CN114931632A

CN114931632A - Cancer vaccine based on antigen presenting cell membrane component and preparation method and application thereof

Info

Publication number: CN114931632A
Application number: CN202210630310.6A
Authority: CN
Inventors: 刘密
Original assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Current assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Priority date: 2022-06-06
Filing date: 2022-06-06
Publication date: 2022-08-23
Also published as: WO2023236330A1

Abstract

The invention relates to a cancer vaccine based on antigen presenting cell membrane components, a preparation method and application thereof, wherein the cancer vaccine comprises one of the following components: (1) preparing nano vesicles from activated antigen-presenting cell membrane components; (2) a second delivery particle internally and/or externally loaded with a cancer cell whole cell component and surface loaded with an activated antigen presenting cell membrane component. The invention realizes that the vaccine derived from the dendritic cells loads broad-spectrum and diverse cancer cell antigens, simultaneously overcomes the difficult problems of the live cell vaccine such as difficult maintenance of the activity of the dendritic cells and incapability of freeze-drying and long-term storage, and can prepare the cancer vaccine loading broad-spectrum cancer cell epitope for preventing and treating cancers.

Description

Cancer vaccine based on antigen presenting cell membrane component and preparation method and application thereof

Technical Field

The invention relates to the field of immunotherapy, in particular to a cancer vaccine based on an antigen presenting cell membrane component and a preparation method and application thereof.

Background

Cancer immunotherapy is one of the most important therapeutic approaches for cancer, and cancer vaccines are one of the important approaches for cancer immunotherapy. The usual mechanism of action of cancer vaccines is: the antigen carried by the cancer vaccine is phagocytosed and degraded by antigen presenting cells, and the antigen epitope degraded into small peptide is combined with Major Histocompatibility Complex (MHC) molecules and then presented on the surface of the antigen presenting cells to activate cancer cell specific T cells. The process of the cancer vaccine activating the Naive T cells into cancer cell specific T cells through antigen presenting cells belongs to the primary activation of T cells, and is completed only by depending on Dendritic Cells (DC) and in lymph nodes. Once T cells are activated by antigen from Naive T cells to cancer cell specific T cells, antigen polypeptides presented by various antigen presenting cells or mutant cancer cells can be identified. In view of the core function of DC in the primary activation of cancer cell-specific T cells, there is a class of cancer vaccines that uses antigen-activated DC-infused patients as a therapeutic vaccine, such as the prostate cancer therapeutic vaccine prevonqi, a live DC cell vaccine, that is already marketed in the united states. However, the DC cell vaccine also faces a plurality of problems, for example, the DC vaccine is very strict in conditions during preparation, storage, transportation and use, the DC vaccine as a live cell vaccine is easily polluted by various microorganisms, and once the cells are subjected to gene pollution or mutation, the live cell vaccine is easily transmitted to other live cells in the body, and the like. In addition, the cancer cell epitopes loaded by the existing dendritic cell vaccine are not diverse enough, and the application of the dendritic cell vaccine is limited. Therefore, the invention aims to develop a cancer vaccine which is loaded on the surface, contains an antigen presenting cell membrane and an epitope combined with a Major Histocompatibility Complex (MHC) molecule, has various cancer cell epitopes, can be directly combined with T cells to activate the Naive T cells, can be phagocytosed by the antigen presenting cells in an organism to indirectly activate the Naive T cells, and is convenient to transport and store.

Disclosure of Invention

In order to solve the technical problems, the invention provides a cancer vaccine based on antigen presenting cell membrane components.

It is a first object of the present invention to provide a cancer vaccine based on antigen presenting cell membrane components, the cancer vaccine comprising one of:

(1) preparing nano vesicles from activated antigen-presenting cell membrane components;

(2) a second delivery particle internally and/or externally loaded with cancer cell whole cell components and surface-loaded with activated antigen-presenting cell membrane components;

wherein, the first and the second end of the pipe are connected with each other,

the activated antigen-presenting cell membrane fraction is prepared from pre-activated antigen-presenting cells by co-incubating antigen-presenting cells with first delivery particles loaded with a tumor tissue and/or cancer cell whole cell fraction;

the first delivery particle and the second delivery particle (hereinafter, "delivery particle" is used to mean the first delivery particle and/or the second delivery particle, and the first delivery particle and the second delivery particle may or may not be subjected to the following treatment process at the same time, or one of them is subjected to the following treatment process) are independently a nanoparticle or a microparticle; the antigen presenting cell membrane component is a membrane component from a cell membrane or an extracellular vesicle membrane.

Further, the cancer cell whole cell component may be a whole cell component obtained by lysing cancer cells and/or tumor tissues and containing a water-soluble component and a water-insoluble component, and preferably is a whole cell antigen contained in tumor tissues and/or whole cell components obtained by lysing cancer cells, the whole cell antigen includes a water-soluble antigen and a water-insoluble antigen obtained by lysing cancer cells and/or tumor tissues, and the water-insoluble antigen is loaded on the delivery particle after being lysed by a lytic agent.

Further, the dissolving agent is at least one selected from urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein degrading enzyme, albumin, lecithin, inorganic salt (0.1-2000mg/mL), Triton, Tween, amino acid, glycoside, and choline.

Further, the antigen-presenting cell may be at least one of a dendritic cell, a B cell and/or a macrophage, preferably, two of the above three cells may be selected, and most preferably, a combination of a dendritic cell, a B cell and a macrophage.

Further, the delivery particle is loaded with an immune enhancing adjuvant inside and/or outside.

Further, immunopotentiating adjuvants include, but are not limited to, at least one of immunopotentiators of microbial origin, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicines, and others; immunopotentiating adjuvants include, but are not limited to, pattern recognition receptor agonists, BCG (BCG), STING agonists, BCG cell wall backbone, BCG methanol extraction residues, BCG muramyl dipeptide, Mycobacterium phlei, polyoxin, mineral oil, virus-like particles, immunopotentiating reconstituted influenza virions, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharide, curcumin, STING agonists, Toll-like receptor agonists, immunoadjuvant CpG, immunoadjuvant poly (I: C), immunoadjuvant poly ICLC, corynebacterium parvum, hemolytic streptococcal preparations, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvants, aluminum adjuvants, calcium adjuvants, various cytokines (e.g., colony stimulating factors), interleukins, interferons, poly inosinic acid, poly adenylic acid, alum, and poly (I: C), Aluminum phosphate, lanolin, squalene, cytokine, vegetable oil, endotoxin, liposome adjuvant, MF59, double-stranded RNA, double-stranded DNA, aluminum-related adjuvant, CAF01, Ginseng radix, and effective components of radix astragali.

Preferably, the immune enhancing adjuvant is a Toll-like receptor agonist; more preferably, two or more Toll-like receptor agonists are combined to ensure that the nanoparticles or microparticles can better activate cancer cell-specific T cells after being phagocytosed by antigen presenting cells.

Further, two or more Toll-like receptor agonists are used in combination with poly (I: C)/Poly (ICLC) and CpG-ODN (CpG oligodeoxynucleotide). Preferably, the CpG-ODN is two or more CpG-ODN.

Further, the delivery particle is also loaded internally and/or externally with a substance that increases lysosomal escape.

Further, the lysosomal escape-increasing substances include, but are not limited to, carrier materials that increase the osmotic pressure within the lysosome, carrier materials that decrease the stability of the lysosome membrane, substances with proton sponge effect.

Further, agents that increase lysosomal escape include, but are not limited to, amino acids, polyamino acids, organic polymers, nucleic acids, polypeptides, lipids, carbohydrates, and the like.

Further, the delivery particle is also loaded internally and/or externally with a target head that actively targets T cells, antigen presenting cells, or NK cells. The target head can be mannose, mannan, CD19 antibody, CD3 antibody, CD56 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, etc.

Further, the cancer vaccine based on the antigen presenting cell membrane component is a freeze-dried preparation prepared by freeze-drying the cancer vaccine based on the antigen presenting cell membrane component, wherein the freeze-drying protective agent comprises, but is not limited to, polyhydroxy compounds, saccharides and amino acids.

Further, the polyol includes, but is not limited to, one or more of glycerol, mannitol, sorbitol, inositol, thiol, polyethylene glycol, and the like.

Further, sugars include, but are not limited to, one or more of trehalose, mannose, lactose, sucrose, dextran, maltose, inulin, heparin, and the like.

Further, amino acids include, but are not limited to, one or more of arginine, histidine, tyrosine, proline, tryptophan, glycine, and the like.

Preferably, the lyoprotectant is a combination of any one of:

based on the mass percentage, the weight percentage of the mixture,

(1) 1-5% of trehalose, 1-5% of mannitol and 1-5% of arginine, and the balance of water;

(2) 1-5% of trehalose, 1-5% of mannitol and 1-5% of glycine, and the balance of water;

(3) 1-5% of trehalose, 1-5% of mannitol, 1-5% of lysine and the balance of water;

(4) 1-5% of mannitol, 1-5% of sucrose, 1-5% of lysine and the balance of water;

(5) 1-5% of trehalose, 1-5% of mannitol and 1-5% of gelatin, and the balance of water;

(6) 1-5% of trehalose, 1-5% of mannitol, 1-5% of polyvinylpyrrolidone and the balance of water;

(7) 1-5% of trehalose, 1-5% of mannitol and 1-5% of albumin, and the balance of water.

Further, the particle size of the nano particles is 1nm-1000 nm; the particle size of the micron particles is 1-1000 μm; the surface of the nano-particle or the micro-particle is electrically neutral, negatively charged or positively charged.

Further, the nanoparticles or microparticles are prepared from organic synthetic polymer materials, natural polymer materials or inorganic materials, and can be prepared by the existing preparation methods, including but not limited to common solvent evaporation methods, dialysis methods, microfluidic methods, extrusion methods and hot melting methods.

Further, the organic synthetic polymer material includes PLGA, PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, etc.; the natural polymer material comprises lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, saccharide, polypeptide, etc.; the inorganic material includes ferric oxide, ferroferric oxide, carbonate, phosphate, etc.

Further, the means for supporting the water-soluble antigen or the water-insoluble antigen on the surface of the nanoparticle or the microparticle includes at least one of adsorption, covalent attachment, charge interaction, hydrophobic interaction, one or more steps of solidification, mineralization and encapsulation.

Furthermore, the nanoparticles or microparticles may not be modified during the preparation process, and suitable modification techniques may also be employed to increase the antigen loading of the nanoparticles or microparticles. Modification techniques include, but are not limited to, biomineralization (e.g., silicidation, calcification, magnesiation), gelation, crosslinking, chemical modification, addition of charged species, and the like.

Further, the antigen is loaded inside the nanoparticle or microparticle in any manner that can load the antigen inside the nanoparticle or microparticle, such as entrapment.

Further, the antigen may be loaded on the surface of the nanoparticle or microparticle by means including, but not limited to, adsorption, covalent attachment, charge interaction (e.g., addition of positively charged species, addition of negatively charged species), hydrophobic interaction, one or more steps of immobilization, mineralization, encapsulation, and the like.

Furthermore, one or more layers of water-soluble antigens and/or water-insoluble antigens loaded on the surfaces of the nano particles or the micro particles are loaded, and when a plurality of layers of water-soluble antigens and/or water-insoluble antigens are loaded on the surfaces, modifiers are arranged between the layers.

The second object of the present invention is to provide a method for preparing the above cancer vaccine based on antigen presenting cell membrane components, comprising the steps of:

s1, co-incubating the antigen-presenting cells with the first delivery particle loaded with the cancer cell whole cell component to activate the antigen-presenting cells;

s2, preparing the cell membrane components of the activated antigen-presenting cells in the S1 into nano vesicles to obtain the cancer vaccine based on the antigen-presenting cell membrane components;

or obtaining cell membrane components of the activated antigen-presenting cells in S1, and allowing the cell membrane components and/or nanovesicles to act together with second delivery particles loaded with cancer cell whole cell components to obtain the cancer vaccine based on the antigen-presenting cell membrane components.

Further, the means for obtaining the cell membrane fraction of the activated antigen-presenting cells in S1 may be mechanical disruption, membrane filtration, gradient centrifugation, or chemical treatment; the mode of co-action is co-incubation, co-extrusion, sonication, stirring, homogenization or homogenization.

Further, the gradient centrifugation is gradient centrifugation with sequentially increasing centrifugation speed; the aperture of the filter membrane used in the membrane filtration is sequentially from large to small; the mechanical disruption can be ultrasonic, homogenizing, high-speed stirring, high-pressure disruption, high shear disruption, swelling; the chemical treatment may be by placing the cells in low osmotic pressure PBS, glucose solution or saline solution containing chemicals, shrinking, or the like. Wherein the ultrasound is low-power ultrasound (less than 500W).

Further, the co-incubation is for at least 4 hours to allow the antigen to be delivered into, processed by and presented to the surface of the antigen-presenting cell.

Furthermore, the incubation liquid contains cell factors and/or antibodies during the co-incubation process.

Further, the cytokine is selected from at least one of interleukins, tumor necrosis factors, interferons, growth factors, and colony stimulating factors.

Further, cytokines include, but are not limited to, interleukin 2(IL-2), interleukin 7(IL-7), interleukin 14(IL-14), interleukin 4(IL-4), interleukin 15(IL-15), interleukin 21(IL-21), interleukin 17(IL-17), interleukin 12(IL-12), interleukin 6(IL-6), interleukin 33(IL-33), interferon gamma (IFN- γ), TNF- α, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and the like.

Further, antibodies include, but are not limited to, α CD-3 antibodies, α CD-4 antibodies, α CD-8 antibodies, α CD-28 antibodies, CD-40 antibodies, CD-80 antibodies, α OX-40L antibodies, CD86 antibodies, and the like.

Preferably, a combination of any one of the following is added during incubation:

(1) granulocyte-macrophage colony stimulating factor, interleukin 2, interleukin 7, and interleukin 12;

(2) granulocyte-macrophage colony stimulating factor, interleukin 4, and tumor necrosis factor alpha;

(3) granulocyte-macrophage colony stimulating factor, interleukin 2, interleukin 7, interleukin 12, CD80 antibody, and CD40 antibody;

(4) granulocyte-macrophage colony stimulating factor, interleukin 12, and CD80 antibodies;

(5) interleukin 2, interleukin 7, and interleukin 15;

(6) interleukin 4, interleukin 6, and interleukin 10;

(7) granulocyte-macrophage colony stimulating factor, interleukin 2, interleukin 7, interleukin 12, and CD86 antibodies;

(8) granulocyte-macrophage colony stimulating factor, interleukin 2, interleukin 7, interleukin 12, albumin, and CD80 antibodies;

(9) granulocyte-macrophage colony stimulating factor, interleukin 2, interleukin 7, interleukin 12, and CD40 antibodies;

(10) granulocyte-macrophage colony stimulating factor, interleukin 2, interleukin 7, interleukin 12, gamma interferon, and CD80 antibodies;

(11) granulocyte-macrophage colony stimulating factor, interleukin 2, interleukin 7, interleukin 12, gamma interferon, CD80 antibody, and CD40 antibody.

Further, in step S1, the method further includes a step of washing the activated antigen-presenting cells, and the washing solution used in the washing step may contain a protease inhibitor and/or a phosphatase inhibitor.

Dendritic cell cancer vaccine is a kind of cancer vaccine, and in the prior art, because of the limitation of the activation mode, the antigen that Dendritic Cell (DC) can load is relatively limited, so the activated types and clone number of T cell after being injected into human body are few, and because DC belongs to living cell preparation, activated DC used as vaccine has many defects in the process of storage, transportation and administration. According to the invention, the nano particles and/or micro particles loaded with cancer cell antigens are used for activating the antigen presenting cells, then the antigen presenting cells are processed by a certain method to prepare the nano vaccine or the micro vaccine, and the obtained vaccine is loaded with the cancer cell whole cell component antigen, so that broad-spectrum and diverse cancer cell specific T cells can be activated after being injected into a human body, and more and better cancer cells can be identified and killed.

