WO2023040127A1 - Utilisation de système de vaccin contre le cancer sur la base de composants cellulaires entiers dans la préparation de médicaments de prévention croisée ou de traitement de cancers hétérogènes - Google Patents

Utilisation de système de vaccin contre le cancer sur la base de composants cellulaires entiers dans la préparation de médicaments de prévention croisée ou de traitement de cancers hétérogènes Download PDF

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WO2023040127A1
WO2023040127A1 PCT/CN2021/143434 CN2021143434W WO2023040127A1 WO 2023040127 A1 WO2023040127 A1 WO 2023040127A1 CN 2021143434 W CN2021143434 W CN 2021143434W WO 2023040127 A1 WO2023040127 A1 WO 2023040127A1
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water
components
cancer
vaccine
whole cell
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PCT/CN2021/143434
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English (en)
Chinese (zh)
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刘密
刁璐
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苏州尔生生物医药有限公司
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Publication of WO2023040127A1 publication Critical patent/WO2023040127A1/fr

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    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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

Definitions

  • the invention belongs to the field of immunotherapy, in particular to a broad-spectrum nano or micro cancer vaccine based on cancer cells or tumor tissues, in particular to a whole cell based on cancer cells and/or tumor tissues of one or more cancers Components of broad-spectrum nano- or micro-cancer vaccines and their use in the cross-prophylaxis of many other different types of cancer.
  • Immunity is a physiological function of the human body.
  • the human body relies on this function to identify "self” and “non-self” components, thereby destroying and repelling antigenic substances (such as viruses and bacteria) entering the human body, or damaged cells produced by the human body itself and tumor cells to maintain human health.
  • Immunotechnology has developed extremely rapidly in recent years, especially in the field of cancer immunotherapy. With the continuous improvement of the understanding of cancer, people have found that the human immune system and various immune cells play a key role in the process of inhibiting the occurrence and development of cancer. By regulating the strength and balance of the body's immune system, it is expected to affect and control the occurrence, development and treatment of cancer.
  • cancer vaccines have shown great potential in the prevention and treatment of cancer.
  • the basis for developing cancer vaccines is to select appropriate cancer antigens to activate the human immune system to recognize abnormally mutated cancer cells, and cancer cells or cancer tumor tissues themselves are the best source of cancer antigens.
  • scientists have used new technologies to analyze and identify cancer-specific or cancer-related antigenic polypeptides from tumor cells of cancer patients, and then artificially synthesize them in vitro to prepare cancer vaccines for cancer treatment.
  • the technology has shown some efficacy in clinical trials of cancer patients, but such methods are time-consuming, laborious and costly.
  • the methods used only extract and analyze the differences between cancer cells and normal cells from the water-soluble components of cancer cells, and then look for the different polypeptides. Therefore, such methods and technologies can only find a limited number of antigens with good water solubility. Peptides, which greatly limit the application of this type of method.
  • many antigenic proteins or polypeptides with strong immunogenicity in the real environment of the human body are insoluble in pure water. Water-insoluble proteins and peptides in water are very important and critical. Therefore, it is a promising approach to use cancer cells or whole cell components of cancer tissues as a source of vaccines for the prevention and treatment of cancer.
  • the prior art discloses a targeted delivery system loaded with whole cell components, which is a nano-sized or micron-sized particle with a target head on the surface, and the particles are loaded with whole cell components of cancer cells or tumor tissues ;
  • the whole cell component is the water-soluble component and the water-insoluble component of the whole cell in the cell or tissue, and the water-insoluble component is dissolved by a solubilizing agent;
  • the target head is combined with molecules on the surface of a specific cell or tissue to help The particles enter cells or tissues; however, the disclosed delivery system is used for the prevention and treatment of the same cancer.
  • the object of the present invention is to address the problems existing in the prior art, and to provide a micro or nano vaccine system loaded with whole cell components of one or more cancer cells or tumor tissues for cross-prevention or cross-treatment of other vaccines. Approaches to different types of cancer.
  • the present invention adopts the following technical solutions: the application of nano cancer vaccine and/or micron cancer vaccine system based on whole cell components in the preparation of cross-prevention or treatment of heterogeneous cancer drugs;
  • the cancer vaccine system of the invention comprises whole cell components, nano and/or micro particles; the whole cell components are cancer cell whole cell components and/or tumor tissue whole cell components.
  • the whole cell components are whole cell components of cancer cells and/or whole cell components of tumor tissues.
  • a vaccine system for cross-prevention or treatment of heterogeneous cancers including cancer cells or tumor tissue whole cell components, nanometers and/or microparticles, said vaccine system for cross-prevention or cross-treatment is different from the cancer cells used to prepare vaccines or other types of cancer of tumor tissue.
  • the invention is inventive in that the cancer for which the whole cell component is provided is different from the cancer for which prevention and treatment is desired.
  • the cancer that provides whole cell components and the cancer that needs prevention and treatment are all the same type of cancer, such as melanoma, lung cancer, breast cancer, etc.
  • the present invention proposes for the first time that the cancer that provides whole cell components and the cancer that needs prevention and treatment Cancers are different.
  • nano- and/or micro-particles loaded with whole cell components of melanoma are used to form nano-vaccine or micro-vaccine systems for the prevention and treatment of diseases such as lung cancer, breast cancer, and liver cancer.
  • the present invention can achieve very good results. The control effect is unpredictable.
  • the whole cell component-based nano cancer vaccine and/or micro cancer vaccine system further includes an immune enhancing adjuvant.
  • the whole cell component-based nano cancer vaccine and/or micro cancer vaccine system also includes an immune enhancing adjuvant in and/or on the surface.
  • the interior and/or surface of the particles further include an immune enhancing adjuvant.
  • the way of adding the immunoenhancing adjuvant includes loading in nanoparticles or microparticles, or loading on the surface of nanoparticles or microparticles, or loading in nanoparticles or microparticles and loading on the surface of nanoparticles or microparticles.
  • the immune-enhancing adjuvants include, but are not limited to, immune-enhancing agents derived from microorganisms, products of the human or animal immune system, innate immune stimulants, adaptive immune stimulants, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicines, and other types of at least one category of .
  • the immune-enhancing adjuvants include but are not limited to pattern recognition receptor agonists, Bacillus Calmette-Guerin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyclonal anti-A, mineral Oil, virus-like particles, immune-enhanced reengineered influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharides, curcumin, immune adjuvant CpG , immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium pumilus vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid , polyadenylic acid, alum,
  • the whole cell components for the preparation of cancer vaccines are derived from cancer cells and/or tumor tissues of one or more solid tumor cancers or non-solid tumor cancers;
  • the heterogeneous cancers for cross-prevention or cross-treatment are different Cancers of cancer cells or tumor tissues from which vaccines are prepared;
  • the heterogeneous cancers are one or more solid tumor cancers or non-solid tumor cancers.
  • cancer is solid tumor cancer or non-solid tumor cancer, such as endocrine system tumor, nervous system tumor, reproductive system tumor, digestive system tumor, urinary system tumor, immune system tumor, circulatory system tumor, respiratory system tumor, blood system tumor Tumors, tumors of the skin system.
  • Cancers are solid tumors or hematological system tumors, such as endocrine system tumors, nervous system tumors, reproductive system tumors, digestive system tumors, urinary system tumors, immune system tumors, circulatory system tumors, respiratory system tumors, hematological system tumors, and skin system tumors.
  • the whole cell components are water-soluble components and/or water-insoluble components.
  • the whole cell component can be divided into two parts according to the solubility in pure water or an aqueous solution without a solubilizer: a water-soluble component and a water-insoluble component.
