) and CpG 2216 (A class) are loaded per 1mgPLGA microparticle.

(3) Preparation and activation of antigen presenting cells

BMDC and spleen-derived B cells were used as antigen presenting cells, and the preparation method was as described above.

2mg of microparticles (microparticles 1, or microparticles 2 or microparticles 3) were co-incubated with 100 ten thousand mixed antigen presenting cells (DC to B cell number ratio 1:19) in 5mL of RPM 1640 complete medium for 12 hours (37 ℃,5% CO ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (20 ng/mL) and GM-CSF (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove particles from the system, and the activated mixed cells were used as cancer vaccines. Wherein, the mixed antigen presenting cell activated by the microparticle 1 is the vaccine 1; using the mixed antigen presenting cells activated by the microparticles 2 as vaccine 2; mixed antigen presenting cells activated using microparticles 3 are vaccine 3.

(4) Vaccine for treating cancer

Female BALB/C of 6-8 weeks is selected as a model mouse to prepare a breast cancer tumor-bearing mouse. Each mouse was subcutaneously vaccinated 1.0X10% on day 0 under the back right ⁶ LLC cells. On day 3, 6, 9, 14 and 20, 20 ten thousand vaccine cells (vaccine 1, or vaccine 2, or vaccine 3) or 100 μl PBS were injected subcutaneously in mice, respectively. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.

(5) Experimental results

As shown in FIG. 10, the PBS group mice showed a rapid tumor volume and the mice died rapidly. Mice using Vaccine 1 (Vaccine 1), vaccine 2 (Vaccine 2) and Vaccine 3 (Vaccine 3) all had significantly slower tumor growth rates and significantly longer survival, with some mice recovering from tumor-free. Furthermore, the effect of the vaccine prepared by the micron particle activated mixed cells loaded with the CpG adjuvant and the Poly ICLC mixed adjuvant is better than that of the vaccine prepared by the micron particle activated mixed cells loaded with the two CpG mixed adjuvants. In addition, the effect of the vaccine prepared by the micron particle activated mixed cells loaded with the B class CpG, the A class CpG and the Poly ICLC mixed adjuvant is better than that of the vaccine prepared by the micron particle activated mixed cells loaded with the two A class CpG and the Poly ICLC mixed adjuvants. This demonstrates that the effect of the vaccine prepared by the microparticle-activated mixed cells loaded with the mixed adjuvant of two different Toll-like receptors is better, and the effect of the vaccine prepared by the microparticle-activated mixed cells containing the mixed CpG of the B class CpG and the Toll-like receptor 3 agonist as the mixed adjuvant is better.

In this embodiment, hydrogen peroxide (hydrogen peroxide) is used as the oxidizing agent to oxidize the antigen component, and any oxidizing agent capable of oxidizing the antigen component of the protein and the polypeptide, such as hypochlorous acid, may be used in practical applications.

Example 9 vaccine for prevention of cancer

(1) Preparation of microparticles

Antigen delivery microparticles were prepared using mRNA encoding two cancer-associated antigens (IO 102: DTLLKALLEIASCLEKALQVF and IO103: FMTYWHLLNAFTVTVPKDL) simultaneously with 4 tumor-specific antigens B16-M20 (Tubb 3), B16-M24 (Dag 1), B16-M46 (Actn 4) and TRP 2:180-188. Mixing 4 tumor specific antigens B16-M20 (Tubb 3), B16-M24 (Dag), B16-M46 (Actn 4) and TRP2:180-188 in a mass ratio of 1:1:1:1, reacting with hypochlorous acid for 2 minutes to oxidize polypeptide antigens, dissolving the 4 oxidized novel antigen polypeptides by using 0.1M potassium guanidinosuccinate aqueous solution respectively, and mixing with mRNA encoding the antigen in a mass ratio of 1:1:1:1:20 to obtain the antigen component 1.

In this example, microparticles 1 (Micronpartcle 1) were prepared by the solvent evaporation method. The PLGA molecular weight of the preparation material of the microparticles 1 is 38KDa-54KDa, the immunoadjuvant is Poly (I: C), cpG2006 (B class) and CpGSL01 (B class), and the loaded substances for increasing lysosome escape are RALA polypeptides (WEARLARALARALARHLARALARALRACEA). The preparation method is as described above, the antigen component 1, the adjuvant and the RALA polypeptide are loaded in the microparticles, 100mg of the microparticles are centrifuged at 10000g for 15 minutes, and 10mL of ultra-pure water containing 4% trehalose is used for resuspension and then freeze drying is carried out for 48 hours, thus obtaining the freeze-dried powder for standby. The average particle diameter of the microparticles 1 is about 5.0 mu m, each 1mg of PLGA microparticles is loaded with about 8 mu g of antigen polypeptide component and 40 mu g of antigen mRNA component, and each 1mg of PLGA microparticles is loaded with 0.02mg of Poly (I: C), cpG2006, cpGSL01 and RALA polypeptide.

The preparation material and the preparation method of the micron particle 2 (micron particle 2) are the same as that of the micron particle 1, the particle size is about 5.0 mu m, the same amount of antigen component 1 is loaded, and each 1mg of PLGA is loaded with 0.02mg of Poly (I: C), cpG1585 (A class), cpG2216 (A class) and RALA polypeptide.

The preparation material and the preparation method of the micron particle 3 (micron particle 3) are the same as that of the micron particle 1, the particle size is about 5.0 mu m, the equivalent amount of antigen component 1 is loaded, and each 1mg of PLGA is loaded with 0.06mg of Poly (I: C) and 0.02mg of RALA polypeptide.

(2) Activation of antigen presenting cells

BMDC and spleen-derived B cells were used as antigen presenting cells and were prepared as in example 1.

50mg of microparticles (microparticles 1, or microparticles 2, or microparticles 3) were co-incubated with 1200 ten thousand mixed antigen presenting cells (DC to B cell number ratio 2:1) in 25mL of RPM 1640 complete medium for 18 hours (37 ℃,5% CO ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (5 ng/mL), IL-21 (30 ng/mL) and GM-CSF (20 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove particles from the system, and the activated mixed cells were used as cancer vaccines. Wherein, the mixed antigen presenting cell activated by the microparticle 1 is the vaccine 1; using the mixed antigen presenting cells activated by the microparticles 2 as vaccine 2; mixed antigen presenting cells activated using microparticles 3 are vaccine 3.