Moreover, because the surface of the nano-vaccine or the micro-vaccine is loaded with cell membrane components and the cell membrane contains broad-spectrum polypeptide antigen combined with MHC, the nano-vaccine or the micro-vaccine can be used as an artificial antigen presenting cell to be directly connected with the MHC

T cell action activates it into cancer cell specific T cells; in addition, the nano-vaccine or the micro-vaccine can be phagocytized by antigen presenting cells, and then cancer cell specific T cells are indirectly activated through the antigen presenting cells. The existing vaccine can only indirectly activate T cells through antigen presenting cells, while the nano-vaccine or the micro-vaccine can activate the T cells through two modes of directly activating the T cells and indirectly activating the T cells through the antigen presenting cells. The present invention is therefore based on the above cancer vaccine, and provides a method for directly activating T cells using an antigen presenting cell derived cancer vaccine, wherein the cancer vaccine is co-incubated with T cells in vitro to induce activation to give cancer cell specific T cells.

The third purpose of the invention is to provide the application of the cancer vaccine based on the antigen presenting cell membrane component in preparing the medicine for treating or preventing cancer.

By the scheme, the invention at least has the following advantages:

(1) the invention can integrate the membrane components of a plurality of antigen presenting cells, the loaded MHC molecules and the antigens into one nano vaccine or micro vaccine, therefore, one vaccine can contain the membrane components of a plurality of antigen presenting cells including DC cells, B cells or macrophages, and the like, so that one nano vaccine particle can have the functions and advantages of a plurality of antigen presenting cells.

(2) The vaccine of the invention can contain membrane components of a plurality of antigen presenting cells including DC cells, and the protein on the membrane of the antigen presenting cells has special affinity to lymph nodes and immune cells in the lymph nodes, so the vaccine has the characteristic of homing the lymph nodes after being injected into a body, has the efficacy of naturally targeting the lymph nodes and can better activate cancer cell specific immune response.

(3) The nano-particle and/or micro-particle loaded with cancer cell antigens are used for activating the antigen presenting cells, and then the antigen presenting cells are used for preparing the vaccine, so that the prepared nano-vaccine can load all antigen components loaded by the prepared nano-particle and/or micro-particle, and the nano-vaccine can load broad-spectrum and diverse cancer cell specific antigens.

(4) The vaccine is derived from micron-sized antigen presenting cells, all components are biocompatible and degradable, the safety is good, the problem that the living cell vaccine has strict requirements on conditions during storage, transportation and injection is solved, and the curative effect of the vaccine is better than that of the living cell vaccine.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to make the technical solutions of the present invention practical in accordance with the contents of the specification, the following description is made with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a schematic diagram of the preparation process and application of the nano-or micro-vaccine of the present invention; wherein a is a schematic diagram of collecting and preparing nano particles or micro particles of water-soluble antigens and water-insoluble antigens respectively; b is a schematic diagram of dissolving cancer cell whole cell antigen and preparing nano particles or micro particles by using a dissolving solution containing a dissolving agent; c is a schematic diagram of using the particles prepared in the step a or the step b to activate antigen presenting cells, preparing the antigen presenting cells into nano vaccines or micro vaccines, and using the nano vaccines or micro vaccines to prevent or treat cancers;

FIGS. 2 to 20 are experimental results of the growth rate and survival time of mouse tumor in the case of cancer prevention or treatment using nano-or micro-vaccine in examples 1 to 19, respectively; a, experimental results of tumor growth speed (n is more than or equal to 8) when preventing or treating cancer; b, the experimental result of the survival period of the mouse when preventing or treating the cancer (n is more than or equal to 8), wherein each data point is the mean value plus or minus standard error (mean plus or minus SEM); c and d are CD8 for nano-vaccine activation using flow cytometry analysis ⁺ And CD4 ⁺ The total CD8 of the cancer cell-specific T cells ⁺ And CD4 ⁺ The proportion of T cells; e is a nano vaccine Transmission Electron Microscope (TEM) scanning image; the significant difference of the tumor growth inhibition experiment in the graph a is analyzed by an ANOVA method, and the significant difference in the graph b is analyzed by Kaplan-Meier and log-rank test; indicates a significant difference of p < 0.005 compared to PBS blank control; indicates a significant difference with p < 0.01 compared to PBS blank control;

the comparison p of the nano vaccine control group representing the antigen presenting cells prepared after adding specific cell factors for incubation in the process of activating the antigen presenting cells by the nano particles/micron particles is less than 0.05, and the significant difference exists;&&&shows that compared with a nano vaccine control group prepared by blank nano particles/micron particles and antigen presenting cells activated by free lysate, p is less than 0.005, and has significant difference;&&shows that compared with a nano vaccine control group prepared by blank nano-particles/micro-particles and free lysis activated antigen presenting cells, p is less than 0.01, and the control group has significant difference; delta represents that the p is less than 0.005 and has significant difference compared with the nano vaccine prepared by the antigen presenting cells activated by the polypeptide nanoparticles/micron particles; delta represents the comparison with the nano vaccine group prepared by the polypeptide nanoparticle/micron particle activated antigen presenting cellsp is less than 0.01, and the difference is significant; delta represents a significant difference compared with the group using the mixed antigen presenting cell live cell vaccine activated by the nanoparticles loaded with the whole cell component, wherein p is less than 0.01; the second channel is represented by a remarkable difference compared with a nano vaccine or a micro vaccine prepared by using 1% trehalose, 2% mannitol and 2% PVP aqueous solution as a freeze-drying protective agent and freeze-drying, wherein p is less than 0.05; psi represents that p is less than 0.05 compared with the nano vaccine group prepared by the DC + B cells activated by the nanoparticles/the micron particles, and has significant difference; omega represents that the p is less than 0.05 and has significant difference compared with a nano vaccine group prepared by B cells activated by nano/micron particles; chi represents that compared with a nano vaccine or a micro vaccine prepared by using the antigen presenting cells activated by the specific cytokine combination auxiliary micro particles, the p is less than 0.01, and has significant difference; omega represents a significant difference compared with the nano-or micro-vaccine which only internally loads cancer cell whole cell components but does not load activated antigen presenting cell membrane components on the surface, wherein p is less than 0.05; m represents a significant difference with p < 0.05 compared to the nano-vaccine group prepared from the macrophage activated by the nano/micro particles; theta represents that compared with the T cell group which is not loaded with adjuvant and is assisted by nano particles/micro particles for separation, p is less than 0.005, and the significant difference exists; theta represents a significant difference compared with the nanoparticle/microparticle-assisted separation T cell group without adjuvant, wherein p is less than 0.01; pi represents that the p is less than 0.01 and has significant difference compared with a nano vaccine group prepared by nano/micron activated antigen presenting cells which are not loaded with lysosome escaping substances; pi represents that the p is less than 0.05 and has significant difference compared with a nano vaccine group prepared by the nano/micron particle activated antigen presenting cells which are not loaded with lysosome escaping substances; xi represents that the p is less than 0.01 and has significant difference compared with the nano vaccine prepared by the nano/micron particle activated antigen presenting cell only loading one CpG + Poly (I: C) mixed adjuvant; sigma-delta represents that compared with a nano vaccine or a micro vaccine prepared by only using the antigen presenting cell membrane component, the p is less than 0.01, and the significant difference exists; beta represents that the p is less than 0.05 and has significant difference compared with the nano vaccine or the micro vaccine which is prepared by using 2 percent of sucrose, 2 percent of mannitol and 1 percent of lysine water solution as a freeze-drying protective agent through freeze drying; xi represents andcompared with the p of less than 0.05, the nano vaccine prepared by the antigen presenting cells activated by the nano particles/the micron particles loaded with the CpG + Poly (I: C) mixed adjuvant has significant difference; mu represents that the p is less than 0.05 compared with the nano vaccine group prepared by using the nano/micron particle activated antigen presenting cells of the mixed adjuvant of two A-class CpG and toll-like receptor 3, and has significant difference; rho represents that the p is less than 0.05 and has significant difference compared with a nano vaccine group prepared by the nano/micron particle activated antigen presenting cells loaded with only one adjuvant (two classes of CpG); tau represents that compared with the nano vaccine prepared by not adding IL-2 and IL-7 for incubation in the process of activating antigen presenting cells by nano particles/micron particles, p is less than 0.01, and the tau has significant difference; the # indicates that the p is less than 0.005 and has significant difference compared with the nano vaccine control group prepared by the antigen presenting cells which are not activated by any nano particles/micron particles; # indicates that there is a significant difference compared to the control group of nano-vaccine prepared from antigen-presenting cells that are not activated by any nanoparticles/microparticles, with p < 0.01; # shows a significant difference with p < 0.05 compared to a control group of nano-vaccines prepared from antigen presenting cells that are not activated by any nanoparticles/microparticles.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The cancer vaccine based on the antigen presenting cell membrane component for preventing or treating cancer comprises nano-vaccine or micro-vaccine prepared by antigen presenting cells activated by nano-particles and/or micro-particles loaded with cancer cells and/or tumor tissue whole cell components. The nanoparticles and/or microparticles support a cancer cell whole cell component or a mixture thereof. The whole cell fraction contains whole cell antigens. The preparation process and the application field of the nano vaccine for preventing or treating cancer are shown in figure 1.

When preparing the nano-particle or the micro-particle loaded with cancer cell and/or tumor tissue whole cell components for activating antigen presenting cells, the water-soluble antigen and the water-insoluble antigen are respectively collected and a nano-particle system or a micro-particle system is respectively prepared after the cells or the tissues are cracked; or directly using a dissolving solution containing a dissolving agent to directly crack cells or tissues and dissolve cancer cell whole cell antigens and prepare a nano or micro particle system. The cancer cell whole cell antigen can be subjected to treatments including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment and the like before or (and) after lysis, and then nano particles or micro particles are prepared; the nano-particles or the micro-particles can also be directly prepared before cell lysis or (and) after cell lysis without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification and nuclease treatment. In some embodiments of the present invention, the tumor tissue cells are inactivated or (and) denatured before being lysed, or inactivated or (and) denatured after being lysed during the actual use process; in some embodiments of the present invention, the inactivation or (and) denaturation treatment before or (and) after cell lysis is ultraviolet irradiation and high temperature heating, and during actual use, treatment methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, freeze drying, etc. may also be used. Those skilled in the art can understand that in the practical application process, the technical personnel can make appropriate adjustment according to specific situations.

In the in vitro activation of antigen-presenting cells using nanoparticles or microparticles, cytokines and/or antibodies may be used to help increase the efficiency of activation, and the antigen-presenting cells may be autologous or allogeneic, or derived from cell lines or stem cells. The antigen presenting cells can be DC cells, B cells, macrophages or any mixture of the three, and can also be other cells with antigen presenting functions.

After the antigen presenting cells are activated, the antigen presenting cells are prepared into nano-sized vaccines or micro-sized vaccines by using methods such as low-frequency ultrasound, gradient centrifugation and the like. Other methods for preparing nanometer-sized or micrometer-sized live antigen-presenting cells into nanometer-sized or micrometer-sized nano-or micrometer-sized vaccines without cell activity may also be used in the actual preparation.

In some embodiments, the specific preparation method for preparing nano-or micro-vaccines using cancer cell whole cell antigen loaded nanoparticle or microparticle activated antigen presenting cells 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 an organic phase containing a prepared particle raw material of a second predetermined concentration.

In some embodiments, the aqueous phase solution may contain components of the cancer cell lysate and an immune enhancing adjuvant; the components of the cancer cell lysate are water-soluble antigens or original water-insoluble antigens dissolved in a lytic agent such as urea or guanidine hydrochloride. The aqueous solution contains a concentration of water-soluble antigen or a concentration of antigen that is not water-soluble, i.e., a first predetermined concentration that requires a protein polypeptide concentration of greater than 1ng/mL, sufficient to carry cancer cell whole cell antigen to activate the relevant cells. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.

In some embodiments, the aqueous phase solution contains components of the tumor tissue lysate and an immunopotentiating adjuvant; the components in the tumor tissue lysate are water-soluble antigens or original water-insoluble antigens dissolved in a dissolving agent such as urea or guanidine hydrochloride during preparation. The aqueous solution contains a concentration of water-soluble antigen or a concentration of antigen that is not water-soluble, i.e., a first predetermined concentration that requires a protein polypeptide concentration of greater than 0.01ng/mL, sufficient to carry cancer cell whole cell antigen to activate the relevant cells. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.

In some embodiments, the particle is prepared from PLGA and the organic solvent is dichloromethane. Additionally, in some embodiments, the second predetermined concentration of the starting material for preparing the particles ranges from 0.5mg/mL to 5000mg/mL, preferably 100 mg/mL.

In the present invention, PLGA or modified PLGA is selected because the material is biodegradable and has been approved by the FDA for use as a drug dressing. Research shows that PLGA has certain immunoregulation function, so that it is suitable for use as supplementary material for preparing nanometer particle and micron particle. In practical application, suitable materials can be selected according to practical situations.

In practice, the second predetermined volume of organic phase is set according to its ratio to the first predetermined volume of aqueous phase, and in the present invention, the ratio of the first predetermined volume of aqueous phase to the second predetermined volume of organic phase ranges from 1:1.1 to 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 nanoparticles or microparticles produced during the implementation.

Preferably, when the aqueous phase solution is a lysate component solution, the concentration of the protein and the polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL; when the aqueous phase solution is lysate component/immune adjuvant solution, the concentration of protein and polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL, and the concentration of immune adjuvant is more than 0.01ng/mL, preferably 0.01 mg/mL-20 mg/mL. In the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5mg/mL to 5000mg/mL, preferably 100 mg/mL.

And 2, carrying out ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or homogenizing treatment or microfluidic treatment on the mixed solution obtained in the step 1. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is more than 5W, and the time is more than 0.1 second, such as 2-200 seconds; the homogenizing treatment is carried out by using a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 5psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 100rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates of greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-grade and/or micron-grade particles are subjected to ultrasonic treatment, stirring treatment, homogenizing treatment or microfluidic treatment, the size of the prepared micro-nano particles can be controlled by the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the particle size can be changed when the particles are too large or too small.

And 3, adding the mixture obtained after the treatment in the step 2 into a third preset volume of aqueous solution containing a third emulsifier with a preset concentration, and performing ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing homogenization treatment or microfluidic treatment. Adding the mixture obtained in the step 2 into an emulsifier aqueous solution, and continuing to carry out ultrasonic treatment or stirring for nano-crystallization or micro-crystallization. In the invention, the ultrasonic time is more than 0.1 second, such as 2-200 seconds, the stirring speed is more than 50rpm, such as 50-500 rpm, and the stirring time is more than 1 minute, such as 60-6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.5 hour to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W-500W, and the time is more than 0.1 second, such as 2-200 seconds; a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer is used during homogenization treatment, the pressure is more than 20psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 1000rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-or micro-scale treatment is carried out by ultrasonic treatment, stirring, homogenizing treatment or micro-fluidic treatment, the size of the prepared nano-or micro-particles can be controlled by the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the change of the particle size can be brought by the over-large or over-small of the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time.

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

And 4, adding the liquid obtained after the treatment in the step 3 into a fourth preset volume of emulsifier aqueous solution with a fourth preset concentration, and stirring until preset stirring conditions are met.

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

The fourth predetermined concentration is 5mg/mL, and the fourth predetermined concentration is selected based on obtaining nanoparticles or microparticles of a suitable size. The fourth predetermined volume is selected based on a ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5 to 1:2000, preferably 1: 10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted during implementation to control the size of the nanoparticles or microparticles.

In the present invention, the predetermined stirring condition in this step is until the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in step 1 is completed.

And 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition at the rotating speed of more than 100RPM for more than 1 minute, removing the supernatant, and resuspending the remaining precipitate in a fifth preset volume of aqueous solution with a freeze-drying protective agent at a fifth preset concentration or in a sixth preset volume of PBS (or physiological saline).

In some embodiments of the present invention, the pellet obtained in step 5 is resuspended in the sixth predetermined volume of PBS (or physiological saline) without lyophilization, and the subsequent experiments related to the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles can be directly performed.

In some embodiments of the present invention, the pellet obtained in step 5 is re-suspended in the aqueous solution containing the lyoprotectant, and then freeze-dried, followed by the subsequent experiments related to the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles.