  • the water-soluble component is the original water-soluble part that is soluble in pure water or an aqueous solution without a solubilizer
  • the water-insoluble component is the original non-water-soluble part that is insoluble in pure water.
  • the part that is insoluble in water or an aqueous solution containing no solubilizing agent becomes soluble in an aqueous solution containing a solubilizing agent or an organic solvent.
  • Both the water-soluble part and the water-insoluble part in the whole cell fraction can be dissolved by a solubilizing aqueous solution containing a solubilizing agent or an organic solvent.
  • the solubilizer is at least one of the solubilizers that can increase the solubility of proteins or polypeptides in aqueous solution;
  • the organic solvent is an organic solvent that can dissolve proteins or polypeptides.
  • the solubilizer includes but not limited to urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerin, alkaline solution with pH greater than 7, acidic solution with pH less than 7, various protein degrading enzymes, albumin, lecithin, high Concentration Inorganic salt, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, propanol, isopropanol, acetic acid, cholesterol, amino acid, glycoside, choline, Brij TM -35, Octaethylene glycol monododecyl ether, CHAPS, Digitonin , lauryldimethylamine oxide, IGEPAL® CA-630.
  • the water-insoluble components can also be changed from insoluble in pure water to soluble by using other methods that can solubilize proteins and polypeptide fragments.
  • the organic solvent includes but not limited to DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, dichloromethane, ethyl acetate.
  • the organic solvent can also use other organic solvent-containing methods that can solubilize proteins and polypeptide fragments.
  • whole cell components are loaded inside and/or on the surface of nanoparticles or microparticles, specifically, water-soluble components and/or water-insoluble components of whole cells are respectively or simultaneously loaded on nanoparticles and/or inside the micron particle, and/or separately or simultaneously loaded on the surface of the nanometer and/or micron particle.
  • the water-soluble components and/or water-insoluble components of the whole cells are separately or simultaneously loaded inside the particle, and/or separately or simultaneously loaded on the surface of the particle.
  • the loading method is that the water-soluble components and non-water-soluble components of the whole cell are separately or simultaneously loaded inside the particles, and/or are separately or simultaneously loaded on the surface of the particles; specifically, including but not limited to, the water-soluble components are simultaneously loaded on the Particles are neutralized and loaded on the surface of particles, water-insoluble components are loaded in particles and on the surface of particles at the same time, water-soluble components are loaded in particles but not water-soluble components are loaded on the surface of particles, water-insoluble components are loaded in particles and water-soluble The active ingredient is loaded on the particle surface, the water-soluble ingredient and the water-insoluble ingredient are loaded in the particle and only the water-insoluble ingredient is loaded on the particle surface, the water-soluble ingredient and the water-insoluble ingredient are loaded in the particle, and only the water-soluble ingredient is loaded on the particle On the surface, the water-soluble components are loaded in the particles, while the water-soluble components and the water-insoluble components are loaded on the particle surface at the same time, the water-insoluble components are loaded in the particles
  • the surface of the whole cell component-based cancer vaccine system may not be connected with a target head with active targeting function or may be connected with a target head with active targeting function.
  • the target head can lead the delivery system to target specific cells; the specific cells or tissues are dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils One or more of cells, basophils, lymph nodes, thymus, spleen, bone marrow.
  • the particle diameter of the nanoparticles is 1 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 100 nm to 600 nm; the particle diameter of the micron particles is 1 ⁇ m to 1000 ⁇ m, preferably 1 ⁇ m to 100 ⁇ m, more preferably 1 ⁇ m ⁇ 10 ⁇ m, more preferably 1 ⁇ m to 5 ⁇ m.
  • a cancer vaccine system based on whole cell components constructed from nanoparticles is a nano vaccine, and a cancer vaccine system based on whole cell components constructed from micron particles is called a micro vaccine.
  • the particle size of the nano vaccine is 1 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 100 nm to 600 nm; the particle size of the micron vaccine is 1 ⁇ m to 1000 ⁇ m, preferably 1 ⁇ m to 100 ⁇ m, more preferably 1 ⁇ m to 10 ⁇ m, more preferably 1 ⁇ m to 5 ⁇ m.
  • the surface of the nano-sized or micro-sized particles of the present invention can be neutral, negatively charged or positively charged.
  • the preparation materials of the nano and/or micro particles include but not limited to organic synthetic polymer materials, natural polymer materials or inorganic materials.
  • the organic synthetic polymer materials include but are not limited to PLGA, PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, synthetic lipid, Synthetic nucleic acids;
  • the natural polymer materials include but not limited to lecithin, cholesterol, lipids, sodium alginate, protein, nucleic acid, gelatin, cell membrane components, starch, sugars, polypeptides;
  • the inorganic materials include but not Limited to ferric oxide, ferric oxide, calcium carbonate, calcium phosphate.
  • the shape of the nano and/or micron particles is any common shape
  • the shape of the prepared nano-vaccine or micro-vaccine is any common shape, including but not limited to spherical, ellipsoidal, barrel-shaped, polygonal, Rods, flakes, wires, worms, squares, triangles, butterflies or discs.
  • the cancer vaccine based on cancer cells or tumor tissue nanometers or micrometers disclosed by the present invention can cross-prevent many other different types of cancers.
  • the vaccine system loaded with whole cell components is used for cross-treatment or cross-prevention of various other cancers, Nano-sized or micron-sized particles and whole cell components loaded on the particles, or composed of nano-sized or micron-sized particles, whole cell components loaded on the particles, and an immune-enhancing adjuvant, said
  • the whole cell fraction refers to the water-soluble and/or water-insoluble components of whole cells in cancer cells or tumor tissues.
  • the present invention uses a cancer vaccine system derived from cancer cells and/or tumor tissue whole cell components for cross-prevention or cross-treatment of other different types of cancer, and the other types of cancer used for cross-prevention or cross-treatment can be one or more than one.
  • the nano-vaccine or micro-vaccine can be prepared according to the published preparation methods of nano-sized particles and micron-sized particles, including but not limited to common solvent evaporation method, dialysis method, extrusion method, and hot-melt method.
  • the nano-vaccine or micro-vaccine is prepared by the double emulsion method in the solvent evaporation method.
  • the vaccine system based on the whole cell components of cancer cells and/or tumor tissues of the present invention can simultaneously use nanoparticles or microparticles loaded with only water-soluble components and nanoparticle or microparticles loaded with only water-insoluble components when preventing or treating diseases.
  • the present invention provides a vaccine system that utilizes nanoscale or micronscale particles to deliver cell water-soluble components and/or water-insoluble components, and is used to prepare drugs for the prevention and treatment of non-same cancers .
  • the whole cell components of relevant cells or tissues are divided into two parts according to their solubility in pure water, the water-soluble part soluble in pure water and the insoluble part insoluble in pure water, and the water-soluble part and The water-insoluble part is loaded inside and/or on the surface of nanoparticles or microparticles, so most of the mutated proteins or polypeptides produced by cancer in cell components are loaded inside and/or on the surface of nanoparticles or microparticles .
  • the water-soluble part and the water-insoluble part in the cell component include the components of the whole cell; the water-soluble part and the water-insoluble part in the cell component can also be dissolved by the aqueous solution containing the solubilizer at the same time.
  • the unmutated proteins, polypeptides and genes that are the same as normal cell components will not cause immune response due to the immune tolerance produced during the development of the autoimmune system; while the mutations of genes, proteins and polypeptides produced by cancer, etc. will not cause immune responses due to the lack of autoimmunity Immune tolerance developed during phylogeny is thus immunogenic and can activate immune responses.