(3) Vaccine for prevention of cancer

Female C57BL/6 of 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice, and 300 ten thousand cell vaccines (vaccine 1, or vaccine 2, or vaccine 3), or 100. Mu.L PBS were injected subcutaneously into the mice on days-35, day-28, day-21, and day-14, respectively, before the mice were vaccinated with tumors. Day 0, 1.5X10 s were subcutaneously inoculated on the lower right back of each mouse ⁵ B16F10 cells. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.

(4) Experimental results

The results showed that the tumors of the PBS control mice grew very fast, whereas the tumor growth rate of the vaccine treated mice was significantly slower, and the tumors disappeared after the inoculation of the cancer cells of the majority of mice, as shown in fig. 11. Vaccine 1 works best. This demonstrates that the effect of the microparticle-activated mixed antigen presenting cell vaccine loaded with two classes of B CpG and Poly (I: C) as mixed adjuvants is better than the microparticle-activated mixed antigen presenting cell vaccine loaded with two classes of A CpG and Poly (I: C) as mixed adjuvants and the microparticle-activated mixed antigen presenting cell vaccine loaded with Poly (I: C) alone as adjuvants.

Example 10 vaccine for treatment of T lymphoma

(1) Preparation of antigenic components

The cultured E.G7-OVA cell lines were collected and centrifuged at 350g for 5 minutes, the supernatant was discarded and washed twice with PBS, and the cells were resuspended in ultrapure water and freeze-thawed 5 times repeatedly with sonication. After cell lysis, trypsin (Trypsin, 0.5 mg/mL) and Chymotrypsin (Chymotorypsin, 0.5 mg/mL) are added for co-incubation for 15 minutes to carry out enzymolysis on lysate components, then heating is carried out for 10 minutes at 75 ℃, then the lysate is centrifuged for 5 minutes at 10000g, and supernatant is taken to obtain water-soluble components which are soluble in pure water; adding 0.2M methyl guanidine hydrochloride and 0.1M arginine water solution into the obtained precipitation part to dissolve the precipitation part, so as to convert the insoluble components insoluble in pure water into the soluble components in 0.2M methyl guanidine hydrochloride and 0.1M arginine water solution.

Mixing all the water-insoluble components, the water-soluble components, the cancer-related antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF) and the cancer-related antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) in the lysate dissolved by the 0.2M methyl guanidine hydrochloride and the 0.1M arginine water solution according to the mass ratio of 5:1:0.01:0.02, namely the antigen component 1.

(2) Lysis and lysis of BCG

BCG was collected and cleaved using 0.2M methyl guanidine hydrochloride and 0.1M arginine in water followed by dissolution of the cleaved components of BCG using 0.2M methyl guanidine hydrochloride and 0.1M arginine in water.

(3) Preparation of nanoparticles

And (3) mixing the antigen component 1 with the BCG lysate component dissolved by the 0.2M methyl guanidine hydrochloride and the 0.1M arginine water solution in the step (2) according to the mass ratio of 10:1, namely the antigen component 2.

Nanoparticle 1 (Nanoparticle 1) was prepared by solvent evaporation in this example. The molecular weight of the PLA material prepared by the nano-particle 1 is 20kDa, the antigen component 2 and the immunoadjuvant are loaded in the nano-particle, and the cancer cell lysate component is loaded on the surface. The immunoadjuvant used was poly ICLC. Preparation method As previously described, antigen component 2 and adjuvant were loaded inside the nanoparticle, and then 100mg of the nanoparticle was centrifuged at 11000g for 20 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose and lyophilized for 48 hours. Before use, 20mg of the nanoparticle was resuspended in 0.9mL of PBS and incubated with 0.1mL of antigen component 1 (80 mg/mL) sample at room temperature for 5 minutes. The average particle size of the nano particles 1 is about 400nm, and about 400 mug protein and polypeptide components are loaded per 1mg of PLA nano particles 1, and 0.005mg of Poly ICLC immunoadjuvant is loaded.

(4) Activation of antigen presenting cells

20mg of nanoparticle 1 were co-incubated with 1000 ten thousand mixed antigen presenting cells (DC and B cell number ratio 1:1) in 10mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (10 ng/mL), IL-21 (10 ng/mL) and IL-6 (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 1.

Alternatively, 800mg of nanoparticle 1 was co-incubated with 1000 ten thousand mixed antigen presenting cells (DC to B cell number ratio 1:1) in 10mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (10 ng/mL), IL-21 (10 ng/mL) and IL-6 (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 min to removeThe activated mixed cells are then used as cancer vaccine 2, except for particles in the system.

Alternatively 20ng of nanoparticle 1 were co-incubated with 1000 ten thousand mixed antigen presenting cells (DC to B cell number ratio 1:1) in 10mL of RPM 1640 complete medium for 48 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (10 ng/mL), IL-21 (10 ng/mL) and IL-6 (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 3.

Alternatively, 20mg of nanoparticle 1 was co-incubated with 1000 ten thousand mixed antigen presenting cells (DC to B cell number ratio 1:1) in 10mL of RPM 1640 complete medium for 0.5 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (10 ng/mL), IL-21 (10 ng/mL) and IL-6 (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 4.

Alternatively, 20mg of nanoparticle 1 was co-incubated with 1000 ten thousand mixed antigen presenting cells (DC and B cell number ratio 1:1) in 10mL of RPM 1640 complete medium for 14 days (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (10 ng/mL), IL-21 (10 ng/mL) and IL-6 (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 5.

(8) Vaccine for the treatment of cancer

Female C57BL/6 for 6-8 weeks was selected as a model mouse to prepare tumor-bearing mice. Subcutaneous inoculation of 5X 10 on day 0 for each mouse ⁵ Mice were injected with 100 μlpbs or 50 ten thousand mixed cell vaccines (vaccine 1, or vaccine 2, or vaccine 3, or vaccine 4, or vaccine 5) on day 5, day 8, day 13, and day 20, respectively. The tumor volume and survival monitoring method of the mice are the same as those described above.

(9) Experimental results

As shown in fig. 12, tumors of mice in PBS control group grew very fast and mice survived very short, and mice treated with several vaccines survived significantly longer. Among them, vaccine 1 works best. The effect of the vaccine 1 is obviously better than that of the vaccine 2 and the vaccine 3, which indicates that when the antigen delivery nano particles activate the mixed antigen presenting cells, the mixed antigen presenting cells need to be incubated together within a certain concentration range; the effect of the vaccine 1 is obviously better than that of the vaccine 4 and the vaccine 5, which indicates that when the antigen delivery nano particles activate the mixed antigen presenting cells, a certain incubation time is needed, and the incubation time is too long or too short, so that the effect cannot be optimal.

