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WO2024212380A1 - Vaccin contre le cancer basé sur une partie de composants de cellules cancéreuses ou de tissu tumoral, et son procédé de préparation - Google Patents

Vaccin contre le cancer basé sur une partie de composants de cellules cancéreuses ou de tissu tumoral, et son procédé de préparation Download PDF

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Publication number
WO2024212380A1
WO2024212380A1 PCT/CN2023/106259 CN2023106259W WO2024212380A1 WO 2024212380 A1 WO2024212380 A1 WO 2024212380A1 CN 2023106259 W CN2023106259 W CN 2023106259W WO 2024212380 A1 WO2024212380 A1 WO 2024212380A1
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components
water
cancer
vaccine
dissolving
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Chinese (zh)
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刘密
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Suzhou Ersheng Biopharmaceutical Co Ltd
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Suzhou Ersheng Biopharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/303—Extraction; Separation; Purification by precipitation by salting out
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the field of immunotherapy, and in particular to a cancer vaccine based on a portion of components in cancer cells or tumor tissues and a preparation method thereof.
  • Cancer vaccine is one of the important methods of cancer immunotherapy. Cancer vaccine is mainly composed of antigen and adjuvant, among which antigen is the main component that can induce specific immune response and is also the most important component in cancer vaccine. Cancer cells or tumor tissues contain all cancer cell specific antigens and cancer cell related antigens, and are the best raw materials for preparing cancer vaccines. Cancer cells or tumor tissues contain a variety of cancer antigens, among which the most important antigen components are protein and polypeptide components.
  • cancer vaccines such as cancer nano vaccines or micro vaccines
  • the relative content of protein and polypeptide antigens in whole cell components is reduced due to the variety of cancer cell antigens and the presence of various substances such as DNA, RNA, lipids and sugars in the whole cell components of cancer cells.
  • the antigen content loaded by nano vaccines or micro vaccines is high, it is possible that the immune response it can activate will be stronger, so appropriate enrichment and purification of antigens may be able to improve the efficacy of cancer nano vaccines or micro vaccines. How to properly purify and enrich cancer vaccines is very important.
  • the first object of the present disclosure is to provide a cancer vaccine, which includes a skeleton structure formed by a particle material and loaded with a portion of components of cancer cells and/or tumor tissues, wherein the interior and/or surface of the skeleton structure is loaded with one or two of the following: (1) protein and polypeptide components in water-soluble components of cancer cells and/or tumor tissues, and water-insoluble components of cancer cells and/or tumor tissues or protein and polypeptide components in water-insoluble components, (2) directly obtained antigen components.
  • components in the cancer cells or tumor tissues are proteins and polypeptide components in water-soluble components in lysates of cancer cells and/or tumor tissues, and water-insoluble components in lysates of cancer cells and/or tumor tissues, or proteins and polypeptide components in water-insoluble components in lysates, or directly obtained antigen components;
  • the method for preparing the protein and polypeptide components in the water-soluble components in the lysate comprises: treating all the water-soluble components by at least one of salting out, heating and enzymatic hydrolysis, and then re-dissolving the precipitated components using a dissolving solution containing a dissolving agent;
  • the method for preparing the water-insoluble component in the lysate comprises: directly dissolving the water-insoluble part using a dissolving solution containing a dissolving agent;
  • the method for preparing the protein and polypeptide components in the water-insoluble components in the lysate comprises: dissolving the water-insoluble part with a dissolving solution containing a dissolving agent, treating it with at least one of salting out, heating and enzymolysis, and then dissolving the obtained precipitated components again with a dissolving solution containing a dissolving agent;
  • the method for preparing the directly obtained antigen component comprises: lysing cancer cells and/or tumor tissues using a dissolving solution containing a dissolving agent, dissolving the obtained lysate component using the dissolving solution containing a dissolving agent, and then subjecting the obtained dissolved lysate component to at least one of salting out, heating and enzymatic hydrolysis, and then re-dissolving the obtained precipitate component using the dissolving solution containing a dissolving agent;
  • the dissolving agent is independently selected from one or more of a compound containing a structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, a protein degrading enzyme, a polypeptide, an amino acid, a glycoside and choline; wherein structural formula 1 is as follows:
  • R 1 is C, N or O, and R 2 to R 5 are independently selected from hydrogen, alkyl, amino, carboxyl, and substituted or unsubstituted guanidine.
  • heating is performed at 70-120°C.
  • the cancer cells are cancer cells from one or more organisms, or are from one or more cancer cell lines; the tumor tissues are tumor tissues from one or more organisms; and the protein and polypeptide components in the water-soluble components of the cancer cells and/or tumor tissues and the water-insoluble components of the cancer cells and/or tumor tissues/the protein and polypeptide components in the water-insoluble components contain antigen components.
  • a protease inhibitor or a protease may be added to the cancer cell or tumor tissue lysate component.
  • the process of separating and purifying the antigen component in the water-soluble component or the antigen component in the whole cell lysate component may include salting out.
  • the cations 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 the reagent used for salting out include , but are not limited to, Cl- , SO4 2- , NO3- , CO3 2- , SiO3 2- , S2O7 2- , B4O7 2- , PO4 3- , RCOO- , NO2- , S2O8 2- , S2- , CrO4 2- , MnO4- , P2O7 4- , and the like.
  • the salting out process can be carried out by segmented salting out or one-time salting out.
  • the process of separating and purifying the antigen component in the water-soluble fraction or the antigen component in the whole cell lysate fraction may include heating.
  • the cancer vaccine is a nano vaccine or a micro vaccine.
  • the particle size of the cancer vaccine is nano or micro scale, which can ensure that the cancer vaccine can be efficiently phagocytosed by antigen presenting cells.
  • the particle size of the nano vaccine is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, more preferably, the particle size is 50nm-600nm; more preferably, the particle size is 50-500nm; more preferably, the particle size is The size is 100-400nm.
  • the particle size of the nano vaccine is 10nm, 50nm, 100nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 400nm, 500nm, etc.
  • the particle size of the micron vaccine is 1 ⁇ m-1000 ⁇ m, more preferably, the particle size is 1 ⁇ m-100 ⁇ m, more preferably, the particle size is 1 ⁇ m-10 ⁇ m, more preferably, the particle size is 1 ⁇ m-5 ⁇ m, more preferably 1-10 ⁇ m; more preferably 1-2 ⁇ m.
  • the particle size of the micron vaccine is 1 ⁇ m, 1.1 ⁇ m, 1.2 ⁇ m, 1.3 ⁇ m, 1.4 ⁇ m, 1.5 ⁇ m, 1.6 ⁇ m, 1.7 ⁇ m, 1.8 ⁇ m, 1.9 ⁇ m, 2 ⁇ m, 2.5 ⁇ m, 3 ⁇ m, 5 ⁇ m, 10 ⁇ m, and the like.
  • cancer vaccine of the present disclosure wherein the cancer vaccine is further loaded with at least one component as shown below:
  • a positively charged substance selected from a positively charged amino acid, a positively charged polypeptide, a positively charged lipid, a positively charged protein, a positively charged polymer, and/or a positively charged inorganic substance;
  • the immune adjuvant comprises at least one of the following: pattern recognition receptor agonists, BCG (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG cell wall acyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhancing reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn calf liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I: C), immune adjuvants poly ICLC, bacillus brevis vaccine, hemolytic streptococcus preparation, coenzyme QIO, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, cytokine,
  • BCG
  • the immune adjuvant includes at least one of a Toll-like receptor 3 agonist and a Toll-like receptor 9 agonist; further, the immune enhancing adjuvant includes (1) Poly(I:C) and/or Poly(ICLC); (2) CpG-ODN; wherein the CpG-ODN is at least one of Class A CpG-ODN, Class B CpG-ODN and Class C CpG-ODN; preferably at least two, and at least one of them is Class B CpG-ODN or Class C CpG-ODN.
  • CpG or “CpG-ODN” (CpG oligonucleotide, CpG oligodeoxynucleotide) is a synthetic oligodeoxynucleotide (ODN) containing unmethylated cytosine guanine dinucleotide (CpG).
  • ODN oligodeoxynucleotide
  • CpG-ODN oligodeoxynucleotide
  • Different types of CpG-ODN have different structural characteristics and immune effects, and are generally divided into three categories: A, B, and C.
  • CpG-ODN includes, but is not limited to, CpG 1018 (Class B), CpG 7909 (Class B), CpG 2006 (Class B), CpG-BW006 (Class B), CpG 2395 (Class C), CpG SL01, CpG 1585 (Class A), CpG 2216 (Class A), CpG SL03, CpG 2395 (Class C), CpG M362 (Class C), and CpG 2336 (Class A).
  • the positively charged substance includes but is 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, and positively charged inorganic substances.
  • the positively charged substance includes bee venom peptide, RALA polypeptide, KALA polypeptide, Any one or any combination of R8 polypeptide, arginine, histidine, lysine, polyarginine, polylysine, polyhistidine and NH 4 HCO 3 .
  • 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.
  • positively charged amino acids include, but are not limited to, arginine, histidine, lysine, and the like.
  • the positively charged high molecular polymer includes, but is not limited to, polyarginine, polylysine, polyhistidine, and the like.
  • positively charged lipids include, but are not limited to, DOTAP and the like.
  • positively charged proteins include, but are not limited to, protamine, histones, and the like.
  • the positively charged inorganic substance includes, but is not limited to, NH 4 HCO 3 , aluminum hydroxide, and the like.
  • the cancer nano-vaccine or micro-vaccine surface may also be loaded with targeting molecules.
  • the targeting molecules include at least one of the following: mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, etc.
  • the particle material forming the cancer vaccine is formed of natural polymer materials and/or synthetic polymer materials.
  • the antigen delivery particles are formed of natural polymer materials and/or synthetic polymer materials.
  • organic synthetic polymer materials include, but are not limited to, PLGA, PLA, PEG-PLGA, PEG-PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acids, synthetic polypeptides, and the like.
  • natural polymer materials include, but are not limited to, lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, polypeptides, and the like.
  • the inorganic material includes, but is not limited to, ferric oxide, ferrosin, carbonates, phosphates, and the like.
  • the shape of the cancer nanovaccine or microvaccine described in the present disclosure is any common shape, including but not limited to sphere, ellipsoid, barrel, polygon, rod, sheet, line, worm, square, triangle, butterfly, disc, vesicle, etc.
  • the mass ratio of the skeleton material, protein and polypeptide components prepared by the particles is 1:0.001-10; preferably, the mass ratio of the skeleton material, protein and polypeptide components prepared by the particles is 1:0.01-2; most preferably, the mass ratio of the skeleton material, protein and polypeptide components prepared by the particles is 1:0.05-1.
  • the cancer vaccine disclosed herein further comprises at least one component as shown below on the surface or inside of the cancer vaccine:
  • cancer cell membrane components derived from tumor tissues and/or tumor cells (a) cancer cell membrane components derived from tumor tissues and/or tumor cells;
  • the membrane component When the membrane component is located on the surface of the nano-vaccine or micro-vaccine, the membrane component is loaded onto the surface of the nano-vaccine or micro-vaccine.
  • the methods include, but are not limited to, one or more of sonication, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bond connection, stirring, dialyzing, homogenization and homogenization.
  • the bacteria include at least one of the following: BCG, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus gestatum, Lactobacillus reuteri, and Lactobacillus rhamnosus.
  • the nano-vaccine or micro-vaccine prepared from the components in the cancer cells and/or tumor tissues is selected from one or two of the following: (1) protein and polypeptide components in the water-soluble components + water-insoluble components; (2) protein and polypeptide components in the water-soluble components + protein components or polypeptides in the water-insoluble components).
  • the mass ratio of the protein and polypeptide in the water-soluble components to the protein and polypeptide in the water-insoluble components/water-insoluble components is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2).
  • the mass ratio of the protein and polypeptide components in the water-soluble components to the protein and polypeptide in the water-insoluble components/water-insoluble components 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 immunogenic protein and/or polypeptide is derived from a portion of the components in cancer cells/tumor tissues and extracellular vesicle lysates.
  • the extracellular vesicle lysate is selected from extracellular vesicle lysates of cancer cells and/or extracellular vesicle lysates of bacteria.
  • the mass ratio of a portion of the components in the cancer cells and/or tumor tissues to the extracellular vesicle lysate components is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2).
  • 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 immunogenic protein and/or polypeptide is derived from a portion of the components in the cancer cell/tumor tissue and a bacterial lysate.
  • the mass ratio of the portion of the components in the cancer cell/tumor tissue to the bacterial lysate is (0.1-10): (0.1-10); preferably (0.5-2): (0.5-2).
  • 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.
  • adding an appropriate amount of PEG-modified PLGA or PLA to the main materials such as PLGA or PLA for preparing nanovaccines or micron vaccines can better achieve the effects of long circulation and passive targeting; the mass ratio of PEG-modified PLGA or PLA to unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.
  • the particle material is PEG-modified or not PEG-modified.
  • the mass ratio of the particle material not modified with PEG to the particle material modified with PEG is 25-200:1.
  • the cancer vaccine disclosed in the present invention wherein the preparation steps of the protein and polypeptide components in the water-soluble components in the cancer cells and/or tumor tissues are as follows: first lyse the cancer cells and/or tumor tissues to obtain their lysates; then use one or more of the methods such as centrifugation, filtration, dialysis, ultrafiltration, etc.
  • the water-soluble components in the obtained lysates are subjected to salting out and/or heating treatment to precipitate the protein and polypeptide components therein; then the protein and polypeptide components in the precipitated part are dissolved using a dissolving agent to obtain the protein and polypeptide components in the water-soluble components.
  • the dissolving agent is selected from compounds containing the structure of structural formula 1 (including but not limited to metformin hydrochloride, metformin sulfate, polyhexamethylene guanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidinoacetic acid, One or more of guanidine phosphate, guanidine sulfamate, guanidine succinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compounds (gliben
  • R 1 is C, N or O, and R 2 to R 5 are independently selected from hydrogen, alkyl, amino, carboxyl, and substituted or unsubstituted guanidine.
  • the cancer vaccine disclosed in the present invention comprises the following steps for preparing the protein and polypeptide components in the water-insoluble components in the cancer cells and/or tumor tissues: firstly lyse the cancer cells and/or tumor tissues to obtain their lysates; then separate the water-soluble components and the water-insoluble components in the lysates by one or more methods such as centrifugation, filtration, dialysis, ultrafiltration, etc., to obtain the water-soluble components and the water-insoluble components in the lysates respectively; then subject the obtained water-insoluble components in the lysates to salting out and/or heating treatment to precipitate the protein and polypeptide components therein; then dissolve the precipitated protein and polypeptide components using a solvent to obtain the protein and polypeptide components in the water-insoluble components.
  • the dissolving agent is selected from a compound containing the structure of structural formula 1 (including but not limited to metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, guanidine sulfonate, guanidine salt, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide,
  • the cancer vaccine disclosed in the present invention wherein the preparation steps of the protein and polypeptide components in the cancer cells and/or tumor tissues are as follows: first lyse the cancer cells and/or tumor tissues to obtain their lysates; then use a dissolving agent to dissolve all the lysate components; then the lysate components dissolved by the dissolving agent are subjected to salting out and/or heating treatment to precipitate the protein and polypeptide components therein; then the protein and polypeptide components of the precipitated part are dissolved by a dissolving agent to obtain the protein and polypeptide components in all the lysates.
  • the dissolving agent 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 and the like containing the structure of formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, polypeptide, amino acid, glycoside and choline.
  • the bacterial lysate and/or the extracellular vesicle lysate is obtained by lysing bacteria and/or extracellular vesicles with a lysing solution containing a lysing agent; preferably, the lysing agent is selected from metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine sulfamate,
  • the bacterial lysate and/or extracellular vesicle lysate is obtained by lysing bacteria and/or extracellular vesicles with a lysing solution containing a lysing agent; preferably, the lysing agent is selected from metformin hydrochloride, polyhexamethylene guanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other compounds containing guanidine or urea, guanidine carbonate, arginine , guanidine acetic acid, guanidine phosphate, guanidine sulfamate
  • the cancer vaccine disclosed herein wherein the water-insoluble antigen, the bacterial lysate or the extracellular vesicle lysate are independently dissolved in a dissolving solution comprising at least one of the following dissolving agents: metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other compounds containing guanidine or urea, guanidine carbonate, or Amino acid, guanidine acetic acid, guanidine phosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydro
  • the cancer vaccine described in the present disclosure wherein the particle material is selected from natural polymer materials, synthetic polymer materials and/or inorganic materials.
  • nucleic acid delivery particles and antigen delivery particles are any shapes, including but not limited to sphere, ellipsoid, barrel, polygon, rod, sheet, line, worm, square, triangle, butterfly, disc, vesicle, etc.;
  • cancer vaccine disclosed herein, wherein the cancer vaccine is a nano vaccine and/or a micro vaccine;
  • the particle size of the nano vaccine is 1nm-1000nm; preferably 50-500nm; more preferably 100-400nm;
  • the particle size of the micron vaccine is 1 ⁇ m-1000 ⁇ m, preferably 1-10 ⁇ m, and more preferably 1-5 ⁇ m.
  • the third object of the present disclosure is to provide a pharmaceutical composition comprising the cancer nanovaccine and/or microvaccine provided by the present disclosure.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition provided by the present disclosure can exert a significant effect in preventing or treating diseases.
  • nucleic acid delivery particles or nucleic acid delivery systems disclosed herein can target antigen presenting cells, effectively activate the body's immune response, and efficiently exert the preventive or therapeutic effects of nucleic acid vaccines.
  • the cancer vaccine or pharmaceutical composition disclosed herein is used in at least one of the following:
  • the disease is cancer or a tumor
  • the cancer or tumor is a solid tumor and a blood tumor
  • the solid tumor or blood tumor includes but is not limited to squamous cell carcinoma, myeloma, lung cancer, glioma, liver cancer, lymphoma, acute myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, leukemia, colon cancer, rectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, sarcoma, cell tumor, pancreatic cancer, cervical cancer, brain cancer, bladder cancer, breast cancer and head and neck cancer.