In the present invention, the fifth predetermined concentration of the lyoprotectant in this step is 4% by mass, and is set so as not to affect the lyophilization effect in the subsequent lyophilization.

And 6, freeze-drying the suspension containing the freeze-drying protective agent obtained in the step 5, and then keeping the freeze-dried substance for later use.

Step 7, directly using the suspension containing the nanoparticles, which is obtained in the step 5 and is resuspended in PBS (or normal saline) in a sixth predetermined volume, or using PBS (or normal saline) in a sixth predetermined volume to resuspend the freeze-dried substance containing the nanoparticles or microparticles and the freeze-drying protective agent obtained in the step 6; or mixing the above sample with a seventh predetermined volume of water-soluble antigen or dissolved original water-insoluble antigen.

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

In some embodiments, the volume of the resuspended nanoparticle suspension is 10mL, and the volume of the cancer cell lysate or the tumor tissue lysate containing the water-soluble antigen or the solubilized original water-insoluble antigen is 1 mL. When in actual use, the volume and the proportion of the two can be adjusted according to the needs.

And 8, incubating the antigen presenting cells and the prepared nano particles and/or micro particles for a certain time. The tumor tissue and/or cancer cells from which the nanoparticles and/or microparticles are prepared and the antigen-presenting cells may be autologous or allogeneic.

Step 9, collecting the antigen presenting cells after co-incubation, and performing mechanical disruption or chemical addition to disrupt the cell structure and preserve the cell membrane components, with or without washing, wherein the mechanical disruption or chemical addition is performed by one or more of ultrasound at less than 500W, high pressure homogenization, stirring, swelling, shrinking, high shear disruption, small pore size extrusion disruption, extrusion, and addition of low osmotic pressure PBS or glucose solution containing chemical or saline solution.

And step 10, centrifuging the sample subjected to mechanical damage or specific chemical substance addition, and/or filtering and extruding the sample through a filter membrane with a specific pore size, and/or performing coaction with the nano-particles or the micro-particles loaded with the whole cell components to prepare the nano-vaccine or the micro-vaccine. The means by which the sample, either mechanically disrupted or with the addition of a particular chemical, interacts with the whole cell component loaded nanoparticles or microparticles include, but are not limited to, co-incubation, sonication, extrusion, mechanical agitation, high pressure homogenization, extrusion, and the like, in which the membrane component can be loaded onto the surface of the nanoparticles or microparticles.

In other embodiments, the specific method of preparation of the antigen loaded nanoparticle or microparticle is as follows:

the steps 1-4 are the same as above.

And 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition at the rotating speed of more than 100RPM for more than 1 minute, removing the supernatant, and re-suspending the remaining precipitate in a fifth preset volume of solution containing water-soluble and/or non-water-soluble antigens in the cancer cell whole cell antigens at a fifth preset concentration, or re-suspending the remaining precipitate in a fifth preset volume of solution containing water-soluble and/or non-water-soluble antigens in the cancer cell whole cell antigens and adjuvants at a fifth preset concentration.

And 6, centrifuging the mixed solution which is processed in the step 5 and meets the preset stirring condition for more than 1 minute at the rotating speed of more than 100RPM, removing the supernatant, resuspending the remaining precipitate in a sixth preset volume of a solidification treatment reagent or a mineralization treatment reagent, performing centrifugal washing after acting for a certain time, and then adding a seventh preset volume of a substance with positive charge or negative charge and performing action for a certain time.

In some embodiments of the present invention, the pellet obtained in step 6 may be resuspended in the seventh predetermined volume of the charged substance without lyophilization, and then the subsequent experiments related to the loading of cancer cell/tissue lysate on the surface of the nanoparticles or microparticles can be directly performed.

In some embodiments of the present invention, the precipitate obtained in step 6 is re-suspended in an aqueous solution containing a desiccation protectant, and then is dried in vacuum or freeze-dried at room temperature, and then is dried and then subjected to the related experiments of adsorbing cancer cell lysate on the surface of nano-or micro-particles.

In the invention, the freeze-drying protective agent is Trehalose (Trehalose) or a mixed solution of mannitol and sucrose. In the present invention, the concentration of the drying protective agent in this step is set so as not to affect the drying effect in the subsequent drying, because the concentration is 4% by mass.

And 7, drying the suspension containing the drying protective agent obtained in the step 6, and then keeping the dried substance for later use.

8, resuspending the suspension containing the nanoparticles in PBS (or physiological saline) obtained in the step 6 with an eighth predetermined volume or resuspending the dried substance containing the nanoparticles or microparticles and the dry protective agent obtained in the step 7 with PBS (or physiological saline) with an eighth predetermined volume for direct use; or mixed with a ninth predetermined volume of water-soluble antigen or water-insoluble antigen.

In the present invention, the modification and antigen loading steps of steps 5-8 can be repeated multiple times to increase the antigen loading. When a substance having a positive or negative charge is added, a substance having the same charge may be added a plurality of times or a substance having different charges may be added alternately.

In some embodiments, the volume of the resuspended nanoparticle suspension is 10mL, and the volume of the cancer cell lysate or the tumor tissue lysate containing water-soluble antigen or the original water-insoluble antigen is 0.1-100 mL. The volume and the proportion of the two can be adjusted according to the needs when in actual use.

And 9, incubating the antigen presenting cells and the prepared nano particles and/or micro particles for a certain time. The tumor tissue and/or cancer cells from which the nanoparticles and/or microparticles are prepared and the antigen-presenting cells may be autologous or allogeneic.

Step 10, collecting the antigen presenting cells after co-incubation, and performing mechanical disruption or chemical addition to disrupt the cell structure and preserve the cell membrane components, with or without washing, wherein the mechanical disruption or chemical addition is performed by one or more of ultrasound at less than 500W, high pressure homogenization, stirring, extrusion, swelling, shrinking, high shear disruption, small pore size extrusion disruption, addition of low osmotic pressure PBS or glucose solution containing chemicals, or saline solution.

And 11, centrifuging the sample subjected to mechanical disruption or specific chemical substance addition, and/or filtering the sample through a filter membrane with a specific pore size, and/or performing coaction with the nanoparticles or the microparticles loaded with the whole cell components to prepare the nano vaccine or the micro vaccine. The means by which the sample, either mechanically disrupted or added with a specific chemical, interacts with the nanoparticles or microparticles loaded with whole cell components include, but are not limited to, co-incubation, sonication, mechanical agitation, extrusion, high pressure homogenization, extrusion, and the like, which can load the membrane components onto the surface of the nanoparticles or microparticles.

Example 1 DC-derived Nanoprotein for prevention of melanoma

This example illustrates how a DC-derived nano-vaccine can be used to prevent melanoma using mouse melanoma as a cancer model. In this embodiment, a B16F10 melanoma tumor tissue is lysed to prepare a water-soluble antigen and a water-insoluble antigen of the tumor tissue, then a nanoparticle system loaded with the water-soluble antigen and the water-insoluble antigen of the tumor tissue is prepared by a solvent evaporation method using organic polymer material PLGA as a nanoparticle backbone material and Polyinosinic-polycytidylic acid (poly (I: C)) as an immunoadjuvant, then a DC cell is activated by using the nanoparticles, and then the DC cell is used to prepare a nano vaccine for preventing and treating cancer.

(1) Lysis of tumor tissue and Collection of fractions

Each C57BL/6 mouse was inoculated subcutaneously into the back of 1.5X 10 mice ⁵ B16F10 cells, which were about 1000mm in tumor length to volume ³ Mice were sacrificed and tumor tissue was harvested. Cutting tumor tissue into pieces, grinding, and adding into the obtained product through cell filter screenAn appropriate amount of ultrapure water was added and freeze-thawing was repeated 5 times with sonication to destroy lysed cells. After the cells are lysed, centrifuging the lysate for 5 minutes at the rotating speed of 5000g, and taking supernatant fluid, namely the water-soluble antigen which can be dissolved in pure water; the precipitation fraction is dissolved by adding 8M urea to the obtained precipitation fraction, thereby converting the water-insoluble antigen insoluble in pure water into a soluble antigen in an 8M urea aqueous solution. Mixing water-soluble antigen and water-insoluble antigen according to the mass ratio of 1:1 to obtain the antigen raw material source for preparing the nano particles.

(2) Preparation of nanoparticle systems

In this example, the nanoparticles were prepared by a multiple emulsion method in a solvent evaporation method. The molecular weight of PLGA used as a material for preparing the nano particles is 24-38 KDa, and the adopted immunologic adjuvant is poly (I: C) which is only loaded in the nano particles. As mentioned above, during the preparation process, the whole cell lysate component and the adjuvant are loaded inside the nanoparticles by using the multiple emulsion method, after the cell lysate component and the adjuvant are loaded inside the nanoparticles, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are resuspended by using 10mL of ultrapure water containing 4% trehalose, and then are freeze-dried for 48 hours. The average particle diameter of the nano particles is about 250nm, and the surface potential of the nano particles is about-3 mV; each 1mg PLGA nanoparticle is loaded with about 100 μ g protein or polypeptide component, and each 1mg PLGA nanoparticle uses 0.02mg poly (I: C) immunoadjuvant. The blank nano preparation material and the preparation method are the same, the particle size is about 240nm, and the same amount of adjuvant is adopted to replace whole cell components.

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

This example illustrates how to prepare BMDCs by taking dendritic cells prepared from mouse bone marrow cells as an example. Firstly, 1C 57 mouse with age of 6-8 weeks is taken out and killed by dislocation of cervical vertebra, tibia and femur of hind leg are taken out by operation and put into PBS, and muscle tissue around the bone is removed by scissors and tweezers. The two ends of the bone are cut off by scissors, the PBS solution is extracted by a syringe, the needles are respectively inserted into the marrow cavity from the two ends of the bone, and the marrow is repeatedly washed into a culture dish. Bone marrow solution was collected, centrifuged at 400g for 3min, and then 1mL of erythrocyte lysate was added to lyse red blood. Lysis was stopped by addition of 3mL RPMI1640 (10% FBS) medium and centrifugation at 400g for 3miAnd n, discarding the supernatant. Cells were plated in 10mm culture dishes using RPMI1640 (10% FBS) medium with recombinant mouse GM-CSF (20ng/mL), 37 degrees, 5% CO ₂ The culture was carried out for 7 days. The flask was gently shaken on day 3 and supplemented with the same volume of RPMI1640 medium containing GM-CSF (20ng/mL) (10% FBS). On day 6, medium was subjected to half-volume change. On day 7, a small number of suspended and semi-adherent cells were collected and tested by flow assay for CD86 ⁺ CD80 ⁺ The cells were in CD11c ⁺ The proportion of cells is between 15 and 20 percent, and the BMDCs cultured by induction can be used for the next experiment.

(4) Activation of antigen presenting cells

Nanoparticles (500. mu.g) or blank nanoparticles (500. mu.g) + free lysate loaded with cancer cell whole cell components derived from tumor tissue were incubated with BMDC (1000 ten thousand) in 15mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained cytokine combination 1: granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(1000U/mL), or a mixture comprising cytokine component 2: GM-CSF (1000U/mL), IL-4(100U/mL), tumor necrosis factor alpha (TNF-alpha, 200U/mL).

(5) Preparation of DC-derived nano-vaccine

The post-incubation DCs were collected by centrifugation at 400g for 5 minutes, followed by washing the cells twice with physiological saline, resuspending the cells in physiological saline followed by sonication at 7.5W for 20 minutes. And centrifuging the sample at 2000g for 20 minutes, collecting supernatant, centrifuging the supernatant at 7000g for 20 minutes, collecting supernatant, centrifuging at 15000g for 120 minutes, collecting the supernatant, discarding the precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine with the particle size of 120 nm.

(6) Nano-vaccine for cancer prevention

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Each mouse was subcutaneously injected with 500. mu.g of the nano-vaccine on days-42, 35, 28, 21, 14 and 7, respectively, before the mice were inoculated with cancer cells. On day 0, each mouse was given subcutaneous to the lower right backInoculation 1.5X 10 ⁵ And B16F10 cells. The tumor growth rate and survival time of the mice were monitored. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 3. Tumor volume is calculated by the formula v ═ 0.52 × a × b ² Calculation, where v is tumor volume, a is tumor length, and b is tumor width. From animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in the life test of the mouse ³ I.e. the mice were considered dead and were euthanized.

(7) Results of the experiment

As shown in FIG. 2, the mice in the PBS control group showed a rapid tumor growth rate and a short survival time. The tumor growth rate of mice treated by the nano vaccine prepared by the DC activated by the blank nanoparticles and the free lysate is reduced. The tumor growth rate of mice treated with the nano-vaccine prepared from the DC activated by the nanoparticles loaded with the cancer cell whole cell lysate is significantly slower than that of the two groups, and the survival time is longest. Wherein, the effect of adding the cytokine combination 1 in the DC cell activation process is better than that of adding the cytokine combination 2. In conclusion, the nano vaccine provided by the invention has a good prevention effect on melanoma.

Example 2 DC-based cancer vaccine for melanoma prevention

In this example, mouse melanoma was used as a cancer model to illustrate how to use nanoparticles to help activate DCs, and then prepare DC cells into cancer vaccines for preventing melanoma. In the embodiment, B16F10 melanoma tumor tissue is cracked to prepare water-soluble antigen and water-insoluble antigen of the tumor tissue, then PLGA is used as a nanoparticle framework material, poly (I: C), CpG2216 and CpG2395 which are Toll-like receptor agonists are used as mixed immunologic adjuvants to prepare a nanoparticle system loaded with the water-soluble antigen and the water-insoluble antigen of the tumor tissue by a solvent volatilization method, then the DC is activated by the nanoparticles, and the DC is properly treated to prepare the DC-derived nano vaccine to be injected into a body to prevent melanoma.

(1) Lysis of tumor tissue and Collection of fractions

Each C57BL/6 mouse was inoculated subcutaneously into the back of 1.5X 10 mice ⁵ B16F10 cellsGrowth to volume of the tumor was about 1000mm ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, and a proper amount of pure water is added through a cell filter screen and freeze thawing is repeated for 5 times, and ultrasonic waves are accompanied to destroy the lysed cells. After the cells are lysed, centrifuging the lysate for 5 minutes at the rotating speed of 5000g, and taking supernatant fluid, namely the water-soluble antigen which can be dissolved in pure water; the precipitation fraction is dissolved by adding 8M urea to the obtained precipitation fraction, thereby converting the water-insoluble antigen insoluble in pure water into a soluble antigen in an 8M urea aqueous solution. The above is the source of the antigen raw material for preparing the nanoparticle system.

(2) Preparation of nanoparticle systems

The nano vaccine and the blank nanoparticles used as the control in the embodiment are prepared by a solvent volatilization method. The nano vaccine loaded with the water-soluble antigen in the cancer cell whole-cell antigen and the nano particle loaded with the water-insoluble antigen in the cancer cell whole-cell antigen are prepared respectively and then used together when in use. The molecular weight of PLGA used as a material for preparing the nano particles is 7Da-17KDa, the adopted immunologic adjuvants are poly (I: C), CpG2216 and CpG2395, and the adjuvants are loaded in the nano particles. As mentioned above, in the preparation process, the antigen and adjuvant are loaded inside the nanoparticles by using a multiple emulsion method, after the antigen is loaded inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and then suspended in 10mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours. The average particle size of the nano particles is about 280 nm; each 1mg PLGA nano particle is loaded with about 100 mug protein and polypeptide components, and each 1mg PLGA nano particle uses 0.025mg of poly (I: C), CpG2216 and CpG2395 immune adjuvant. In the embodiment, nanoparticles loaded with four polypeptide neoantigens B16-M20(Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24(Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46(Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188(SVYDFFVWL) in equal mass are used as control nanoparticles, the particle diameter of the control nanoparticles is about 260nm, 100 mu g of polypeptide component is loaded, and the same amount of adjuvant is loaded. The blank nanoparticle has the grain diameter of about 250nm, and only the same amount of immunologic adjuvant is loaded, but no antigen component is loaded.