Example 11 vaccine for treatment of melanoma

(1) Preparation of antigenic components

The two melanoma cell lines of the cultured B16F10 melanoma cancer cell line and S91 melanoma cancer cell line were mixed at a quantitative ratio of 1:1 after collection, and then centrifuged at 500g for 5 minutes, and the supernatant was discarded and washed twice with PBS, and then the cells were resuspended with ultrapure water and repeatedly freeze-thawed 5 times to lyse the cells. Centrifuging the lysate at 10000g for 5 min, and collecting supernatant to obtain water soluble component soluble in pure water; adding 3M guanidine sulfate aqueous solution to the obtained precipitation part to dissolve the precipitation part, so as to convert the insoluble water-insoluble component insoluble in pure water into the water-soluble 3M guanidine sulfate aqueous solution.

Dropwise adding a saturated ammonium sulfate aqueous solution into a water-soluble component in a lysate, centrifuging 12000g of the obtained sample for 5 minutes after complete precipitation, dissolving the precipitate in a 3M guanidine sulfate aqueous solution for later use, heating the supernatant for 2 minutes at 100 ℃ after the supernatant and DNA digestive enzyme are subjected to co-action for 5 minutes, centrifuging 3000g of the obtained sample for 5 minutes, discarding the supernatant, and dissolving the precipitate in the 3M guanidine sulfate aqueous solution; the salted-out precipitate dissolved using 3M guanidine sulfate and the heated precipitate were then combined and used as a part of the water-soluble components.

And mixing all water-insoluble components in the lysate dissolved in the 3M guanidine sulfate solution and part of components in the water-soluble components dissolved in the 3M guanidine sulfate solution according to a mass ratio of 1:5 to obtain the antigen component 1 for preparing the nano particles.

(2) Preparation of nanoparticles

In this example, nanoparticle 1 (Nanoparticle 1) was prepared by a multiple emulsion method. The nanoparticle 1 is prepared from PLGA with molecular weight of 24-38 kDa. The immunological adjuvants used were poly (I: C) and CpG1018, and the substances that increased the escape of lysosome immunity were KALA polypeptides (WEAKLAKALAKALAKHLAKALAKALKACEA). Preparation method As previously described, antigen component 1, adjuvant, KALA polypeptide were loaded inside the nanoparticle, and then 100mg of the nanoparticle was centrifuged at 12000g for 25 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose and lyophilized for 48 hours. The average particle diameter of the nano particles is about 250 nm; about 200. Mu.g of antigen protein or polypeptide component was loaded per 1mg of PLGA nanoparticles, 0.05mg each of poly (I: C) and CpG1018, and 0.03mg of KALA polypeptide were loaded.

(3) Activation of antigen presenting cells

5mg of nanoparticle 1 were co-incubated with 900 ten thousand mixed antigen presenting cells (DC to B cell number ratio 5:1) in 5mL of RPM 1640 complete medium for 18 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (10 ng/mL), IL-21 (10 ng/mL) and GM-CSF (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 1.

Alternatively, 5mg of nanoparticle 1 was co-incubated with 900 ten thousand mixed antigen presenting cells (DC to B cell number ratio 44:1) in 5mL of RPM 1640 complete medium for 18 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (10 ng/mL), IL-21 (10 ng/mL) and GM-CSF (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 2.

Alternatively, 5mg of nanoparticle 1 was co-incubated with 900 ten thousand mixed antigen presenting cells (DC and B cell number ratio 1:89) in 5mL of RPM 1640 complete medium for 18 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (10 ng/mL), IL-21 (10 ng/mL) and GM-CSF (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 3.

5mg of nanoparticle 1 was co-incubated with 900 ten thousand mixed antigen presenting cells (DC and B cell number ratio 5:1) in 5mL of RPM 1640 complete medium for 1 hour (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-15 (10 ng/mL), IL-21 (10 ng/mL) and GM-CSF (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 4.

(4) Vaccine for treating cancer

Female C57BL/6 after 6-8 weeks is selected as a model mouse to prepare melanoma tumor-bearing mice. Each mouse was subcutaneously vaccinated 1.5X10 lower right back on day 0 ⁵ B16F10 cells. 100 μl of PBS or 150 ten thousand mixed cell vaccines (vaccine 1, or vaccine 2, or vaccine 3, or vaccine 4) were subcutaneously injected on days 4, 7, 10, 15, and 20, respectively, after melanoma inoculation. In the experiment, the tumor volume and the survival monitoring method of the mice are the same as those described above.

(5) Experimental results

As shown in FIG. 13, the results showed that the tumors of the PBS control group grew very rapidly. Compared with the control group, the tumor growth speed of the mice treated by the vaccine is obviously slowed down and the survival period is obviously prolonged. Moreover, vaccine 1 is better than nanovaccine 2 and nanovaccine 3, which means that DC and B cells need to be within a certain ratio to achieve optimal effect when mixed antigen presenting cells are activated using antigen delivery particles; moreover, the co-incubation needs a certain time to achieve better effect. In conclusion, the nano vaccine disclosed by the disclosure has a good therapeutic effect on cancers.

EXAMPLE 12 micron vaccine for cancer prevention

(1) Preparation of cancer cell and bacterial exovesicle component

Cultured E.G7-OVA mouse T-lymphoma cells were centrifuged at 400g for 5 minutes, and then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells are respectively subjected to inactivation and denaturation treatment by ultraviolet rays and high-temperature heating, and then the cancer cells are lysed by using a 6M guanidine hydrochloride aqueous solution, and then the lysate component is lysed by using an 8M urea aqueous solution.

Lactobacillus acidophilus was centrifuged at 5000g for 30 minutes, then the precipitate was discarded, the supernatant was collected, the supernatant was filtered using a 1 μm filter membrane, then centrifuged at 16000g for 90 minutes, the precipitate obtained after discarding the supernatant was the bacterial outer vesicle fraction, and the precipitate was lysed using an 8M aqueous urea solution to dissolve the bacterial outer vesicle fraction.

And mixing the cancer cell lysate component dissolved in the 8M urea solution and the bacterial outer vesicle component dissolved in the 8M urea solution according to the mass ratio of 8:1 to obtain the antigen component for preparing the microparticles.