  • the use of these immunogenic substances produced by disease mutations in the whole cell components can be used for the treatment of cancer.
  • the broad-spectrum cancer vaccine system of the whole cell components of the present invention is used to prepare vaccines for cross-prevention and/or treatment of other different kinds of cancers.
  • the vaccine described in the present invention can be administered multiple times before or after the occurrence of cancer or after surgical resection of tumor tissue to activate the body's immune system, thereby delaying the progression of cancer, Treat cancer or prevent cancer from coming back.
  • Figures 1-8 are schematic structural diagrams of nano-sized particles or micron-sized vaccines loaded with water-soluble and water-insoluble cell components, wherein 1: water-soluble components in cell or tissue components; 2, cell or tissue components 3, the immune enhancing adjuvant; 4, nanoparticles or microparticles; 5: the core part of the nanoparticles.
  • Figure 9- Figure 19 is a schematic diagram of the structure of a nano-vaccine or a micro-vaccine loaded with water-soluble and water-insoluble cell components modified actively targeting the target head, wherein 1: the water-soluble component in the cell or tissue component; 2: Water-insoluble components in cells or tissue components; 3: immune adjuvants; 4: nanoparticles or microparticles; 5: the core part of nanoparticles; 6: targets that can target specific cells or tissues.
  • Figures 20-29 are the experimental results of tumor growth rate and survival period in mice when nano-vaccine or micro-vaccine prepared from one or more cancer tumor tissues in Examples 1-10 are used for cross-prevention or cross-treatment of other types of cancer; a, the experimental results of tumor growth rate when nano-vaccine or micro-vaccine cross-prevention or cross-treatment of other cancers (n ⁇ 8); b, the experimental results of mouse survival time when nano-vaccine or micro-vaccine cross-prevention or cross-treatment of other cancers ( n ⁇ 8), each data point is the mean ⁇ standard error (mean ⁇ SEM); the significant difference in the tumor growth inhibition experiment in figure a was analyzed by ANOVA method, and the significant difference in figure b was analyzed by Kaplan-Meier and log- rank test analysis; ** indicates that this group has a significant difference with p ⁇ 0.005 compared with the PBS blank control group; ## indicates that this group has a significant difference with p ⁇ 0.005 compared with the blank nanoparticle + cell lysate control
  • Fig. 30 represents the preparation process and application field schematic diagram of the vaccine of the present invention
  • a water-soluble components and non-water-soluble components are respectively collected and prepared schematic diagrams of nano vaccines or micro vaccines
  • b adopting a solubilizing solution containing a solubilizing agent to dissolve Schematic diagram of whole cell components and preparation of nanovaccine or microvaccine.
  • the invention discloses a nano-vaccine or micro-vaccine system based on whole cell components of cancer cells and/or tumor tissues, and is used for cross-prevention or cross-treatment of other different types of cancers.
  • Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the methods and products of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention.
  • Invent technology is a nano-vaccine or micro-vaccine system based on whole cell components of cancer cells and/or tumor tissues, and is used for cross-prevention or cross-treatment of other different types of cancers.
  • the whole cell components of the nano or micro vaccine system based on cancer cells and/or tumor tissue whole cell components of the present invention are prepared from one or more cancer cells or tumor tissues, and used for cross-prevention or cross-treatment is different from preparation
  • the used cancer cells or other cancer types of tumor tissue can be one or more types of other cancers used for cross-prevention or cross-treatment.
  • the water-soluble components that are soluble in pure water or an aqueous solution without a solubilizing agent are firstly obtained, and then the water-insoluble components are dissolved by a solubilizing aqueous solution containing a solubilizing agent
  • the solubilization solution all the cell components can be converted into components that can be dissolved in the aqueous solution and then loaded inside and outside the nanoparticles or microparticles to prepare nano-vaccine or micro-vaccine for the prevention and treatment of cancer treat.
  • cells or tissues can also be lysed directly with a solubilizing aqueous solution containing a solubilizing agent to dissolve the whole cell components without collecting the water-soluble components and water-insoluble components separately, and the whole cell components dissolved in the solubilizing aqueous solution can be used
  • Cellular components make nanovaccine or microvaccine.
  • the present invention converts components insoluble in pure water or aqueous solutions without solubilizers in cells into soluble in specific solubilizing solutions and can be used to prepare nanoparticles and microparticles by using an aqueous solution containing a solubilizing agent, thereby improving The comprehensiveness and immunogenicity of the antigenic substances or components loaded by nano-vaccine or micro-vaccine.
  • the present invention divides the whole cell components in cancer cells and/or tumor tissues into water-soluble parts that can be dissolved in pure water or aqueous solutions without solubilizers and water-insoluble parts that can be dissolved in aqueous solutions with certain solubilizers, and
  • the water-soluble part and the water-insoluble part are entrapped in the nanoparticles or micro-particles and loaded on the surface thereof, thereby ensuring that most of the antigenic substances are loaded in the prepared vaccine.
  • the whole cell components of the present invention can be inactivated or (and) denatured before or (and) after lysis to prepare nano-vaccine or micro-vaccine, or can not be processed before or (and) after lysis Any inactivation or (and) denaturation treatment can directly prepare nano-vaccine or micro-vaccine.
  • the tumor tissue cells have been inactivated or (and) denatured before lysing. In actual use, they can also be inactivated or (and) denatured after cell lysis, or the cells can be lysed.
  • Inactivation or (and) denaturation treatment before and after lysis; the inactivation or (and) denaturation treatment method before or (and) after lysis of cells in some embodiments of the present invention is ultraviolet irradiation and high temperature heating. Inactivation or denaturation treatment methods such as radiation irradiation, high pressure, freeze-drying and formaldehyde can also be used in the process. Those skilled in the art can understand that during actual application, the skilled person can make appropriate adjustments according to specific conditions.
  • FIG. 1-19 The structural schematic diagrams of the nano-vaccine or micro-vaccine system of the whole cell component of the present invention are shown in Figures 1-19. In actual use, only nanoparticles or microparticles of a specific structure may be used, or two or more nanoparticles or microparticles of different structures may be used simultaneously. In Fig. 1 and Fig. 2, the surface and interior of nanoparticles or microparticles all contain immune enhancing adjuvants; in Fig. 3-Fig.
  • Microparticles only contain immunoenhancing adjuvants on the outer surface;
  • Figure 7-8 have no immune enhancing adjuvants on the inner and outer surfaces of nanoparticles or microparticles;
  • Figure 1A, Figure 3A, Figure 5A and Figure 7A When the water-soluble or non-water-soluble components in the loaded cells or tissue components are distributed inside the nanoparticles or microparticles, no obvious inner core is formed;
  • Figure 1B, Figure 3B, Figure 5B and Figure 7B show that the nanoparticles or microparticles
  • the water-soluble or non-water-soluble components of the loaded cell or tissue components form a core part when they are distributed inside the nanoparticles or microparticles.
  • the core can be generated during the preparation process or formed by using polymers or inorganic salts, etc.