  • the antigen component loaded by the vaccine is derived from cells or tissues related to the disease. Further, the antigen component is derived from a part of the whole cell component of cancer cells and/or tumor tissues.
  • the present disclosure provides a method for preventing or treating a disease, wherein the method comprises administering a preventively or therapeutically effective amount of a cancer vaccine to a subject.
  • the fourth object of the present disclosure is to provide a method for preparing the above-mentioned cancer vaccine.
  • the preparation method may include the following steps:
  • lysis cancer cells and/or tumor tissues to obtain their lysates.
  • Ultrapure water, aqueous solution, etc. can be used to lyse cancer cells or tumor tissues.
  • Cancer cells are one or more cancer cells or cancer cell lines; tumor tissues are tumor tissues derived from one or more organisms.
  • the lysis method is a commonly used lysis method for cancer cells or tumor tissues, including but not limited to repeated freezing and thawing, swelling, ultrasonic treatment, high pressure treatment, homogenization, extrusion, homogenization, high-speed stirring, chemical treatment, high shear force treatment, ultrafiltration treatment, shrinkage and other treatment methods.
  • the water-soluble components in the lysate are subjected to salting out and/or heating treatment to obtain a precipitate, and then the precipitate is dissolved using a dissolving solution containing a dissolving agent; the water-insoluble components in the lysate are dissolved using a dissolving solution containing a dissolving agent, and then subjected to salting out and/or heating treatment, and then dissolved using a dissolving solution containing a dissolving agent; or the water-insoluble components in the lysate are directly dissolved using a dissolving solution containing a dissolving agent without treatment.
  • the dissolving agent in each step is selected separately.
  • Dissolving agents include, but are not limited to, metformin hydrochloride, metformin sulfate, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glime
  • the protein and polypeptide components in the water-soluble components obtained in S3 and/or the water-insoluble components or the protein and polypeptide components therein are loaded separately or simultaneously into the interior and/or surface of nanoparticles or microparticles to obtain the nanovaccine Or micro vaccine.
  • the surface of nano vaccine or micro vaccine can also be loaded with membrane components.
  • the method for preparing the cancer vaccine disclosed herein may also include the following steps:
  • lyse cancer cells and/or tumor tissue to obtain lysates thereof may be used to lyse cancer cells or tumor tissue.
  • Cancer cells are one or more cancer cells or cancer cell lines; tumor tissue is tumor tissue derived from one or more organisms.
  • a dissolving solution containing a dissolving agent is directly used to dissolve the cancer cells and/or tumor tissue to obtain its lysate.
  • the dissolving agent includes but is not limited to metformin hydrochloride, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, aminosulfonate guanidine, guanidine succinic acid, semicarbazide hydrochloride, aminoform
  • the whole cell lysate component dissolved in a dissolving solution containing a dissolving agent is subjected to salting out and/or heating treatment to obtain a precipitate, and then the precipitate is dissolved in a dissolving solution containing a dissolving agent to obtain the desired antigen component.
  • Dissolving agents include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone, glime
  • preparation method may also include the following steps:
  • lysis cancer cells and/or tumor tissues to obtain their lysates.
  • Ultrapure water, aqueous solution, etc. can be used to lyse cancer cells or tumor tissues.
  • Cancer cells are one or more cancer cells or cancer cell lines; tumor tissues are tumor tissues derived from one or more organisms.
  • the lysis method is a commonly used lysis method for cancer cells or tumor tissues, including but not limited to repeated freezing and thawing, swelling, ultrasonic treatment, high pressure treatment, homogenization, extrusion, homogenization, high-speed stirring, chemical treatment, high shear force treatment, ultrafiltration treatment, shrinkage and other treatment methods.
  • the water-soluble components in the lysate are subjected to salting out and/or heating treatment to obtain a precipitate and a supernatant.
  • the precipitate is then dissolved in a dissolving solution containing a dissolving agent, and the mRNA component in the supernatant is separated and extracted using an appropriate method.
  • the mRNA component in the supernatant is then mixed with the precipitate dissolved in the lysate containing the lysate and set aside; the water-insoluble component in the lysate is dissolved in the lysate and then treated with salting out and/or heating before being dissolved in the lysate containing the lysate; or the water-insoluble component in the lysate is directly dissolved in the lysate containing the lysate without treatment.
  • the lysate in each step is selected separately.
  • Dissolving agents include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone,
  • the obtained components and/or water-insoluble components or the protein and polypeptide components in the water-soluble components obtained in S3 are loaded into the interior and/or surface of nanoparticles or microparticles respectively or simultaneously to obtain the nanovaccine or microvaccine.
  • the surface of the nanovaccine or microvaccine can also be loaded with a membrane component.
  • the method for preparing the cancer vaccine disclosed herein may also include the following steps:
  • lyse cancer cells and/or tumor tissue to obtain lysates thereof may be used to lyse cancer cells or tumor tissue.
  • Cancer cells are one or more cancer cells or cancer cell lines; tumor tissue is tumor tissue derived from one or more organisms.
  • a dissolving solution containing a dissolving agent is directly used to dissolve the cancer cells and/or tumor tissue to obtain its lysate.
  • the dissolving agent includes but is not limited to metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, guanidine salt, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, aminosulfonate guanidine, guanidine succinic acid, semicarbazide hydrochloride, aminoform
  • the whole-cell lysate component dissolved in a lytic solution containing a lytic agent is subjected to salting-out and/or heating treatment to obtain two parts, a precipitate and a supernatant, and then the precipitate part is dissolved using a lytic solution containing a lytic agent; an mRNA component is separated from the supernatant using an appropriate method, and then the mRNA component is mixed with the precipitate part dissolved in the lytic solution containing a lytic agent to obtain the desired antigen component.
  • the present invention uses specific means to separate and purify antigen components in whole cell components of cancer cells and/or tumor tissues, which can enrich protein and polypeptide antigen components and avoid potential toxic side effects that may be caused by substances such as sugars and DNA.
  • salting out, heating, enzymatic hydrolysis and the like are used to purify antigen components in cancer cell/tumor tissue lysates.
  • Other methods such as phase separation, chromatographic separation, etc. that can remove DNA components in the lysate or separate and purify protein polypeptides and mRNA components therein may also be used.
  • Figure 2 is a schematic diagram of the preparation process and application of nucleic acid delivery particles in the present disclosure
  • a is a schematic diagram of collecting some components of water-soluble antigen components and water-insoluble antigen components/partial antigen components and preparing nanovaccines/micron vaccines
  • b is a schematic diagram of the process of using a dissolving solution containing a dissolving agent to directly lyse and dissolve the whole cell antigens of cancer cells, then separating and purifying some components and preparing nanovaccines or micron vaccines.
  • Figures 3-19 are respectively the experimental results of tumor growth rate and survival period when nano-vaccine or micro-vaccine is used to prevent or treat cancer in Example 1-17; in Figure 3-19, a is the experimental result of tumor growth rate when preventing or treating cancer (n ⁇ 8); b is the experimental result of mouse survival period when preventing or treating cancer (n ⁇ 8), and each data point is mean ⁇ standard error (mean ⁇ SEM); wherein, the significant difference of tumor growth inhibition experiment in Figure a is analyzed by ANOVA method, and the significant difference in Figure b is analyzed by Kaplan-Meier and log-rank test; *** indicates that there is a significant difference compared with the PBS control group at p ⁇ 0.005; ** indicates that there is a significant difference compared with the PBS control group at p ⁇ 0.01; ### indicates that there is a significant difference between the two groups at p ⁇ 0.005; ## indicates that there is a significant difference between the two groups at p ⁇ 0.01; # indicates that there is a significant difference between the two groups at
  • the word “a” or “an” or “the” may mean “one”, but may also mean “one or more”, “at least one” and “one or more than one”.
  • treatment means that after suffering from a disease, a subject is exposed to (e.g., administered) a nucleic acid delivery particle, a nucleic acid delivery system, a nucleic acid vaccine, a drug loaded with nucleic acid, or a pharmaceutical composition, thereby alleviating the symptoms of the disease compared to when the subject is not exposed, and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means that the body has symptoms of the disease.
  • prevention means that before a subject develops a disease, by exposing the subject to (e.g., administering) the nucleic acid delivery particles, nucleic acid delivery systems, nucleic acid vaccines, nucleic acid-loaded drugs, and pharmaceutical compositions of the present disclosure, the symptoms after the disease is alleviated compared to when the subject has not been exposed to the disease, and does not necessarily mean that the disease must be completely suppressed.
  • pharmaceutically acceptable excipient or “pharmaceutically acceptable carrier” refers to auxiliary materials widely used in the field of drug production.
  • the main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionality, and also to provide a method so that after the drug is administered to the subject, the active ingredient can be dissolved at a desired rate, or to promote the effective absorption of the active ingredient in the subject receiving the drug.
  • Pharmaceutically acceptable excipients can be inert fillers or functional ingredients that provide a certain function to the pharmaceutical composition (for example, stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient 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, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.
  • compositions of the present disclosure can be prepared using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, pulverizing, encapsulating, embedding and/or lyophilizing processes.
  • the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the properties of the drug, the convenience of the patient and the medical staff, and other relevant factors.
  • mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domestic animals e.g., cows, sheep, cats, dogs and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats.
  • tumor and “cancer” are used interchangeably herein to encompass both solid and liquid tumors.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • the vaccine system disclosed in the present invention internally carries a portion of the whole cell components of cancer cells, and its preparation process and application field are shown in FIG1 .
  • the cells or tissues When preparing nano or micro vaccines, the cells or tissues may be lysed, and then the proteins and polypeptides in the water-soluble components and the proteins and polypeptides in the water-insoluble components/water-soluble components may be collected separately, and the nano or micro vaccines may be prepared separately; or the cells or tissues may be directly lysed using a dissolving solution containing a dissolving agent, and the whole cell antigens of the cancer cells may be dissolved, and then the protein and polypeptide components therein may be extracted and separated using an appropriate method, and the obtained components may be loaded onto the nano or micro vaccines.
  • the cancer cells and/or tumor tissues described in the present disclosure may be subjected to the following steps before or (and) after lysis: It is not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nucleic acid separation and purification, protease endo-cleavage or degradation, nuclease treatment and the like before and after the extraction and separation of protein and polypeptide components; it is also possible to directly extract and separate the protein and polypeptide components before or after cell lysis without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, protease endo-cleavage or degradation, nuclease treatment.
  • tumor tissue cells are inactivated or (and) denatured before lysis. In actual use, they can also be inactivated or (and) denatured after cell lysis, or they can be inactivated or (and) denatured before and after cell lysis.
  • the inactivation or (and) denaturation treatment methods before or after cell lysis are ultraviolet irradiation and high temperature heating. In actual use, treatment methods including but not limited to radiation irradiation, high pressure, nucleic acid separation and purification, solidification, biomineralization, ionization, chemical modification, nuclease treatment, protease endo-cutting or degradation, collagenase treatment, freeze-drying, etc. can also be used. It can be understood by those skilled in the art that in actual application, the technicians can make appropriate adjustments according to the specific circumstances.
  • Urea and guanidine hydrochloride contain the structure in Structural Formula 1.
  • the inventors have found that substances having the structure in Structural Formula 1 can be used as solvents in a dissolving solution to dissolve water-insoluble components in cells or tumor tissues. Therefore, in addition to common compounds containing guanidine structures such as urea, guanidine hydrochloride, and metformin, other compounds containing modified structures also have the ability to be used as solvents to dissolve water-insoluble components.
  • adding an appropriate amount of PEG-modified PLGA or PLA to the main materials such as PLGA or PLA for preparing nanovaccines or micron vaccines can better achieve the effects of long circulation and passive targeting; the mass ratio of PEG-modified PLGA or PLA to unmodified PLGA or PLA is 0.05% to 20%, preferably 0.1% to 10%.
  • PEG is used to modify the surface of the nano-vaccine or micro-vaccine.
  • amphiphilic molecules, zwitterionic ionized substances, etc. can also be used to modify the surface of the nano-vaccine or micro-vaccine.
  • the surface of the nanovaccine or microvaccine may also be loaded with membrane components, which may be derived from one or more of antigen presenting cells, cancer cells, bacteria or extracellular vesicles.
  • Antigen presenting cells used to prepare the biofilm components loaded on the surface of nano- or micro-vaccines can be derived from autologous or allogeneic, or from cell lines or stem cells.
  • Antigen presenting cells can be DC cells, B cells, macrophages, or any mixture of the above three, or other cells with antigen presenting function.
  • Antigen presenting cells can be activated by nanoparticles or microparticles loaded with antigens.
  • biomembrane components carried on the surface of cancer nano- or micro-vaccines are derived from extracellular vesicles, they may be one or more of the extracellular vesicles of cancer cells, extracellular vesicles of bacteria, or extracellular vesicles of antigen-presenting cells.
  • any method for preparing nanoparticles or micron particles known to those skilled in the art can be used to prepare the nanovaccine or micron vaccine described in the present disclosure, including but not limited to solvent evaporation method, dialysis method, microfluidics method, ultrafiltration method, homogenization emulsification method, dispersion method, precipitation method, etc.
  • the present disclosure takes the solvent volatilization method as an example and provides the following exemplary preparation method:
  • Step 1 lyse cancer cells and/or tumor tissue to obtain a lysate thereof.
  • Ultrapure water, aqueous solution, etc. can be used to lyse cancer cells or tumor tissue.
  • Cancer cells are one or more cancer cells or cancer cell lines; tumor tissue is derived from an organism. or tumor tissues of multiple organisms.
  • the lysis method is a commonly used lysis method for cancer cells or tumor tissues, including but not limited to one or more of repeated freezing and thawing, swelling, ultrasonic treatment, high pressure treatment, homogenization, extrusion, homogenization, high-speed stirring, chemical treatment, high shear force treatment, ultrafiltration treatment, shrinkage and the like.
  • a dissolving solution containing a dissolving agent can be used to lyse cancer cells or tumor tissues.
  • Cancer cells are one or more cancer cells or cancer cell lines; tumor tissues are tumor tissues derived from one or more organisms.
  • Step 2 After lysing cancer cells or tumor tissues using ultrapure water or aqueous solution, separate the water-soluble components and water-insoluble components in the lysate using one or more methods such as centrifugation, filtration, dialysis, ultrafiltration, etc. to obtain water-soluble components and water-insoluble components, respectively.
  • Dissolving agents include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine
  • Step 3 The water-soluble components in the lysate are subjected to salting out and/or heating treatment to obtain a precipitate, and then the precipitate is dissolved using a dissolving solution containing a dissolving agent; the water-insoluble components in the lysate are dissolved using a dissolving solution containing a dissolving agent, and then subjected to salting out and/or heating treatment, and then dissolved using a dissolving solution containing a dissolving agent; or the water-insoluble components in the lysate are directly dissolved using a dissolving solution containing a dissolving agent without treatment.
  • the dissolving agent in each step is selected separately.
  • Dissolving agents include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, guanidine salts, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone,
  • the water-soluble components in the lysate are subjected to salting out and/or heating treatment to obtain a precipitate and a supernatant, and then the precipitate is dissolved in a solvent containing a solvent, and the mRNA component in the supernatant is separated and extracted using an appropriate method, and then the mRNA component in the supernatant is mixed with the precipitate dissolved in the solvent containing a solvent for later use; the water-insoluble components in the lysate are dissolved in a solvent, and then subjected to salting out and/or heating treatment, and then dissolved in a solvent containing a solvent; or the water-insoluble components in the lysate are directly dissolved in a solvent containing a solvent without treatment.
  • the solvent in each step is selected separately.
  • the solvent includes but is not limited to metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, urea, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, One or more of methylguanidine hydrochloride, tetramethylguanidine hydrochloride, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine sulfamate, guanidine succinic acid, semicarbazide hydrochloride, carbamoyl urea, acetylurea, sulfonylurea compounds (glibenclamide,
  • the whole cell lysate component dissolved in a dissolving solution containing a dissolving agent is subjected to salting out and/or heating treatment to obtain a precipitate, and then the precipitate is dissolved in a dissolving solution containing a dissolving agent to obtain the desired antigen component.
  • Dissolving agents include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, guanidine salts, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone,
  • the whole-cell lysate component dissolved in a lytic solution containing a lytic agent is subjected to salting-out and/or heating treatment to obtain two parts, a precipitate and a supernatant, and then the precipitate part is dissolved using a lytic solution containing a lytic agent; an mRNA component is separated from the supernatant using an appropriate method, and then the mRNA component is mixed with the precipitate part dissolved in the lytic solution containing a lytic agent to obtain the desired antigen component.
  • Dissolving agents include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, polyhexamethyleneguanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine aminosulfonate, guanidine succinic acid, semicarbazide hydrochloride, aminoformyl urea, acetylurea, sulfonylurea compounds (glibenclamide, gliclazide, gliquidone,
  • salting out, heating and other methods are used to purify the antigen components in the cancer cell/tumor tissue lysate.
  • other methods such as enzymatic hydrolysis, phase separation, chromatographic separation, etc. that can remove the DNA component in the lysate or separate and purify the protein polypeptide and mRNA components therein can also be used.
  • Step 4 Load the protein and polypeptide components and/or the water-insoluble components or the protein and polypeptide components in the water-soluble components obtained in step 3 into the interior and/or surface of nanoparticles or microparticles, respectively or simultaneously, to obtain the nanovaccine or microvaccine; or load the antigen components obtained in step 3 into the interior and/or surface of nanoparticles or microparticles, respectively or simultaneously, to obtain the nanovaccine or microvaccine.
  • the initial aqueous phase and the organic phase are mixed, specifically, a first predetermined volume of aqueous solution containing a first predetermined concentration of antigen components is added to a second predetermined volume of organic phase containing a second predetermined concentration of raw materials for preparing particles.
  • the aqueous solution may contain at least one of the following i) to iii): i) components in cancer cell lysates, ii) components in tumor tissue lysates, and immunoenhancing adjuvants.