(2) Preparation of microparticles

In this example, microparticle 1 (Micronpartcle 1) was prepared by the multiple emulsion method. The molecular weight of the PLA skeleton material of the microparticles 1 is 20kDa-40kDa, and the immunological adjuvants used are CpG1018 (B class) and Poly ICLC. When the preparation is carried out, firstly, a double emulsion method is adopted to prepare microparticles internally loaded with antigen components and adjuvants, then 100mg of microparticles are centrifuged at 9000g for 15 minutes, 10mL of ultrapure water containing 4% trehalose is used for resuspension and then dried for 48 hours, thus obtaining microparticles 1, the average particle size is about 3.5 mu m, 100 mu g of protein or polypeptide components are loaded per 1mg of PLGA microparticles 1, and 0.07mg of CpG1018 and Poly ICLC are loaded.

Microparticle 2 (microparticle 2) was prepared from the same materials and methods as microparticle 1, with a particle size of about 3.5 μm, loaded with an equivalent amount of antigen components, but not with CpG1018 and Poly ICLC.

(3) Activation of antigen presenting cells

This example uses BMDC and B derived from peripheral blood as antigen presenting cells, and was prepared as described above.

1mg of microparticles 1 or microparticles 2 were co-incubated with 1500 ten thousand mixed antigen presenting cells (300 of BMDC, 1200 ten thousand B cells) in 10mL of high sugar DMEM complete medium for 12 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained IL-15 (20 ng/mL), IL-6 (10 ng/mL) and GM-CSF (10 ng/mL). After incubation, the particles are removed by centrifugation at 400g for 5 minutes, and then the mixed antigen presenting cells are collected to obtain the cancer vaccine. Using the mixed antigen presenting cells activated by the microparticles 1 as vaccine 1; mixed antigen presentation using microparticle 2 activationThe cells were vaccine 2.

Alternatively, 1mg of microparticles 1 were co-incubated with 1500 ten thousand mixed antigen presenting cells (300 ten thousand DC, 1200 ten thousand B cells) in 10mL of high sugar DMEM complete medium for 12 hours (37 ℃,5% CO) ₂ ). After the incubation is completed, the mixed antigen presenting cells are collected after centrifugation at 400g for 5 minutes to remove particles, and then the cancer vaccine 3 is obtained.

(4) Vaccine for prevention of cancer

Female C57BL/6 for 6-8 weeks was selected as a model mouse to prepare tumor-bearing mice. Mice were injected with 100 μlpbs or 50 ten thousand cell vaccines (vaccine 1, or vaccine 2, or vaccine 3) on day-35, day-28, day-21, day-14, and day-7, respectively, prior to tumor inoculation, and each mouse was subcutaneously vaccinated 5×10 on day 0 ⁵ E.G7-OVA cells. The tumor volume and survival monitoring method of the mice are the same as those described above.

(5) Experimental results

As shown in fig. 14, the tumor growth rate was significantly slowed and the survival time of mice was significantly prolonged in the vaccine-treated mice compared to the PBS control group. Moreover, vaccine 1 is significantly better than vaccine 2 and vaccine 3, demonstrating that the addition of cytokines during co-incubation of antigen presenting cells with antigen delivery particles is beneficial to improving the effectiveness of the vaccine, and that the use of mixed adjuvants in antigen delivery particles helps to improve the effectiveness of the antigen delivery particles in activating mixed antigen presenting cells to prepare the vaccine. It follows that the vaccine described in the present disclosure can be used to prevent or treat cancer.

Example 13 vaccine for treatment of colon cancer

(1) Preparation of cancer cell lysate fraction and cancer extracellular vesicle fraction

Collecting MC38 cancer cell line and its culture medium, centrifuging at 500g for 5 min, collecting cell precipitate, and completely dissolving the cell precipitate after cracking with 6M guanidine hydrochloride aqueous solution to obtain the collected cancer cell lysate component; collecting culture supernatant, filtering the supernatant with a 1 μm filter membrane, centrifuging at 160000g for 90 min, discarding the supernatant, and completely dissolving the precipitate after lysing with 6M guanidine hydrochloride aqueous solution to obtain the extracellular vesicle lysate component of cancer cells. The two are combined, then saturated magnesium sulfate aqueous solution is added until the precipitation is complete, the supernatant is discarded, and the precipitate is secondarily dissolved in 0.2M sodium guanidinoacetate and 0.2M arginine aqueous solution. Namely, the antigen component 1 for preparing the nano-particles 1.

Collecting MC38 cancer cell line and its culture medium, centrifuging at 500g for 5 min, collecting cell precipitate, and completely dissolving the cell precipitate after lysing with 2% Triton X100 aqueous solution to obtain the fraction of cancer cell lysate; collecting culture supernatant, filtering the supernatant with a 1 μm filter membrane, centrifuging at 160000g for 90 min, discarding the supernatant, and dissolving the precipitate after lysing with 2% Triton X100 aqueous solution to obtain the extracellular vesicle lysate component of the collected cancer cells. Mixing the above two, adding saturated magnesium sulfate aqueous solution until precipitation is completed, discarding supernatant, and solubilising the precipitate with 2% Triton X100 aqueous solution to obtain antigen component 2 for preparing nanoparticle 2.

(2) Preparation of nanoparticles

In this example, nanoparticle 1 (Nanoparticle 1) was prepared by a multiple emulsion method. The PLGA molecular weight of the preparation material of the nanoparticle 1 is 7KDa-17KDa, and the antigen component 1 and the immune adjuvant are loaded in the nanoparticle by taking Poly (I: C) and CpG22395 as adjuvants. The preparation method is as described above, firstly, antigen component 1 and adjuvant are loaded in the nano particles, then 100mg of nano particles are centrifugated at 18000g for 40 minutes, and 10mL of ultrapure water containing 4% trehalose is used for resuspension and then freeze drying is carried out for 48 hours for later use; the average particle size of the nanoparticle 1 is about 110nm, each 1mg of PLGA nanoparticle is loaded with about 2 mug of protein and polypeptide components, and each 1mg of PLGA nanoparticle is loaded with 0.01mg of poly (I: C) and CpG 2395.

The Nanoparticle 2 (Nanoparticle 2) is prepared from the same materials and method as the Nanoparticle 1, but the antigen component 2 and the adjuvant are internally loaded, the particle size is about 110nm, 2 mug of protein and polypeptide components are loaded per 1mg of PLGA Nanoparticle, and 0.01mg of poly (I: C) and CpG2395 are loaded per 1mg of PLGA Nanoparticle.