  • the water-soluble component or the non-water-soluble component in the cell or tissue component loaded by nanoparticle or microparticle form multiple inner cores when they are distributed inside the nanoparticle or microparticle
  • the inner core can be generated during the preparation process or formed by using polymers or inorganic salts
  • Figure 2B, Figure 4B, Figure 6B and Figure 8B The water-soluble components of cells or tissue contained in nanoparticles or microparticles When the active component or water-insoluble component is distributed inside the nanoparticle or microparticle, it is located in the outer layer of the formed inner core;
  • a The inside and surface of the nanoparticle or microparticle are all water-soluble components in the cell or tissue components ;
  • c Nanoparticles or microparticles contain water-insoluble components in cells or tissue components The components loaded on the surface are all water
  • FIG 9- Figure 10 the surface and interior of nanoparticles or microparticles contain immune adjuvants; in Figure 11- Figure 12, immune adjuvants are only distributed in the interior of nanoparticles or microparticles; Particles only contain immune adjuvant on the outer surface; Figure 15- Figure 16 has no immune adjuvant inside and outside the nanoparticle or microparticle; Figure 17 cell components and/or immune adjuvant are only distributed inside the nanoparticle or microparticle; Figure 18 Cell components and/or immune adjuvants are only distributed outside the nanoparticles or microparticles; Figure 19 Cell components and immune adjuvants are distributed inside or outside the nanoparticles or microparticles, respectively.
  • the water-soluble or non-water-soluble components in the cells or tissue components are distributed in the outer layer of the inner core formed inside the nanoparticles or micro-particles; a: both the internal and surface loading of nanoparticles or micro-particles are cells or tissue groups the water-soluble components in the component; b: the nanoparticle or microparticle internally and surface-loaded are all water-insoluble components in the cell or tissue components; c: the nanoparticle or microparticle internally encapsulated is the cell or tissue
  • the water-insoluble components in the components are all water-soluble components in the cell or tissue components; d: the water-soluble components in the cells or tissue components are loaded on the surface of the nanoparticles or microparticles are the water-insoluble components in the cell or tissue components; e: the water-soluble components and the water-insoluble components in the cells or
  • the water-soluble or water-insoluble components of the cells or tissue components loaded by nanoparticles or microparticles in a, b and c do not form an obvious inner core when they are distributed inside the nanoparticles or microparticles ;
  • the water-soluble components or water-insoluble components in the cells or tissue components loaded by nanoparticles or microparticles in d, e and f are distributed in a core part inside the nanoparticles or microparticles; g, h and i
  • the water-soluble components or water-insoluble components in the cells or tissue components loaded by particles or microparticles are distributed in multiple core parts inside nanoparticles or microparticles; j, k and l are contained in nanoparticles or microparticles
  • the water-soluble or non-water-soluble components in the cell or tissue components are distributed in the outer layer of the inner core formed by nanoparticles or microparticles; a, d,
  • the method for preparing the vaccine system based on whole cell components described in the present invention is a common preparation method.
  • the double emulsion method in the solvent evaporation method is used to prepare nano or micro vaccines.
  • the nanoparticle preparation material used is organic polymer polylactic acid-glycolic acid copolymer (PLGA), and the immune adjuvant used is poly(I:C), Bacillus Calmette-Guerin (BCG), manganese adjuvant, or CpG.
  • PLGA organic polymer polylactic acid-glycolic acid copolymer
  • the immune adjuvant used is poly(I:C), Bacillus Calmette-Guerin (BCG), manganese adjuvant, or CpG.
  • the specific preparation method of the double-emulsion method used in the present invention is as follows: Step 1, adding a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of a medical solution containing a second predetermined concentration. In the organic phase of polymer materials.
  • the aqueous phase solution may contain the components in the cancer cell and/or tumor tissue lysate and the immunoenhancing adjuvant poly(I:C), BCG, manganese adjuvant or CpG; each component in the lysate
  • the components are respectively water-soluble components or original water-insoluble components dissolved in a solubilizing agent; or whole cell components dissolved in a solubilizing agent.
  • the predetermined concentration requires that the concentration of protein and peptide is greater than 1 ng/mL, enough antigen can be loaded to activate the relevant immune response.
  • the concentration of the immune enhancing adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.
  • the aqueous phase solution contains each component in the cancer cell and/or tumor tissue lysate and the immunoenhancing adjuvant poly(I:C), BCG, manganese adjuvant or CpG; each group in the lysate
  • the components are respectively water-soluble components or original water-insoluble components dissolved in the solubilizing agent during preparation.
  • the concentration of the water-soluble components contained in the aqueous phase solution or the concentration of the original water-insoluble components dissolved in the solubilizer, that is, the first predetermined concentration requires that the concentration of the protein polypeptide be greater than 1 ng/mL, can load enough cancer antigens to activate relevant immune responses.
  • the concentration of the immune enhancing adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.
  • the medical polymer material is dissolved in an organic solvent to obtain a second predetermined volume of an organic phase containing a second predetermined concentration of the medical polymer material.
  • the medical polymer material is PLGA
  • the organic solvent is dichloromethane.
  • the range of the second predetermined concentration of the medical polymer material is 0.5mg/mL-5000mg/mL , preferably 100 mg/mL.
  • PLGA or modified PLGA is selected because the material is a biodegradable material and has been approved by the FDA as a drug excipient. Studies have shown that PLGA has a certain immune regulation function, so it is suitable as an auxiliary material for vaccine preparation.
  • the second predetermined volume of the organic phase is set according to its ratio with 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 ranges 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 required to adjust the size of the prepared nanoparticles or microparticles.
  • step 2 the mixed solution obtained in step 1 is subjected to ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or homogenization treatment or microfluidic treatment.
  • This step is for nanometerization or micronization.
  • the length of ultrasonic time or the stirring speed and time can control the size of the prepared nanoparticles. Too long or too short will bring about changes in particle size. Therefore, it is necessary to select an appropriate ultrasonic time. .
  • the ultrasonic time is greater than 0.1 second, such as 2-200 seconds
  • the stirring speed is greater than 50 rpm, such as 50 rpm-500 rpm
  • the stirring time is greater than 1 minute, such as 60-600 seconds.
  • Step 3 adding the mixture obtained after the treatment in step 2 into a third predetermined volume of an aqueous solution containing an emulsifier of a third predetermined concentration and performing ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing homogenization treatment or using micro Flow control processing.
  • the mixture obtained in step 2 is added to the emulsifier aqueous solution to continue ultrasonication or stirring or homogenization treatment or microfluidic treatment for nanometerization or micronization.
  • the emulsifier aqueous solution is polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined volume is 5 mL
  • the third predetermined concentration is 20 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is 1:1.1 -1:1000 for setting, preferably 2:5.
  • the ratio of the second predetermined volume to the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time in this step the volume and concentration of the emulsifier aqueous solution are all based on the purpose of obtaining nanoparticles or microparticles of appropriate size.
  • Step 4 adding the liquid obtained after the treatment in step 3 into a fourth predetermined volume of an emulsifier aqueous solution of a fourth predetermined concentration, and performing stirring and/or vacuum treatment until predetermined conditions are met.
  • the emulsifier aqueous solution is still PVA
  • the fourth predetermined concentration is 5 mg/mL
  • the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of appropriate size.
  • the selection of the fourth predetermined volume is determined according to 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 between the third predetermined volume and the fourth predetermined volume can be adjusted.
  • the predetermined condition of this step is until the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in step 1 is completed.
  • Step 5 after centrifuging the mixed solution that meets the predetermined conditions in step 4 at a speed greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in the fifth predetermined volume of the first Five predetermined concentrations of the aqueous solution containing the lyoprotectant or the sixth predetermined volume of PBS (or physiological saline).
  • step 5 when the precipitate obtained in step 5 is resuspended in the sixth predetermined volume of PBS (or physiological saline), freeze-drying is not required, and subsequent nanoparticle or microparticle surface adsorption of cancer cells and/or Related experiments on tumor tissue lysates.
  • PBS physiological saline
  • the precipitate obtained in step 5 when the precipitate obtained in step 5 is resuspended in an aqueous solution containing a lyoprotectant, it needs to be lyophilized, and after lyophilization, the subsequent adsorption of cancer cells and/or tumors on the surface of nanoparticles or microparticles Related experiments on tissue lysates.