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

This example illustrates how to prepare BMDCs by taking dendritic cells prepared from mouse bone marrow cells as an example. Firstly, 1C 57 mouse with age of 6-8 weeks is taken out and killed by dislocation of cervical vertebra, tibia and femur of hind leg are taken out by operation and put into PBS, and muscle tissue around the bone is removed by scissors and tweezers. The two ends of the bone are cut off by scissors, the PBS solution is extracted by a syringe, the needles are respectively inserted into the marrow cavity from the two ends of the bone, and the marrow is repeatedly washed into a culture dish. Bone marrow solution was collected, centrifuged at 400g for 3min, and then 1mL of erythrocyte lysate was added to lyse red blood. Lysis was stopped by adding 3mL of RPMI1640 (10% FBS) medium, centrifugation at 400g for 3min and discarding the supernatant. Cells were plated in 10mm dishes using RPMI1640 (10% FBS) medium with recombinant mouse GM-CSF (20ng/mL), 37 degrees, 5% CO ₂ The culture was carried out for 7 days. The flasks were gently shaken on day 3 and supplemented with the same volume of RPMI1640 medium containing GM-CSF (20ng/mL) (10% FBS). On day 6, medium was subjected to half-volume change. On day 7, a small number of suspended and semi-adherent cells were collected and tested by flow assay for CD86 ⁺ CD80 ⁺ The cells were in CD11c ⁺ The proportion of cells is between 15 and 20 percent, and the BMDCs cultured by induction can be used for the next experiment.

(4) Activation of DC

Nanoparticles (500. mu.g, wherein the nanoparticles are 250. mu.g loaded with a water-soluble component and 250. mu.g loaded with a water-insoluble component) or polypeptide nanoparticles (500. mu.g) or blank nanoparticles (500. mu.g) + free lysate loaded with cancer cell whole cell components derived from tumor tissue were incubated with BMDC (1000 ten thousand) in 15mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ) The incubation system contained granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(500U/mL), IL-7(1000U/mL), IL-12(500U/mL) and CD86 antibody (20 ng/mL).

(5) Preparation of DC-derived nano-vaccine

The post-incubation DCs were collected by centrifugation at 400g for 5 minutes, followed by washing the cells twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by 1 minute of sonication at 4 ℃ with low power (22.5W). And centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting the supernatant, centrifuging at 16000g for 90 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, resuspending the precipitate in PBS, and filtering the sample by using a membrane filter to obtain the nano vaccine with the particle size of 120 nanometers.

(6) Nano-vaccine for cancer prevention

Female C57BL/6 of 6-8 weeks is selected as a model mouse to prepare a melanoma-bearing mouse, and 500 mu g of nano-vaccine is respectively inoculated to each mouse at the 35 th day, the 28 th day, the 21 st day, the 14 th day and the 7 th day before the mouse is inoculated with cancer cells. On day 0, each recipient mouse was subcutaneously inoculated at 1.5X 10 on the lower right back ⁵ And B16F10 cells. The tumor growth rate and survival time of the mice were monitored. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 3. Tumor volume is calculated by the formula v ═ 0.52 × a × b ² Calculation, where v is tumor volume, a is tumor length, and b is tumor width. From animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in the life test of the mouse ³ I.e. the mice were considered dead and were euthanized.

(7) Analysis of Nano-vaccine activated cancer cell-specific T cells

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Mice were each injected subcutaneously with 100 μ g of nano-vaccine or PBS on

days

0, 7, 14, 28 and 42, respectively. On day 45, mice were sacrificed, spleens were harvested and single cell suspensions of splenocytes were prepared, and B cells and T cells were sorted from mouse splenocytes using magnetic bead sorting. Mu.g of whole-cell fraction-loaded nanoparticles, 500 ten thousand B cells and 100 ten thousand T cells were CO-incubated in 5mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ). The incubated cells were then collected and labeled with live and dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody and IFN-. gamma.antibody, and then analyzed by flow cytometry for IFN-. gamma.in T cells ⁺ The proportion of T cells. The cancer cell whole cell antigen loaded by the nano particles can be degraded into antigen epitope after being phagocytized by B cells of antigen presenting cells to be presented toThe specific T cell capable of recognizing the cancer cell whole cell antigen on the surface of the antigen presenting cell can be activated and secrete killer cell factors after recognizing the cancer cell whole cell antigen epitope. IFN-gamma is the most prominent cytokine secreted by antigen-specific T cells upon recognition of the antigen. IFN-gamma analysis using flow cytometry ⁺ The T cell is a cancer cell specific T cell which can recognize and kill cancer cells.

(8) Results of the experiment

As shown in a and b in fig. 3, the tumor growth rate of mice receiving PBS control group and blank nanoparticle control group was very fast, and the survival time of mice was very short. Compared with the two groups of control groups, the tumor growth speed of the mice treated by the nano vaccine prepared by the DC activated by the nanoparticles is obviously reduced, and part of the mice tumors disappear and are cured. Moreover, the effect of the nano vaccine prepared by the DC activated by the nano particles loaded with the cancer cell whole cell antigen is better than that of the nano vaccine prepared by the DC activated by the nano particles loaded with four antigen polypeptides. This shows that the antigen epitope species treated and presented by the DC activated by the loaded four new antigen polypeptide nanoparticles, so the cancer cell-specific T antigen presenting cell cancer vaccine activated by the nano vaccine prepared by the method has few T cell clones, and few cancer cells can be identified and killed. The DC activated by the nano-particles loaded with the cancer cell whole cell antigen has wider spectrum of cancer cell antigens, so that the prepared nano vaccine can activate wider range of T cell clone number, more cancer cells can be identified and killed, and better effect of treating or preventing cancer is achieved.

As shown in c and d in fig. 3, the ratio of CD8+ IFN- γ + T cells and CD4+ IFN- γ + T cells activated by the whole-cell component-loaded nanoparticle-activated DC cells to CD8+ T cells and CD4+ T cells is significantly higher than the ratio of activated by the polypeptide-loaded nanoparticles and the blank nanoparticle + free lysate-activated DC cells. Therefore, the nano vaccine prepared by the DC cell activated by the nano particle loaded with the whole cell component can better activate the cancer cell specific T cell with the capacity of identifying and killing the cancer cell.

Example 3

This example illustrates how to treat cancer using nano-vaccines, using mouse melanoma as a cancer model. In this example, B16F10 melanoma tumor tissue and cancer cells were first lysed to prepare a water-soluble antigen mixture (mass ratio 1:1) and a water-insoluble antigen mixture (mass ratio 1:1) of tumor tissue and cancer cells, and the water-soluble antigen mixture and the water-insoluble antigen mixture were mixed in a mass ratio 1:1. Then, PLGA is used as a nanoparticle framework material, Poly (I: C) and CpG2006 are used as adjuvants to prepare nanoparticles loaded with lysate components, the nanoparticles and T antigen presenting cells are incubated for a certain time in vitro, and then activated antigen presenting cells are used to prepare the nano vaccine for treating cancer.

(1) Lysis of tumor tissue and cancer cells and collection of fractions

Tumor tissue was collected by subcutaneous inoculation of 1.5X 10 cells into the dorsal area of each C57BL/6 mouse ⁵ B16F10 cells, which were about 1000mm in tumor length to volume ³ Killing mice and picking tumor tissues, cutting the tumor tissues into blocks, grinding, adding a proper amount of pure water through a cell filter screen, repeatedly freezing and thawing for 5 times, and destroying samples obtained by lysis with ultrasound; when the cultured B16F10 cancer cell line was collected, the medium was removed by centrifugation, washed twice with PBS, and centrifuged to collect cancer cells, the cancer cells were resuspended in ultrapure water, freeze-thawed repeatedly 3 times, and lysed with ultrasonic disruption. After the tumor tissue or the cancer cells are cracked, centrifuging the lysate for 5 minutes at the rotating speed of 5000g and taking supernatant fluid as water-soluble antigen which can be dissolved in pure water; the precipitation fraction is dissolved by adding 8M urea to the obtained precipitation fraction, thereby converting the water-insoluble antigen insoluble in pure water into a soluble antigen in an 8M urea aqueous solution. Mixing water-soluble antigens of tumor tissues and water-soluble antigens of cancer cells according to the mass ratio of 1: 1; the water-insoluble antigen of the tumor tissue and the water-insoluble antigen of the cancer cell are mixed in a mass ratio of 1:1. And mixing the water-soluble antigen mixture and the water-insoluble antigen mixture according to the mass ratio of 1:1 to obtain the antigen raw material source for preparing the nano particles.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by a multiple emulsion method. The molecular weight of PLGA used as a material for preparing the nano particles is 7-17 KDa, the adopted immunologic adjuvant is poly (I: C) and CpG2006, and the adjuvant is loaded in the nano particles. The preparation method is as described above, in the preparation process, firstly, a lysate component and an adjuvant are loaded inside the nano particles by a multiple emulsion method, then 100mg of the nano particles are centrifuged for 20 minutes at 10000g, and 10mL of ultrapure water containing 4% trehalose is used for resuspension and then is frozen and dried for 48 hours; before use, it was resuspended in 9mL PBS and then 1mL of the lysate fraction (protein concentration 80mg/mL) was added and allowed to act at room temperature for 10min, resulting in a lysate loaded nanoparticle system both inside and outside. The average particle size of the nano-particle is about 270nm, the surface potential is about-5 mV, about 130 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano-particle, and 0.02mg of each 1mg of PLGA nano-particle is loaded with poly (I: C) and CpG2006 immunoadjuvant.

(3) Isolation of B cells

After C57BL/6 mice were sacrificed, spleens of the mice were harvested to prepare single cell suspensions of splenocytes of the mice, and CD19 among live cells in the splenocytes (dead cells were labeled with a live-dead cell dye to remove dead cells) was separated by magnetic bead sorting ⁺ B cells.

(4) Activation of antigen presenting cells

Nanoparticles (500. mu.g) or polypeptide nanoparticles (500. mu.g) loaded with cancer cell whole cell components were mixed with DC2.4(1000 ten thousand) and B cells (1000 ten thousand) in 15mL of RPMI1640 complete medium and incubated for 48 hours (37 ℃, 5% CO) ₂ ) The incubation system contained granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), albumin (50ng/mL), and CD80 antibody (10 ng/mL). After the incubation is finished, centrifuging the incubated cells for 5 minutes at 400g, removing supernatant, and washing the cells twice by using PBS to obtain activated antigen presenting cells, wherein the activated mixed antigen presenting cells can be used as antigen presenting cell vaccines.

(5) Preparation of antigen presenting cell derived nano vaccine

The DC and B cells (2000 ten thousand each, 1000 ten thousand each of DC and B cells) after the incubation in step (4) were collected by centrifugation at 400g for 5 minutes, and then the cells were washed three times using PBS, and the mixed cells were sonicated for 30 minutes at low power (10W) after being resuspended in PBS water. And centrifuging the sample at 500g for 5 minutes, collecting supernatant, filtering the supernatant by sequentially passing through membranes with the pore diameters of 30 microns, 10 microns, 5 microns, 0.45 microns and 0.22 microns, collecting filtrate samples, incubating the collected filtrate samples with the nanoparticles (20mg) prepared in the step (2) for 15 minutes, co-extruding the filtrate samples through a filter membrane with the pore diameter of 0.45 microns, centrifuging the filtrate samples for 25 minutes at 13000g, discarding the supernatant, re-suspending the precipitate by using a freeze-drying protective agent aqueous solution (containing 1% trehalose, 2% mannitol and 1% arginine), and freeze-drying the precipitate to obtain the nano vaccine with the cancer cell whole cell components loaded inside and the mixed antigen presenting cell membrane components loaded on the surface, wherein the particle size of the nano vaccine is 280 nanometers.

(6) Nano-vaccine for cancer treatment

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 ⁵ And B16F10 cells. Mice were injected subcutaneously with 100 μ g of nano-vaccine or 1000 ten thousand of the mixed antigen presenting cells activated in step (4) (500 ten thousand D C +500 ten thousand B cells) or with 100 μ L PBS on

day

4, 7, 10, 15, 20 and 25, respectively, after melanoma inoculation. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 3. Tumor volume is determined by the formula v 0.52 × a × b ² Calculation, where v is tumor volume, a is tumor length, and b is tumor width. From animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in the life test of the mouse ³ I.e. the mice were considered dead and were euthanized.

(7) Analysis of Nano-vaccine activated cancer cell-specific T cells

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Mice were inoculated with 2X 10 on day 0 ⁵ And (3) injecting 2mg of the nanoparticles loaded with the cancer cell whole cell components prepared in the step (2) into each mouse subcutaneously on the 7 th day, the 12 th day and the 17 th day of the B16F10 cells. In thatMice were sacrificed on day 20, spleens were harvested and single cell suspensions of splenocytes were prepared, and B cells and T cells were separated from mouse splenocytes using magnetic bead sorting. Mu.g of the nano-vaccine prepared in step (5) and 100 ten thousand T cells were CO-incubated in 5mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ) (ii) a Alternatively, 1000 ten thousand of the mixed antigen-presenting cells activated in step (4) and 100 ten thousand of T cells were CO-incubated in 5mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ). The incubated cells were then collected and labeled with live and dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody and IFN-. gamma.antibody, and then analyzed by flow cytometry for IFN-. gamma.in T cells ⁺ The proportion of T cells. Specific T cells that recognize cancer cell antigens are activated and secrete killer cytokines upon recognition of cancer cell epitopes. IFN-gamma is the most prominent cytokine secreted by antigen-specific T cells upon recognition of the antigen. IFN-gamma analysis using flow cytometry ⁺ The T cell is a cancer cell specific T cell which can recognize and kill cancer cells.

(8) Results of the experiment

As shown in a and b in FIG. 4, the tumor growth rate of the PBS control group mice is very high, and the antigen presenting cell vaccine activated by the nanoparticles or the nano vaccine prepared in step (6) can significantly slow down the tumor growth of the mice and prolong the survival time of the tumor of the mice, and cure part of the mice. Moreover, the effect of the nano vaccine prepared by the mixed antigen presenting cells activated by the nanoparticles loaded with the whole cell components is better than that of the live antigen presenting cell vaccine activated by the nanoparticles. In conclusion, the cancer vaccine based on the antigen presenting cell has an excellent therapeutic effect on cancer.

As shown in c and d of fig. 4, CD8 activated by the nano-vaccine prepared in step (6) ⁺ IFN-γ ⁺ T cells and CD4 ⁺ IFN-γ ⁺ T cell occupancy CD8 ⁺ T cells and CD4 ⁺ The proportion of T cells is significantly higher than that which can be activated by the nanoparticle activated live mixed antigen presenting cells. Therefore, compared with the live antigen presenting cells activated by the nanoparticles, the nano-vaccine can better activate the cellsCancer cell-specific T cells that recognize cancer cells and have the ability to kill cancer cells.

Example 4 Nanoprotein for prevention of melanoma Lung metastasis

This example illustrates how to use an antigen presenting cell derived nano-vaccine to prevent cancer metastasis in a mouse melanoma lung model. In this example, the B16F10 melanoma tumor tissue was first lysed to prepare water-soluble and water-insoluble antigens of the tumor tissue; then, a nanoparticle system loaded with water-soluble and water-insoluble antigens of tumor tissues was prepared. In this example, the method of silicification and addition of charged substances was used to increase the antigen loading, and only one cycle of mineralization treatment was performed. In this example, the nanoparticles are used to activate antigen-presenting cells, and then the antigen-presenting cells are used to prepare a nano-vaccine for preventing cancer metastasis.

(1) Lysis of tumor tissue and Collection of fractions

Each C57BL/6 mouse was inoculated subcutaneously on the back with 1.5X 10 ⁵ B16F10 cells, which were about 1000mm in tumor length to volume ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, collagenase is added to the pieces to be incubated in RPMI1640 medium for 30min, and then a proper amount of pure water is added through a cell filter screen and freeze thawing is repeated for 5 times, and ultrasonic waves can be accompanied to destroy the lysed cells. After the cells are cracked, the lysate is centrifuged for 5 minutes at the rotating speed of 5000g, and the supernatant is taken as the water-soluble antigen which can be dissolved in pure water; and adding 10% sodium deoxycholate into the obtained precipitate to dissolve the precipitate, so that the water-insoluble antigen which is insoluble in pure water can be converted into the antigen which is soluble in a 10% sodium deoxycholate aqueous solution, and mixing the water-soluble antigen and the water-insoluble antigen according to the mass ratio of 2:1 to obtain the antigen raw material source for preparing the particles.