(3) Preparation of nanoparticles carrying antigen presenting cell membrane fraction and cancer cell extracellular vesicle membrane fraction

This embodimentBMDCs and B were used as antigen presenting cells. BMDC was prepared as above. B cells were derived from mouse peripheral blood PBMC and were isolated using magnetic beads. And mixing BMDC and B cells according to the number ratio of 1:2 to obtain the mixed antigen presenting cell. 1mg of nanoparticle 1 or nanoparticle 2 was co-incubated with 1500 ten thousand mixed antigen presenting cells (DC 1000 ten thousand and B cells 500 ten thousand) in 15mL of high sugar DMEM complete medium for 4 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained vinblastine (10 ng/mL) and GM-CSF (10 ng/mL).

Collecting 2000 ten thousand of the incubated mixed antigen presenting cells, centrifuging for 5 minutes at 400g, collecting supernatant after discarding the precipitate, centrifuging for 60 minutes at 16000g, collecting the precipitate after discarding the supernatant, and re-suspending the precipitate in PBS to obtain the activated extracellular vesicles of the antigen presenting cells.

Collecting 2000 thousands of cultured MC38 cells, centrifuging at 400g for 5 min, collecting supernatant after discarding the precipitate, centrifuging at 15000g for 60 min, collecting the precipitate after discarding the supernatant, and re-suspending the precipitate in PBS to obtain the extracellular vesicles of cancer cells.

Mixing the collected extracellular vesicles of activated antigen presenting cells with the extracellular vesicles of cancer cells, performing low-power (20W) ultrasound at 4 ℃ for 2 minutes, repeatedly performing coextrusion by using a filter membrane with the diameter of 0.22 mu m, mixing the extrusion liquid with the nano particles 1 or 2 prepared in the step (2), performing treatment by using a high-pressure homogenizer (10000 bar) for 1 minute, repeatedly performing coextrusion by using a filter membrane with the diameter of 0.22 mu m, centrifuging at 15000g for 30 minutes, discarding supernatant to collect precipitate, re-suspending the precipitate in a water solution with the diameter of 4% of trehalose, and performing freeze drying for 48 hours to obtain the nano vaccine. Wherein, the nanoparticle prepared by coaction of the antigen presenting extracellular vesicle membrane component activated by the nanoparticle 1 and the nanoparticle 1 is nanoparticle 3, the particle size is 120 nanometers, each 1mg of PLGA nanoparticle is loaded with about 2 mug antigen protein and polypeptide components, and each of poly (I: C) and CpG2395 is loaded with 10 mug, and each of the two is loaded with 80 mug membrane components. The nanoparticles prepared by the coaction of the antigen presenting extracellular vesicle membrane component activated by the nanoparticles 2 and the nanoparticles 2 are nanoparticles 4 with the particle size of 120 nanometers, and each 1mg of PLGA nanoparticles is loaded with about 2 mug of antigen protein and polypeptide components, 10 mug of poly (I: C) and CpG2395 respectively, and 80 mug of membrane component.

(4) Activation of antigen presenting cells

This example uses BMDCs and B as antigen presenting cells. BMDC was prepared as above. B cells were derived from mouse peripheral blood PBMC and were isolated using magnetic beads. And mixing BMDC and B cells according to the number ratio of 1:2 to obtain the mixed antigen presenting cell. 0.5mg of nanoparticle 3 or nanoparticle 4 was co-incubated with 1500 ten thousand mixed antigen presenting cells (DC 1000 ten thousand and B cells 500 ten thousand) in 20mL of high sugar DMEM complete medium for 8 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained vinblastine (10 ng/mL) and GM-CSF (10 ng/mL). After incubation, the particles are removed by centrifugation at 400g for 5 minutes, and then the mixed antigen presenting cells are collected to obtain the cancer vaccine. Wherein, the mixed antigen presenting cell activated by the nanoparticle 3 is used as a vaccine 1, and the mixed antigen presenting cell activated by the nanoparticle 4 is used as a vaccine 2.

(7) Vaccine for treating cancer

Female C57BL/6 for 6-8 weeks was selected as model mice to prepare colon cancer mice. Each mouse was subcutaneously inoculated 2×10 on day 0 under the back right ⁶ And MC38 cells. 100 ten thousand mixed cell vaccines (vaccine 1, or vaccine 2) or 100 μl of PBS were injected subcutaneously on day 3, 6, 9, 12, 15, 20, and 25 after inoculation of colon cancer cells, respectively. The methods for monitoring tumor growth and survival of mice are the same.

(8) Experimental results

As shown in fig. 15, the tumor growth rate of the vaccine treated mice was significantly slowed and the survival time of the mice was significantly prolonged compared to the PBS control group. Furthermore, vaccine 1 is better effective than vaccine 2. It follows that it is indicated that a suitable dissolution solution containing a lytic agent must be used to produce an antigen delivery particle-activated mixed cell vaccine for good therapeutic efficacy.

Example 14 vaccine for prevention of cancer

(1) Preparation of cancer cell-related components

The cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water, and then the cancer cells were lysed using 0.2M arginine and 0.2M polyhexamethylene guanidine hydrochloride aqueous solution and the lysate fractions were lysed to obtain protein and polypeptide fractions in the cancer cells dissolved in 0.2M arginine and 0.2M polyhexamethylene guanidine hydrochloride aqueous solution. The protein and polypeptide components, the cancer-related antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF) and the cancer-related antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) which are dissolved in the cancer cells in the 0.2M arginine and the 0.2M polyhexamethylene guanidine hydrochloride aqueous solution are mixed according to the mass ratio of 1:1:1, and the mixture is the antigen component 1.

The cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water, and then the cancer cells were lysed using a 3% Tween 80 aqueous solution and the lysate fraction was solubilized to obtain protein and polypeptide fractions in the cancer cells dissolved in the 3% Tween 80 aqueous solution. The protein and polypeptide components, the cancer-related antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF) and the cancer-related antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) which are dissolved in the 3% Tween 80 aqueous solution are mixed according to the mass ratio of 1:1:1, and the mixture is the antigen component 2.

The cancer related antigen polypeptide IO102 (DTLLKALLEIASCLEKALQVF) and the cancer related antigen polypeptide IO103 (FMTYWHLLNAFTVTVPKDL) are mixed according to the mass ratio of 1:1, and the mixture is an antigen component 3.