  • the components in the lysate are respectively the protein and polypeptide components in the water-soluble antigen during preparation, or the original water-insoluble antigen or some of its components dissolved in a dissolving solution containing a dissolving agent such as urea or guanidine hydrochloride.
  • the first predetermined concentration is the concentration of proteins and polypeptides contained in the aqueous solution, or the concentration of water-soluble antigens and/or the concentration of original water-insoluble antigens contained in the aqueous solution.
  • the first predetermined concentration requires that the protein and polypeptide concentration content be greater than 1 ng/mL so that sufficient antigen components can be loaded to activate related cells.
  • the concentration of the immunoenhancing adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.
  • the organic solvent is selected from dichloromethane.
  • the second predetermined concentration of the raw material for preparing particles ranges from 0.5 mg/mL to 5000 mg/mL, and is selected as 100 mg/mL.
  • the second predetermined volume of the organic phase is set according to the ratio of the second predetermined volume of the organic phase to the first predetermined volume of the aqueous phase.
  • the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is in the range of 1:1.1-1:5000, preferably 1:10.
  • the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.
  • the concentration of proteins and polypeptides is greater than 1 ng/mL, preferably 1 mg/mL to 100 mg/mL. In some embodiments, when the aqueous phase solution is a solution containing lysate components and immune adjuvants, the concentration of proteins and polypeptides is greater than 1 ng/mL, preferably 1 mg/mL to 100 mg/mL, and the concentration of immune adjuvants is greater than 0.01 ng/mL, preferably 0.01 mg/mL to 20 mg/mL.
  • the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane;
  • the concentration of the organic phase is 0.5 mg/mL to 5000 mg/mL, preferably 100 mg/mL.
  • the concentration of the protein and polypeptide components is greater than 0.01 ng/mL, preferably 1 ⁇ g/mL to 1 mg/mL. In some embodiments, when the aqueous phase solution is a solution containing protein and polypeptide components and an immune adjuvant, the concentration of the protein and components is greater than 1 ng/mL, preferably 1 ⁇ g/mL to 1 mg/mL, and the concentration of the immune adjuvant is greater than 0.01 ng/mL, preferably 0.01 mg/mL to 20 mg/mL.
  • the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane;
  • the concentration of the organic phase is 0.5 mg/mL to 5000 mg/mL, preferably 100 mg/mL.
  • Step 5 subjecting the mixed solution obtained in step 1 to any of the following treatments: i) ultrasonic treatment for more than 2 seconds; ii) stirring for more than 1 minute; iii) homogenization treatment; iv) microfluidic treatment.
  • the stirring is mechanical stirring or magnetic stirring
  • the stirring speed is greater than 50 rpm
  • the stirring time is greater than 1 minute, such as the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.1 hour to 24 hours
  • the ultrasonic power is greater than 5 W
  • the time is greater than 0.1 second, such as 2 to 200 seconds
  • a high pressure/ultra-high pressure homogenizer or a high shear homogenizer is used, and when a high pressure/ultra-high pressure homogenizer is used, the pressure is greater than 5 psi, such as 20 psi to 100 psi, and when a high shear homogenizer is used, the speed is greater than 100 rpm,
  • Ultrasound or stirring or homogenization or microfluidics treatment for nano- and/or micronization the length of ultrasound time or stirring speed or homogenization pressure Force and time can control the size of the prepared nanoparticles or micron particles. Too large or too small will lead to changes in particle size.
  • Step 6 adding the mixture obtained after the treatment in step 5 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of an emulsifier and performing any of the following treatments: i) ultrasonic treatment for more than 2 seconds; ii) stirring for more than 1 minute; iii) homogenization; iv) microfluidic treatment.
  • the mixture obtained in step 2 is added to the emulsifier aqueous solution and continues to be ultrasonicated or stirred or homogenized or mixed for nano- or micron-ization.
  • the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds
  • the stirring speed is greater than 50 rpm, such as 50 rpm to 500 rpm
  • the stirring time is greater than 1 minute, such as 60 to 6000 seconds.
  • the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, such as the stirring speed is 50rpm ⁇ 1500rpm, and the stirring time is 0.5 hour ⁇ 5 hours; during ultrasonic treatment, the ultrasonic power is 50W ⁇ 500W, and the time is greater than 0.1 second, such as 2 ⁇ 200 seconds; during homogenization, a high pressure/ultra-high pressure homogenizer or a high shear homogenizer is used, and the pressure is greater than 20psi when using a high pressure/ultra-high pressure homogenizer, such as 20psi ⁇ 100psi, and the speed is greater than 1000rpm when using a high shear homogenizer, such as 1000rpm ⁇ 5000rpm; the flow rate of microfluidics treatment is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasound or stirring or homogenization or microfluidics treatment is carried out for nano- or micronization, and the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared nanoparticles or micron particles. Too large or too small will bring about changes in particle size.
  • the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined volume is 5mL
  • the third predetermined concentration is 20mg/mL.
  • the third predetermined volume is adjusted according to its ratio with the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5.
  • the ratio of the second predetermined volume to the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time or homogenization time of this step the volume of the emulsifier aqueous solution and the concentration are based on the values all in order to obtain nanoparticles or micron particles of appropriate size.
  • Step 7 adding the liquid obtained after the treatment in step 6 to a fourth predetermined volume of an aqueous emulsifier solution with a fourth predetermined concentration, and stirring until a predetermined stirring condition is met.
  • the emulsifier aqueous solution is a PVA solution or other solutions.
  • the fourth predetermined concentration is 5 mg/mL.
  • the selection of the fourth predetermined concentration is based on obtaining nanoparticles or micron particles of suitable size.
  • the selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume.
  • 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 can be adjusted in order to control the size of the nanoparticles or micron particles.
  • the predetermined stirring condition of this step is until the organic solvent is completely volatilized, that is, the dichloromethane in step 1 is completely volatilized.
  • Step 8 after the mixed solution that meets the predetermined stirring conditions in step 7 is centrifuged at a speed greater than 100 RPM for more than 1 minute, the supernatant is removed, and the remaining precipitate is re-suspended in a fifth predetermined volume of an aqueous solution containing a fifth predetermined concentration of a lyophilized protective agent or a sixth predetermined volume of PBS (or physiological saline); or ultrafiltration centrifugation or dialysis that can remove substances with a specific molecular weight is used to remove free PVA and other substances, and at the same time, the solution in the system is replaced with a fifth predetermined volume of an aqueous solution containing a fifth predetermined concentration of a lyophilized protective agent or a sixth predetermined volume of PBS (or physiological saline). volume of PBS (or normal saline).
  • Step 9 freeze-drying the suspension containing the lyophilization protectant obtained in step 8, and keeping the lyophilized material for later use.
  • Step 10 directly use the suspension containing nanoparticles obtained in step 9 and resuspended in PBS (or physiological saline) with a sixth predetermined volume, or directly use the freeze-dried material containing nanoparticles or micron particles and lyoprotectant obtained in step 6 and resuspended with a sixth predetermined volume of PBS (or physiological saline); or use the above sample after mixing with a seventh predetermined volume of water-soluble antigen or dissolved original water-insoluble antigen.
  • the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferred volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1.
  • the nano vaccine or micro vaccine has 0.1-1000 ⁇ g of protein or polypeptide components per 1 mg of particle material. In some embodiments, or the nano vaccine or micro vaccine also has an immune enhancing adjuvant, wherein 1-400 ⁇ g of immune enhancing adjuvant is loaded per 1 mg of particle material. In some embodiments, the nano vaccine or micro vaccine also has a positively charged molecule, wherein 1-800 ⁇ g of positively charged molecules are loaded per 1 mg of particle material.
  • the interior and/or surface of the nanovaccine or microvaccine also contains membrane components, and the membrane components inside and/or on the surface of the nanovaccine or microvaccine are one or more mixed membranes selected from the cell membrane of antigen-presenting cells, the extracellular vesicles of antigen-presenting cells, the cell membrane of cancer cells, the extracellular vesicles of cancer cells, the cell membrane of bacteria, and the extracellular vesicles of bacteria.
  • the method of loading the membrane component onto the surface of the nanovaccine or microvaccine includes but is not limited to one or more of ultrasound, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bond connection, stirring, dialysis, homogenization and homogenization.
  • Step 11 using the nano vaccine or micro vaccine prepared in step 10 to prevent or treat diseases such as cancer.
  • experimental techniques and methods used in this example are all conventional technical methods unless otherwise specified.
  • the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • Example 1 Nanovaccine loaded with partially water-soluble components and water-insoluble components for the treatment of melanoma
  • ultrapure water is first used to lyse B16F10 melanoma tumor tissue to prepare protein and polypeptide components and water-insoluble antigens in the water-soluble components of the tumor tissue.
  • the nanovaccine is prepared by the solvent volatilization method using the organic polymer material PLGA as the nanoparticle skeleton material and Polyinosinic-polycytidylic acid (poly(I:C)) as the immune adjuvant.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mouse was killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells.
  • the lysate was centrifuged at 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into soluble in 8M urea aqueous solution. Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble component in the lysate, and after the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes, and the precipitate was dissolved in 8M urea aqueous solution for standby use.
  • the supernatant was heated at 95°C for 10 minutes and the obtained sample was centrifuged at 3000g for 5 minutes. After the supernatant was discarded, the precipitate was dissolved in 8M urea aqueous solution; then the precipitate after salting out and the precipitate after heating dissolved in 8M urea were combined and used as part of the water-soluble component.
  • the components obtained by salting out and heating the precipitate in the non-water-soluble component in the lysate dissolved in the above 8M urea aqueous solution and the water-soluble component dissolved in 8M urea are the antigen component 1 for preparing cancer nanovaccine 1.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mouse was killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells.
  • the lysate was centrifuged at 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble antigen insoluble in pure water was converted into soluble in 8M urea aqueous solution.
  • the components obtained by salting out and heating the precipitate in the non-water-soluble component in the lysate dissolved in the above 8M urea aqueous solution and the water-soluble component dissolved in 8M urea are the antigen component 2 for preparing cancer nanovaccine 2.
  • the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; adding 8M urea aqueous solution to the obtained precipitate to dissolve the precipitate can convert the water-insoluble antigen insoluble in pure water into soluble in 8M urea aqueous solution.
  • the water-insoluble component and water-soluble component in the lysate dissolved by the above 8M urea aqueous solution are the antigen component 3 for preparing cancer nanovaccine 3.
  • B16F10 cells 1.5 ⁇ 10 5 B16F10 cells were inoculated subcutaneously on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mice were killed and the tumor tissues were removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells.
  • the lysate was centrifuged at 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water can be converted into a soluble component in 8M urea aqueous solution. Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble component in the lysate. After the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes. After 10 minutes, the precipitate was solubilized with a 5% PEG5000 aqueous solution and set aside.
  • the supernatant was heated at 95°C for 10 minutes and the obtained sample was centrifuged at 3000g for 5 minutes. After the supernatant was discarded, the precipitate was solubilized with a 5% PEG5000 aqueous solution; then the precipitate after salting out and the precipitate after heating solubilized with a 5% PEG5000 aqueous solution were combined and used as part of the water-soluble component.
  • the components obtained by salting out and heating and precipitating in the water-soluble component dissolved in the 8M urea aqueous solution and the 5% PEG5000 aqueous solution are the antigen component 4 for preparing the cancer nanovaccine 4.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mouse was killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells.
  • the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 5% PEG5000 aqueous solution was added to the obtained precipitate to solubilize the precipitate to obtain the component of the water-insoluble component soluble in 5% PEG5000 aqueous solution. Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble components in the lysate. After the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes. The precipitate was solubilized with 5% PEG5000 aqueous solution and then set aside.
  • the supernatant was heated at 95°C for 10 minutes and the obtained sample was centrifuged at 3000g for 5 minutes. After the supernatant was discarded, the precipitate was solubilized with 5% PEG5000 aqueous solution; then the precipitate after salting out and the precipitate after heating solubilized with 5% PEG5000 aqueous solution were combined and used as part of the water-soluble components.
  • the components obtained by salting out and heating and precipitating the non-water-soluble components in the lysate dissolved with 5% PEG5000 aqueous solution and the water-soluble components dissolved with 5% PEG5000 aqueous solution are antigen component 5 for preparing cancer nanovaccine 5.
  • nanovaccine 1 (Nanovaccine 1) is prepared by the emulsion method in the solvent volatilization method. During the preparation, some of the water-soluble components in the tumor tissue lysate are components dissolved in 8M urea aqueous solution after ammonium sulfate salting out and heating separation purification.
  • nanoparticles loaded with water-soluble components of cancer cell lysates (components dissolved in 8M urea aqueous solution after ammonium sulfate salting out and heating separation purification) and nanoparticles loaded with water-insoluble antigens in whole cell antigens of cancer cells (dissolved in 8M urea aqueous solution) are prepared separately, and then used together as nanovaccine 1.
  • the PLGA molecular weight of the antigen delivery nanoparticle preparation material used is 10KDa-20KDa
  • the immune adjuvant used is poly(I:C) and poly(I:C) is loaded inside the nanoparticles.
  • the cell antigen components and adjuvants were first loaded inside the nanoparticles by the double emulsion method, and then 100 mg of nanoparticles were centrifuged at 15000g for 25 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanoparticle 1 was about 100 nm, and each 1 mg of PLGA nanoparticles was loaded with about 15 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA nanoparticles was loaded with 0.2 mg of poly(I:C).
  • Nanovaccine 2 The preparation method and preparation steps of Nanovaccine 2 are the same as those of Nanovaccine 1.
  • some of the water-soluble components in the tumor tissue lysate are components that are dissolved in 8M urea aqueous solution after being purified by ammonium carbonate salting out and heated separation purification.
  • nanoparticles loaded with the water-soluble parts of the cancer cell lysate (components that are dissolved in 8M urea aqueous solution after being purified by ammonium carbonate salting out and heated separation purification) and nanoparticles loaded with the water-insoluble antigens in the whole cell antigens of the cancer cells (dissolved in 8M urea aqueous solution) are prepared separately, and then used together as Nanovaccine 2.
  • the PLGA material used for the preparation of the antigen delivery nanoparticles has a molecular weight of 10KDa-20KDa, and the immune adjuvant used is poly(I:C), and poly(I:C) is loaded inside the nanoparticles.
  • the preparation method is as described above. In the preparation process, a complex is first used. The cell antigen components and adjuvants were loaded inside the nanoparticles by the emulsion method, and then 100 mg of the nanoparticles were centrifuged at 15000g for 25 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours.
  • the average particle size of the nanoparticle 2 was about 100 nm, and each 1 mg of PLGA nanoparticles loaded about 15 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA nanoparticles loaded 0.2 mg of poly(I:C).
  • Nanovaccine 3 The preparation method and preparation steps of Nanovaccine 3 are the same as those of Nanovaccine 1. During the preparation, all water-soluble components in the tumor tissue lysate are used directly without any treatment. During the preparation, nanoparticles loaded with water-soluble components in cancer cell lysate and nanoparticles loaded with water-insoluble antigens (dissolved in 8M urea aqueous solution) in whole cell antigens of cancer cells are prepared separately, and then used together as Nanovaccine 3.
  • the PLGA molecular weight of the antigen delivery nanoparticle preparation material used is 10KDa-20KDa, and the immune adjuvant used is poly(I:C) and poly(I:C) is loaded inside the nanoparticles.
  • the preparation method is as described above.
  • the cell antigen components and adjuvants are first loaded inside the nanoparticles by the double emulsion method, and then 100mg of nanoparticles are centrifuged at 15000g for 25 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of the nanoparticle 3 is about 100 nm.
  • Each 1 mg of PLGA nanoparticle is loaded with approximately 15 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA nanoparticle is loaded with 0.2 mg of poly(I:C).
  • Nanovaccine 4 The preparation method and preparation steps of Nanovaccine 4 are the same as those of Nanovaccine 1.
  • some of the water-soluble components in the tumor tissue lysate are components dissolved in a 5% PEG aqueous solution after purification by ammonium sulfate salting out and heating separation purification.
  • nanoparticles loaded with water-soluble components of cancer cell lysates (components dissolved in a 5% PEG5000 aqueous solution after purification by ammonium sulfate salting out and heating separation purification) and nanoparticles loaded with water-insoluble antigens of whole cell antigens of cancer cells (dissolved in an 8M urea aqueous solution) are prepared separately, and then used together as Nanovaccine 4.
  • the PLGA molecular weight of the antigen delivery nanoparticle preparation material used is 10KDa-20KDa
  • the immune adjuvant used is poly(I:C) and poly(I:C) is loaded inside the nanoparticles.
  • Preparation method As described above, during the preparation process, the cell antigen components and adjuvants were first loaded inside the nanoparticles by the double emulsion method, and then 100 mg of nanoparticles were centrifuged at 15000g for 25 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanoparticles 4 was about 100 nm, and each 1 mg of PLGA nanoparticles was loaded with about 15 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA nanoparticles was loaded with 0.2 mg of poly(I:C).
  • nano vaccine 5 (Nanovaccine 5) are the same as those of nano vaccine 1.
  • some of the water-soluble components in the tumor tissue lysate are components dissolved in a 5% PEG aqueous solution after being purified by ammonium sulfate salting out and heated separation purification.
  • nanoparticles loaded with water-soluble components of cancer cell lysate (components dissolved in a 5% PEG5000 aqueous solution after ammonium sulfate salting out and heated separation purification) and nanoparticles loaded with water-insoluble antigens in whole cell antigens of cancer cells (dissolved in a 5% PEG5000 aqueous solution) are prepared separately, and then used together as nano vaccine 5.
  • the PLGA molecular weight of the antigen delivery nanoparticle preparation material used is 10KDa-20KDa
  • the immune adjuvant used is poly(I:C) and poly(I:C) is loaded inside the nanoparticles.