(2) Preparation of nanoparticles

In the embodiment, the nanoparticles and the blank nanoparticles serving as a reference are prepared by a solvent volatilization method, appropriate modification and improvement are performed, and two modification methods of low-temperature silicification and charged substance addition are adopted in the preparation process of the nanoparticles to improve the antigen loading capacity. The molecular weight of the adopted nano particle preparation material PLGA is 24KDa-38KDa, and the adopted immunologic adjuvant is poly (I: C). Preparation method As described above, in the preparation process, the antigen and adjuvant are loaded inside the nanoparticles by the double emulsion method, after the antigen (lysis component) is loaded inside, 100mg of nanoparticles are centrifuged at 10000g for 20 minutes, then 7mL of PBS is used for resuspending the nanoparticles and mixed with 3mL of PBS solution containing cell lysate (60mg/mL), then centrifuged at 10000g for 20 minutes, then 10mL of silicate solution (containing 150mM NaCl, 80mM tetramethyl orthosilicate and 1.0mM HCl, pH 3.0) is used for resuspending, and fixed at room temperature for 10 minutes, then-80 ℃ is used for fixing for 24 hours, after ultra pure water centrifugation washing, 3mL of PBS containing protamine (5mg/mL) and polylysine (10mg/mL) is used for resuspension and action for 10 minutes, then 10000g is used for centrifugation and 20 minutes washing, 10mL of PBS solution containing lysate (50mg/mL) is used for resuspension and action for 10 minutes, then centrifuging at 10000g for 20 minutes, using 10mL ultrapure water containing 4% trehalose for resuspending, and freeze-drying for 48 h; before using, the particles are resuspended by 7mL PBS and then added with 3mL cancer tissue lysate component (protein concentration is 50mg/mL) containing adjuvant and acted for 10min at room temperature, and a modified nano particle system which is loaded with lysate inside and outside and is subjected to frozen silicification and cationic substance addition is obtained. The average particle diameter of the nano particles is about 350nm, and the surface potential of the nano particles is about-3 mV; each 1mg PLGA nanoparticle is loaded with about 300 mug protein or polypeptide component, and each 1mg PLGA nanoparticle uses about 0.02mg poly (I: C) immunologic adjuvant inside and outside and inside and outside halves.

The contrast nanoparticles replace the loaded cancer cell whole cell antigen with four melanoma antigen polypeptides with equal mass, and the rest are the same as the cancer cell whole cell antigen loaded nanoparticles. The control nanoparticle uses 0.02mg of poly (I: C) per 1mgPLGA nanoparticle, has an average particle diameter of about 350nm and a surface potential of about-3 mV. The four loaded polypeptide neoantigens are B16-M20(Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24(Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46(Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL).

(3) Preparation of dendritic cells

This example prepares dendrites from mouse bone marrow cellsThe term "dendritic cells" is used as an example to illustrate how bone marrow-derived dendritic cells (BMDCs) are prepared. Firstly, 1C 57 mouse with age of 6-8 weeks is taken out and killed by dislocation of cervical vertebra, tibia and femur of hind leg are taken out by operation and put into PBS, and muscle tissue around the bone is removed by scissors and tweezers. The two ends of the bone are cut off by scissors, PBS solution is extracted by a syringe, needles are respectively inserted into the marrow cavity from the two ends of the bone, and the marrow is repeatedly washed into a culture dish. Bone marrow solution was collected, centrifuged at 400g for 3min, and then 1mL of erythrocyte lysate was added to lyse red blood. Lysis was stopped by adding 3mL of RPMI1640 (10% FBS) medium, centrifugation at 400g for 3min and discarding the supernatant. Cells were plated in 10mm culture dishes using RPMI1640 (10% FBS) medium with recombinant mouse GM-CSF (20ng/mL), 37 degrees, 5% CO ₂ The culture was carried out for 7 days. The flasks were gently shaken on day 3 and supplemented with the same volume of RPMI1640 medium containing GM-CSF (20ng/mL) (10% FBS). On day 6, medium was subjected to half-exchange treatment. On day 7, a small number of suspended and semi-adherent cells were collected and tested by flow assay for CD86 ⁺ CD80 ⁺ Cells in CD11c ⁺ The proportion of cells is between 15 and 20 percent, and the BMDCs cultured by induction can be used for the next experiment.

(4) Preparation of bone marrow-derived macrophages (BMDM)

Anaesthetizing C57 mice, dislocating, killing the mice with 75% ethanol, cutting a small opening on the back of the mice with scissors, directly tearing the skin to the joint of the lower leg of the mice with hands, and removing the foot joints and the skin of the mice. The hind limb was removed with scissors along the greater trochanter at the base of the mouse thigh, the muscle tissue was removed and placed in a 75% ethanol-containing petri dish for 5min, and the 75% ethanol-containing petri dish was replaced with a new one and transferred to a clean bench. The leg bone soaked in ethanol is transplanted into cold PBS for soaking, and the ethanol on the surfaces of the tibia and the femur is washed off, and the process can be repeated for 3 times. The cleaned femur and tibia are separated, both ends of the femur and tibia are cut off by scissors, the cold induction medium is sucked by a 1mL syringe to blow out bone marrow from the femur and tibia, and the flushing is repeated for 3 times until no obvious red color is seen in the leg bone. Repeatedly blowing the culture medium containing the bone marrow cells by using a 5mL pipette to disperse cell aggregates, then sieving the cells by using a 70-micron cell filter, transferring the cells into a 15mL centrifugal tube, centrifuging the cells at 1500rpm/min for 5min, discarding the supernatant, adding erythrocyte lysate, resuspending the cells, standing the cells for 5min, centrifuging the cells at 1500rpm/min for 5min, discarding the supernatant, resuspending the cells by using a cold prepared bone marrow macrophage induction medium (a DMEM high-sugar medium containing 15% L929 culture medium), and plating the cells. Cells are cultured overnight to remove other contaminating cells that adhere faster such as fibroblasts and the like. Collecting non-adherent cells, and seeding into a dish or cell culture plate according to the experimental design. Macrophage colony-stimulating factor (M-CSF) was used at a concentration of 40ng/mL to stimulate differentiation of bone marrow cells to mononuclear macrophages. Culturing for 8 days, and observing the macrophage morphological change under a light microscope. After 8 days the cells were harvested by digestion and incubated with anti-mouse F4/80 and anti-mouse CD11b antibodies at 4 ℃ for 30min in the dark, the proportion of induced successful macrophages was identified using flow cytometry.

(5) Activation of antigen presenting cells

Nanoparticles (500. mu.g) or polypeptide nanoparticles (500. mu.g) loaded with cancer cell whole cell components were incubated with prepared BMDCs (1000 ten thousand) and BMDM cells (1000 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) The incubation system contained granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL) and CD40 antibody (10 ng/mL).

(6) Preparation of antigen presenting cell derived nano vaccine

After incubation, BMDCs and BMDMs were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing a protease inhibitor, and then resuspended in PBS water and sonicated at low power (22.5W) for 1 minute at 4 ℃. The sample was then centrifuged at 3000g for 15 minutes and the supernatant collected, the supernatant collected after centrifugation at 8000g for 15 minutes, then the supernatant collected after centrifugation at 16000g for 90 minutes was discarded and the pellet collected, resuspended in PBS and filtered by extrusion using a 0.22 μm membrane filter. Then, freeze-drying the sample in a mixed freeze-drying protective agent aqueous solution 1 (1% trehalose, 2% mannitol and 2% gelatin) for 48 hours to obtain a nano vaccine 1, wherein the particle size of the nano vaccine 1 is 150 nanometers; or freeze-drying the sample in a mixed freeze-drying protective agent aqueous solution 2 (1% trehalose, 2% mannitol and 2% PVP) for 48 hours to obtain the nano vaccine 2, wherein the particle size of the nano vaccine 2 is 150 nanometers.

(7) Nano vaccine prepared from antigen presenting cell for preventing cancer metastasis

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. 120 μ g of the nano-vaccine was injected subcutaneously per mouse on days-35, 28, 21, 14 and 7 before the mice cancer model. Each mouse was inoculated intravenously at day 0 with 0.5X 10 ⁵ Individual B16F10 cells, mice were sacrificed on day 14 and the number of melanoma foci in the lungs of the mice was visually recorded.

(8) Results of the experiment

As shown in fig. 5, the control mice had more and larger foci of cancer, while the nano-vaccine treated mice had reduced numbers of cancer. Moreover, the effect of the nano vaccine prepared from the DC activated by the nanoparticles loaded with the cancer cell whole cell antigen and the macrophage on preventing cancer lung metastasis is better than that of the nano vaccine prepared from the DC activated by the nanoparticles loaded with four antigen polypeptides and the macrophage. This shows that the nano vaccine prepared by the antigen presenting cell activated by the nano particle loaded with the cancer cell whole cell antigen can activate wider and diversified cancer cell specific T cells, so that the more cancer cells can be identified and killed, and the better the effect of preventing cancer metastasis. And the effect of the vaccine prepared by using the mixed freeze-drying protective agent 1 through freeze drying is better than that of the vaccine prepared by using the mixed freeze-drying protective agent 2 through freeze drying.

Example 5 preparation of micron vaccines for cancer prevention by antigen presenting cells activated by micron particles

In this example, the B16F10 melanoma cancer cell whole cell fraction was first lysed using 6M guanidine hydrochloride. Whole cell antigens are included in the whole cell fraction. Then, PLGA is used as a microparticle framework material, poly (I: C), CpGM362 and CpGBW006 are used as immune adjuvants to prepare microparticles loaded with cancer cell whole cell antigens, the microparticles are used for activating antigen presenting cells, and then the antigen presenting cells are used for preparing the micron vaccine for preventing cancers.

(1) Lysis of cancer cells

The cultured B16F10 melanoma cancer cell line is collected and then centrifuged for 5 minutes at 350g, then the supernatant is discarded and washed twice by PBS, then 6M guanidine hydrochloride is adopted to re-suspend and crack the cancer cells, and the antigen of the whole cancer cell is cracked and dissolved in 6M guanidine hydrochloride, thus obtaining the antigen raw material source for preparing the micron particle system.

(2) Preparation of microparticle systems

In the embodiment, the micro-particles are prepared by a multiple emulsion method, the molecular weight of PLGA used as a material for preparing the micro-particles is 38KDa-54KDa, and the adopted immunologic adjuvants are poly (I: C), CpGM362 and CpG BW 006. The preparation method is as described above, in the preparation process, firstly, the cancer cell whole cell antigen and the adjuvant are loaded inside the microparticles by the double emulsion method, then 100mg of the microparticles are centrifuged at 10000g for 15 minutes, and then 10mL of 4% trehalose aqueous solution is used for resuspending the microparticles, and then the microparticles are frozen and dried for 48 times for later use. The average particle diameter of the micron particles is about 2.50 mu m, and the surface potential of the micron particles is about-2 mV; each 1mg PLGA microparticle was loaded with about 100. mu.g of protein or polypeptide component, and each 1mg PLGA microparticle was loaded with 0.02mg of each of the immunological adjuvants poly (I: C), CpGM362 and CpG BW 006. This microparticle was defined as a control micrometer vaccine 3 when subjected to the cancer prevention experiment of the following step (6).

(3) Preparation of antigen-presenting cells

This example uses BMDC and B cells mixed antigen presenting cells. BMDC was prepared as in example 1. Killing mice, taking spleens of the mice, shearing the spleens, preparing a splenocyte single cell suspension through a cell screen, and then separating CD19 from the splenocyte single cell suspension by using a magnetic bead sorting method ⁺ B cells. And mixing the BMDC and the B cells according to the number ratio of 1:1 to obtain the mixed antigen presenting cells.

(4) Activation of antigen presenting cells

The cancer cell whole cell fraction-loaded microparticles (500. mu.g) were incubated with mixed antigen-presenting cells (1000 ten thousand) in 20mL of high-glucose DMEM complete medium for 72 hours (37 ℃, 5% CO) ₂ ). The incubation system contained cytokine fraction 1: IL-2(500U/mL), IL-7(200U/mL), IL-15(200U/mL), activatedThe antigen presenting cell is an activated mixed antigen presenting cell 1; or the incubation system contains the control cytokine combination 2: IL-4(500U/mL), IL-6(200U/mL), IL-10(200U/mL), the activated antigen presenting cells are activated mixed antigen presenting cells 2.

(5) Preparation of antigen presenting cell derived micron vaccine

Incubated mixed antigen-presenting cells were collected by centrifugation at 400g for 5 minutes, followed by washing twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by low power (22.5W) sonication for 2 minutes at 4 ℃. And centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting the supernatant, centrifuging at 16000g for 90 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, resuspending the precipitate in PBS, and filtering and extruding by using a 0.22 mu m membrane to obtain the mixed antigen presenting cell membrane component. 10mL of the mixed antigen presenting cell membrane fraction (4mg) was mixed with 100mg of the microparticles prepared in step (2), followed by sonication at 4 ℃ for 1 minute with low power (22.5W), followed by 5 minutes of stirring using a homogenizer, followed by 10 minutes of co-incubation, followed by centrifugation at 12000g for 10 minutes, and the supernatant was discarded to resuspend the pellet in 10mL of a lyoprotectant (2% trehalose + 2% mannitol + 1% arginine in water) and freeze-dried for 48 hours before use. Wherein the micro vaccine prepared by the activated mixed antigen presenting cell 1 is the micro vaccine 1, and the particle size is 2.51 mu m; the micro-vaccine prepared from the activated mixed antigen presenting cells 2 is the micro-vaccine 2, and the particle size is 2.51 mu m.

(6) Micron vaccine for preventing cancer

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Mice were each vaccinated with 100 μ g of the micro-vaccine (micro-vaccine 1, or micro-vaccine 2, or the micro-particles prepared in step (2) defined as micro-vaccine 3) or PBS on days-42, 35, 28, 21, 14 and 7, respectively, before they were vaccinated with cancer cells. Each mouse was inoculated subcutaneously at day 0 with 1.5X 10 ⁵ B16F10 cells, the size of the tumor volume of the mice was recorded every 3 days from day 3. Tumor volume adoptionFormula v is 0.52 × a × b ² Calculation, where v is tumor volume, a is tumor length, and b is tumor width. From animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in the life test of the mouse ³ I.e. the mice were considered dead and were euthanized.

(7) Results of the experiment

As shown in FIG. 6, the tumors of the PBS control mice grew rapidly and the mice died rapidly. The growth rate of the tumor of the mice treated by the micron vaccine is obviously slowed down, and the survival period is obviously prolonged. Moreover, the effect of the micro-vaccine 1 prepared by using the mixed antigen presenting cell 1 activated by the cytokine combination 1 is obviously better than that of the micro-vaccine 2 prepared by using the mixed antigen presenting cell 2 activated by the cytokine component 2. Moreover, the effect of the micro-vaccine 2 is obviously better than that of the micro-vaccine 3 which only internally loads cancer cell whole cell components and does not load any antigen presenting cell membrane components on the surface. Therefore, the surface loaded antigen presenting cell membrane component can obviously improve the curative effect of the micron vaccine; moreover, the addition of specific cytokines when the microparticles activate antigen-presenting cells can improve the efficacy of the micro-vaccine prepared from the activated antigen-presenting cells.

Example 6 Nanoprotein for prevention of cancer

In this example, B16F10 melanoma tumor tissue was first lysed using 8M urea and the tumor tissue lysate fraction was solubilized. Then, PLGA is used as a nanoparticle framework material, Poly (I: C) and CpG2395 are used as immune adjuvants to prepare nanoparticles loaded with cancer cell whole cell antigens, and the nanoparticles are used for activating antigen presenting cells and then preparing the nano vaccine for preventing cancers by utilizing the antigen presenting cells.

(1) Collection and lysis of tumor tissue

Each C57BL/6 mouse was inoculated subcutaneously into the back of 1.5X 10 mice ⁵ B16F10 cells, which were about 1000mm in tumor length to volume ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, and a proper amount of 8M urea is added through a cell filter screen to lyse cells and lyse cell lysates. The above is the source of the antigen raw material for preparing the nanoparticle system.