(2) Preparation of nanoparticles

In this example, the Nanoparticle 1 (Nanoparticle 1) was prepared by the multiple emulsion method. The nanoparticle 1 skeleton material is PLGA (molecular weight 40 kDa) and mannose-PEG 2000-PLGA (molecular weight 10-20 kDa), and the mass ratio of PLGA (molecular weight 10-20 kDa) to mannose-PEG 2000-PLGA (molecular weight 40 kDa) is 9:1. The immunological adjuvants are CpG2395 (B class), cpG2216 (A class) and Poly ICLC, and bee venom peptide (GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 6) is used as a substance for increasing lysosome escape, wherein nano particles of antigen component 1, adjuvant and bee venom peptide are firstly loaded in a double emulsion method, 100mg of nano particles are centrifuged for 20 minutes at 10000g, 10mL of ultra-pure water containing 4% trehalose is used for resuspension, and then the nano particles are dried for 48 hours, so that the nano particles 1 with the average particle size of about 200nm are obtained, and about 500 mug of cancer cell antigen protein and polypeptide component are loaded per 1mg of PLGA nano particles, and 0.005mg of CpG2395, cpG2216 and Poly ICLC are loaded.

Nanoparticle 2 (Nanoparticle 2) was prepared in the same manner as Nanoparticle 1. But internally loaded are antigen component 2, adjuvant and melittin. The average particle size of the nanoparticle 2 is about 200nm, and each 1mg of PLGA nanoparticle is loaded with about 500 mug of protein and polypeptide components of cancer cell antigen, 0.005mg of CpG2395, cpG2216 and Poly ICLC, and 0.05mg of melittin.

Nanoparticle 3 (Nanoparticle 3) was prepared in the same manner as Nanoparticle 1. But internally loaded are antigen component 3, adjuvant and melittin. The average particle size of the nanoparticle 3 is about 200nm, and about 500 mug of antigen polypeptide components are loaded per 1mg of PLGA nanoparticle, 0.005mg of CpG2006, cpG2216 and Poly ICLC are loaded, and 0.05mg of melittin is loaded.

(3) Preparation and activation of antigen presenting cells

Nanoparticles (nanoparticle 1, or nanoparticle 2 or nanoparticle 3) were co-incubated with 100 ten thousand mixed antigen presenting cells (DC and B cell number ratio 9:1) in 5mL RPM 1640 complete medium for 12 hours (37 ℃,5% CO ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained cytokines IL-2 (10 ng/mL) and IL-7 (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 min to remove particles from the system, and the activated mixed cells were used as cancer vaccines. Wherein, the mixed antigen presenting cell activated by the nanoparticle 1 is used as the vaccine 1; mixed antigen presenting cells activated with nanoparticle 2 are vaccine 2; mixed antigen presenting cells activated using nanoparticle 3 are vaccine 3.

(4) Vaccine for prevention of cancer

Female C57BL/6 for 6-8 weeks was selected as a model mouse to prepare tumor-bearing mice. Mice were injected with 100 μlpbs or 100 ten thousand cell vaccine (vaccine 1, orVaccine 2, or vaccine 3), each mouse was subcutaneously vaccinated 5 x 10 on day 0 ⁵ E.G7-OVA cells. The tumor volume and survival monitoring method of the mice are the same as those described above.

(4) Experimental results

As shown in fig. 16, the tumor growth rate was significantly slowed and the survival time of mice was significantly prolonged in the cell vaccine-treated mice compared to the PBS control group. Furthermore, vaccine 1 is better than vaccine 2 and vaccine 3, indicating that appropriate solubilizers are necessary. Mannose is used as the active targeting target for targeting dendritic cells in this example, and other targeting molecules including, but not limited to, mannans, CD20 antibodies, CD11c antibodies, CD103 antibodies, CD32 antibodies, CD11b antibodies, CD19 antibodies, CD38 antibodies, etc. may be used in practical applications.

Example 15 vaccine for pancreatic cancer prevention

(1) Preparation of antigenic components

The cultured Pan02 cell line was collected, washed twice with PBS, and then the cells were resuspended with ultrapure water and freeze-thawed repeatedly 5 times to lyse the cancer cells. Centrifuging the lysate at 10000g for 5 min, and collecting supernatant to obtain water soluble component soluble in pure water; adding 8M urea aqueous solution into the obtained precipitation part to dissolve the precipitation part, so as to convert the insoluble component insoluble in pure water into soluble component in the 8M urea aqueous solution. Mixing the water-soluble component in the lysate and the water-insoluble component dissolved by 8M urea according to the mass ratio of 1:1 to obtain the antigen component 1 for preparing the nano particles 1.

(2) Preparation of nanoparticles

Nanoparticle 1 (Nanoparticle 1) or Nanovaccine 1 (nanovaccinee 1) in this example was prepared by a multiple emulsion method. The nanoparticle 1 is prepared from PLGA with molecular weight of 7-17 kDa. The immunoadjuvants used were poly (I: C) and CpG1018. Preparation method As previously described, antigen component 1 and adjuvant were loaded inside the nanoparticle, and then 200mg of the nanoparticle was centrifuged at 12000g for 25 minutes, and resuspended in 10mL of ultra-pure water containing 4% trehalose, followed by lyophilization for 48 hours. The average particle diameter of the nanoparticle 1 (nano vaccine 1) is about 250 nm; about 300. Mu.g of the antigen protein or polypeptide component was loaded per 1mg of PLGA nanoparticles, with 0.05mg of each of poly (I: C) and CpG1018.

(3) Activation of antigen presenting cells

5mg of nanoparticle 1 were co-incubated with 1000 ten thousand mixed antigen presenting cells (DC to B cell number ratio 1:1) in 5mL of RPM 1640 complete medium for 18 hours (37 ℃,5% CO) ₂ ) The incubation system contained the cytokine IL-15 (10 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 2.

(4) Vaccine for preventing cancer

Female C57BL/6 for 6-8 weeks was selected as a model mouse to prepare tumor-bearing mice. The experimental mice were given a 100mg/kg dose of cyclophosphamide intraperitoneally injected once on day-7 before the mice were vaccinated with cancer cells to clear immune cells in the recipient mice. Mice were injected subcutaneously with 100 μl of PBS or nanovaccine 1 (2 mg) or 50 ten thousand mixed cell vaccine (vaccine 2) on day-6 before inoculating the mice with cancer cells, respectively. Each mouse was inoculated subcutaneously 1.0×10 back on day 0 ⁶ Pan02 pancreatic cancer cells. In the experiment, the tumor volume and the survival monitoring method of the mice are the same as those described above.