  • the lyoprotectant is selected from trehalose (Trehalose).
  • the fifth predetermined volume of the lyoprotectant in this step is 20 mL, and the fifth predetermined concentration is 4% by mass.
  • the reason for this setting is to not affect the effect of lyophilization in subsequent lyophilization. .
  • step 6 the suspension containing the lyoprotectant obtained in step 5 is lyophilized, and the lyophilized substance is used for future use.
  • Step 7 resuspend the sixth predetermined volume of the nanoparticle/microparticle-containing suspension obtained in step 5 in PBS (or normal saline) or resuspend with the sixth predetermined volume of PBS (or normal saline)
  • the freeze-dried freeze-dried substance containing nanoparticles/microparticles and freeze-drying protective agent obtained in step 6 is mixed with the seventh predetermined volume of water-soluble components or the original water-insoluble components dissolved in 8M urea.
  • Nano vaccines or micro vaccines are vaccine systems based on whole cell components.
  • the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferential volume ratio is 1:100 to 100:1, and the optimum volume ratio is 1:30 to 30:1 .
  • the resuspended nanoparticle or microparticle suspension has a volume of 10 mL, contains cancer cell lysate or contains water-soluble components in tumor tissue lysate or original non-alcohol dissolved in 8M urea.
  • the volume of the water-soluble component is 1 mL. The required volume and ratio of the two can be adjusted in the application.
  • the water-soluble components in the cancer cell and/or tumor tissue lysates or the original non-water-soluble components dissolved in 8M urea contain poly(I:C), Bacillus Calmette-Guerin (BCG), Manganese adjuvant or CpG, and the concentration of poly(I:C), BCG or CpG is greater than 1 ng/mL.
  • the particle size of nano-vaccine or micro-vaccine is nano-scale or micron-scale, which can ensure that the vaccine is phagocytized by antigen-presenting cells and activates the immune response. In order to improve the phagocytosis efficiency, the particle size should be within an appropriate range.
  • the particle size of the nano vaccine is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, most preferably, the particle size is 100nm-600nm; 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, most preferably, the particle size is 1 ⁇ m-5 ⁇ m.
  • the particle size of the nanoparticle vaccine is 100nm-600nm
  • the particle size of the micron vaccine is 1 ⁇ m-5 ⁇ m.
  • urea and guanidine hydrochloride are used to solubilize the original water-insoluble components in the cancer cell and/or tumor tissue lysate, and any other components that can make the cancer cell and/or tumor
  • the original water-insoluble components in the tissue lysate are dissolved in the solubilizing substances of the aqueous solution, such as sodium deoxycholate, SDS, alkaline solution with pH greater than 7, acidic solution with pH less than 7, albumin, lecithin, high concentration Inorganic salts, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, acetic acid, cholesterol, amino acids, glycosides, choline, Brij TM -35, Octaethylene glycol monododecyl ether, CHAPS, Digitonin, lauryldimethylamine oxide, IGEPAL® CA-630; or the above-mentioned solub
  • 8M urea and 6M guanidine hydrochloride aqueous solution are used to solubilize the original water-insoluble components in cancer cells and/or tumor tissue lysates, and any other components that can make cancer cells
  • the original water-insoluble components in cell and/or tumor tissue lysates are dissolved in the urea concentration or guanidine hydrochloride concentration of the aqueous solution; sex component.
  • the preparation of nano-vaccine and micro-vaccine adopts the double emulsion method, but any other commonly used method for preparing nanoparticles or micro-particles can also be used in practice.
  • the preparation material of the nano-vaccine and the micro-vaccine is PLGA, but any other material capable of preparing nanoparticles or micro-particles can also be used in practice.
  • the water-soluble components in the cancer cell and/or tumor tissue lysates or the original water-insoluble components dissolved in 8M urea are respectively entrapped inside the nano/micro particles and adsorbed on the nano/micro particles.
  • the water-soluble components in cancer cell and/or tumor tissue lysates and the original water-insoluble components dissolved in 8M urea can also be mixed and then packed into the particles or adsorbed to the particles surface; or 8M urea can also be used to simultaneously dissolve the water-soluble component and the water-insoluble component and then entrapped inside the nanoparticle or microparticle and/or adsorbed on the surface of the nanoparticle or microparticle.
  • poly(I:C), Bacillus Calmette-Guerin (BCG), manganese adjuvant and CpG are used as immune adjuvants.
  • Immune adjuvants such as pattern recognition receptor agonists, BCG cell wall skeleton, BCG methanolic extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyantibody A, mineral oil, virus-like particles, reconstitution for immune enhancement Influenza virion, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant poly ICLC, corynebacterium brevis vaccine, hemolytic chain Bacillus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum
  • the vaccines used in some embodiments are nano vaccines, and some embodiments use micro vaccines.
  • nano-vaccine or micro-vaccine according to the actual situation, that is, a vaccine system based on whole cell components of cancer cells and/or tumor tissues.
  • the vaccine system based on whole cell component of the present invention is composed of whole cell component, nano/micro particle, or composed of whole cell component, nano/micro particle and immune enhancing adjuvant.
  • the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. . Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
  • the methods used in the examples of the present invention are conventional methods; the materials and reagents used can be obtained from commercial sources.
  • the nanoparticle or microparticle structure, preparation method, and use strategy for disease treatment involved in the embodiments of the present invention are only representative methods, and the use of other nanoparticle or microparticle structures, preparation methods, and disease prevention or treatment
  • the strategy and the combination strategy with other drugs can also adopt the method described in the present invention.
  • the examples only list the application of the present invention in some cancers, but the present invention can also be used in any other types of cancer.
  • those skilled in the art can make conventional replacements based on the technical ideas of the present invention and existing technologies, and are not limited to the specific descriptions of the embodiments of the present invention.
  • the specific administration time, administration frequency, administration regimen, and combination with other drugs can be adjusted according to the actual situation.
  • Example 1 The whole cell components of lung cancer tumor tissue are loaded inside and on the surface of nanoparticles for the prevention of melanoma: this example uses B16F10 mouse melanoma as a cancer model to illustrate how to prepare the whole cell components of lung cancer tumor tissue Nano-vaccine, and apply the vaccine to prevent melanoma. First, the tumor block of LLC lung cancer tissue is cracked to prepare water-soluble components and water-insoluble components of lung cancer tumor tissue.
  • the organic polymer material PLGA was used as the nanoparticle framework material, and Polyinosinic-polycytidylic acid (poly(I:C)) was used as the immune adjuvant to prepare nanoparticles loaded with water-soluble components and water-insoluble components by solvent evaporation method. vaccine.
  • the nanovaccine was then employed to prevent melanoma.
  • the component is the raw material source of the nano vaccine used to prevent melanoma prepared from melanoma tumor tissue.
  • the preparation of nano-vaccine and the blank nano-particles used as a control adopt the double emulsion method in the solvent evaporation method, and the molecular weight of the nano-particle preparation material PLGA used is 24KDa-38KDa.
  • the adjuvant is poly(I:C) And poly(I:C) is not only distributed inside the nanoparticles but also adsorbed on the surface of the nanoparticles.
  • the preparation method is as described above.
  • the average particle size of nanoparticles is 320nm, and the average particle size of nano-vaccine after adsorption of cell components and immune adjuvant on the surface of nanoparticles is 340nm, and the surface potential of nano-vaccine is about -5mV .