  • the cell antigen components and adjuvants are first loaded inside the nanoparticles by the double emulsion method, and then 100 mg of nanoparticles are centrifuged at 15000g for 25 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanoparticles 5 is about 100 nm, and each 1 mg of PLGA nanoparticles is loaded with about 15 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.2 mg of poly(I:C).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated at the lower right back of each mouse. On the 3rd, 6th, 9th, 14th and 20th days before the mice were inoculated with tumors, 2 mg of nanovaccine 1 (1 mg of nanoparticles loaded with purified components in water-soluble components + 1 mg of nanoparticles loaded with non-water-soluble components) or 2 mg of nanovaccine 2 (1 mg of nanoparticles loaded with purified components in water-soluble components + 1 mg of nanoparticles loaded with non-water-soluble components) or 2 mg of nanovaccine 3 (1 mg of nanoparticles loaded with water-soluble components + 1 mg of nanoparticles loaded with non-water-soluble components) or 2 mg of nanovaccine 4 (1 mg of nanoparticles loaded with purified components in water-soluble components + 1 mg of nanoparticles loaded with non-water-soluble components) or 2 mg
  • the tumor growth rate and survival of the mice were monitored.
  • the size of the tumor volume of the mice was recorded every 3 days starting from the 3rd day.
  • v 0.52 ⁇ a ⁇ b 2
  • v 0.52 ⁇ a ⁇ b 2
  • mice in the PBS group in Figure 3 grew rapidly and the mice died soon.
  • the tumor growth rate of mice using nano vaccine 1 (Nanovaccine 1), nano vaccine 2 (Nanovaccine 2), nano vaccine 3 (Nanovaccine 3), nano vaccine 4 (Nanovaccine 4) and nano vaccine 5 (Nanovaccine 5) was significantly slowed down, and the survival period was significantly prolonged. Some mice recovered without tumors.
  • nano vaccine 1 was better than nano vaccine 2, nano vaccine 3, nano vaccine 4 and nano vaccine 5, indicating that the effect of using ammonium sulfate salting out to separate and purify the protein and polypeptide components in the water-soluble components is better than using ammonium carbonate, and the effect of separating and purifying some components in the water-soluble components is better than using the water-soluble components directly.
  • the precipitation produced by using urea to dissolve the water-soluble components after salting out and heating treatment is better than using PEG.
  • Example 2 Micronized vaccine for the treatment of melanoma
  • ultrapure water is first used to lyse cancer cells isolated from B16F10 melanoma tumor tissue. After the separated cancer cells are cultured, the protein and polypeptide components in the water-soluble components of the tumor tissue and the water-insoluble antigen dissolved in 6M guanidine hydrochloride are prepared, and then the two are mixed and loaded on the micron vaccine.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mouse was killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and a single cell suspension was prepared through a cell filter.
  • the cancer cells in the tumor tissue were separated and cultured in RPMI1640 (containing 10% FBS) complete medium for 7 days (37°C, 5% CO 2 ). After the culture was completed, the cancer cells were collected and centrifuged at 400g for 5 minutes to remove the medium.
  • the precipitated cancer cells were resuspended in an appropriate amount of ultrapure water and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy and lyse the cancer cells. After lysis, the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 6M guanidine hydrochloride aqueous solution was added to the obtained precipitate to dissolve the precipitate, so that the water-insoluble antigen insoluble in pure water could be converted into a soluble antigen in 6M guanidine hydrochloride aqueous solution.
  • the water-soluble components in the lysate are mixed with the water-insoluble components in the lysate dissolved in 6M guanidine hydrochloride aqueous solution at a mass ratio of 1:1 without any treatment to obtain antigen component 3 for preparing micron vaccine 3 (Micronvaccine 3).
  • the micron vaccine 1 (Micronvaccine 1) is prepared by the emulsion method in the solvent evaporation method. During the preparation, the antigen component 1 is loaded on the micron vaccine 1.
  • the molecular weight of the micron particle preparation material PLGA used is 38KDa-54KDa
  • the immune adjuvant used is poly(I:C) and poly(I:C) and CpG ODN7909, and the antigen component and adjuvant are loaded inside the micron particles.
  • the preparation method is as described above.
  • the cell antigen component and adjuvant are first loaded inside the micron particles by the emulsion method, and then 100mg of micron particles are centrifuged at 8000g for 30 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of the micron vaccine 1 is about 1.2 ⁇ m, and each 1mg PLGA micron particle is loaded with about 10 ⁇ g of protein or polypeptide components, and each 1mg PLGA micron particle is loaded with 0.01mg of poly(I:C) and Cp G ODN.
  • micron vaccine 2 (Micronvaccine 2) in this embodiment are the same as those of micron vaccine 1.
  • antigen component 2 is loaded on micron vaccine 2.
  • the molecular weight of the micron particle preparation material PLGA used is 38KDa-54KDa
  • the immune adjuvant used is poly(I:C) and poly(I:C) and CpG ODN7909, and the antigen component and adjuvant are loaded inside the micron particle.
  • the preparation method is as described above.
  • the cell antigen component and adjuvant are first loaded inside the micron particle by the double emulsion method, and then 100mg of micron particles are centrifuged at 8000g for 30 minutes, and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of the micron vaccine 2 is about 1.2 ⁇ m, and each 1mg PLGA micron particle is loaded with about 10 ⁇ g of protein or polypeptide components, and each 1mg PLGA micron particle is loaded with 0.01mg of poly(I:C) and Cp G ODN.
  • micron vaccine 3 in this embodiment are the same as those of micron vaccine 1.
  • antigen component 3 is loaded on micron vaccine 3.
  • the molecular weight of the micron particle preparation material PLGA used is 38KDa-54KDa
  • the immune adjuvant used is poly(I:C) and poly(I:C) and CpG ODN7909
  • the antigen component and adjuvant are loaded on the inside of the micron particle.
  • the preparation method is as described above.
  • the cell antigen component and adjuvant are first loaded on the inside of the micron particle by the double emulsion method, and then 100mg of micron particles are centrifuged at 8000g for 30 minutes, and 10mL of ultrapure water containing 4% trehalose is used for resuspending and freeze drying for 48h.
  • the average particle size of the micron vaccine 3 is about 1.2 ⁇ m, and each 1mg of PLGA micron particles is loaded with about 10 ⁇ g of protein or polypeptide components, and each 1mg of PLGA micron particles is loaded with 0.01mg of poly(I:C) and Cp G ODN.
  • micron vaccine 4 in this embodiment are the same as those of micron vaccine 1.
  • equal amounts of four melanoma polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) are loaded simultaneously.
  • the molecular weight of the micron particle preparation material PLGA used is 38KDa-54KDa
  • the immune adjuvants used are poly(I:C) and CpG ODN7909, and the polypeptide and adjuvant are simultaneously loaded inside the micron particles.
  • Preparation method As described above, in the preparation process, the cell antigen components and adjuvants are first loaded inside the nanoparticles by the double emulsion method, and then 100 mg of micronized particles are centrifuged at 10000g for 20 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the micronized vaccine 4 is about 1.2 ⁇ m, and each 1 mg of PLGA micronized particles is loaded with about 10 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA micronized particles is loaded with 0.01 mg of poly(I:C) and Cp G ODN.
  • mice in the PBS group grew rapidly and the mice died soon.
  • the tumor growth rate of mice using Micronvaccine 1, Micronvaccine 2, Micronvaccine 3 and Micronvaccine 4 was significantly slowed down, the survival period was significantly prolonged, and some of the mice recovered.
  • Micronvaccine 1 was better than Micronvaccine 2, Micronvaccine 3 and Micronvaccine 4, indicating that the effect of using magnesium sulfate salting out to separate and purify the protein and polypeptide components in the water-soluble components is better than using sodium silicate, and the effect of separating and purifying a part of the water-soluble components is better than using the water-soluble components directly.
  • using a part of the water-soluble components in the lysate is better than using the new antigen polypeptide.
  • ultrapure water was first used to lyse B16F10 melanoma cancer cells to prepare protein and polypeptide components and water-insoluble components in the water-soluble components of the cancer cells, and then PLA and PEG-modified PLA were used as nanoparticle skeleton materials to prepare nanovaccines.
  • the cultured B16F10 cells were collected, the supernatant was discarded, and an appropriate amount of ultrapure water was added to the cancer cell pellet to resuspend and freeze-thaw repeatedly for 5 times, accompanied by ultrasound to lyse the cells.
  • the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 10% sodium deoxycholate aqueous solution was added to the obtained precipitate to dissolve the precipitate, so that the water-insoluble antigen insoluble in pure water can be converted into a soluble antigen in 10% sodium deoxycholate aqueous solution.
  • the water-soluble components in the lysate were heated at 100°C for 10 minutes, and the obtained sample was centrifuged at 3000g for 5 minutes. The supernatant was discarded and the precipitate was dissolved in a 10% sodium deoxycholate aqueous solution.
  • the components obtained by heating and precipitating the water-soluble components dissolved in the 10% sodium deoxycholate aqueous solution and the water-insoluble components in the lysate dissolved in the 10% sodium deoxycholate aqueous solution were mixed at a mass ratio of 10:1 to prepare the antigen component 1 of the nanovaccine 1.
  • the water-soluble components in the lysate are used directly without any treatment.
  • the water-soluble components and the water-insoluble components in the lysate dissolved in 10% sodium deoxycholate aqueous solution are mixed at a mass ratio of 10:1 to prepare the antigen component 2 of the nano vaccine 2.
  • the nano vaccine 1 (Nanovaccine 1) is prepared by the double emulsion method in the solvent evaporation method.
  • the nano vaccine preparation materials used are PLA (molecular weight of 10KDa-20KDa) and PEG5000-PLA (PLA molecular weight of 10KDa-20KDa), and the mass ratio of PLA and PEG5000-PLA used is 200:1;
  • the immune adjuvants used are poly (I:C), CpG SL01 (class B) and CpG SL03 (class C), and the adjuvants are loaded inside the nano vaccine.
  • the cell antigen component 1 and the adjuvant are first loaded inside the nanoparticles by the double emulsion method, and then 100mg of nanoparticles are centrifuged at 14000g for 20 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of the nanoparticle 1 is about 500 nm.
  • Each 1 mg of PLA nanoparticles is loaded with approximately 50 ⁇ g of protein or polypeptide components, and 0.2 mg each of poly(I:C), CpG SL01 (type B) and CpG SL03 (type C).
  • nano vaccine 2 (Nanovaccine 2) in this embodiment are the same as those of nano vaccine 1.
  • the nano vaccine preparation materials used are PLA (molecular weight of 10KDa-20KDa) and PEG5000-PLA (PLA molecular weight of 10KDa-20KDa), and the molar ratio of PLA and PEG5000-PLA used is 200:1;
  • the immune adjuvants used are poly (I:C), CpG SL01 (class B) and CpG SL03 (class C), and the adjuvants are loaded inside the nano vaccine.
  • the preparation method is as described above.
  • the cell antigen component 2 and the adjuvant are first loaded inside the nanoparticles by the emulsion method, and then 100 mg of nanoparticles are centrifuged at 14000g for 20 minutes, and resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanoparticle 2 is about 500 nm.
  • Each 1 mg of PLA nanoparticles is loaded with approximately 50 ⁇ g of protein or polypeptide components, and 0.2 mg each of poly(I:C), CpG SL01 (type B) and CpG SL03 (type C).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse. On the 3rd, 6th, 9th, 14th and 20th days before the mice were inoculated with tumors, 100 ⁇ L of 0.5 mg nanovaccine 1 (0.25 mg nanoparticles loaded with purified components in water-soluble components + 0.25 mg nanoparticles loaded with non-water-soluble components) or 100 ⁇ L of 0.5 mg nanovaccine 2 (0.25 mg nanoparticles loaded with water-soluble components + 0.25 mg nanoparticles loaded with non-water-soluble components) or 100 ⁇ L of PBS were subcutaneously injected into the mice.
  • 0.5 mg nanovaccine 1 (0.25 mg nanoparticles loaded with purified components in water-soluble components + 0.25 mg nanoparticles loaded with non-water-soluble components
  • 0.5 mg nanovaccine 2 0.25 mg nanop
  • the tumor growth rate and survival of the mice were monitored.
  • the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day.
  • Nanovaccines 1 was more effective than Nanovaccines 2, indicating that the use of heated purification of protein and peptide components in water-soluble components can improve the vaccine effect, and the effect of separating and purifying some components in water-soluble components is better than using water-soluble components directly.
  • LLC mouse lung cancer cells were first lysed using ultrapure water to prepare protein and polypeptide components and water-insoluble components in the water-soluble components of the cancer cells, and then the nano-vaccine was prepared using PLGA as the nanoparticle skeleton material.
  • the supernatant was discarded and an appropriate amount of ultrapure water was added to the cell pellet to resuspend the cells and then freeze-thawed repeatedly 5 times.
  • the lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was taken as the water-soluble component soluble in pure water; 0.1% octyl glucoside aqueous solution was added to the obtained precipitate to dissolve the precipitate, thereby converting the water-insoluble antigen insoluble in pure water into one soluble in 0.1% octyl glucoside aqueous solution.
  • a saturated sodium chloride aqueous solution is added dropwise to the water-soluble component in the lysate, and after the precipitation is complete, the obtained sample is centrifuged at 3000g for 5 minutes, and the precipitate is dissolved in a 0.1% octyl glucoside aqueous solution for standby use, and the supernatant is heated at 80°C for 30 minutes and then the obtained sample is centrifuged at 3000g for 5 minutes, and the supernatant is discarded and the precipitate is dissolved in a 0.1% octyl glucoside aqueous solution; then the precipitate after salting out and the precipitate after heating dissolved with the 0.1% octyl glucoside aqueous solution are combined and used as part of the water-soluble component.
  • the non-water-soluble component in the lysate dissolved with the 0.1% octyl glucoside aqueous solution and the component obtained by salting out and heating precipitation in the water-soluble component dissolved with 0.1% octyl glucoside are mixed at a mass ratio of 10:1 to prepare the antigen component 1 of the cancer nanovaccine 1.
  • the non-water-soluble component in the lysate dissolved with 0.1% octyl glucoside aqueous solution and the component obtained by salting out and heating precipitation in the water-soluble component dissolved with 0.1% octyl glucoside are mixed at a mass ratio of 10:1 to prepare the antigen component of cancer nanovaccine 2.
  • Nano vaccine 1 adopts the multiple emulsion method in the solvent evaporation method to prepare in the present embodiment.
  • the nano vaccine preparation material PLGA molecular weight adopted is 7KDa-17KDa
  • the immunoadjuvant adopted is poly (I: C)
  • adjuvant and antigen component are loaded in the nano vaccine inside.
  • Preparation method As mentioned above, in the preparation process, first adopt the multiple emulsion method in nano particle internal load cell antigen component 1 and adjuvant, then 100mg nano particle is centrifuged at 12000g for 20 minutes, and use 10mL to contain 4% trehalose ultrapure water resuspended back freeze drying 48h.
  • the nano vaccine 1 has an average particle size of about 280 nm, and each 1 mg of PLGA nanoparticles is loaded with about 200 ⁇ g of protein and polypeptide components, and 0.02 mg of poly(I:C), CpG ODN 1018 (type B) and CpG ODN M362 (type C) are loaded.
  • Nanovaccine 2 The preparation method and preparation steps of Nanovaccine 2 are the same as those of Nanovaccine 1.
  • the PLGA molecular weight of the nanovaccine preparation material used is 7KDa-17KDa
  • the immune adjuvants used are poly(I:C), CpG ODN 1018 (Class B) and CpG ODN M362 (Class C)
  • the adjuvant and antigen components are co-loaded inside the nanovaccine.
  • the preparation method is as described above.
  • the cell antigen component 2 and adjuvant are first loaded inside the nanoparticles by the double emulsion method, and then 100 mg of nanoparticles are centrifuged at 12000g for 20 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanovaccine 2 is about 280 nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 200 ⁇ g of protein or polypeptide components, and 0.02 mg each of poly(I:C), CpG ODN 1018 (Type B) and CpG ODN M362 (Type C).
  • Nanovaccine 3 The preparation materials and methods of Nanovaccine 3 are basically the same as those of Nanovaccine 1.
  • the average particle size of Nanovaccine 3 is about 280 nm. 1 mg of PLGA nanoparticles is loaded with 0.02 mg each of poly(I:C), CpG ODN 1018 (Type B) and CpG ODN M362 (Type C). The difference is that Nanovaccine 3 does not load any antigen components.
  • BMDC Bone marrow-derived dendritic cells
  • B cells were used as antigen presenting cells.
  • the preparation of BMDC was the same as in Example 1.
  • the B cell extraction process was as follows: the mouse spleen was removed after the mouse was killed, and then a mouse spleen cell single cell suspension was prepared, and then CD19 + B cells were sorted from the spleen cell single cell suspension using a magnetic bead sorting method.
  • BMDC and B cells were mixed in a quantitative ratio of 1:1 and used as mixed antigen presenting cells.
  • Nanovaccine 1 500 ⁇ g or nanovaccine 2 (500 ⁇ g) or nanovaccine 3 (500 ⁇ g) + free lysate were co-incubated with 20 million mixed antigen-presenting cells (10 million BMDCs + 10 million B cells) in 15 mL RPMI1640 complete culture medium for 48 hours (37°C, 5% CO2 ); the incubation system contained a cytokine combination: IL-15 (500 U/mL), IL-7 (500 U/mL), and IL-21 (1000 U/mL).
  • IL-15 500 U/mL
  • IL-7 500 U/mL
  • IL-21 1000 U/mL
  • the nanovaccine 1 was co-incubated with 20 million BMDCs in 15 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contained a cytokine combination: IL-15 (500 U/mL), IL-7 (500 U/mL), and IL-21 (1000 U/mL).
  • IL-15 500 U/mL
  • IL-7 500 U/mL
  • IL-21 1000 U/mL
  • the sample was then filtered through a filter membrane with a pore size of 50 ⁇ m, 10 ⁇ m, 5 ⁇ m, 1 ⁇ m, 0.45 ⁇ m, and 0.22 ⁇ m, and the filtrate was collected and co-incubated with the corresponding nano vaccine (50mg) prepared in step (2) for 10 minutes, and then co-extruded repeatedly using a 400nm filter membrane, and the extrudate was centrifuged at 15000g for 60 minutes, and the supernatant was discarded and the precipitate was resuspended with saline to obtain nanoparticles.