(5) Experimental results

As shown in FIG. 17, the results showed that the tumors of the PBS control group grew very rapidly. Compared with the control group, the tumor growth speed of the mice treated by the vaccine is obviously slowed down and the survival period is obviously prolonged. Furthermore, vaccine 2 is better than nanovaccine 1, which means that the effect of using mixed cell vaccine is better than that of using cancer nanovaccine by direct injection when there are fewer immune cells in the body. In summary, the vaccine of the present disclosure has a good therapeutic effect on cancer.

Example 16 vaccine for treatment of liver cancer

(1) Preparation of antigenic components

The Hepa 1-6 cell line was co-incubated with low concentration of doxorubicin (1 nM) in RPMI1640 complete medium for 2h (37 ℃,5% CO) ₂ ). Co-incubationDoxorubicin can stimulate cancer cells during the course of incubation. Then, the cultured Hepa 1-6 hepatoma carcinoma cell line was collected and centrifuged at 350g for 5 minutes, the supernatant was discarded and washed twice with PBS, then the cells were resuspended in ultrapure water and then lysed by repeated freeze thawing 5 times, followed by oxidation with hypochlorous acid for 10 minutes, and then the sample after oxidation of the lysate component was dissolved using 6M aqueous guanidine hydrochloride solution. Then adding saturated ammonium sulfate aqueous solution dropwise into the sample, centrifuging the sample at 10000g for 25 minutes, discarding supernatant, and dissolving the precipitate in 6M guanidine hydrochloride aqueous solution to obtain antigen component 1 for preparing antigen delivery microparticles.

(2) Preparation of microparticles

In this example, microparticle 1 (Micronpartcle 1) was prepared by the multiple emulsion method. The PLGA molecular weight of the material for preparing the microparticles 1 is 35KDa-54KDa, and the immunological adjuvants used are CpG ODN 7909 and Poly (I: C). Antigen component 1 and immunoadjuvant were co-supported in microparticles, and then centrifuged at 10000g for 15 minutes, and resuspended in 10mL of ultrapure water containing 4% trehalose, followed by lyophilization for 48 hours, to give microparticles 1. The average particle size of the microparticles was 3.0. Mu.m, and about 850. Mu.g of protein or polypeptide component was loaded per 1mg of PLGA microparticles, with 0.02mg of each of the loaded CpG ODN 7909 and Poly (I: C).

(3) Preparation and activation of antigen presenting cells

BMDC and B cells from peripheral blood were used as antigen presenting cells, and the preparation method was as described above.

5mg of microparticles 1 were co-incubated with 1000 ten thousand mixed antigen presenting cells (DC to B cell number ratio 1:2) in 5mL of DMEM complete medium for 24 hours (37 ℃,5% CO) ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the The incubation system contained the cytokine IL-15 (50 ng/mL). After incubation, the mixed cells were centrifuged at 400g for 4 minutes to remove particles in the system, and the activated mixed cells were used as cancer vaccine 1.

(4) Vaccine for treating cancer

Female C57BL/6 for 6-8 weeks was selected as a model mouse to prepare tumor-bearing mice. Each mouse was subcutaneously vaccinated 1.0X10% on day 0 under the back right ⁶ Liver cancer of Hepa 1-6. On day 3, day 650 ten thousand cell vaccine 1 or 100 μl PBS was injected subcutaneously in mice on days 10, 15 and 22, respectively. The method for monitoring the growth speed and the survival period of the tumor of the mice is the same as that of the method.

(5) Experimental results

As shown in fig. 18, the PBS group mice quickly grew in tumor volume and the mice died quickly. Mice treated with Vaccine 1 (Vaccine 1) had significantly slower tumor growth rates and significantly longer survival, with some mice recovering from tumor-free. The cancer vaccine disclosed by the disclosure has excellent cancer treatment effect.

In this example, 1nM doxorubicin is used to stimulate cancer cells, and other concentrations, such as 0.01nM to 2. Mu.M doxorubicin, may be used in practical applications.

In this embodiment, low concentration of doxorubicin is used to stimulate cancer cells, and in practical use, paclitaxel, vincristine, bacterial secretion, bacterial membrane component, retinoic acid, arsenic trioxide, cytokine, plant extract (such as Chinese medicinal extract and plant rhizome extract), growth factor, and chemokine can be used to stimulate cancer cells.

In this example, hypochlorous acid is used to oxidize the antigen component, and any other oxidizing agent that can oxidize the antigen component may be used in practical applications.

The amino acid sequences involved in the present invention are shown below:

r8 polypeptides, RRRRRRRRRR

B16-M20 antigen polypeptide, FRRKAFLHWYTGEAMDEMEFTEAESNM

B16-M24 antigen polypeptide, TAVITPPTTTTKKARVSTPKPATPSTD

B16-M46 antigen polypeptide, NHSGLVTFQAFIDVMSRETTDTDTADQ

TRP2:180-188 antigen polypeptide, SVYDFFVWL

Melittin, GIGAVLKVLTTGLPALISWIKRKRQQ

KALA polypeptide, WEAKLAKALAKALAKHLAKALAKALKACEA

IO102 antigen polypeptide, DTLLKALLEIASCLEKALQVF

IO103 antigen polypeptide, FMTYWHLLNAFTVTVPKDL

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications will be apparent to persons skilled in the art from the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present disclosure.

Claims

1. An in vitro activated mixed cell cancer vaccine comprising dendritic cells and B cells, characterized in that: the mixed cell cancer vaccine comprises mixed cells obtained by in vitro simultaneous activation of dendritic cells and B cells by antigen delivery particles; wherein the antigen delivery particles are nanoparticles and/or microparticles carrying an antigen component;

the antigenic component is derived from one or more of the following: (1) A water-soluble component and/or a water-insoluble component of cells in cancer cells and/or tumor tissue; (2) Protein and polypeptide components of cells in cancer cells and/or tumor tissue; (3) RNA components in cancer cells and/or tumor tissue; (4) In vitro synthesized protein and/or polypeptide containing antigen polypeptide epitope; (5) Nucleic acids capable of expressing epitopes of antigenic polypeptides are synthesized in vitro.

2. The mixed cell cancer vaccine of claim 1, wherein: the number ratio of the dendritic cells to the B cells is 50:1-1:100; in the co-incubation system, the antigen delivery particle concentration is 0.002 μg/mL-80mg/mL.