(2) Preparation of nanoparticle systems

In this embodiment, the nanoparticles and the blank nanoparticles used as a control were prepared by a solvent evaporation method. The molecular weight of PLGA used as a material for preparing the nano particles is 7-17 KDa, the adopted immunologic adjuvant is poly (I: C) and CpG2395, and a lysate component and the adjuvant are loaded in the nano particles. The preparation method is as described above, in the preparation process, firstly, a lysate component and an adjuvant are loaded inside the nanoparticles by a multiple emulsion method, then, 100mg of the nanoparticles are centrifuged at 12000g for 20 minutes, and are subjected to freeze drying for 48 hours after being resuspended by 10mL of ultrapure water containing 4% trehalose, so as to obtain freeze-dried powder for later use. The average particle diameter of the nano particles is about 270nm, and the surface potential of the nano particles is about-3 mV; each 1mg PLGA nanoparticle is loaded with about 110 μ g protein or polypeptide component, and each 1mg PLGA nanoparticle uses 0.02mg of poly (I: C) and CpG2395 immunoadjuvants.

The preparation material and the preparation method of the contrast nano-particle are the same, each 1mg PLGA nano-particle is loaded with 0.02mg of poly (I: C) and CpG2395 respectively, the particle size is about 270nm, the surface potential of the nano-particle is about-3 mV, and each 1mg PLGA nano-particle is loaded with about 110 mu g of polypeptide component. The four loaded polypeptide neoantigens are B16-M20(Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24(Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46(Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL).

(3) Preparation of DC and B cells

Mouse lymph nodes are harvested after C57BL/6 sacrifice, a single cell suspension of mouse lymph nodes is prepared, and then CD11C is separated from the single cell suspension of lymph nodes cells by flow cytometry ⁺ DC and CD19 ⁺ B cells.

(4) Activation of antigen presenting cells

Nanoparticles loaded with cancer cell whole cell fractions (1mg) or polypeptide nanoparticles (1mg) were incubated with DCs (2000 ten thousand) and B cells (2000 ten thousand) in 20mL of high-glucose DMEM complete medium for 72 hours (37 ℃, 5% CO ₂ ) Or, nanoparticles (500. mu.g) loaded with cancer cell whole cell components were incubated with DCs (1000 ten thousand) in 20mL of high-glucose DMEM complete medium for 72 hours (37 ℃, 5% C)O ₂ ) (ii) a The incubation system contained granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), and CD86 antibody (10 ng/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated DC and B cells (1000 ten thousand DC cells + 1000 ten thousand B cells) or only DC cells (2000 ten thousand) were collected by centrifugation at 400g for 5 minutes, then the cells were washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing a protease inhibitor, and after resuspension in PBS water, the cells were disrupted at 4 ℃ for 25 minutes with stirring at 2000rpm using a homogenizer. And centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, mixing the obtained supernatant with the corresponding nano particles (30mg) for activating the antigen presenting cells prepared in the step 3, performing ultrasonic treatment at 50W for 3 minutes, filtering and extruding by using a 0.45-micrometer membrane, centrifuging at 12000g for 30 minutes, removing supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine with the particle size of 310 nm.

(6) Antigen presenting cell derived nano vaccine for preventing cancer

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Mice were subcutaneously injected with 40 μ g of nano-vaccine or PBS on days-42, 28, and 14, respectively, prior to inoculation of the mice with cancer cells. Each mouse was inoculated subcutaneously at day 0 with 1.5X 10 ⁵ The individual B16F10 cells, mouse tumor volume and survival monitoring methods were the same as in example 5.

(7) Results of the experiment

As shown in FIG. 7, the tumor growth rate of the control mice was very fast, while the tumor growth rate of the nano-vaccine treated mice was significantly slow or the tumor disappeared. In addition, the prevention effect of the nano vaccine prepared by DC and B cells activated by the nano particles loaded with cancer cell whole cell antigens is superior to that of the nano vaccine prepared by DC and B cells activated by the nano particles loaded with four antigen polypeptides. Moreover, the effect of the nano vaccine prepared by mixing the DC activated by the nano particles loaded with the cancer cell whole cell antigen and the B cell is better than that of the nano vaccine prepared by the DC activated by the nano particles loaded with the cancer cell whole cell antigen, which indicates that the effect of the nano vaccine prepared by a plurality of antigen presenting cells activated by the nano particles is better. This is probably because a part of the components within activated B cells can help enhance the activation of cancer cell-specific T cells by the nano-vaccine after inclusion into the nano-vaccine prepared by the antigen presenting cell.

EXAMPLE 7 Nanoprotein preparation of antigen presenting cells for treatment of Colon cancer

This example uses MC38 mouse colon cancer as a cancer model to illustrate how a nano-vaccine prepared using antigen presenting cells can be used to treat colon cancer. Colon cancer tumor tissue and lung cancer cells are first lysed to prepare a mixture of water-soluble antigens (mass ratio 1:1) and a mixture of water-insoluble antigens (mass ratio 1:1), and the mixture of water-soluble antigens and the mixture of water-insoluble antigens are mixed in a mass ratio 1:1. Then, PLA is used as a nanoparticle framework material, CpGSL03 and BCG (BCG) are used as immune adjuvants to prepare nanoparticles, and the nanoparticles are used for activating antigen presenting cells in vitro to prepare a nano vaccine for treating colon cancer.

(1) Lysis of tumor tissue and cancer cells and collection of fractions

Each C57BL/6 mouse was inoculated subcutaneously on the back 2X 10 ⁶ The MC38 cells grow to a tumor volume of about 1000mm ³ Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, and a proper amount of pure water is added through a cell filter screen and freeze thawing is repeated for 5 times, and ultrasonic waves are accompanied to destroy the lysed cells. After the cells are cracked, centrifuging the lysate for 5 minutes at the rotating speed of more than 5000g and taking supernatant fluid as water-soluble antigen which can be dissolved in pure water; the precipitate is dissolved by adding 5% sodium dodecyl benzene sulfonate (SDS) aqueous solution to the precipitate to convert the water-insoluble antigen insoluble in pure water into soluble antigen in aqueous solution.

The cultured LLC lung cancer cell lines were harvested and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, then the cells were resuspended with ultrapure water and freeze-thawed repeatedly 5 times, possibly with sonication to disrupt the lysed cells. After the cells are cracked, the lysate is centrifuged for 6 minutes at the rotating speed of 3000g, and the supernatant is taken as the water-soluble antigen which can be dissolved in pure water; the precipitate is dissolved by adding 5% SDS aqueous solution to the precipitate to convert the water-insoluble antigen insoluble in pure water to soluble in SDS aqueous solution.

Mixing water-soluble antigens from colon cancer tumor tissue and lung cancer cells according to the mass ratio of 1: 1; the water-insoluble antigens dissolved in 5% SDS were also mixed in a mass ratio of 1:1. Then mixing the water-soluble antigen mixture and the water-insoluble antigen mixture according to the mass ratio of 1:1, wherein the mixture is a raw material source for preparing the nano particles.

(2) Lysis of BCG and Collection of fractions

The lysis method of BCG and the collection method of each component are the same as the lysis method of cancer cells and the collection method of each component, and the water-soluble antigen and the solubilized water-insoluble antigen are mixed in a mass ratio of 1:1.

(3) Preparation of nanoparticles

In this example, the nanoparticles were prepared by a solvent evaporation method. The molecular weight of PLA which is used as a material for preparing the nanoparticles is 20KDa, the adopted immune adjuvants are CpGSL03 and BCG, and the adjuvants are distributed in and on the surfaces of the nanoparticles simultaneously. Preparation method As mentioned above, in the preparation process, firstly, the lysate mixture and the adjuvant are loaded inside the nanoparticles by using the multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and after being resuspended by using 10mL of ultrapure water containing 4% trehalose, the nanoparticles are freeze-dried for 48 hours. Before use, 20mg of nanoparticles were resuspended in 0.9mL of PBS and incubated for 5 minutes with 0.1mL of a sample containing the lysate mixture (80mg/mL) and adjuvant mixed at room temperature. The average particle diameter of the nano particles is about 280nm, and the surface potential of the nano particles is about-3 mV; about 140. mu.g of protein or polypeptide component is loaded per 1mg of PLGA nanoparticle, and each 1mg of PLGA nanoparticle contains 0.04mg of CpGSL03 and BCG immunoadjuvant.

(3) Preparation of antigen-presenting cells

Peripheral blood was collected from mice after sacrifice of C57BL/6, Peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood, and CD11C was selected from PBMC using flow cytometry ⁺ DC and CD19 ⁺ B cells. In this example, BMDC and BMDC were used simultaneouslyBMDM acts as an antigen presenting cell. BMDC was prepared as in example 2 and BMDM was prepared as in example 4.

(4) Activation of antigen presenting cells

Nanoparticles (1000. mu.g) loaded with cancer cell whole cell components were incubated with peripheral blood-derived DCs (500 ten thousand), BMDCs (500 ten thousand), BMDM (500 ten thousand) and B cells (500 ten thousand) in 20mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ) Or, nanoparticles (500. mu.g) loaded with cancer cell whole cell components were incubated with DCs of peripheral blood origin (1000 ten thousand) and BMDCs (1000 ten thousand) in RPMI1640 complete medium for 72 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), and CD86 antibody (10 ng/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated peripheral blood-derived DCs, B cells and BMDMs (500 ten thousand DC +500 ten thousand B cells +500 ten thousand BMDMs) or only DC and B cells (750 ten thousand DC + 750 ten thousand B cells) were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing a protease inhibitor, resuspended in PBS water, and then treated in a high pressure homogenizer (5000bar) for 5 minutes. Centrifuging the sample at 2000g for 15 minutes, collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, co-incubating the supernatant and the corresponding nanoparticles (50mg) prepared in the step 3 at normal temperature for 6 hours, filtering and extruding by using a 0.45-micrometer membrane, centrifuging at 13000g for 20 minutes, collecting the supernatant, discarding the precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine with the particle size of 320 nanometers.

(6) Nano-vaccine for cancer treatment

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare colon cancer tumor-bearing mice. Each mouse was inoculated subcutaneously 2X 10 on day 0 ⁶ MC38 cells were injected subcutaneously with 50 μ g of nano-vaccine per mouse on

days

4, 7, 10, 15 and 20, respectively. Mice tumor volumes were recorded every 3 days starting on day 3Size. Tumor volume is calculated by the formula v ═ 0.52 × a × b ² Calculation, where v is tumor volume, a is tumor length, and b is tumor width. From animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in mouse survival period test ³ I.e. the mice were considered dead and were euthanized.

(7) Results of the experiment

As shown in FIG. 8, the tumor growth rate of the control mice was very fast, while the tumor growth rate of the nano-vaccine treated mice was significantly slow or the tumor disappeared. Moreover, the effect of the nano vaccine prepared by mixing the DC activated by the nano particles, the B cells and the macrophages is better than that of the nano vaccine prepared by the DC activated by the nano particles and the B cells, which shows that the effect of the nano vaccine prepared by a plurality of antigen presenting cells activated by the nano particles is better. In conclusion, the nano vaccine prepared by the antigen presenting cell has a good treatment effect on colon cancer.

EXAMPLE 8 Nanoprotein preparation of antigen presenting cells for the treatment of melanoma

This example uses melanoma as a cancer model to illustrate how to activate antigen-presenting cells using nanoparticles loaded with cancer cell whole-cell antigens derived from melanoma and lung cancer tumor tissues, and then use the antigen-presenting cells to prepare a nano-vaccine and use the nano-vaccine to treat melanoma. In this example, B16F10 melanoma tumor tissue and LLC lung cancer tumor tissue were first lysed to prepare a water-soluble antigen mixture (3:1 mass ratio) and a water-insoluble antigen mixture (3:1) of tumor tissue. PLGA is used as a nanoparticle framework material, Poly ICLC and CpG2395 are used as immune adjuvants to prepare nanoparticles loaded with the mixture, then the nanoparticles are used for activating antigen presenting cells, and the antigen presenting cells are used for preparing the nano vaccine for treating cancer.

(1) Lysis of tumor tissue and Collection of fractions

Each C57BL/6 mouse was inoculated subcutaneously into the back of 1.5X 10 mice ⁵ B16F10 cells or 2X 10 cells ⁶ LLC lung cancer cells with tumor growth volume of about 1000mm ³ Mice were sacrificed and tumor tissue was removed. Tumor lysis and fraction collection methodThe procedure is as in example 1. Mixing water soluble antigen from melanoma tumor tissue and lung cancer tumor tissue with original water insoluble antigen dissolved in 8M urea at ratio of 3:1 to obtain respective mixture. And then mixing the water-soluble antigen mixture and the water-insoluble antigen mixture according to the mass ratio of 1:1 to obtain the antigen source for preparing the nano particles.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by a multiple emulsion method. The molecular weight of PLGA used as a nano particle preparation material is 24-38 KDa, and the adopted immunologic adjuvant is Poly ICLC and CpG 2395. The preparation method is as described above. Firstly, loading lysate components and adjuvant inside PLGA nano particles, then centrifuging 100mg of nano particles for 25 minutes at 10000g, resuspending the nano particles by using 10mL of ultrapure water containing 4% trehalose, and freeze-drying the nano particles for 48 hours for later use. The average particle diameter of the nano particles is about 300nm, and the surface potential of the nano particles is about-5 mV; about 80 mug of protein and polypeptide components are loaded on each 1mg of PLGA nano particles, and each 1mg of PLGA nano particles is loaded with 0.04mg of poly ICLC and CpG 2395.

(3) Preparation of antigen-presenting cells

The DC is a mixed DC of a DC derived from peripheral blood and a BMDC. BMDC was prepared as above. Peripheral blood was collected from mice after sacrifice of C57BL/6, Peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood, and CD11C was selected from PBMC using flow cytometry ⁺ The DC used in this example was a mixed DC obtained by mixing BMDC and peripheral blood-derived DC at a ratio of 1:1.

(4) Activation of antigen presenting cells

Nanoparticles loaded with cancer cell whole cell components (800 μ g) were incubated with mixed DCs (1000 ten thousand) in 15mL high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) The incubation system contained granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL) and CD86 antibody (10 ng/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

Post-incubation DCs (1000 ten thousand) were harvested by centrifugation at 400g for 5 minutes, followed by washing the cells twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by low power (20W) sonication at 4 ℃ for 1 minute and then treatment with a homogenizer at 1000rpm for 3 minutes. And (3) centrifuging the sample at 3000g for 10 minutes, collecting supernatant, centrifuging the supernatant at 8000g for 5 minutes, collecting supernatant, mixing the supernatant with the nanoparticles (50mg) prepared in the step (2) and the DSPE-PEG-CD32 monoclonal antibody (50 mu g), performing ultrasonic treatment at 100W for 2 minutes, filtering by using a 0.45 mu m membrane, centrifuging at 15000g for 30 minutes, collecting the supernatant, discarding the precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine 1, wherein the particle size of the nano vaccine is 280 nanometers.

Post-incubation DCs (1000 ten thousand) were harvested by centrifugation at 400g for 5 minutes, followed by washing the cells twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by low power (20W) sonication at 4 ℃ for 1 minute and then treatment with a homogenizer at 1000rpm for 3 minutes. And centrifuging the sample at 3000g for 10 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 5 minutes, collecting supernatant, mixing the supernatant with DSPE-PEG-CD32 monoclonal antibody (50 mu g), performing ultrasonic treatment at 100W for 2 minutes, centrifuging at 18000g for 90 minutes, collecting the supernatant, discarding the precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine 2, wherein the particle size of the nano vaccine is 150 nanometers.

(6) Nano-vaccine for cancer treatment

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Each mouse was inoculated subcutaneously 1.5X 10 on day 0 ⁵ B16F10 cells, mice were injected subcutaneously with 100 μ g of nano-vaccine on

days

4, 7, 10, 15 and 20, respectively. Mouse tumor volume and survival were monitored as above.