  • the nanoparticles obtained by the co-action of the mixed antigen presenting cell membrane components activated by nanovaccine 1 and nanovaccine 1 are nanoparticles 4 (Nanovaccine 4), with a particle size of 300nm and a surface potential of -6mV; each 1mg of PLGA nanoparticles is loaded with about 200 ⁇ g of protein or polypeptide components, and 0.02mg of poly(I:C), CpG ODN 1018 (class B) and CpG ODN M362 (class C) are loaded; each 1mg of PLGA nanoparticles is loaded with about 100 ⁇ g of cell membrane components.
  • the nanoparticles obtained by the co-action of the mixed antigen presenting cell membrane components activated by nanovaccine 2 and nanovaccine 2 are nanoparticles 5 (Nanovaccine 5), with a particle size of 300nm and a surface potential of -6mV; each 1mg of PLGA nanoparticles is loaded with about 100 ⁇ g of cell membrane components. About 200 ⁇ g of protein or peptide components are loaded, and 0.02mg of poly(I:C), CpG ODN 1018 (B type) and CpG ODN M362 (C type) are loaded; about 100 ⁇ g of cell membrane components are loaded per 1mg of PLGA nanoparticles.
  • nanoparticles prepared by the co-action of the mixed antigen presenting cell membrane components activated by nanovaccine 3 and nanovaccine 3 are nanovaccine 6, with a particle size of 300nm and a surface potential of -6mV; about 200 ⁇ g of protein or peptide components are loaded per 1mg of PLGA nanoparticles, and 0.02mg of poly(I:C), CpG ODN 1018 (B type) and CpG ODN M362 (C type) are loaded; about 100 ⁇ g of cell membrane components are loaded per 1mg of PLGA nanoparticles.
  • nanovaccine 7 prepared with antigen-presenting cell membranes, with a particle size of 200nm and a surface potential of -4mV.
  • BMDCs incubated with nanovaccine 1 were collected by centrifugation at 400 g for 5 minutes, and then the BMDCs were washed twice with physiological saline, and the cells were resuspended in physiological saline and then subjected to low-power 7.5 W ultrasound at 4°C for 10 minutes to destroy the cells and prepare a sample containing cell membrane components.
  • the sample was then filtered through a filter membrane with a pore size of 50 ⁇ m, 10 ⁇ m, 5 ⁇ m, 1 ⁇ m, 0.45 ⁇ m, and 0.22 ⁇ m.
  • nanovaccine 1 50 mg
  • step (2) The filtrate was collected and incubated with the nanovaccine 1 (50 mg) prepared in step (2) for 10 minutes, and then co-extruded repeatedly using a 400 nm filter membrane.
  • the extrudate was centrifuged at 15000 g for 60 minutes, the supernatant was discarded, and the supernatant was resuspended in ultrapure water containing 4% trehalose and freeze-dried for 48 hours for use. That is, nanovaccine 8, with a particle size of 300 nm and a surface potential of -6 mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 200 ⁇ g of protein and peptide components, and 0.02 mg each of poly(I:C), CpG ODN 1018 (Type B) and CpG ODN M362 (Type C); each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of cell membrane components.
  • the sample was then filtered through a filter membrane with a pore size of 50 ⁇ m, 10 ⁇ m, 5 ⁇ m, 1 ⁇ m, 0.45 ⁇ m, and 0.22 ⁇ m, and the filtrate was collected and co-incubated with the corresponding nanovaccine 1 (50mg) prepared in step (2) for 10 minutes, and then co-extruded repeatedly using a 400nm filter membrane, and the extrudate was centrifuged at 15000g for 60 minutes, and the supernatant was discarded and the precipitate was resuspended with saline to obtain the nanovaccine 9.
  • the nanovaccine is Nanovaccine 9, with a particle size of 300nm and a surface potential of -6mV.
  • Each 1mg of PLGA nanoparticles is loaded with approximately 200 ⁇ g of protein or polypeptide components, and 0.02mg each of poly(I:C), CpG ODN 1018 (Class B) and CpG ODN M362 (Class C). Each 1mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of cell membrane components.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare lung cancer tumor-bearing mice. On day 0, 1.5 ⁇ 10 6 LLC lung cancer cells were subcutaneously inoculated in the lower right back of each mouse. On the 3rd, 6th, 9th, 14th and 20th days before the mice were inoculated with tumors, 100 ⁇ L of 0.2 mg nanovaccines (nanovaccines 1, 2 or 3) were subcutaneously injected into the mice. The mice were treated with 100 ⁇ L of PBS (100 ⁇ L of PBS or 100 ⁇ L of 5 ⁇ L of 5 ⁇ L of 1 ...
  • the results in Figure 6 show that the tumor growth rate of mice in the PBS control group is very fast and the survival period of mice is very short. Compared with the control group, the tumor growth rate of mice in the vaccine group is significantly slowed down, and some mice have disappeared and recovered.
  • nano vaccine 4 has the best effect, and nano vaccine 4 is significantly better than other nano vaccines.
  • the effects of nano vaccine 4 and nano vaccine 5 are better than nano vaccine 9; and nano vaccine 1, nano vaccine 5 and nano vaccine 8 are significantly better than nano vaccine 3, nano vaccine 6 and nano vaccine 7.
  • nano vaccine is loaded with antigen presenting cell membrane components on the surface, which is beneficial to improve the effect of nano vaccines with some cancer cell components loaded inside; and the membrane components of mixed antigen presenting cells using dendritic cells (DC) and B cells are better than the membrane components of single antigen presenting cells DC. Moreover, the membrane components of antigen presenting cells activated by nanoparticles loaded with cancer cell antigens are better than the membrane components of antigen presenting cells not activated by nanoparticles.
  • DC dendritic cells
  • B cells single antigen presenting cells
  • the cell membrane fraction of antigen presenting cells is used.
  • the membrane fraction of extracellular vesicles secreted by antigen presenting cells may also be used; or the cell membrane fraction of bacteria or cancer cells may also be used at the same time.
  • the membrane components of antigen presenting cells are located on the surface of the nano-vaccine or micro-vaccine.
  • the membrane components of antigen presenting cells, bacteria or cancer cells can also be loaded inside the nano-vaccine or micro-vaccine.
  • ultrapure water is first used to lyse MC38 mouse lung and colon cancer cells to prepare protein and polypeptide components and water-insoluble components in the water-soluble components of cancer cells, and then a nano vaccine is prepared to treat colon cancer.
  • the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells are loaded on the surface of the nano vaccine.
  • the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells can also be loaded inside the nano vaccine as needed; or loaded on the surface of the nano vaccine and inside the nano vaccine at the same time.
  • the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells used in this embodiment can also use the outer membrane components of bacteria or the membrane components of cancer cells in practical applications; or the membrane components (cell membranes or extracellular vesicle membranes) of antigen-presenting cells loaded with cancer cell antigens and any mixed membrane components derived from cancer cell membrane components and/or membrane components derived from bacteria can also be used.
  • the supernatant was discarded and an appropriate amount of ultrapure water was added to the cell pellet to resuspend the cells and then freeze-thawed repeatedly 5 times.
  • the lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was taken as the water-soluble component soluble in pure water; 10% sodium dodecyl sulfate (SDS) aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble antigen that was insoluble in pure water was converted into a soluble antigen in 10% SDS aqueous solution.
  • SDS sodium dodecyl sulfate
  • the non-water-soluble components in the lysate dissolved in the above 10% SDS aqueous solution and the components obtained by salting out and heating and precipitating in the water-soluble components dissolved in 10% SDS are mixed in a mass ratio of 1:1 to prepare the antigen for the cancer nanovaccine.
  • nano vaccine 1 (Nanovaccine 1) is prepared by the emulsion method in the solvent evaporation method. During the preparation, the water-insoluble components in the cancer cell lysate (dissolved in 10% SDS aqueous solution) and some components in the water-soluble components in the cancer cell lysate (components dissolved in 10% SDS aqueous solution after salting out purification) are mixed in a mass ratio of 1:1 and used as antigen components.
  • the molecular weight of the nano vaccine preparation material PLGA used is 7KDa-17KDa
  • the immune adjuvants used are poly (I: C), CpG ODN 7909 (Class B) and CpG ODN 2395 (Class C)
  • the substance used to promote lysosomal escape is bee venom peptide, and the adjuvant, bee venom peptide and antigen components are co-loaded inside the nano vaccine.
  • the cell antigen components and adjuvants are first loaded inside the nanoparticles by the double emulsion method, and then 100 mg of nanoparticles are centrifuged at 12000g for 20 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanovaccine 1 is about 380 nm, and each 1 mg of PLGA nanoparticles is loaded with about 400 ⁇ g of protein or polypeptide components of cancer cells, 0.05 mg of poly (I: C), CpG ODN 7909 (B type) and CpG ODN 2395 (C type), and 0.1 mg of bee venom peptide.
  • nano vaccine 2 in this embodiment is the same as that of nano vaccine 1.
  • the water-insoluble components in the cancer cell lysate dissolved in 10% SDS aqueous solution
  • some components in the water-soluble components in the cancer cell lysate are mixed in a mass ratio of 1:1 and used as antigen components.
  • the molecular weight of the nano vaccine preparation material PLGA used is 7KDa-17KDa
  • the immune adjuvants used are poly (I: C), CpG ODN 7909 (Class B) and CpG ODN 2395 (Class C), and the adjuvant and antigen components are co-loaded inside the nano vaccine.
  • the preparation method is as described above. In the preparation process, the cell antigen components and adjuvants are first loaded inside the nanoparticles by the double emulsion method, and then 100mg of nanoparticles are centrifuged at 12000g for 20 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of the nanovaccine 2 is about 380 nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 400 ⁇ g of protein or polypeptide components of cancer cells, and 0.05 mg each of poly(I:C), CpG ODN 7909 (Class B) and CpG ODN 2395 (Class C).
  • Nanovaccine 3 The preparation materials and methods of Nanovaccine 3 are the same as those of Nanovaccine 1.
  • the average particle size of Nanovaccine 3 is about 380 nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 400 ⁇ g of protein or polypeptide components, without any adjuvant.
  • Bifidobacterium longum was centrifuged at 5000g for 30 minutes, and the precipitate was discarded to collect the supernatant, which was filtered using a 1 ⁇ m filter membrane, treated with 20W ultrasound at 4°C for 5 minutes, and then centrifuged at 16000g for 90 minutes. The precipitate was resuspended in PBS to obtain the bacterial extracellular vesicle membrane component.
  • the cancer cells are centrifuged at 5000g for 30 minutes, and the precipitate is discarded and the supernatant is collected.
  • the supernatant is filtered using a 1 ⁇ m filter membrane, treated with 20W ultrasound at 4°C for 5 minutes, and then centrifuged at 16000g for 90 minutes.
  • the precipitate is resuspended in PBS to obtain the extracellular vesicle membrane component of the collected cancer cells.
  • the bacterial extracellular vesicle membrane component and the cancer cell extracellular vesicle membrane component are mixed in a mass ratio of 1:1 and set aside.
  • nano vaccine 1, or nano vaccine 2, or nano vaccine 3 prepared in step (2) was mixed with 5 mg of the mixed membrane component of cancer cells and bacterial extracellular vesicles prepared in step (3), and then allowed to stand for 1 minute. Then, low-power 7.5 W ultrasound was used at 4°C for 1 minute, and then the mixture was repeatedly co-extruded through a filter membrane with a pore size of 0.45 ⁇ m. The filtrate was collected and freeze-dried to obtain the nano vaccine.
  • the nano vaccine 4 obtained by co-acting the nano vaccine 1 with the mixed membrane component of cancer cells and bacterial extracellular vesicles had a particle size of 400 nm and a surface potential of -6 mV; each 1 mg of PLGA nanoparticles was loaded with approximately 400 ⁇ g of protein or polypeptide components, 0.05 mg of poly (I: C), CpG ODN 7909 (class B) and CpG ODN 2395 (class C), and 0.1 mg of bee venom peptide; each 1 mg of PLGA nanoparticles was loaded with approximately 20 ⁇ g of membrane components.
  • Nanovaccine 5 was prepared by co-acting nanovaccine 2 with the mixed membrane components of cancer cells and bacterial extracellular vesicles. The particle size was 400nm and the surface potential was -6mV. Each 1mg PLGA nanoparticle was loaded with about 400 ⁇ g of protein or peptide components, and 0.05mg of poly(I:C), CpG ODN 7909 (B type) and CpG ODN 2395 (C type) were loaded. Each 1mg PLGA nanoparticle was loaded with about 20 ⁇ g of membrane components.
  • Nanovaccine 6 was prepared by co-acting nanovaccine 3 with the mixed membrane components of cancer cells and bacterial extracellular vesicles. The particle size was 400nm and the surface potential was -6mV. Each 1mg PLGA nanoparticle was loaded with about 400 ⁇ g of protein or peptide components, and no adjuvant was loaded. Each 1mg PLGA nanoparticle was loaded with about 20 ⁇ g of membrane components.
  • nanovaccine 4 was better than that of nanovaccine 1; the effect of nanovaccine 5 was better than that of nanovaccine 2; the effect of nanovaccine 6 was better than that of nanovaccine 3; the effects of nanovaccine 4 and nanovaccine 5 were better than those of nanovaccine 6; the effects of nanovaccine 1 and nanovaccine 2 were better than those of nanovaccine 3; the effect of nanovaccine 4 was better than that of nanovaccine 5; and the effect of nanovaccine 1 was better than that of nanovaccine 2.
  • the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells are loaded on the surface of the nanovaccine.
  • the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells can also be loaded inside the nanovaccine as needed; or loaded on the surface of the nanovaccine and inside the nanovaccine at the same time.
  • the extracellular vesicles of bacteria and the extracellular vesicles of cancer cells used in this embodiment can also use the outer membrane components of bacteria or the membrane components of cancer cells in practical applications; Alternatively, a mixed membrane fraction of a membrane fraction (cell membrane or extracellular vesicle membrane) of an antigen-presenting cell loaded with a cancer cell antigen and a membrane fraction derived from cancer cells and/or a membrane fraction derived from bacteria may be used.
  • ultrasound, co-incubation and co-extrusion methods were used to load the membrane components onto the surface of the nanovaccine.
  • any method that can load the membrane components onto the surface of the nanovaccine or micron vaccine through co-action can also be used, including but not limited to one or more of ultrasound, co-incubation, co-extrusion, ultrafiltration, centrifugation, dialysis, chemical bond connection, stirring, dialysis, homogenization and homogenization.
  • the membrane components of bacteria and cancer cells are located on the surface of the nano-vaccine or micro-vaccine.
  • the membrane components of bacteria or cancer cells can also be loaded inside the nano-vaccine or micro-vaccine.
  • the vaccine disclosed in the present invention has excellent therapeutic effects on cancer.
  • 6M guanidine hydrochloride was first used to lyse the whole-cell antigens of 4T1 breast cancer cancer cells. After the antigens were purified by salting out and heating, micron vaccines were prepared using CpG BW006 (class B), CPG2216 (class A) and Poly ICLC as immune adjuvants.
  • the cultured 4T1 breast cancer cell line was collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, then the cells were resuspended in ultrapure water containing 0.1% protease inhibitors and repeatedly frozen and thawed 5 times to lyse the cancer cells, and then the lysate components were dissolved using a 6M guanidine sulfate aqueous solution.
  • micron vaccine 1 (Micronvaccine 1) is prepared by the double emulsion method.
  • the molecular weight of the PLGA material used for preparing micron vaccine 1 is 50KDa-75KDa, and the immune adjuvants used are CpG ODN BW006, CpG ODN 2216 and Poly ICLC.
  • Poly ICLC is a toll-like receptor 3 agonist, and various types of CpG are toll-like receptor 9 agonists, and both Toll-like receptor 3 and Toll-like receptor 9 are located in the endosomal membrane structure in the cell.
  • the antigen component and the immune adjuvant are co-loaded in the micron particles, and then centrifuged at 10000g for 15 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h; before the particles are used, they are resuspended with 7mL PBS and then 3mL of antigen component (protein concentration 50mg/mL) is added and allowed to act at room temperature for 10min to obtain a micron vaccine 1 loaded with lysate inside and outside.
  • the average particle size of the micron particles is about 2.50 ⁇ m; each 1mg PLGA micron particles1 is loaded with about 140 ⁇ g protein or polypeptide components, and the loaded CpG BW006 (Class B), CpG 2216 (Class A) and Poly ICLC are each 0.03mg.
  • Micronvaccine 2 is prepared by the same materials and methods, and the immune adjuvants loaded are CpG2336 (Class A), CpG 2216 (Class A) and Poly ICLC.
  • the particle size of Micronvaccine 2 is about 2.50 ⁇ m, and each 1mg PLGA micron particle is loaded with about 140 ⁇ g of protein or polypeptide components, and each 1mg PLGA micron particle is loaded with 0.03mg of CpG2336 (Class A), CpG 2216 (Class A) and Poly ICLC immune adjuvants.
  • Micronvaccine 3 is prepared with the same materials and methods, and the loaded immune adjuvants are CpG BW006 (Class B) and CpG 2216 (Class A).
  • the particle size of each 1 mg PLGA micron particle of Micronvaccine 3 is about 2.50 ⁇ m.
  • Each 1 mg of PLGA microparticles is loaded with approximately 140 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA microparticles is loaded with 0.045 mg of CpG BW006 (type B) and CpG 2216 (type A).
  • mice Female BALB/C mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. On day 0, 1.5 ⁇ 10 5 4T1 cells were subcutaneously inoculated in the lower right back of each mouse. On the 4th, 7th, 10th, 15th, 20th and 25th days after inoculation of cancer cells, 0.8 mg of micro vaccine (micro vaccine 1, or micro vaccine 2, or micro vaccine 3) or 100 ⁇ L PBS was subcutaneously injected. The methods for monitoring the tumor growth rate and survival of mice were the same as above.