3. The mixed cell cancer vaccine of claim 1, wherein: the activation is by co-incubating antigen delivery particles with dendritic cells and B cells in the same system.

4. The mixed cell cancer vaccine of claim 3, wherein: macrophages are also included in the co-incubation system.

5. The mixed cell cancer vaccine of claim 3, wherein: the co-incubation system also contains substances for assisting activation; the substances that aid in activation include one or more of cytokines, growth factors, antibodies, and chemokines.

6. The mixed cell cancer vaccine of claim 3, wherein: the incubation time is 2 hours to 14 days.

7. The mixed cell cancer vaccine of claim 1, wherein the antigen component preparation process comprises:

firstly, cracking cancer cells or tumor tissues, then respectively collecting water-soluble components and non-water-soluble components in the cracking liquid, and using the water-soluble components together with the water-soluble components after the non-water-soluble components are dissolved by using a dissolving liquid containing a dissolving agent;

Or lysing cancer cells or tumor tissue with a lysing solution containing a lysing agent, and then lysing the lysate component with a lysing solution containing a lysing agent;

or firstly cracking cancer cells or tumor tissues, then respectively collecting water-soluble components and non-water-soluble components in the cracking liquid, and after the water-soluble components are treated by one or more methods of salting out, heating, enzyme treatment, oxidation, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, mineralization, radiation and irradiation, re-dissolving the precipitation part by using a dissolving liquid containing a dissolving agent to obtain protein and polypeptide components in the water-soluble components; the non-water-soluble part is directly dissolved by using a dissolving solution containing a dissolving agent, or is dissolved by using a dissolving solution containing a dissolving agent, and is subjected to one or more methods of salting out, heating, enzyme treatment, oxidation, reduction, mineralization, radiation and irradiation, and then the obtained precipitate component is secondarily dissolved by using the dissolving solution containing the dissolving agent, so that protein and polypeptide components in the non-water-soluble component are obtained; mixing the protein polypeptide component in the water-soluble component with the water-insoluble component or mixing the protein and polypeptide components in the water-insoluble component; or separating RNA component from water soluble component and/or non-water soluble component, and using as antigen component alone or in combination with the above protein and polypeptide components;

Or lysing cancer cells and/or tumor tissues by using a lysis solution containing a lysis agent, dissolving lysate components by using the lysis solution containing the lysis agent, and then subjecting the obtained lysate components after dissolution to one or more methods of salting out, heating, enzyme treatment, oxidation, fixation, chromatography, electrophoresis, chromatography, recrystallization, precipitation, extraction, dialysis, reduction, mineralization, radiation and irradiation, and then re-dissolving the obtained precipitate components by using the lysis solution containing the lysis agent for a second time; wherein one or more of the extracted protein and polypeptide components and the RNA component are separated and used as antigen components;

wherein the lytic agent is independently selected from one or more of a compound comprising the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, a protein degrading enzyme, a polypeptide, an amino acid, a glycoside, and choline; wherein, structural formula 1 is as follows:

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

8. The mixed cell cancer vaccine of claim 7, wherein: the compound containing the structure shown in the structural formula 1 is selected from one or more of metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, urea salt, guanidine carbonate, arginine, guanidinoacetic acid, guanidinosporic acid, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compound and thiourea compound.

9. According to claim 7The mixed cell cancer vaccine is characterized in that: the cations contained in the reagent used for salting-out include Al ³⁺ 、Fe ³⁺ 、Fe ²⁺ 、Mg ²⁺ 、Sn ²⁺ 、Zn ²⁺ 、Ca ²⁺ 、Li ⁺ 、Na ⁺ 、NH ₄ ⁺ 、K ⁺ 、Cu ²⁺ 、Ag ⁺ 、Ba ²⁺ The anions contained include Cl ^- 、SO ₄ ^2- 、NO ₃ ^- 、CO ₃ ^2- 、SiO3 ^2- 、S ₂ O ₇ ^2- 、B ₄ O ₇ ^2- 、PO4 ^3- 、COO ^- 、NO ₂ ^- 、S ₂ O ₈ ^2- 、S ^2- 、CrO ₄ ^2- 、MnO ₄ ^- 、P ₂ O ₇ ^4- 。

10. The mixed cell cancer vaccine of claim 1, wherein: the antigen component is supported within and/or on the surface of the delivered particle, either separately or simultaneously; the means for supporting the antigenic component on the surface of the delivery particle includes at least one of adsorption, covalent attachment, charge interactions, hydrophobic interactions, one or more steps of solidification, mineralization and encapsulation.

11. The mixed cell cancer vaccine of claim 1, wherein the antigen delivery particles are further loaded with at least one component as follows:

(i) RNA component in the water-soluble component and/or the non-water-soluble component;

(ii) An immunoadjuvant;

(iii) A positively charged substance selected from positively charged amino acids, positively charged polypeptides, positively charged lipids, positively charged proteins, positively charged polymers, and/or positively charged inorganics.

12. The mixed cell cancer vaccine of claim 1, wherein: in the antigen delivery particles, the mass ratio of the particle preparation framework material to the antigen component is 1:0.001-10.

13. The mixed cell cancer vaccine of claim 1, wherein: the antigen delivery particles further comprise at least one component as shown below:

(a) Cancer cell membrane fraction derived from tumor tissue and/or tumor cells;

(c) Bacterial membrane fractions derived from bacterial lysates.

14. The mixed cell cancer vaccine of claim 13, wherein: the bacterial lysate and/or extracellular vesicle lysate is obtained by lysing bacteria and/or extracellular vesicles with lysis solution containing a lytic agent.

15. The mixed cell cancer vaccine of claim 1, wherein: the antigen delivery particles are prepared from natural polymer materials, synthetic polymer materials and/or inorganic materials.

16. The mixed cell cancer vaccine of claim 1, wherein: the cancer cells or tumor tissue may be co-incubated with a specific chemical to stimulate the cancer cells or tumor tissue prior to being lysed.

17. The mixed cell cancer vaccine of claim 1, wherein: the surface of the antigen delivery particle is connected with a target head for actively targeting dendritic cells and/or B cells.

18. A pharmaceutical composition characterized by: the pharmaceutical composition comprising the mixed cell cancer vaccine of any one of claims 1-17.

19. Use of the mixed cell cancer vaccine of any one of claims 1-17 or the pharmaceutical composition of claim 18 in at least one of the following (1) - (3):

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

(2) Preparing a medicament for inducing an immune response;

(3) Preparing a cancer vaccine.