  • Each 1mg PLGA nanoparticle is loaded with 180 ⁇ g protein or polypeptide component, and the poly(I:C) immune adjuvant used inside and outside each 1mg PLGA nanoparticle is about 0.01mg in total, and the inside and outside are divided in half.
  • the particle size of blank nanoparticles is 290nm.
  • Nano-vaccine for the treatment of cancer The control groups in this study were PBS group, blank nanoparticles + tumor tissue lysate group. Select 6-8-week-old female C57BL/6 as model mice to prepare melanoma-bearing mice.
  • the dosage regimen of the lung cancer tumor tissue nano-vaccine group is as follows: 200 ⁇ L of lung cancer tumor tissue lysates were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. 2 mg PLGA nanoparticles of water-soluble components and 200 ⁇ L of 2 mg PLGA nanoparticles loaded with original water-insoluble components dissolved in 8M urea in 200 ⁇ L; on day 0, 1.5 ⁇ 10 5 were subcutaneously inoculated in the lower right lower back of each mouse B16F10 cells.
  • the dosing regimen of the melanoma tumor tissue nanovaccine group is as follows: 200 ⁇ L of melanoma tumor tissue was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. 2 mg PLGA nanoparticles of the water-soluble component in the lysate and 200 ⁇ L of 2 mg PLGA nanoparticles of the original water-insoluble component dissolved in 8 M urea were loaded on the inside and on the surface; on day 0, 1.5 ⁇ 10 5 B16F10 cells.
  • the protocol of the PBS blank control group was as follows: 400 ⁇ L of PBS was injected subcutaneously on the 49th day, 42th day, 35th day, 28th day and 14th day before melanoma inoculation. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • Blank nanoparticles + tissue lysate control group 400 ⁇ L of blank nanoparticles and free lysate (equal to nanovaccine group) were subcutaneously injected 49 days, 42 days, 35 days, 28 days and 14 days before melanoma inoculation; Nanoparticles and free lysates were injected at different sites; on day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated on the lower right back of each mouse.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 2 The water-soluble components of lung cancer cells are loaded inside and on the surface of microparticles for the prevention of melanoma:
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare microparticles loaded with only the water-soluble components of LLC lung cancer cell components Micron vaccine, and the use of the vaccine to prevent melanoma.
  • LLC lung cancer cells were firstly lysed to prepare water-soluble components and water-insoluble components of LLC lung cancer cells. Then, the micron vaccine loaded with the water-soluble components of LLC cells is prepared by using the polymer material as the micron particle skeleton material and CpG as the immune adjuvant. And use the vaccine to prevent melanoma.
  • the LLC lung cancer cells were replaced with B16F10, and the water-soluble fraction derived from the melanoma cell lysate was prepared by the same method as above, which was the raw material source of the micron vaccine prepared by the melanoma cells.
  • micron vaccines In this example, the preparation of micron vaccines and the blank micron particles used as a control adopt the double emulsion method in the solvent evaporation method, and the micron particle preparation material used is an organic polymer material PLGA with a molecular weight of 38KDa-54KDa , the immune adjuvant used is CpG, and CpG is not only distributed inside the microparticles but also adsorbed on the surface of the microparticles.
  • the preparation method is as described above.
  • the micron vaccine particle size obtained after adsorbing cell components and immune adjuvant on the surface of the micron particle is about 1.30 ⁇ m, and the average surface potential of the micron particle is about -5mV.
  • Each 1 mg of PLGA microparticles is loaded with 200 ⁇ g of protein or polypeptide components, and the CpG immune adjuvant used inside and outside of each 1 mg of PLGA microparticles is 0.01 mg, and the inside and outside are divided into half.
  • the particle size of the blank microparticles is about 1.25 ⁇ m, and the corresponding water-soluble components are replaced by pure water containing the same amount of CpG when the blank microparticles are prepared.
  • Micron vaccine for cancer prevention select 6-8 week old female C57BL/6 to prepare melanoma tumor-bearing mice.
  • the micro-vaccine group scheme is as follows: 400 ⁇ L of 4 mg PLGA micro-particles loaded with water-soluble components in cancer cell lysates were subcutaneously injected on the 28th, 21st, and 14th days before inoculation of melanoma, respectively. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the protocol of the PBS blank control group was as follows: 400 ⁇ L of PBS were subcutaneously injected on the 28th, 21st, and 14th days before melanoma inoculation. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • Blank microparticles+cell lysate control group 400 ⁇ L of blank microparticles and cancer cell lysate equal to that in the vaccine were subcutaneously injected on the 28th day, 21st day, and 14th day before melanoma inoculation.
  • Example 3 Lung cancer tumor tissue lysate components loaded inside and on the surface of nanoparticles for the prevention of liver cancer: This example describes how to prepare a nano-vaccine loaded with lung cancer tumor tissue lysate components and apply the vaccine to prevent liver cancer Cross-prophylaxis against liver cancer using a vaccine prepared from lung cancer tumor tissue.
  • the lysed components of mouse LLC lung cancer tumor tissue were loaded inside and on the surface of nanoparticles to prepare nanovaccine.
  • the mouse LLC lung cancer tumor tissue was obtained and lysed to prepare the water-soluble fraction of the tumor tissue and the original water-insoluble fraction dissolved in 8M urea.
  • using PLGA as the nanoparticle framework material and poly(I:C) as the immune adjuvant to prepare nanovaccine loaded with water-soluble components and non-water-soluble components of the lysate, and use the vaccine to prevent Hepatocellular carcinoma 1-6 Liver cancer.
  • the nano-vaccine loaded with lung cancer tumor tissue is used for the prevention of liver cancer: select 6-8 week old female C57BL/6 to prepare Hepa 1-6 liver cancer tumor-bearing mice.
  • liver cancer cells On days 49, 42, 35, 28, and 14 before inoculation of liver cancer cells, 200 ⁇ L of 2 mg PLGA nanoparticles loaded with water-soluble components in tissue lysates and 200 ⁇ L of internal and surface Both were loaded with 2mg of PLGA nanoparticles dissolved in 8M urea of the original water-insoluble component. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • the protocol of the PBS blank control group was as follows: 400 ⁇ L of PBS was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • Blank nanoparticles + free lysate control group Subcutaneously inject 400 ⁇ L of blank nanoparticles and the same amount of vaccine-loaded free lysate. Blank nanoparticles and free tissue lysates were injected at different sites. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 4 Whole cell components of lung cancer and melanoma tumor tissues loaded inside nanoparticles for the prevention of liver cancer: This example uses mouse liver cancer as a cancer model to illustrate how to prepare whole cell components loaded with lung cancer and melanoma tumor tissues Nano-vaccine, and apply the vaccine to prevent liver cancer.
  • lung cancer and melanoma tumor tissues were first lysed to prepare the water-soluble and water-insoluble fractions of the whole cell fraction. Then, using PLGA as the nanoparticle framework material and poly(I:C) as the immune adjuvant, a nanovaccine loaded with water-soluble components or water-insoluble components of lung cancer tumor mass and melanoma tumor mass was prepared by solvent evaporation method. , and use the nano-vaccine to prevent liver cancer.
  • the preparation of nano-vaccine adopts the double emulsion method in the solvent evaporation method
  • the molecular weight of the nanoparticle preparation material PLGA used is 7KDa-14KDa
  • the immune adjuvant used is poly(I:C) and poly(I:C) are distributed inside the nanoparticles.
  • the water-soluble component is a mixture (equal mass ratio) of the water-soluble component of lung cancer tumor tissue and the water-soluble component of melanoma tumor tissue
  • the water-insoluble component is the water-insoluble component of lung cancer tumor tissue and melanin A mixture of tumor tissue water-insoluble components (equal mass ratio).