  • the tumors of the mice in the control group grew larger, while the tumor growth rate of the mice treated with nano-vaccines was significantly slowed down and the survival period was significantly prolonged.
  • the micro-vaccine loaded with CpG adjuvant and Poly ICLC mixed adjuvant was better than the micro-vaccine loaded with two CpG mixed adjuvants.
  • the micro-vaccine loaded with one type B CpG, one type A CpG and Poly ICLC mixed adjuvant was better than the micro-vaccine loaded with two type A CpG and Poly ICLC mixed adjuvant.
  • micro-vaccine loaded with mixed adjuvants of two different toll-like receptors is more effective, and the micro-vaccine containing mixed CpG of type B CpG and Toll-like receptor 3 agonist as a mixed adjuvant is more effective.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mouse was killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter.
  • the tumor tissue was repeatedly frozen and thawed 5 times to lyse the tumor tissue.
  • the lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was taken as the water-soluble component soluble in pure water; 6M guanidine sulfonate aqueous solution was added to the obtained precipitate to dissolve the precipitate, so that the water-insoluble component insoluble in pure water could be converted into a component soluble in 6M guanidine sulfonate aqueous solution.
  • a saturated magnesium sulfate aqueous solution is added dropwise to the water-soluble component in the lysate, and after the precipitation is complete, the obtained sample is centrifuged at 12000g for 5 minutes, and the precipitate is dissolved in a 6M guanidine sulfonate aqueous solution for standby use.
  • the obtained sample is centrifuged at 3000g for 5 minutes, and the supernatant is discarded and the precipitate is dissolved in a 6M guanidine sulfonate aqueous solution; then the precipitate after salting out and the precipitate after heating dissolved in the 6M guanidine sulfonate aqueous solution are combined and used as part of the water-soluble component.
  • a saturated magnesium sulfate aqueous solution is added dropwise to the water-insoluble component in the lysate dissolved in the 6M guanidine sulfonate aqueous solution, and after the precipitation is complete, the obtained sample is centrifuged at 12000g for 5 minutes, and then the precipitate component is dissolved in the 6M guanidine sulfonate aqueous solution for a second time.
  • the partial components of the water-insoluble component in the lysate dissolved in the 6M guanidine sulfonate aqueous solution and the partial components of the water-soluble component dissolved in the 6M guanidine sulfonate aqueous solution are mixed in a mass ratio of 1:2 to prepare the antigen component 1 of the cancer micron vaccine.
  • 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the mice were killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter.
  • the tumor tissue was repeatedly frozen and thawed 5 times to lyse the tumor tissue.
  • the lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was taken as the water-soluble component soluble in pure water; 5% PEG5000 aqueous solution was added to the obtained precipitate to solubilize the precipitate to obtain a component in which the water-insoluble component was dissolved in 5% PEG5000 aqueous solution. Saturated magnesium sulfate aqueous solution was added dropwise to the water-soluble component in the lysate. After the precipitation was complete, the obtained sample was centrifuged at 12000g for 5 minutes to dissolve the precipitate. The supernatant was heated at 100°C for 2 minutes and then the obtained sample was centrifuged at 3000g for 5 minutes.
  • the precipitate was solubilized with a 5% PEG5000 aqueous solution; then the precipitate after salting out and the precipitate after heating dissolved with a 5% PEG5000 aqueous solution were combined and used as part of the water-soluble component.
  • a saturated magnesium sulfate aqueous solution was added dropwise to the non-water-soluble component in the lysate dissolved with a 5% PEG5000 aqueous solution.
  • the obtained sample was centrifuged at 12000g for 5 minutes, and then the precipitate component was solubilized with a 5% PEG5000 aqueous solution for the second time.
  • the non-water-soluble component in the lysate dissolved with the above 5% PEG5000 aqueous solution and the water-soluble component dissolved with the 5% PEG5000 aqueous solution were mixed in a mass ratio of 1:2 to prepare the antigen component 2 of the cancer micron vaccine.
  • 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mouse was killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter.
  • the tumor tissue was repeatedly frozen and thawed 5 times to lyse the tumor tissue.
  • the lysate was then centrifuged at 5000g for 5 minutes, and the supernatant was taken as the water-soluble component soluble in pure water; 6M guanidine sulfonate aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a soluble component in 6M guanidine sulfonate aqueous solution. All water-insoluble components and all water-soluble components in the obtained tumor tissue lysate were mixed at a mass ratio of 1:2 to prepare the antigen component 3 of the control micro vaccine.
  • micron vaccine 1 (Micronvaccine 1) is prepared by solvent evaporation method.
  • the molecular weight of the preparation material PLGA used in micron vaccine 1 is 38KDa-54KDa
  • the immune adjuvants used are Poly (I: C), CpG2006 (Class B) and CpGSL01 (Class B)
  • the substance loaded to increase lysosomal escape is RALA polypeptide (WEARLARALARALARHLARALARALRACEA).
  • Antigen component 1, adjuvant and RALA polypeptide are co-loaded inside the micron particles.
  • the preparation method is as described above.
  • micron particles After the lysate antigen component and adjuvant are loaded inside the micron particles, 100mg of micron particles are centrifuged at 10000g for 15 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h to obtain freeze-dried powder for use.
  • the average particle size of the micron vaccine 1 is about 5.0 ⁇ m, and the surface potential of the micron particles is about -13mV; each 1mg of PLGA micron particles is loaded with approximately 8 ⁇ g of tumor tissue protein and polypeptide components, and each 1mg of PLGA micron particles is loaded with 0.02mg each of Poly(I:C), CpG2006, CpGSL01 and RALA polypeptides.
  • micron vaccine 2 The preparation method of micron vaccine 2 is the same as that of micron vaccine 1.
  • Antigen component 2, adjuvant and RALA polypeptide are co-loaded inside micron particles.
  • the average particle size of micron vaccine 2 is about 5.0 ⁇ m, and the surface potential of micron particles is about -13 mV; each 1 mg PLGA micron particle is loaded with about 8 ⁇ g tumor tissue protein and polypeptide components, and each 1 mg PLGA micron particle is loaded with 0.02 mg of Poly (I: C), CpG2006, CpGSL01 and RALA polypeptide.
  • Micronvaccine 3 The preparation materials and methods of Micronvaccine 3 are the same as those of Micronvaccine 1.
  • the particle size is about 5.0 ⁇ m, and it is loaded with an equal amount of antigen component 1.
  • the loaded immune adjuvant is Poly(I:C). Every 1 mg of PLGA is loaded with 0.06 mg of Poly(I:C) and 0.02 mg of RALA polypeptide.
  • Micronvaccine 4 The preparation materials and methods of Micronvaccine 4 are the same as those of Micronvaccine 1.
  • the particle size is about 5.0 ⁇ m, and it is loaded with an equal amount of antigen component 1.
  • Each 1 mg of PLGA is loaded with 0.02 mg each of Poly(I:C), CpG1585 (Class A), CpG2216 (Class A), and RALA polypeptide.
  • Micronvaccine 5 The preparation method of Micronvaccine 5 is the same as that of Micronvaccine 1.
  • Antigen component 3, adjuvant and RALA polypeptide The micron vaccine 5 has an average particle size of about 5.0 ⁇ m, and the surface potential of the micron particles is about -13 mV; each 1 mg of PLGA micron particles is loaded with about 8 ⁇ g of tumor tissue protein and polypeptide components, and each 1 mg of PLGA micron particles is loaded with 0.02 mg of Poly(I:C), CpG2006, CpGSL01 and RALA polypeptide.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On days -35, -28, -21 and -14 before tumor inoculation, 3 mg of micro vaccine (micro vaccine 1, or micro vaccine 2, or micro vaccine 3, or micro vaccine 4, or micro vaccine 5) were subcutaneously injected into the mice, or 100 ⁇ L of PBS was inoculated. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse. The methods for monitoring the tumor growth rate and survival period of mice were the same as above.
  • Micron vaccine 1 was better than micron vaccine 2, micron vaccine 3, micron vaccine 4 and micron vaccine 5.
  • the vaccine prepared by using a part of the protein and polypeptide components of the lysate separated and purified as the antigen component is better than the vaccine prepared by directly using the lysate as the antigen component; moreover, the micron vaccine loaded with two types of B CpG and Poly (I: C) as a mixed adjuvant is better than the micron vaccine loaded with two types of A CpG and Poly (I: C) as a mixed adjuvant or the micron vaccine loaded with Poly (I: C) as an adjuvant.
  • the micron vaccine prepared by using guanidine sulfonate to solubilize the non-water-soluble components in the lysate and the precipitation of the water-soluble components by salting out and the like is better than PEG5000.
  • E.G7-OVA cell line After the cultured E.G7-OVA cell line is collected, centrifuged at 350g for 5 minutes, the supernatant is discarded and washed twice with PBS, and then the cells are resuspended in ultrapure water and repeatedly frozen and thawed 5 times, and ultrasound can be used to destroy the lysed cells.
  • trypsin Trypsin
  • Chymotrypsin chymotrypsin
  • a saturated aqueous solution of ammonium sulfate is added dropwise to the water-soluble component in the lysate.
  • the obtained sample is centrifuged at 12000g for 5 minutes, and the precipitate is dissolved in a 0.5M metformin hydrochloride aqueous solution for later use.
  • the supernatant is heated at 100°C for 2 minutes, and the obtained sample is centrifuged at 3000g for 5 minutes. After the supernatant is discarded, the precipitate is dissolved in a 0.5M metformin hydrochloride aqueous solution; then the precipitate after salting out dissolved with 0.5M metformin hydrochloride and the heated precipitate are combined and used as part of the water-soluble component.
  • BCG was collected and lysed with 0.5M metformin hydrochloride aqueous solution and then washed with 0.5M metformin hydrochloride aqueous solution. The solution dissolves the lysed components of BCG for later use.
  • nano vaccine 1 (Nanovaccine 1) was prepared by solvent evaporation method.
  • the nanoparticles are loaded with cancer cell lysates, bacterial lysates and immune adjuvants, and the surface is loaded with cancer cell lysate components.
  • the immune adjuvants used are CpG2395 (Class C), CpGM362 (Class C) and poly ICLC, and the adjuvants are loaded inside the nanoparticles.
  • the mass ratio of the cancer cell lysate antigen component to the bacterial lysate component used in the preparation of the nanoparticles is 1:1.
  • the multiple emulsion method is first used to load the cancer cell lysate antigen, bacterial lysate component and adjuvant inside the nanoparticles, and then 100mg of nanoparticles are centrifuged at 10000g for 20 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h. Before use, resuspend 20 mg of nanoparticles in 0.9 mL PBS and mix with 0.1 mL of sample containing cancer cell lysate antigen component (80 mg/mL) at room temperature for 5 minutes before use. The average particle size of nanoparticle 1 is about 400 nm.
  • Each 1 mg of PLA nanoparticle 1 is loaded with about 400 ⁇ g of protein or peptide component.
  • Each 1 mg of PLA nanoparticle is loaded with 0.005 mg of CpG2395 (C type), CpGM362 (C type) and Poly ICLC immune adjuvant.
  • Nanovaccine 2 (Nanovaccine 2) Preparation materials and preparation methods Nanoparticle 1, the particle size is about 400nm, each 1mg PLA nanoparticles loads about 140 ⁇ g protein or polypeptide components, each 1mg PLA loads 0.005mg of CpG1585 (Class A), CpG2336 (Class A) and Poly ICLC.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. 5 ⁇ 10 5 E.G7-OVA cells were subcutaneously inoculated into each mouse on day 0, and 100 ⁇ L PBS or 0.1 mg of nanovaccine (nanovaccin 1 or nanovaccine 2) were injected into the mice on days 5, 8, 13, and 20, respectively. The methods for monitoring the tumor volume and survival of mice were the same as above.
  • the tumors of mice in the PBS control group grew very fast and the mice had a short survival period.
  • the tumor growth rate of mice treated with several nano-vaccines was significantly slowed down and the survival period was significantly prolonged.
  • nano-vaccine 1 was more effective.
  • Example 9 Vaccine for the treatment of melanoma
  • the cultured B16F10 melanoma cancer cell line and S91 melanoma cancer cell line were collected and mixed at a quantity ratio of 1:1, and then centrifuged at 500g for 5 minutes, and then the supernatant was discarded and washed twice with PBS, and then the cells were resuspended in ultrapure water and repeatedly frozen and thawed 5 times, accompanied by ultrasound to lyse the cells.
  • the lysate was centrifuged at a speed of 10000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 0.3M metformin sulfate aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into soluble in 0.3M metformin sulfate aqueous solution.
  • Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble components in the lysate. After the precipitation was complete, the obtained sample was centrifuged at 12000g for 5 minutes. The precipitate was dissolved in 0.3M metformin sulfate aqueous solution for later use. The supernatant was treated with DNA digestion enzyme for 5 minutes, heated at 100°C for 2 minutes, and the obtained sample was centrifuged at 3000g for 5 minutes.
  • nano vaccine 1 (Nanovaccine 1) is prepared by the double emulsion method.
  • the preparation material of the nano vaccine 1 used is PLGA with a molecular weight of 24KDa-38KDa.
  • the immune adjuvants used are poly(I:C), CpG1018 and CpG2216, and the substance that increases lysosomal immune escape is KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA), and the adjuvant and KALA polypeptide are encapsulated in the nanoparticles.
  • the preparation method is as described above. In the preparation process, the double emulsion method is first used to load the lysate components, adjuvants, and KALA polypeptide inside the nanoparticles.
  • nanoparticles are centrifuged at 12000g for 25 minutes, and resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanoparticles is about 250nm; each 1mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or polypeptide components, and each 1mg of PLGA nanoparticles is loaded with 0.04mg of poly(I:C), CpG1018 and CpG2216, and 0.3mg of KALA polypeptide.
  • Nanovaccine 2 was prepared using the same materials and methods. Its particle size was about 250 nm, and its surface potential was about -5 mV. It did not carry KALA polypeptide, but carried an equal amount of adjuvant and cell lysis antigen components.
  • Nanovaccine 3 The preparation materials and preparation methods of Nanovaccine 3 are the same, with a size of about 250nm and a surface potential of about -5mV.
  • Each 1mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein and peptide components.
  • Each 1mg of PLGA nanoparticles is loaded with 0.04mg of poly(I:C), 0.08mg of CpG1018, and 0.3mg of KALA peptide.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse. On days 4, 7, 10, 15, and 20 after melanoma inoculation, 100 ⁇ L PBS or 0.4 mg of the corresponding nanovaccine was subcutaneously injected. In the experiment, the tumor volume and survival monitoring methods of mice were the same as above.
  • the results show that the tumors in the PBS control group grew up quickly. Compared with the control group, the tumor growth rate of mice treated with nanovaccines was significantly slowed down and the survival period was significantly prolonged. Moreover, nanovaccine 1 was better than nanovaccine 2 and nanovaccine 3, which shows that the nanovaccine with substances that promote lysosomal escape is more effective; the nanovaccine using two CpG and Poly (I: C) as mixed adjuvants has a better therapeutic effect than the nanovaccine using only one CpG and Poly (I: C) mixed adjuvant. In summary, the nanovaccine described in the present disclosure has a good therapeutic effect on cancer.
  • the cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, washed twice with PBS and resuspended in ultrapure water.
  • the obtained cancer cells were inactivated and denatured by ultraviolet light and high temperature heating, respectively, and then lysed with 6M guanidine hydrochloride aqueous solution and then dissolved with 8M urea aqueous solution.
  • Lactobacillus acidophilus was centrifuged at 5000g for 30 minutes, and the precipitate was discarded to collect the supernatant.
  • the supernatant was filtered using a 1 ⁇ m filter membrane and then centrifuged at 16000g for 90 minutes.
  • the precipitate obtained after discarding the supernatant was the bacterial extracellular vesicle component.
  • the precipitate was lysed with 8M urea aqueous solution to dissolve the bacterial extracellular vesicle component.
  • the cancer cell lysate component dissolved in 8M urea solution and the bacterial extracellular vesicle component dissolved in 8M urea solution were mixed in a mass ratio of 8:1, and then a saturated ammonium sulfate aqueous solution was added dropwise to the mixed sample for salting out. After the precipitation was complete, the obtained sample was centrifuged at 12000g for 5 minutes, the supernatant was discarded, and the precipitate obtained by salting out was dissolved in a 6M guanidine hydrochloride aqueous solution to obtain the antigen component 1 for preparing the micro vaccine 1.
  • the cancer cell lysate component dissolved in 8M urea solution and the bacterial extracellular vesicle component dissolved in 8M urea solution are mixed in a mass ratio of 8:1, and then a saturated copper sulfate aqueous solution is added dropwise to the mixed sample for salting out. After the precipitation is complete, the obtained sample is centrifuged at 12000g for 5 minutes, the supernatant is discarded, and the salted-out precipitate is dissolved in a 6M guanidine hydrochloride aqueous solution to obtain the antigen component 2 for preparing micron vaccine 2.
  • micron vaccine 1 (Micronvaccine 1) is prepared by double emulsion method.
  • the skeleton materials of micron vaccine 1 are PLGA (molecular weight of 20KDa-40KDa) and PEG2000-PLGA (molecular weight of PLGA part is 20KDa-40KDa); wherein the mass ratio of PLGA to PEG2000-PLGA is 9:1.
  • the immune adjuvants used are CpG1018 and Poly ICLC.
  • the double emulsion method is first used to prepare micron particles loaded with antigen components and adjuvants.
  • micron particles are centrifuged at 8000g for 15 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours to obtain micron vaccine 1, with an average particle size of about 3.0 ⁇ m.
  • Each 1 mg of PLGA micron particles 1 is loaded with about 100 ⁇ g of protein or polypeptide components (antigen component 1), and loaded with 0.15 mg of CpG 1018 and Poly ICLC.