(7) Results of the experiment

As shown in FIG. 9, the tumors of the PBS control mice grow rapidly, and compared with the control mice, the growth rates of the tumors of the mice treated by the nano vaccine 1 and the nano vaccine 2 prepared by the DC activated by the nanoparticles are obviously slowed down, and part of the tumors of the mice disappear and are cured. Moreover, the effect of the nano vaccine 1 which is loaded with the DC cell membrane component on the surface of the internal loaded cancer cell whole cell component is obviously better than that of the nano vaccine 2 which is prepared only from the DC cell membrane component. In the nano vaccine of the present embodiment, the CD32 monoclonal antibody is used as the target head for active targeting, and in practical applications, any target head having the ability to target cells, such as mannose, CD3 antibody, CD56 antibody, mannan, CD205 monoclonal antibody, CD19 monoclonal antibody, and the like, may be used.

Example 9 use of the micron vaccine for the prevention of Breast cancer

This example uses 4T1 mouse triple negative breast cancer as a cancer model to illustrate how to use 8M urea to lyse cancer cell whole cell antigen and prepare microparticles loaded with cancer cell whole cell antigen, and use the microparticles to activate antigen-presenting cells to prepare cancer vaccines for preventing breast cancer.

(1) Lysis of cancer cells

Cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells are respectively inactivated and denatured by ultraviolet rays and high-temperature heating, and then a proper amount of 8M urea is adopted to crack the breast cancer cells and dissolve a lysate, namely the raw material source for preparing the particle system.

(2) Preparation of microparticle systems

In the embodiment, the preparation of the microparticles adopts a multiple emulsion method, the molecular weight of PLGA serving as a microparticle framework material is 38KDa-54KDa, and the adopted immunologic adjuvant is CpG2395 and Poly (I: C). The preparation method comprises the steps of firstly preparing the micron particles internally loaded with the lysate component and the adjuvant by a multiple emulsion method, then centrifuging 100mg of the micron particles for 20 minutes at 9000g, using 10mL of ultrapure water containing 4% trehalose for resuspension, and drying for 48 hours for later use. The average particle size of the micron particle system is about 2.18 mu m, and the surface potential is about-6 mV; each 1mg PLGA microparticle was loaded with about 110. mu.g of protein or polypeptide components, 0.03mg each of CpG2395 and Poly (I: C).

(3) Preparation of BMDC and B cells

BMDC was prepared as in example 2. B cells were prepared as in example 3.

(4) Activation of antigen presenting cells

Microparticles loaded with cancer cell whole cell components (1000. mu.g) were incubated with BMDCs (1000 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) (ii) a Alternatively, cancer cell whole cell fraction-loaded microparticles (1000. mu.g) were incubated with B cells (1000 ten thousand) in 15mL of high-glucose DMEM complete medium for 72 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), and CD86 antibody (10 ng/mL).

(5) Preparation of antigen presenting cell derived micron vaccine

Incubated DC (1000 ten thousand) or B cells (1000 ten thousand) were collected by centrifugation at 400g for 5 minutes, and then washed twice with Phosphate Buffered Saline (PBS) containing protease inhibitors at 4 ℃, and the cells were resuspended in PBS water and then sonicated at low power (10W) for 10 minutes at 4 ℃. And then filtering the sample through membranes with the pore diameters of 10 microns, 5 microns, 2 microns, 1 micron, 0.45 microns and 0.22 microns in sequence, collecting filtrate, mixing the filtrate with the micron particles (40mg) prepared in the step 2, carrying out ultrasonic treatment for 1 minute at 50W, incubating for 10 minutes, centrifuging for 15 minutes at 9000g, removing supernatant, collecting precipitate, and carrying out resuspension on the precipitate in PBS to obtain the micron vaccine with the particle diameter of 2.20 microns.

(6) Antigen presenting cell derived micro-vaccine for cancer prevention

Female BALB/c of 6-8 weeks is selected as a model mouse to prepare a breast cancer tumor-bearing mouse. The mice were inoculated with 80 μ g of the micro-vaccine per mouse on days-35, 28, 21, 14 and 7, respectively, before the mice were inoculated with cancer cells; the PBS control group was inoculated with 100. mu.L of PBS. Each mouse was inoculated subcutaneously with 1X 10 injection on day 0 ⁶ 4T1 cells, the size of the tumor volume of the mice was recorded every 3 days from day 3. Mouse tumor monitoring was as above.

(7) Results of the experiment

As shown in fig. 10, the tumor growth rate and survival time of the mice treated with the vaccine prepared from the micron-sized activated antigen-presenting cells was significantly slower than those of the PBS control group. Moreover, the effect of the micrometer vaccine prepared by the micrometer activated DC is better than that of the micrometer vaccine prepared by the micrometer activated B cell. Therefore, the micro vaccine provided by the invention has a prevention effect on breast cancer.

Example 10 Nanoprotein for prevention of cancer metastasis

(1) Lysis of tumor tissue and cancer cells

After collecting mouse B16F10 melanoma tumor tissues and cultured cancer cells, 8M urea is adopted to crack and dissolve cancer cell whole cell components from the tumor tissues and the cancer cells, and then the tumor tissue components and the cancer cell components are mixed and dissolved according to the mass ratio of 1: 2.

(2) Preparation of nanoparticles

In the embodiment, the nanoparticles are prepared by a solvent volatilization method, the molecular weight of PLGA used as a nanoparticle preparation material is 24KDa-38KDa, and poly ICLC and CpG1018 are used as immunoadjuvants. As mentioned above, in the preparation process, the cell component and adjuvant are loaded inside the nanoparticles by using a multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and then 10mL of ultrapure water containing 4% trehalose is used for resuspension, and then the mixture is frozen and dried for 48 hours for later use. The average particle diameter of the nano particles is about 250 nm; about 90. mu.g of protein and polypeptide components were loaded per 1mg of PLGA nanoparticle, 0.03mg each of poly ICLC and CpG 1018.

(3) Preparation of DC

This example used mixed DCs of BMDCs and peripheral blood DCs. BMDC was prepared as in example 2. Peripheral blood DC is prepared by collecting peripheral blood of mice after sacrifice, isolating PBMC of the mice, and isolating CD11c from the PBMC of the mice using flow cytometry ⁺ The DCs from peripheral blood and BMDCs were mixed in a quantitative ratio of 1:1.

(4) Activation of antigen presenting cells

Nanoparticles loaded with cancer cell whole cell components (1000. mu.g) and DCs (1000 ten thousand) were incubated in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contains granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL),IL-2(500U/mL), IL-7(200U/mL), IL-15(200U/mL) and CD86 antibody (10 ng/mL).

(4) Preparation of antigen presenting cell derived nano vaccine

The incubated peripheral blood-derived DCs and BMDCs were collected by centrifugation at 400g for 5 minutes, followed by washing the mixed cells twice with Phosphate Buffered Saline (PBS) containing protease inhibitors at 4 ℃, resuspending the cells in PBS water and then sonicating for 5 minutes at 4 ℃ and 20W. And (2) centrifuging the sample at 3000g for 5 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, discarding the precipitate, collecting the supernatant, centrifuging at 18000g for 90 minutes, filtering with a 0.22 mu m filter membrane, discarding the supernatant, suspending the precipitate in PBS to obtain an antigen presenting cell membrane component, mixing 10mL of the cell membrane component (5mg) with 100mg of the nanoparticles prepared in the step (2), incubating at room temperature for 15 minutes, filtering and extruding through the 0.45 mu m filter membrane, and centrifuging at 14000g for 25 minutes to obtain the nano vaccine with the surface loaded with the antigen presenting cell membrane component. Resuspending the nano vaccine with 10mL of a freeze-drying protective agent 1 (2% trehalose, 2% mannitol and 1% arginine aqueous solution), and freeze-drying for 48 hours to obtain the nano vaccine 1 with the particle size of 260 nm; the nano vaccine is subjected to resuspension by using 10mL of a freeze-drying protective agent 2 (2% trehalose, 2% mannitol and 1% glycine), and then freeze-drying for 48 hours to obtain the nano vaccine 2 with the particle size of 260 nm. The nano vaccine 1 and the nano vaccine 2 were used after being stored at room temperature for 360 days.

The particle size of the nano-particles is analyzed for the new freeze-dried nano-vaccine 1 and the nano-vaccine 1 stored at room temperature for 360 days, and the result shows that the particle size of the nano-vaccine 1 is 260nm after the nano-vaccine 1 is stored at room temperature for 360 days, which indicates that the stability of the nano-vaccine 1 is good. In addition, the newly prepared nano vaccine 1 and the nano vaccine 1 stored at room temperature for 360 days were used for the analysis of the efficacy of preventing cancer metastasis, respectively.

(5) Nano-vaccine for preventing cancer metastasis

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Mice were inoculated with each vaccine at-42, -35, -28, -21, -14 and-7 days before inoculation of cancer cells100 μ g of nano-vaccine (freshly prepared nano-vaccine 1, or nano-vaccine 1 after 360 days of storage at room temperature, or nano-vaccine 2 after 360 days of storage at room temperature) or PBS. Each mouse was inoculated intravenously at 0.5X 10 day ⁵ Individual B16F10 cells, mice were sacrificed on day 14 and the number of melanoma foci in the lungs of the mice was visually recorded.

(6) Results of the experiment

As shown in fig. 11, the nano vaccine prepared from the DC activated by nanoparticles can effectively prevent cancer metastasis. Moreover, after the nano vaccine is placed for 360 days at room temperature, the nano vaccine has stable property and consistent effect. Moreover, the effect of the nano vaccine 1 prepared by using 2% of trehalose, 2% of mannitol and 1% of arginine as a freeze-drying protective agent is better than that of the nano vaccine 2 prepared by using 2% of trehalose, 2% of mannitol and 1% of glycine as a freeze-drying protective agent; the nano vaccine 1 stored at room temperature for 360 days has no significant difference in curative effect with the newly prepared nano vaccine 1, which shows that the nano vaccine of the invention can keep long-term stability, and the freeze drying process can damage the structure of the vaccine to make the vaccine unstable and reduce the activity. The specific freeze-drying protective agent can improve the curative effect of the prepared nano vaccine subjected to freeze drying and long-term storage.

Example 11 Nanoprotein for treatment of pancreatic cancer

In the embodiment, the mouse Pan02 pancreatic cancer tumor tissue and MC38 colon cancer tumor tissue lysis component are loaded on the nanoparticles according to the ratio of 3:1, and the antigen presenting cells activated by the nanoparticles are used for preparing the nano vaccine for treating pancreatic cancer. In the experiment, mouse pancreatic cancer and colon cancer tumor tissues were first harvested and lysed to prepare water-soluble antigens and the original water-insoluble antigen dissolved in 6M guanidine hydrochloride. When the particles are prepared, PLGA is used as a nanoparticle framework material, BCG is used as an adjuvant to prepare the nanoparticles, and then the nanoparticles are used for activating antigen presenting cells to prepare the nano vaccine.

(1) Lysis of tumor tissue and Collection of fractions

Each C57BL/6 mouse was inoculated subcutaneously in the axilla of a 2X 10 strain ⁶ MC38 Colon cancer cells or inoculated with 1 × 10 ⁶ Pan02 pancreatic cancer cellCells, the tumors inoculated in each mouse grew to a volume of about 1000mm ³ Mice were sacrificed and tumor tissue was removed. After mechanical disruption of tumor tissue, the cells were digested in RPMI1640 complete medium containing 5mg/mL collagenase type IV, 5mg/mL collagenase type I, 1.5mg/mL hyaluronidase, and 1.5mg/mL DNase for 20min at 37 deg.C, the cell solution was collected and filtered through a 300 mesh sterilized nylon mesh to remove small debris. Then, CD45 magnetic beads are used for negative sorting to obtain purer tumor cells. Repeatedly freezing and thawing the obtained tumor cells in ultrapure water for 3 times, performing ultrasonic lysis on the tumor cells, centrifuging at 3000g for 5 minutes to obtain a supernatant part which is a water-soluble component, and dissolving a precipitate part by using 6M guanidine hydrochloride to obtain a water-insoluble component. The water-soluble component is a mixture of 3:1 of water-soluble components of pancreatic cancer tumor tissues and water-soluble components of colon cancer tumor tissues; the water insoluble component is a mixture of water insoluble components of pancreatic cancer tumor tissue and water insoluble components of colon cancer tumor tissue in a ratio of 3: 1. The water-soluble component mixture and the water-insoluble component mixture are mixed in a mass ratio of 1:1. The lysis and dissolution method of BCG is the same as the tumor tissue lysis method, and the water-soluble component and the water-insoluble component are mixed according to the mass ratio of 1:1. The whole-cell water-soluble component contains whole-cell water-soluble antigen, and the whole-cell water-insoluble component contains whole-cell water-insoluble antigen.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by multiple emulsion method. The molecular weight of PLGA used as a material for preparing the nanoparticles is 7-17 KDa, the adopted immunologic adjuvant is BCG, and the BCG is encapsulated in the nanoparticles. The preparation method is as described above, in the preparation process, firstly, a lysate component and an adjuvant are loaded inside the nanoparticles by a multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 12000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is used for resuspension, and then the lyophilized powder is obtained for later use after being frozen and dried for 48 hours. 20mg of nanoparticles were dissolved in 0.9mL PBS before nanoparticle injection, mixed with 0.1mL of lysate (80mg/mL) containing sample and used after 10min at room temperature. The average particle diameter of the nano particles is about 260nm, and the surface potential of the nano particles is about-4 mV; about 130 mug of protein and polypeptide components are loaded in each 1mg of PLGA nano particles, and 0.08mg of BCG immunoadjuvant is used in each 1mg of PLGA nano particles.

(3) Preparation of antigen-presenting cells

This example uses BMDC or BMDM as an antigen presenting cell. BMDC was prepared as in example 2 and BMDM was prepared as in example 4.

(4) Activation of antigen presenting cells

Nanoparticles loaded with cancer cell whole cell components (1000. mu.g) were incubated with BMDCs (1000 ten thousand) or with BMDM (1000 ten thousand) in 15mL high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), M-CSF (2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), IFN- γ (500U/mL), and CD80 antibody (10 ng/mL).

(4) Preparation of antigen presenting cell derived nano vaccine

Incubated BMDCs or BMDM were collected by centrifugation at 400g for 5 minutes, then washed three times by centrifugation at 1200rpm for 3min in 30mM pH 7.0Tris-HCl buffer containing 0.0759M sucrose and 0.225M mannitol, then applied using a homogenizer at 2000rpm for 10 minutes in the presence of phosphatase and protease inhibitors, and then mechanically disrupted at 20W for 1 minute. After centrifugation, the resulting cell membranes were washed with a solution of 10mM Tris-HCl pH 7.5 and 1mM EDTA. Then filtering the sample by membranes with the aperture of 30 μm, 10 μm, 5 μm, 2 μm and 0.45 μm in sequence, centrifuging the filtrate for 25 minutes at 12000g, removing supernatant, collecting precipitate, resuspending the precipitate in physiological saline containing 4% mannitol, and freeze-drying to obtain the nano vaccine with the particle size of 260 nm.

(5) Nano-vaccine for cancer treatment

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare a pancreatic cancer-bearing mouse. Each mouse was inoculated subcutaneously 1X 10 on day 0 ⁶ A Pan02 cell was injected subcutaneously into mice with 400. mu.g of nano-vaccine or PBS on

days

4, 7, 10, 15, 20 and 25, respectively. The size of the tumor volume of the mice was recorded every 3 days from day 3. The tumor volume calculation method and the mouse survival monitoring method are the same as above.

(6) Results of the experiment

As shown in fig. 12, compared to the PBS control group, the nano vaccine prepared from the nanoparticle-activated DC or the macrophage can effectively treat pancreatic cancer, and the nano vaccine prepared from the nanoparticle-activated DC has better effect.

Example 12 Nanoprotein for prevention of cancer

This example targets mannose to demonstrate how to use actively targeted nanoparticle-activated antigen-presenting cells to prepare a nano-vaccine and use for the prevention of cancer. The specific dosage form, adjuvant, administration time, administration frequency and administration scheme can be adjusted according to the actual application. The nanoparticle system can enter dendritic cells through the uptake of mannose receptors on the surface of the DC, and then the nanometer vaccine is prepared for preventing cancers after the DC is activated.