  • the particle size of the nano-vaccine is about 300nm, and the average surface potential of the nano-particles is about -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with about 200 ⁇ g of protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside of each 1 mg of PLGA nanoparticles is 0.01 mg.
  • the particle size of blank nanoparticles is about 240nm. When preparing blank nanoparticles, pure water containing poly(I:C) or 8M urea are used to replace the corresponding water-soluble components and non-water-soluble components. Blank nanoparticles are loaded with nano Vaccine equivalent poly(I:C).
  • the nano-vaccine is used for the prevention of cancer: the specific administration scheme and tumor growth monitoring scheme of the vaccine group and the control group are as in Example 3.
  • Example 5 Lysis components of melanoma tumor tissue and colon cancer tumor tissue are loaded inside and on the surface of nanoparticles for the treatment of pancreatic cancer:
  • This example uses mouse pancreatic cancer as a cancer model to illustrate how to prepare melanoma tumor tissue and colon cancer tumor tissue lysate components, and apply the vaccine to treat pancreatic cancer.
  • mouse B16F10 melanoma tumor tissue and MC38 colon cancer tumor tissue lysate components were loaded on the interior and surface of nanoparticles to prepare nanovaccine.
  • mouse melanoma and colon cancer tumor tissues were obtained and lysed to prepare the water-soluble fraction and the original water-insoluble fraction dissolved in 8M urea.
  • the water-soluble component is a 2:1 mass ratio mixture of the water-soluble component of the colon cancer tumor tissue and the water-soluble component of the melanoma tumor tissue; the water-insoluble component is the water-insoluble component of the colon cancer tumor tissue and a 2:1 mass ratio mixture of water-insoluble components of melanoma tumor tissue.
  • nanovaccine loaded with water-soluble and water-insoluble components of tumor tissue lysate was prepared. The vaccine was then used to treat Pan02 pancreatic cancer tumor-bearing mice.
  • Nano-vaccine for cancer treatment select 6-8 week old female C57BL/6 to prepare pancreatic cancer tumor mice. On day 0, 1 ⁇ 106 Pan02 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the vaccine group was subcutaneously injected with 200 ⁇ L of 2 mg PLGA nanoparticles loaded with water-soluble components in the lysate and 200 ⁇ L of lysate loaded with lysate on the 4th day, 7th day, 10th day, 15th day and 20th day, respectively. 2mg PLGA nanoparticles of the original water-insoluble component in 8M urea.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L of PBS on the 4th day, 7th day, 10th day, 15th day and 20th day.
  • Blank nanoparticles + lysate control group were subcutaneously injected with 400 ⁇ L of blank nanoparticles and the same amount of free lysate loaded with the vaccine on the 4th day, 7th day, 10th day, 15th day and 20th day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 6 Whole cell components of melanoma tumor tissue loaded inside microparticles for the prevention of lung cancer: This example uses mouse lung cancer as a cancer model to illustrate how to prepare a micron vaccine loaded with whole cell components of melanoma tumor tissue, And apply the vaccine to prevent lung cancer.
  • the specific dosage form, adjuvant, administration time, administration frequency, and administration regimen can be adjusted according to the situation.
  • mouse melanoma tumor tissues were obtained and lysed to prepare water-soluble fractions and original water-insoluble fractions dissolved in 8M urea.
  • PLGA 50:50
  • mannose-modified PLGA were used as the microparticle framework material
  • CpG was used as the immune adjuvant to prepare the water-soluble and non-water-soluble components loaded with tumor tissue lysates by the solvent evaporation method.
  • micron vaccine The micron vaccine has the ability to target dendritic cells.
  • micron vaccines In this example, the micron vaccines and the empty micron particles used as a control adopt the double emulsion method in the solvent evaporation method, and the micron particle preparation material PLGA (50:50) is used The molecular weight is 38KDa-54KDa, and the molecular weight of the mannose-modified PLGA (50:50) used is 38KDa-54KDa.
  • the mass ratio of unmodified PLGA to mannose-modified PLGA was 8:2.
  • the non-target modified micro-vaccine group was all prepared by unmodified PLGA.
  • the immune adjuvant used is CpG and the CpG is distributed inside the microparticles.
  • the preparation method is as mentioned above.
  • the average particle size of the micron particles is about 1.20 ⁇ m, and the average surface potential is about -8 mV. per 1 mg PLGA microparticles are loaded with 60 ⁇ g of protein or polypeptide components, and the CpG immune adjuvant used for each 1 mg of PLGA microparticles is 0.01 mg, and the inside and outside are divided in half.
  • the particle size of the blank microparticles is about 1.10 ⁇ m. When the blank microparticles are prepared, pure water or 8M urea containing the same amount of CpG is used to replace the corresponding water-soluble components and non-water-soluble components.
  • Micro-vaccine targeting dendritic cells for the prevention of cancer select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice.
  • the vaccine group received subcutaneous injections of 200 ⁇ L of 2 mg PLGA microparticles loaded with water-soluble components in cancer cell lysates and 200 ⁇ L of internal Both the surface and the surface are loaded with 2mg PLGA micron particles dissolved in 8M urea, which is the original water-insoluble component.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • Blank microparticles + cell lysate control group were subcutaneously injected with 400 ⁇ L of blank microparticles and the same amount of free cells loaded with the vaccine on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation. Lysate.
  • 2 ⁇ 10 6 LLC lung cancer cells were subcutaneously inoculated into the lower right lower back of each mouse. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from the 3rd day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 7 The whole cell components of lung cancer tumor tissue or melanoma tumor tissue are loaded inside and on the surface of nanoparticles, and the nano-vaccine with bacillus Calmette-Guerin (BCG) as an immune adjuvant is used for the prevention of liver cancer: this example takes mouse liver cancer as the cancer Model and use BCG as an immune adjuvant to illustrate how to use nano-vaccine loaded with whole cell components of lung cancer tumor tissue or melanoma tumor tissue to prevent liver cancer.
  • BCG bacillus Calmette-Guerin
  • the water-soluble and water-insoluble components of lung cancer or melanoma tumor tissue are lysed. Then, using PLGA as the nanoparticle framework material and BCG as the immune adjuvant to prepare nanovaccine loaded with water-soluble components and water-insoluble components of lung cancer or melanoma tumor tissue.
  • Nano-vaccine for the prevention of liver cancer Select female C57BL/6 as model mice to prepare Hepa 1-6 liver cancer tumor-bearing mice.
  • the vaccine group was subcutaneously injected with 200 ⁇ L of 2 mg PLGA nanoparticles loaded with water-soluble components in tumor tissue lysate and 200 ⁇ L of the internal surface on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation, respectively. Both the surface and the surface are loaded with the 2mg PLGA nano-vaccine dissolved in the original water-insoluble component in 8M urea.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • Blank nanoparticles + lysate control group were subcutaneously injected with 400 ⁇ L of blank nanoparticles and the same amount of free lysate loaded with the vaccine on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation. .
  • 2 ⁇ 106 Hepa1-6 liver cancer cells were subcutaneously inoculated into the armpit of each mouse. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from the 3rd day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 8 6M guanidine hydrochloride dissolves tumor tissue components of lung cancer and colon cancer and loads them inside and on the surface of microparticles for the treatment of breast cancer.
  • This example uses mouse breast cancer as a cancer model to illustrate how to use 6M guanidine hydrochloride to dissolve whole Cell components and preparation of micron vaccines loaded with whole cell components for the treatment of breast cancer.