  • Micronvaccine 2 The preparation materials and methods of Micronvaccine 2 are the same as those of Micronvaccine 1.
  • the particle size is about 3.0 ⁇ m.
  • Each 1 mg of PLGA micron particle 1 is loaded with approximately 100 ⁇ g of protein or polypeptide component (antigen component 2), and 0.15 mg each of CpG1018 and Poly ICLC.
  • Micronvaccine 3 The preparation materials and methods of Micronvaccine 3 are the same as those of Micronvaccine 1.
  • the particle size is about 3.0 ⁇ m. It is loaded with an equal amount of antigen component 1, but does not load CpG1018 and Poly ICLC.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice.
  • the mice were injected with 100 ⁇ L PBS or 1.5 mg of micro vaccine (micro vaccine 1, micro vaccine 2, or micro vaccine 3) on days -35, -28, -21, -14, and -7 before tumor inoculation, and 5 ⁇ 10 5 E.G7-OVA cells were subcutaneously inoculated into each mouse on day 0.
  • the tumor volume and survival period of the mice were monitored as above.
  • micron vaccine 1 was significantly better than micron vaccine 2 and micron vaccine 3, indicating that the use of a specific salting-out reagent is more effective, and the addition of a mixed adjuvant helps to improve the vaccine effect. It can be seen that the micron vaccine described in the present disclosure can be used to prevent or treat cancer.
  • the MC38 cancer cell line and its culture medium were collected, and then centrifuged at 500g for 5 minutes. After collecting the cell precipitate, the cell precipitate was lysed with a 6M guanidine hydrochloride aqueous solution and completely dissolved to obtain the collected cancer cell lysate component; the culture medium supernatant was collected, the supernatant was filtered with a 1 ⁇ m filter membrane, and then centrifuged at 160000g for 90 minutes. After discarding the supernatant, the precipitate was lysed with a 6M guanidine hydrochloride aqueous solution and completely dissolved to obtain the collected cancer cell extracellular vesicle lysate component.
  • the MC38 cancer cell line and its culture medium were collected, and then centrifuged at 500g for 5 minutes. After collecting the cell precipitate, the cell precipitate was lysed with a 6M guanidine hydrochloride aqueous solution and completely dissolved to obtain the collected cancer cell lysate component; the culture medium supernatant was collected, the supernatant was filtered with a 1 ⁇ m filter membrane, and then centrifuged at 160000g for 90 minutes. After discarding the supernatant, the precipitate was lysed with a 6M guanidine hydrochloride aqueous solution and completely dissolved to obtain the collected cancer cell extracellular vesicle lysate component.
  • the MC38 cancer cell line and its culture medium were collected, and then centrifuged at 500g for 5 minutes. After collecting the cell precipitate, the cell precipitate was lysed with a 5% PEG5000 aqueous solution and then solubilized with a 5% PEG5000 aqueous solution to obtain the collected cancer cell lysate component; the culture medium supernatant was collected, the supernatant was filtered with a 1 ⁇ m filter membrane, and then centrifuged at 160000g for 90 minutes, the supernatant was discarded, and the precipitate was lysed with a 6M guanidine hydrochloride aqueous solution and completely dissolved to obtain the collected cancer cell extracellular vesicle lysate component.
  • nanoparticles 1 are prepared by the double emulsion method.
  • the molecular weight of the preparation material PLGA of nanoparticles 1 is 7KDa-17KDa, with Poly(I:C), CpG2336 and CpG2006 as adjuvants, and the antigen component 1 and the immune adjuvant are both loaded in the nanoparticles.
  • the preparation method is as described above.
  • the antigen component and adjuvant are first loaded inside the nanoparticles, and then 100mg of nanoparticles are centrifuged at 15000g for 30 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h before use; the average particle size of the nanoparticles 1 is about 110nm, and each 1mg of PLGA nanoparticles is loaded with about 2 ⁇ g of protein and polypeptide components, and each 1mg of PLGA nanoparticles is loaded with 0.01mg of poly(I:C), CpG2336 and CpG2006.
  • nanoparticle 2 The preparation materials and preparation methods of nanoparticle 2 are the same as those of nanoparticle 1, but the antigen component 2 and adjuvant are loaded inside.
  • the particle size is about 110 nm.
  • Each 1 mg of PLGA nanoparticle is loaded with approximately 2 ⁇ g of protein and polypeptide components.
  • Each 1 mg of PLGA nanoparticle is loaded with 0.01 mg each of poly(I:C), CpG2336 and CpG2006.
  • nanoparticle 3 The preparation materials and preparation methods of nanoparticle 3 are the same as nanoparticle 1, but the antigen component 3 and adjuvant are loaded inside.
  • the particle size is about 110nm.
  • Each 1mg of PLGA nanoparticle is loaded with about 2 ⁇ g of protein and polypeptide components.
  • Each 1mg of PLGA nanoparticle is loaded with 0.01mg each of poly(I:C), CpG2336 and CpG2006.
  • BMDC and B cells as antigen presenting cells.
  • the preparation method of BMDC is the same as above.
  • B cells are from mice Peripheral blood PBMCs were obtained by magnetic bead sorting.
  • BMDCs and B cells were mixed in a 1:1 ratio to form mixed antigen presenting cells.
  • 1 mg of nanoparticles 1, 2, or 3 was co-incubated with BMDCs (10 million) and B cells (10 million) in 15 mL of high-glucose DMEM complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contained vinblastine (10 ng/mL) and Flt3L (100 ng/mL).
  • the collected activated extracellular vesicles of antigen presenting cells were mixed with extracellular vesicles of cancer cells, and then sonicated at low power (20W) for 2 minutes at 4°C, and then repeatedly co-extruded using a 0.22 ⁇ m filter membrane.
  • the extrudate was mixed with the nanoparticles 1 or nanoparticles 2 prepared in step (2) and treated with a high-pressure homogenizer (10000bar) for 1 minute, and then repeatedly co-extruded using a 0.22 ⁇ m filter membrane, and then centrifuged at 15000g for 30 minutes, the supernatant was discarded, and the precipitate was collected.
  • the nanovaccine prepared by the co-action of the antigen presenting cell extracellular vesicle membrane component activated by nanoparticle 1 and nanoparticle 1 is nanovaccine 4 (Nanovaccine 4), with a particle size of 120 nanometers, 10 ⁇ g of poly(I:C), CpG2336 and CpG2006 immune adjuvants per 1mg PLGA, and about 80 ⁇ g of membrane component per 1mg PLGA.
  • the nanovaccine prepared by using the antigen-presenting extracellular vesicle membrane component activated by nanoparticle 2 and nanoparticle 2 is nanovaccine 5, with a particle size of 120 nanometers, 10 ⁇ g of poly(I:C), CpG2336 and CpG2006 immune adjuvants per 1 mg PLGA, and about 80 ⁇ g of membrane components are loaded per 1 mg PLGA.
  • the nanovaccine prepared by using the antigen-presenting extracellular vesicle membrane component activated by nanoparticle 3 and nanoparticle 3 is nanovaccine 6, with a particle size of 120 nanometers, 10 ⁇ g of poly(I:C), CpG2336 and CpG2006 immune adjuvants per 1 mg PLGA, and about 80 ⁇ g of membrane components are loaded per 1 mg PLGA.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer mice. On day 0, 2 ⁇ 10 6 MC38-OVA cells were subcutaneously inoculated in the lower right back of each mouse. 0.4 mg of nanovaccine (nanovaccine 4, or nanovaccine 5, or nanovaccine 6) or 100 ⁇ L of PBS was subcutaneously injected on days 6, 9, 12, 15, 20, and 25 after inoculation of colon cancer cells. The methods for monitoring mouse tumor growth and survival were the same as above.
  • the tumor growth rate of mice treated with nano vaccines was significantly slowed down and the survival period of mice was significantly prolonged.
  • the effect of nano vaccine 4 was better than that of nano vaccine 5 and nano vaccine 6. It can be seen that the precipitate treated by salting out and other methods must be dissolved or solubilized by an appropriate dissolving agent to prepare a vaccine to achieve a good therapeutic effect.
  • the nano vaccine disclosed in the present invention has an excellent therapeutic effect on cancer.
  • the cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water.
  • a 6M guanidine hydrochloride aqueous solution was then used to lyse the cancer cells and dissolve the lysate components.
  • a saturated ammonium sulfate aqueous solution was then added until the precipitation was complete, and the supernatant was discarded.
  • the precipitate was again dissolved in a 6M guanidine hydrochloride aqueous solution to obtain the protein and polypeptide components in the cancer cells dissolved in the 6M guanidine hydrochloride aqueous solution, which was the antigen component 1.
  • the cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water.
  • a 3% Tween 80 aqueous solution was then used to lyse the cancer cells and solubilize the lysate components.
  • a saturated ammonium sulfate aqueous solution was then added until the precipitation was complete, and the supernatant was discarded.
  • the precipitate was solubilized again with a 3% Tween 80 aqueous solution to obtain the protein and polypeptide components in the cancer cells dissolved in the 3% Tween 80 aqueous solution, which was the antigen component 2.
  • the cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water.
  • a 6M guanidine hydrochloride aqueous solution was then used to lyse the cancer cells and solubilize the lysate components.
  • a saturated ammonium sulfate aqueous solution was then added until the precipitation was complete, and the supernatant was discarded.
  • the precipitate was solubilized again with a 3% Tween 80 aqueous solution to obtain the protein and polypeptide components in the cancer cells dissolved in the 3% Tween 80 aqueous solution, which was the antigen component 3.
  • the cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. A 3% Tween 80 aqueous solution was then used to lyse the cancer cells and solubilize the lysate components. A saturated ammonium sulfate aqueous solution was then added until the precipitation was complete, and the supernatant was discarded. The precipitate was again dissolved with a 6M guanidine hydrochloride aqueous solution to obtain the protein and polypeptide components in the cancer cells dissolved in the 6M guanidine hydrochloride aqueous solution, which was the antigen component 4.
  • the nano vaccine 1 (Nanovaccine 1) was prepared by the double emulsion method.
  • the skeleton materials of the nano vaccine 1 are PLA (molecular weight 10-30KDa) and mannose-PEG2000-PLA (PLA molecular weight 10-30KDa), and the mass ratio of PLGA (molecular weight 10-30KDa) and mannose-PEG2000-PLGA (PLGA molecular weight 10-30KDa) is 9:1.
  • the immune adjuvants used are CpG1018, CpG7909 and Poly ICLC, and the positively charged substance used is R8 polypeptide (RRRRRRRR).
  • the double emulsion method is first used to prepare micron particles loaded with antigen component 1, adjuvant and R8 polypeptide. Then, 100 mg of nanoparticles are centrifuged at 9000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose and dried for 48 hours to obtain nanovaccine 1 with an average particle size of about 200 nm. Each 1 mg of PLGA nanoparticle 1 is loaded with approximately 5 ⁇ g of protein and polypeptide components of cancer cells, 0.02 mg each of CpG1018, CpG7909 and Poly ICLC, and 0.05 mg of R8 polypeptide.
  • Nanovaccin 2 The preparation method of Nanovaccin 2 is the same as that of Nanovaccin 1. However, the antigen component 2, adjuvant and R8 peptide are loaded inside. The average particle size of Nanovaccin 2 is about 200nm. Each 1mg of PLGA Nanovaccin 2 is loaded with about 5 ⁇ g of protein and peptide components of cancer cells, 0.02mg of CpG1018, CpG7909 and Poly ICLC, and 0.05mg of R8 peptide.
  • Nanovaccin 3 The preparation method of Nanovaccin 3 is the same as that of Nanovaccin 1. However, the antigen component 3, adjuvant and R8 peptide are loaded inside. The average particle size of Nanovaccin 3 is about 200nm. Each 1mg PLGA Nanovaccin 3 is loaded with about 5 ⁇ g of protein and peptide components of cancer cells, 0.02mg each of CpG1018, CpG7909 and Poly ICLC, and 0.05mg of R8 peptide.
  • nano vaccine 4 (Nanovaccine 1) is the same as that of micro vaccine 1. However, the antigen component 4, adjuvant and R8 polypeptide are loaded inside.
  • the average particle size of nano vaccine 4 is about 200nm, and each 1mg of PLGA nano vaccine 4 is loaded with about 5 ⁇ g of cancer cells.
  • 0.02 mg each of CpG1018, CpG7909 and Poly ICLC were loaded, and 0.05 mg of R8 peptide was loaded.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. On days -35, -28, -21, -14 and -7 before tumor inoculation, mice were injected with 100 ⁇ L PBS or 1 mg of nanovaccine 1 or 1 mg of nanovaccine 2 or 1 mg of nanovaccine 3 or 1 mg of nanovaccine 4, and 5 ⁇ 10 5 E.G7-OVA cells were subcutaneously inoculated into each mouse on day 0. The methods for monitoring mouse tumor volume and survival were the same as above.
  • micro vaccine described in the present disclosure can be used to prevent or treat cancer.
  • mannose is used as an active targeting target head to target dendritic cells.
  • other targeted molecules can also be used, including but not limited to mannan, CD11c, CD103, CD32, CD11b, CD19, CD38, etc.
  • Example 13 Nano-vaccine loaded with partially water-soluble components and water-insoluble components for the treatment of melanoma
  • ultrapure water is first used to lyse B16F10 melanoma tumor tissue to prepare protein and polypeptide components and water-insoluble antigens in the water-soluble components of the tumor tissue, and then the nanovaccine is prepared by the solvent evaporation method using the organic polymer material PLGA as the nanoparticle skeleton material and poly(I:C) as the immune adjuvant.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mouse was killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells.
  • the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 0.2M hydrochloric acid semicarbazide aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a soluble component in 0.2M hydrochloric acid semicarbazide aqueous solution. Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble component in the lysate.
  • the obtained sample was centrifuged at 3000g for 5 minutes, and the precipitate was dissolved in 0.2M hydrochloric acid semicarbazide aqueous solution for standby use. The supernatant was heated at 100°C for 5 minutes and the obtained sample was centrifuged at 3000g for 5 minutes. After the supernatant was discarded, the precipitate was dissolved in 0.2M hydrochloric acid semicarbazide aqueous solution; then the precipitate after salting out and the precipitate after heating dissolved with 0.2M hydrochloric acid semicarbazide were combined and used as part of the water-soluble component.
  • the components obtained by salting out and heating the non-water-soluble components in the lysate dissolved with 0.2M hydrochloric acid semicarbazide aqueous solution and the water-soluble components dissolved with 0.2M hydrochloric acid semicarbazide are antigen component 1 for preparing cancer nanovaccine 1.
  • B16F10 cells 1.5 ⁇ 10 5 B16F10 cells were inoculated subcutaneously on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mice were killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter and repeated freezing and thawing 5 times, accompanied by ultrasound to destroy the lysed cells.
  • the lysate was centrifuged at 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 0.2M hydrochloric acid semicarbazide aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water can be converted into a 0.2M hydrochloric acid semicarbazide aqueous solution.
  • a saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble component in the lysate, and after the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes, and the precipitate was dissolved in a 0.2M hydrochloric acid semicarbazide aqueous solution and used as a part of the water-soluble component.
  • the components obtained by salting out and precipitating from the water-insoluble components in the lysate dissolved by the 0.2M hydrochloric acid semicarbazide aqueous solution and the water-soluble components dissolved by the 0.2M hydrochloric acid semicarbazide are the antigen component 2 for preparing the cancer nanovaccine 2.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of about 1000 mm 3
  • the mouse was killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell filter and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells.
  • the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 0.2M hydrochloric acid semicarbazide aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the insoluble component in pure water was converted into a soluble component in 0.2M hydrochloric acid semicarbazide aqueous solution.
  • the water-soluble component in the lysate was heated at 100°C for 5 minutes, and the obtained sample was centrifuged at 3000g for 5 minutes.
  • the precipitate was dissolved in 0.2M hydrochloric acid semicarbazide aqueous solution, which was a part of the water-soluble component.
  • the component obtained by heating and precipitating the water-insoluble component in the lysate dissolved by the 0.2M hydrochloric acid semicarbazide aqueous solution and the water-soluble component dissolved by the 0.2M hydrochloric acid semicarbazide is the antigen component 3 for preparing the cancer nanovaccine 3.
  • nano vaccine 1 (Nanovaccine 1) is prepared by the emulsion method in the solvent volatilization method. During the preparation, some of the water-soluble components in the tumor tissue lysate are components dissolved in 0.2M hydrochloric acid semicarbazide aqueous solution after ammonium sulfate salting out and heating separation purification.
  • nanoparticles loaded with water-soluble components in cancer cell lysates (components dissolved in 0.2M hydrochloric acid semicarbazide aqueous solution after ammonium sulfate salting out and heating separation purification) and nanoparticles loaded with water-insoluble antigens in whole cell antigens of cancer cells (dissolved in 0.2M hydrochloric acid semicarbazide aqueous solution) are prepared separately, and then used together as nano vaccine 1.
  • the antigen delivery nanoparticle preparation material PLGA used has a molecular weight of 20KDa-40KDa, and the immune adjuvant used is poly(I:C) and poly(I:C) is loaded inside the nanoparticles.
  • the cell antigen components and adjuvants were first loaded inside the nanoparticles by the double emulsion method, and then 300 mg of nanoparticles were centrifuged at 10000g for 20 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanoparticle 1 was about 100 nm, and each 1 mg of PLGA nanoparticles was loaded with about 250 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA nanoparticles was loaded with 0.01 mg of poly(I:C).
  • nano vaccine 2 (Nanovaccine 2) is prepared by the multiple emulsion method in the solvent evaporation method. During preparation, some components in the water-soluble components in the tumor tissue lysate are components dissolved by 0.2M hydrochloric acid semicarbazide aqueous solution after purification by ammonium sulfate salting out.
  • nanoparticles of water-soluble partial components in the cancer cell lysate (components dissolved by 0.2M hydrochloric acid semicarbazide aqueous solution after separation and purification by ammonium sulfate salting out) and nanoparticles of water-insoluble antigens in the whole cell antigen of the cancer cell (dissolved by 0.2M hydrochloric acid semicarbazide aqueous solution) are prepared separately, and then used together as nano vaccine 2.