  • 4T1 mouse triple-negative breast cancer cells were used as cancer cell models. Firstly, the tumor tissue cells of lung cancer and colon cancer were inactivated and denatured, and the tumor tissue was lysed with 6M guanidine hydrochloride to dissolve the whole cell components. Then, PLGA is used as the framework material of micron particles, and CpG is used as immune adjuvant to prepare the micron vaccine loaded with whole cell components. The microvaccine was then used to treat tumors in breast cancer-bearing mice.
  • the obtained tumor tissue cells were inactivated and denatured by ultraviolet light and high temperature heating respectively, and then the tumor tissue cells of lung cancer and colon cancer were lysed by appropriate amount of 6M guanidine hydrochloride and the tissue lysate was dissolved, and the tumor tissue lysate of lung cancer and colon cancer tumor tissue were lysed After the mixture is mixed, it is the source of raw materials for preparing vaccines.
  • micron vaccine and blank micron particles used PLGA (50:50) with a molecular weight of 38KD-54KD, and the preparation method was as described above.
  • CpG was used as an immune adjuvant.
  • the average particle size of the prepared micron vaccine is about 2.5 ⁇ m, and the surface potential of the micron particle is -4mV.
  • 210 ⁇ g of protein and polypeptide components are loaded inside and outside each 1 mg PLGA nanoparticle, and the CpG immune adjuvant used inside and outside each 1 mg PLGA nanoparticle is 0.01 mg in total, and the inside and outside are divided into half.
  • Micron vaccine for the treatment of cancer Select 6-8 weeks old female BALB/c to prepare 4T1 tumor-bearing mice. On day 0, 4 ⁇ 105 4T1 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the vaccine treatment group was subcutaneously injected with 400 ⁇ L of 4 mg PLGA micron vaccine loaded with whole cell components of tumor tissue inside and on the surface on the 4th, 7th, 10th, 15th and 20th days.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L of PBS on the 4th day, 7th day, 10th day, 15th day and 20th day.
  • Blank microparticles + lysate control group were subcutaneously injected with equal amount of tumor tissue lysate on the 4th day, 7th day, 10th day, 15th day and 20th day, and loaded with the same amount of CpG without any cell lysis 4mg PLGA blank micron particles of the material composition.
  • mice whose tumor volume exceeded 2000 mm3 were considered dead and were euthanized.
  • Example 9 Melanoma tumor tissue lysate components are loaded inside and on the surface of nanoparticles for the prevention of liver cancer: This example is based on how to prepare a nano-vaccine loaded with melanoma tumor tissue lysate components and apply the vaccine to prevent liver cancer To illustrate how to use a vaccine prepared from melanoma tumor tissue for cross-prevention of liver cancer.
  • mouse B16F10 melanoma tumor tissue lysate components were loaded on the inside and surface of nanoparticles to prepare nanovaccine.
  • mouse tumor tissue was obtained and lysed to prepare the water-soluble fraction of the tumor tissue and the original water-insoluble fraction dissolved in 6M guanidine hydrochloride.
  • poly(I:C) as an immune adjuvant or without an immune adjuvant to prepare nanovaccine loaded with water-soluble and water-insoluble components of the lysate, and using the Vaccine to prevent Hepa 1-6 liver cancer.
  • Nanovaccine loaded with tumor tissue is used for the prevention of liver cancer: 6-8 week old female C57BL/6 were selected to prepare Hepa 1-6 liver cancer tumor-bearing mice.
  • liver cancer cells On the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells, 200 ⁇ L of 2 mg PLGA nano-vaccine loaded with water-soluble components in tumor tissue lysates and 200 ⁇ L of internal and The surfaces are all loaded with 2 mg PLGA nano-vaccine dissolved in 8M urea, which is the original water-insoluble component.
  • 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • the protocol of the PBS blank control group was as follows: 400 ⁇ L of PBS was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • Blank nanoparticles + lysate control group Subcutaneously inject 400 ⁇ L of blank nanoparticles and free cell lysate. Blank nanoparticles and free cell lysates were injected at different sites. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 10 Whole cell components of lung cancer cancer cells are loaded on the inside and surface of nanoparticles for the prevention of melanoma: This example illustrates how to prepare a nano-vaccine loaded with whole cell components of lung cancer cancer cells, and apply the vaccine to prevent melanoma tumor.
  • LLC cancer cells were first lysed to prepare the corresponding water-soluble components and water-insoluble components dissolved in 8M urea. Then, using PLGA as the framework material and colloidal manganese as the immune adjuvant to prepare the nano-vaccine.
  • nano-vaccine Preparation of nano-vaccine:
  • the nano-vaccine and the blank nano-particles were prepared by the double emulsion method, and the water-soluble components were loaded inside the nanoparticles instead of the water-soluble components on the surface of the nano-vaccine.
  • the nanoparticles used The molecular weight of the prepared material PLGA is 7KDa-17KDa, the immune adjuvant adopted is colloidal manganese and the colloidal manganese is distributed inside the nanoparticles.
  • the above samples were dissolved in 9 mL of PBS and mixed with 1 mL of non-water-soluble components (80 mg/mL) dissolved in 8M urea, and then used at room temperature for 10 min.
  • the average particle size of the nano-vaccine loaded with whole cell components is about 350nm, and the surface potential of the nano-vaccine is about -5mV; each 1 mg PLGA nano-particle is loaded with about 180 ⁇ g protein or polypeptide component.
  • the particle size of the blank nanoparticles is about 320nm, and the blank nanoparticles are loaded with an equal amount of colloidal manganese.
  • Nano-vaccine for the prevention of cancer mice in the vaccine group were given 400 ⁇ L of nano-vaccine containing 4 mg PLGA each time, and mice in the control group were given 400 ⁇ L of PBS or blank Nanoparticles + free lysate. Dosing regimen for prophylaxis and protocol for mouse tumor inoculation and monitoring Example 1.
  • the vaccine system with whole cell components of the present invention can be used to prepare drugs for cross-prevention and/or treatment of cancer, and its preparation process and application fields are shown in FIG. 30 .
  • the cells or tissues can be lysed, and then the water-soluble components and water-insoluble components can be collected separately to prepare nano-vaccine or micro-vaccine respectively; or directly use a solubilizing solution containing a solubilizing agent to directly lyse cells or tissues and dissolve whole cells Components and preparation of nano-vaccine or micro-vaccine.

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Abstract

La présente invention concerne l'application d'un système de vaccin contre le cancer sur la base de composants cellulaires entiers dans la préparation de médicaments de prévention croisée ou de traitement de cancers hétérogènes, le système de vaccin utilisant des particules de taille nanométrique ou de taille micrométrique pour administrer des composants solubles dans l'eau et/ou des composants non solubles dans l'eau de composants cellulaires entiers. À mesure que la partie soluble dans l'eau et/ou la partie non soluble dans l'eau sont toutes deux chargées à l'intérieur et/ou sur la surface des particules de taille nanométrique ou des particules de taille micrométrique, des protéines ou des polypeptides variants produits par des cancers dans les composants cellulaires sont par conséquent également chargés à l'intérieur et/ou sur la surface des particules de taille nanométrique ou des particules de taille micrométrique. Les substances immunogènes produites par mutation dans les composants cellulaires entiers peuvent être utilisées pour prévenir et traiter des cancers. Par conséquent, le système de vaccin basé sur des composants cellulaires entiers peut préparer des médicaments de prévention croisée et/ou de traitement de cancers hétérogènes.
PCT/CN2021/143434 2021-09-18 2021-12-30 Utilisation de système de vaccin contre le cancer sur la base de composants cellulaires entiers dans la préparation de médicaments de prévention croisée ou de traitement de cancers hétérogènes WO2023040127A1 (fr)

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