  • the antigen delivery nanoparticle preparation material PLGA molecular weight used is 20KDa-40KDa
  • the immune adjuvant used is poly (I: C) and poly (I: C) and is loaded inside the nanoparticle.
  • the cell antigen components and adjuvants are first loaded inside the nanoparticles by the double emulsion method, and then 100 mg of nanoparticles are centrifuged at 10000g for 20 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanoparticles 2 is about 300 nm, and each 1 mg of PLGA nanoparticles is loaded with about 250 ⁇ g of protein or polypeptide components. 1mg PLGA nanoparticles loaded with 0.01mg poly(I:C).
  • nano vaccine 3 (Nanovaccine 3) is prepared by the emulsion method in the solvent volatilization method. During the preparation, some of the water-soluble components in the tumor tissue lysate are components dissolved in 0.2M hydrochloric acid semicarbazide aqueous solution after heating, separation and purification.
  • nanoparticles loaded with water-soluble components of cancer cell lysates (components dissolved in 0.2M hydrochloric acid semicarbazide aqueous solution after heating, separation and purification) and nanoparticles loaded with water-insoluble antigens in whole cell antigens of cancer cells (dissolved in 0.2M hydrochloric acid semicarbazide aqueous solution) are prepared separately, and then used together as nano vaccine 3.
  • the PLGA molecular weight of the antigen delivery nanoparticle preparation material used is 20KDa-40KDa
  • the immune adjuvant used is poly(I:C) and poly(I:C) is loaded inside the nanoparticles.
  • the cell antigen components and adjuvants were first loaded inside the nanoparticles by the double emulsion method, and then 100 mg of nanoparticles were centrifuged at 10000g for 20 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of the nanoparticles 3 was about 300 nm, and each 1 mg of PLGA nanoparticles was loaded with about 250 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA nanoparticles was loaded with 0.01 mg of poly(I:C).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma tumor-bearing mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse. On the 3rd, 6th, 9th, 14th and 20th days before the mice were inoculated with tumors, 2 mg of nanovaccine 1 (1 mg of nanoparticles loaded with purified components in water-soluble components + 1 mg of nanoparticles loaded with non-water-soluble components), or 2 mg of nanovaccine 2 (1 mg of nanoparticles loaded with purified components in water-soluble components + 1 mg of nanoparticles loaded with non-water-soluble components), or 2 mg of nanovaccine 3 (1 mg of nanoparticles loaded with purified components in water-soluble components + 1 mg of nanoparticles loaded with non-water-soluble components), or 100 ⁇ L PBS were subcutaneously injected into the mice.
  • nanovaccine 1 1 mg of nanoparticles loaded with purified components in water-soluble components
  • mice The tumor growth rate and survival of the mice were monitored.
  • the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day.
  • v 0.52 ⁇ a ⁇ b 2
  • v 0.52 ⁇ a ⁇ b 2
  • v 0.52 ⁇ a ⁇ b 2
  • ultrapure water was first used to lyse B16F10 melanoma cancer cells to prepare protein and polypeptide components and water-insoluble components in the water-soluble components of the cancer cells, and then PLGA and PEG-modified PLGA were used as nanoparticle skeleton materials to prepare nanovaccines.
  • the cultured B16F10 cells were collected, the supernatant was discarded, and an appropriate amount of ultrapure water was added to the cancer cell pellet to resuspend and freeze-thaw repeatedly for 5 times, accompanied by ultrasound to lyse the cells.
  • the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 6M guanidine hydrochloride aqueous solution was added to the obtained precipitate to dissolve the precipitate, so that the water-insoluble antigen insoluble in pure water can be converted into a soluble antigen in 6M guanidine hydrochloride aqueous solution.
  • the water-soluble components in the lysate were heated at 100°C for 10 minutes, and the resulting sample was centrifuged at 3000 g for 5 minutes. The supernatant was discarded and the precipitate was dissolved in a 6M guanidine hydrochloride aqueous solution.
  • the components obtained by heating and precipitating the water-soluble components dissolved in the 6M guanidine hydrochloride solution and the water-insoluble components in the lysate dissolved in the 6M guanidine hydrochloride aqueous solution were mixed in a mass ratio of 1:1 to prepare the antigen component 1 of the nanovaccines 1, 2 and 3.
  • the water-soluble components in the lysate are used directly without any treatment.
  • the water-soluble components and the water-insoluble components in the lysate dissolved in 6M guanidine hydrochloride aqueous solution are mixed in a mass ratio of 1:1 to prepare the antigen component 2 of the nanovaccine 4.
  • nano vaccine 1 (Nanovaccine 1) is prepared by the double emulsion method in the solvent evaporation method.
  • the nano vaccine preparation materials used are PGLA (molecular weight of 10KDa-20KDa) and PEG2000-PLGA (PLGA molecular weight of 10KDa-20KDa), and the mass ratio of PLGA and PEG2000-PLGA used is 49:1;
  • the immune adjuvants used are poly (I:C) and CpG7909, and the adjuvants are loaded inside the nano vaccine.
  • the cell antigen component 1 and the adjuvant are first loaded inside the nanoparticles by the double emulsion method, and then 100mg of nanoparticles are centrifuged at 14000g for 20 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of nanovaccine 1 is about 250 nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 500 ⁇ g of protein or polypeptide components, and 0.05 mg each of poly(I:C) and CpG7909.
  • nano vaccine 2 (Nanovaccine 2) is prepared by the double emulsion method in the solvent evaporation method.
  • the nano vaccine preparation materials used are PGLA (molecular weight of 10KDa-20KDa) and PEG2000-PLGA (PLGA molecular weight of 10KDa-20KDa), and the mass ratio of PLGA and PEG2000-PLGA used is 500:1; the immune adjuvants used are poly (I:C) and CpG7909, and the adjuvants are loaded inside the nano vaccine.
  • the cell antigen component 1 and the adjuvant are first loaded inside the nanoparticles by the double emulsion method, and then 100mg of nanoparticles are centrifuged at 14000g for 20 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of nanovaccine 2 is about 250 nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 500 ⁇ g of protein or polypeptide components, and 0.05 mg each of poly(I:C) and CpG7909.
  • nano vaccine 3 (Nanovaccine 3) is prepared by the emulsion method in the solvent evaporation method.
  • the nano vaccine preparation materials used are PGLA (molecular weight is 10KDa-20KDa) and PEG2000-PLGA (PLGA molecular weight is 10KDa-20KDa), and the mass ratio of PLGA and PEG2000-PLGA used is 4:1;
  • the immune adjuvants used are poly (I:C) and CpG7909, and the adjuvants are loaded inside the nano vaccine.
  • the cell antigen component 1 and the adjuvant are first loaded inside the nanoparticles by the emulsion method, and then 100mg of nanoparticles are centrifuged at 14000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is used to resuspend and freeze-dry for 48h.
  • the average particle size of nanovaccine 3 is about 250 nm.
  • Each 1 mg of PLGA nanoparticles is loaded with about 500 ⁇ g of protein or polypeptide components, and 0.05 mg of poly(I:C) and CpG7909 are loaded.
  • the preparation method and materials of nano vaccine 4 (Nanovaccine 4) in this embodiment are the same as those of nano vaccine 1.
  • the materials used for preparing the nano vaccine are PGLA (molecular weight of 10KDa-20KDa) and PEG2000-PLGA (PLGA molecular weight of 10KDa-20KDa), and the mass ratio of PLGA and PEG2000-PLGA used is 49:1; the immune adjuvants used are poly (I:C) and CpG7909, and the adjuvants are loaded inside the nano vaccine.
  • the preparation method is as described above.
  • the cell antigen component 2 and the adjuvant are first loaded inside the nanoparticles by the emulsion method, and then 100 mg of nanoparticles are centrifuged at 14000g for 20 minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours.
  • the average particle size of nanovaccine 4 is about 250 nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 500 ⁇ g of protein or polypeptide components, and 0.05 mg each of poly(I:C) and CpG7909.
  • nano vaccine 1 Nanovaccine 1
  • nano vaccine 2 Nanovaccine 2
  • nano vaccine 3 nano vaccine 3
  • nano vaccine 4 nano vaccine 4
  • the effect of nano vaccine 1 was better than that of nano vaccine 4, indicating that the use of heated purification of protein and polypeptide components in the water-soluble components can improve the vaccine effect, and the effect of separating and purifying a part of the components in the water-soluble components is better than directly using the water-soluble components.
  • the effect of nano vaccine 1 was better than that of nano vaccine 2 and nano vaccine 3, indicating that the use of a specific proportion of PEG to modify the particle surface can improve the efficacy of nano vaccines or micro vaccines.
  • circulating tumor cells in peripheral blood are first separated, and then the circulating tumor cells in peripheral blood are expanded and cultured in vitro and then lysed. After the whole cell antigen components of the circulating tumor cells obtained by lysis are collected, they are loaded into nanovaccines for the prevention and treatment of cancer.
  • the cultured B16F10 cells were collected, and then 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the back of each mouse. Then, 15 days after the inoculation of cancer cells, the mice were killed to collect peripheral blood. After the circulating tumor cells in the peripheral blood were separated, they were cultured and expanded in vitro. After collecting the peripheral circulating tumor cells after expansion culture, an appropriate amount of ultrapure water was added thereto and the cells were repeatedly frozen and thawed 5 times to lyse the circulating tumor cells.
  • the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 0.2M methylguanidine hydrochloride aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a component soluble in 0.2M methylguanidine hydrochloride aqueous solution. Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble component in the lysate. After the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes to dissolve the precipitate.
  • Dissolve in 0.2M methylguanidine hydrochloride aqueous solution for use heat the supernatant at 100°C for 5 minutes, centrifuge the obtained sample at 3000g for 5 minutes, discard the supernatant and dissolve the precipitate in 0.2M methylguanidine hydrochloride aqueous solution; then combine the precipitate after salting out and the precipitate after heating dissolved in 0.2M methylguanidine hydrochloride and use them as part of the water-soluble component.
  • the nano vaccine 1 (Nanovaccine 1) is prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA is 20KDa-40KDa, and the immune adjuvant is poly(I:C).
  • the antigen component 1 and the adjuvant are loaded inside the nanoparticles by the double emulsion method, and then 1000mg of nanoparticles are centrifuged at 12000g for 20 minutes, and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of the nanoparticle 1 is about 300nm, and each 1mg PLGA nanoparticle is loaded with about 250 ⁇ g of protein or polypeptide components and 0.01mg of poly(I:C).
  • nano vaccine 2 (Nanovaccine 2) is prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA is 20KDa-40KDa, and the immune adjuvant is poly(I:C).
  • the antigen component 1 and the adjuvant are loaded inside the nanoparticles by the double emulsion method, and then 1000mg of nanoparticles are centrifuged at 12000g for 20 minutes, and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of the nanoparticle 2 is about 300nm, and each 1mg of PLGA nanoparticles is loaded with about 0.025ng of protein or polypeptide components and 0.01mg of poly(I:C).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse. On the 3rd, 6th, 9th, 14th and 20th days before the mice were inoculated with tumors, 0.5 mg of nanovaccine (nanovaccin 1, or nanovaccine 2) or 100 ⁇ L PBS was subcutaneously injected into the mice. The tumor growth rate and survival of the mice were monitored. The mouse tumor detection method was the same as above.
  • the cultured B16F10 cells were collected, and after the supernatant was discarded, the cancer cells were lysed using 0.2M polyhexamethyleneguanidine hydrochloride and 0.2M agmatine sulfate, and then the lysate components were dissolved using 0.2M polyhexamethyleneguanidine hydrochloride and 0.2M agmatine sulfate aqueous solution.
  • the obtained sample was centrifuged at 3000g for 5 minutes, and the precipitate was dissolved in 0.2M polyhexamethyleneguanidine hydrochloride and 0.2M agmatine sulfate aqueous solution; saturated sodium chloride aqueous solution was added dropwise to the above-obtained supernatant, and after the precipitation was complete, the obtained sample was centrifuged at 3000g for 5 minutes, and the precipitate was dissolved in 0.2M polyhexamethyleneguanidine hydrochloride and 0.2M agmatine sulfate aqueous solution for standby use; then the heated precipitate dissolved by the dissolving solution and the precipitate after salting out by the dissolving solution were combined to prepare the antigen component 1 of the nanovaccine 1.
  • nano vaccine 1 (Nanovaccine 1) is prepared by the double emulsion method in the solvent evaporation method.
  • the nano vaccine preparation materials used are PGLA (molecular weight of 20KDa-40KDa) and PEG5000-PLGA (PLGA molecular weight of 20KDa-40KDa), and the mass ratio of PLGA and PEG5000-PLGA used is 99:1; the immune adjuvants used are poly (I: C) and CpG7909.
  • the preparation method is as described above.
  • the antigen component 1 and adjuvant are loaded inside the nanoparticles by the double emulsion method, and then 100mg of nanoparticles are centrifuged at 14000g for 20 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of nano vaccine 1 is about 250nm, and each 1mg PLGA nanoparticle is loaded with about 200 ⁇ g of protein or polypeptide components, and 0.01mg of poly (I: C) and CpG7909 are loaded.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse. On the 3rd, 6th, 9th, 14th and 20th days before the mice were inoculated with tumors, 100 ⁇ L of 0.1 mg nanovaccine 1 or 100 ⁇ L of PBS were subcutaneously injected into the mice. The tumor growth rate and survival of the mice were monitored. The mouse tumor submission monitoring method was the same as above.
  • mice in the PBS group grew rapidly and the mice died soon.
  • the tumor growth rate of mice using Nanovaccin 1 was significantly slowed down and the survival period was significantly prolonged.
  • Example 17 Micron vaccine for the treatment of melanoma
  • the cultured B16F10 cells were collected, and then 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the back of each mouse.
  • the mice were killed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, and then passed through a cell filter, an appropriate amount of ultrapure water was added, and the cells were repeatedly frozen and thawed 5 times to lyse the circulating tumor cells.
  • the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; the precipitate was dissolved by adding a 0.1M tetramethylguanidine hydrochloride aqueous solution to the obtained precipitate to convert the insoluble component in pure water into a soluble component in a 0.1M tetramethylguanidine hydrochloride aqueous solution. Saturated ammonium sulfate aqueous solution was added dropwise to the water-soluble components in the lysate.
  • the obtained sample was centrifuged at 3000g for 5 minutes, and the precipitate was dissolved in 0.1M tetramethylguanidine hydrochloride aqueous solution for standby use. The supernatant was heated at 100°C for 5 minutes and then the obtained sample was centrifuged at 3000g for 5 minutes. After the supernatant was discarded, the precipitate was dissolved in 0.1M tetramethylguanidine hydrochloride aqueous solution; then the precipitate after salting out and the precipitate after heating dissolved with 0.1M tetramethylguanidine hydrochloride were combined and used as part of the water-soluble components.
  • the non-water-soluble components in the lysate dissolved with the above 0.1M tetramethylguanidine hydrochloride aqueous solution were mixed with the components obtained by salting out and heating precipitation in the water-soluble components dissolved with 0.1M tetramethylguanidine hydrochloride in a mass ratio of 1:1, which was the antigen component 1 of micron vaccine 1.
  • the micron vaccine 1 (Micronvaccine 1) is prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA is 40KDa-60KDa, and the immune adjuvant is poly(I:C).
  • the antigen component 1 and the adjuvant are loaded inside the micron particles by the double emulsion method, and then 200mg of the micron particles are centrifuged at 9000g for 20 minutes, and resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h.
  • the average particle size of the micron particles 1 is about 3.0 ⁇ m, and each 1mg
  • the PLGA microparticles were loaded with approximately 450 ⁇ g of protein or polypeptide components and 0.01 mg of poly(I:C).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse. On the 3rd, 6th, 9th, 14th and 20th days before the mice were inoculated with tumors, 0.2 mg micron vaccine 1 or 100 ⁇ L PBS was injected subcutaneously in the mice. The tumor growth rate and survival of the mice were monitored. The mouse tumor detection method was the same as above.
  • mice in the PBS group grew rapidly and the mice died soon.
  • the tumor growth rate of mice using Micronvaccine 1 was significantly slowed down, and the survival period was significantly prolonged. Some mice recovered without tumor. This shows that cancer micro-vaccines prepared using some components of circulating tumor cell lysates can effectively treat cancer.
  • amino acid sequences involved in the present disclosure are as follows:
  • R8 polypeptide RRRRRRRR (SEQ ID NO: 1)
  • TAVITPPTTTTKKARVSTPKPATPSTD SEQ ID NO: 3
  • KALA peptide WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO:7)
  • RALA peptide WEARLARALARALARHLARARALALRACEA (SEQ ID NO:8)

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Abstract

L'invention concerne un vaccin contre le cancer basé sur une partie de composants de cellules cancéreuses ou de tissu tumoral et son procédé de préparation. Le procédé de préparation comprend : premièrement, la séparation de protéines solubles dans l'eau et de composants non solubles dans l'eau de tous les composants cellulaires au moyen d'un procédé de relargage et similaire, puis le chargement de celles-ci sur des nanoparticules ou des particules microniques, de façon à être utilisées pour prévenir ou traiter des cancers. Un système de vaccin selon l'invention comprend les nanoparticules et/ou les particules microniques, les protéines dans des composants hydrosolubles de cellules cancéreuses et/ou de tissu tumoral, et les composants non hydrosolubles de cellules cancéreuses et/ou de tissu tumoral, et peut activer efficacement des réponses immunitaires spécifiques contre des cellules cancéreuses, et peut par conséquent être utilisé pour prévenir et traiter des maladies telles que des cancers.
PCT/CN2023/106259 2023-04-12 2023-07-07 Vaccin contre le cancer basé sur une partie de composants de cellules cancéreuses ou de tissu tumoral, et son procédé de préparation Pending WO2024212380A1 (fr)

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