(1) Lysis of cancer cells

Cultured B16F10 cancer cells were harvested and the cancer cell whole cell fraction derived from the cancer cells was lysed and lysed using 8M urea.

(2) Preparation of nanoparticle systems

The nanoparticle system in this example was prepared using a multiple emulsion process. The nano particle preparation materials are PLGA and PLGA modified by mannose, and the molecular weights of the PLGA and the PLGA are both 7KDa-17 KDa. When the target head-carrying nano particles are prepared, the mass ratio of the target head-carrying nano particles and the target head-carrying nano particles is 4: 1. The immunological adjuvants used were Poly (I: C) and CpG SL 03. The preparation method is as described above, the lysate component and the adjuvant are co-loaded inside the nanoparticles by the multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are frozen and dried for 48 hours after being re-suspended by 10mL of ultrapure water containing 4% trehalose for later use. The average particle diameter of the target-head-carrying nanoparticles is about 270nm, and each 1mg of PLGA nanoparticles is loaded with about 80 mug of protein and polypeptide components, and contains 0.04mg of Poly (I: C) and CpGSL03 respectively. The particle size of the control nanoparticle without adjuvant and with mannose target is about 270nm, the control nanoparticle is prepared by using the same amount of cell components without any immunologic adjuvant, and each 1mg of PLGA nanoparticle is loaded with about 80 mug of protein and polypeptide components. The blank nanoparticle with the mannose target head has the particle size of about 250nm, and is prepared by adopting the same amount of adjuvant without loading any cell lysis component.

(3) Preparation of antigen-presenting cells

This example used BMDC and DC2.4 as antigen presenting cells. BMDC was prepared as in example 2.

(4) Activation of antigen presenting cells

Nanoparticles (1000. mu.g) or blank nanoparticles (1000. mu.g) + free lysate loaded with cancer cell whole cell components were incubated with BMDCs (500 ten thousand) and DC2.4 cells (500 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), M-CSF (2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), IFN- γ (500U/mL), and CD80 antibody (10 ng/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The post-incubation DCs were collected by centrifugation at 400g for 5 minutes, followed by washing the cells twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by 2 minutes of low power (20W) sonication at 4 ℃. Centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 5000g for 10 minutes, collecting supernatant, filtering the supernatant through a 0.45-micron membrane, performing ultrafiltration centrifugal filtration and concentration by using an ultrafiltration membrane (with the molecular weight cut-off of 50KDa), centrifuging the filtered and concentrated sample at 17000g for 120 minutes, collecting and discarding supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine with the particle size of 120 nanometers.

(6) Nano-vaccine for cancer prevention

Selecting female C57BL/6 of 6-8 weeks as model mice to prepare melanoma-bearing mice, respectively inoculating 400 μ g nanometer vaccine or PBS to each mouse on the 35 th day, 28 th day, 21 st day, 14 th day and 7 th day before inoculating cancer cells, and inoculating 1.5 × 10 subcutaneous part of the back of each recipient mouse on the 0 th day ⁵ And B16F10 cells. The tumor growth rate and survival time of the mice were monitored. Tumor growth and survival monitoring methods were as above.

(5) Results of the experiment

As shown in fig. 13, the tumor growth rate of mice treated with the nano-vaccine prepared from the DC activated by nanoparticles loaded with the whole cell fraction was significantly slower compared to the PBS control group and the nano-vaccine control group prepared from the actively targeted blank nanoparticles + DC activated by free lysate. The nano vaccine prepared by the DC activated by the nano particles can effectively prevent cancer regardless of the adjuvant, but the adjuvant effect is better. And it is demonstrated that the nano-vaccine of the present invention can effectively prevent cancer.

Example 13 Nanoprotein for prevention of liver cancer

In this example, Hepa1-6 hepatoma cells were first lysed, nanoparticles loaded with the whole cell fraction of hepatoma cells were prepared using PLGA as the nanoparticle scaffold, Poly (I: C) and BCG as the immunoadjuvants, and then the nanoparticle-activated DC and B cells were used to prepare the nano-vaccine for preventing hepatoma.

(1) Lysis of cancer cells and collection of fractions

After collecting the cultured hepatoma cells Hepa1-6, they were washed twice with PBS, treated with heat and UV irradiation, and then lysed and lysed with an 8M urea aqueous solution (containing 200MM sodium chloride) of the whole cell fraction of the cancer cells derived from the cancer cells. The lysis fraction was dissolved after lysis of BCG using 8M urea aqueous solution (containing 200MM sodium chloride) and used as an adjuvant.

(3) Preparation of nanoparticle systems

In the embodiment, the nanoparticle system is prepared by a solvent volatilization method, the molecular weight of PLGA (polylactic-co-glycolic acid) serving as a nanoparticle preparation material is 24-38 KDa, the adopted immunologic adjuvant is BCG and Poly (I: C), and the adjuvant is wrapped in the nanoparticles. As mentioned above, in the preparation process, firstly, the cancer cell whole cell component and the adjuvant are loaded inside the nanoparticles by a multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are re-suspended by using 10mL of ultrapure water containing 4% trehalose, and then are frozen and dried for 48 hours for later use. The average particle size of the nano particles is about 285 nm; each 1mg PLGA nanoparticle was loaded with approximately 100. mu.g of protein and polypeptide components, 0.04mg each of BCG and Poly (I: C). The average particle size of the control nanoparticles was about 285nm, and about 100. mu.g of protein and polypeptide components were loaded per 1mg of PLGA nanoparticles, without any adjuvant.

(3) Preparation of antigen-presenting cells

This example uses BMDCs and B as antigen presenting cells. The BMDC was prepared as in example 2. B cells were derived from mouse peripheral blood PBMCs, prepared as described above.

(4) Activation of antigen presenting cells

Nanoparticles loaded with cancer cell whole cell components (1000. mu.g) were incubated with BMDCs (500 ten thousand) and B cells (500 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), IFN- γ (500U/mL), and CD80 antibody (10ng/mL) and CD40 antibody (20 mg/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated DCs and B cells (500 ten thousand DCs +500 ten thousand B cells) were collected by centrifugation at 400g for 5 minutes, then washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspended in PBS water and sonicated at 4 ℃ for 2 minutes with low power (20W). Centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 5000g for 10 minutes and collecting supernatant, filtering the supernatant through a 0.45 mu m membrane, then using an ultrafiltration membrane (with the molecular weight cutoff of 50KDa) to perform ultrafiltration centrifugal filtration and concentration, mixing the filtered and concentrated sample with the nanoparticles (10mg) prepared in the step 2, then using a high-pressure homogenizer (10000bar) to process for 3 minutes, then using a 0.45 mu m filter membrane to perform filtration, then centrifuging at 13000g for 30 minutes, then removing the supernatant and collecting precipitate, re-suspending the precipitate in a mixed freeze-drying protective agent aqueous solution (2% trehalose, 2% mannitol and 1% albumin), and then performing freeze drying for 48 hours to obtain the nano vaccine with the particle size of 300 nanometers.

(4) Prevention of liver cancer

Selecting female C57BL/6 of 6-8 weeks as model mouse to prepare liver cancer tumor-bearing mouse. 80 μ g of nano-vaccine or PBS was injected on days-42, 35, 28, 21 and 7 before the mice were inoculated with cancer cells. Each mouse was also injected subcutaneously on day 01.0X 10 seed ⁶ The recording mode of the tumor growth and the survival period of the mice of the Hepa1-6 liver cancer cells is the same as that of the mice.

(5) Results of the experiment

As shown in fig. 14, the tumor growth rate of the mice treated with the nano-vaccine prepared from the antigen-presenting cells activated by the nanoparticle was significantly slower than that of the PBS control group. Moreover, regardless of the adjuvant, the nano vaccine prepared by the antigen presenting cells activated by the nano particles can effectively prevent cancer, but the effect of the adjuvant is better. This demonstrates that the nano-vaccine of the present invention can effectively prevent cancer.

EXAMPLE 14 Nanoprotein preparation of antigen presenting cells for prevention of cancer

(1) Lysis of tumor tissue and cancer cells

After mouse B16F10 melanoma tumor tissues and cultured cancer cells are collected, 10% sodium deoxycholate aqueous solution (containing 8M arginine) is adopted to crack and dissolve cancer cell whole cell components derived from the tumor tissues and the cancer cells, and then the tumor tissue components and the cancer cell components are mixed and dissolved according to the mass ratio of 1:1.

(2) Preparation of nanoparticles

In the embodiment, the nanoparticles are prepared by a solvent volatilization method, PLGA serving as a nanoparticle preparation material has the molecular weight of 7-17 KDa, the adopted immunoadjuvants are CpG2006, CpG1018 and Poly (I: C), the lysosome escape substance is arginine, and the immunoadjuvant and the arginine are loaded in the nanoparticles. The preparation method is as described above, the antigen, arginine and adjuvant are loaded inside the nanoparticles by a double emulsion method, then 100mg PLGA nanoparticles are centrifuged at 13000g for 20min, the supernatant is discarded and the precipitate is collected, the precipitate is resuspended in 4% trehalose, and the freeze-dried product is ready for use after 48 hours. The average particle diameter of the nano particles is about 240 nm; each 1mg PLGA nanoparticle is loaded with about 80 μ g of protein or polypeptide component, 0.03mg of each of CpG2006, CpG1018 and Poly (I: C), and 0.02mg of arginine.

(3) Preparation of antigen-presenting cells

This example uses BMDCs and B as antigen presenting cells. BMDC was prepared as in example 2. B cells were derived from mouse peripheral blood PBMCs, prepared as described above.

(4) Activation of antigen presenting cells

Nanoparticles loaded with cancer cell whole cell components (1000. mu.g) were incubated with BMDCs (500 ten thousand) and B cells (500 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), IFN- γ (500U/mL), and CD80 antibody (10ng/mL) and CD40 antibody (20 mg/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated DC and B cells were harvested by centrifugation at 400g for 5 minutes, then washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspended in PBS water and sonicated at 4 ℃ for 10 minutes with low power (10W). Then the sample is filtered by membranes of 2 mu m, 1 mu m, 0.45 mu m and 0.22 mu m in sequence, the filtrate is collected, then the filtrate is centrifuged at 15000g for 30 minutes, the supernatant is collected and discarded, and the precipitate is collected and resuspended in PBS, thus obtaining the antigen presenting cell membrane component. Then mixing 10mL of antigen presenting cell membrane components (5mg) with 100mg of the nanoparticles prepared in the step (2), carrying out ultrasonic treatment at low power (20W) for 2 minutes at room temperature, then incubating for 10 minutes, filtering and extruding through a 0.45-micrometer membrane, centrifuging at 15000g for 20 minutes, removing supernatant, then re-suspending the precipitate in 4% trehalose water solution, and then carrying out freeze drying for 48 hours to obtain the nano vaccine 2, wherein the particle size of the nano vaccine 2 is 250 nanometers; or resuspending the precipitate in mixed lyophilized protectant aqueous solution (2% sucrose + 2% mannitol + 1% lysine), and freeze-drying for 48 hr to obtain nanometer vaccine 1 with particle size of 250 nm. The lyophilized nano-vaccine 1 and nano-vaccine 2 were used after being left at room temperature for 180 days, and 1mg of the nano-vaccine was resuspended in 1mL of PBS at the time of use.

(4) Nanovaccine cancer prevention

Selecting female C57BL/6 of 6-8 weeks as model mouse, preparing melanoma-bearing mouse, inoculating each mouse with the vaccine which has been left at room temperature for more than 180 days before inoculating cancer cells on-35 days, -28 days, -21 days, -14 days and-7 days100 μ

g nano vaccine

1 or 100 μ g nano vaccine 2 or PBS. On day 0, each recipient mouse was subcutaneously inoculated at 1.5X 10 in the lower right back ⁵ And B16F10 cells. Mouse tumor growth and survival monitoring methods are as above.

(5) Results of the experiment

As shown in fig. 15, compared with the PBS control group, both the nano-vaccine 1 and the nano-vaccine 2 prepared from the nanoparticle-activated antigen-presenting cells can significantly prolong the lifetime of the mice and effectively prevent cancer. Moreover, the effect of the nano vaccine 1 prepared by freeze drying the mixed freeze-drying protective agent (2% of sucrose, 2% of mannitol and 1% of lysine) is better than that of the nano vaccine 2 prepared by freeze drying the 4% trehalose freeze-drying protective agent.

Example 15 Nanoprotein for treatment of melanoma

This example illustrates the use of a nano-vaccine to treat melanoma in mice as a cancer model.

(1) Lysis of tumor tissue and cancer cells and collection of fractions

Tumor tissue was collected by subcutaneous dorsal vaccination of 1.5X 10 mice per C57BL/6 mouse ⁵ B16F10 cells, which were about 1000mm in tumor length to volume ³ Killing mice and picking tumor tissues, cutting the tumor tissues into blocks, grinding, passing through a cell filter screen to prepare single cell suspension, adding ultrapure water, repeatedly freezing and thawing and carrying out ultrasonic lysis on the cells, adding nuclease for 5 minutes, and then inactivating the nuclease at 95 ℃ for 10 minutes. Then, centrifuging the mixture for 3 minutes at 8000g, and obtaining supernatant which is the water-soluble antigen; the precipitated fraction was dissolved with a 10% aqueous solution of sodium deoxycholate to dissolve the water-insoluble antigen. The water-soluble antigen and the water-insoluble antigen dissolved by the sodium deoxycholate are mixed and dissolved according to the mass ratio of 1:1, and the mixture is the source of the whole cell antigen raw material for preparing the nanoparticle system.

(2) Preparation of nanoparticle systems

In the embodiment, the nanoparticles are prepared by a multiple emulsion method, and have the capacity of targeting dendritic cells. The adopted nano particle preparation materials are PLGA and mannan-modified PLGA, the molecular weights of the PLGA and the mannan-modified PLGA are both 24KDa-38KDa, and the mass ratio of the unmodified PLGA to the mannan-modified PLGA is 9:1 when the nano particle preparation material is used. The adopted immune adjuvants are poly (I: C), CpG1018 and CpG2216, the substance for increasing the immune escape of lysosome is KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA), and the adjuvants and the KALA polypeptide are encapsulated in the nanoparticles. The preparation method is as described above, in the preparation process, firstly, the lysate component, the adjuvant and the KALA polypeptide are loaded inside the nanoparticles by a multiple emulsion method, after the components are loaded inside, 100mg of the nanoparticles are centrifuged at 12000g for 25 minutes, and are resuspended by using 10mL of ultrapure water containing 4% trehalose, and then are frozen and dried for 48 hours. The average particle diameter of the nano particles is about 250nm, and the surface potential is about-5 mV; each 1mg of PLGA nano particle is loaded with about 100 mug of protein or polypeptide component, each 1mg of PLGA nano particle is loaded with 0.02mg of poly (I: C), CpG1018 and CpG2216 immunologic adjuvant, and each 1mg of PLGA nano particle is loaded with 0.05mg of KALA polypeptide. The preparation material and the preparation method of the nanoparticle 2 are the same, the particle size is about 250nm, the surface potential is about-5 mV, KALA polypeptide is not loaded, and the same amount of adjuvant and cell lysis component are loaded. The preparation material and the preparation method of the nano particles 3 are the same, the particle size is about 250nm, and the surface potential is about-5 mV; each 1mg PLGA nanoparticle is loaded with about 100 mug protein and polypeptide components, each 1mg PLGA nanoparticle is loaded with 0.02mg poly (I: C), 0.04mg CpG1018 and 0.05mg KALA polypeptide.

(3) Preparation of antigen-presenting cells

This example uses BMDC and B as antigen presenting cells. BMDC was prepared as in example 2. B cells were derived from mouse peripheral blood PBMCs, prepared as described above.

(4) Activation of antigen presenting cells

(5) Preparation of antigen presenting cell derived nano vaccine

Incubated DC and B cells were collected by centrifugation at 400g for 5 minutes, followed by washing twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by low power (20W) sonication for 2 minutes at 4 ℃. And centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, filtering and extruding the supernatant through a 0.22-micron membrane, centrifuging at 16000g for 60 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine with the particle size of 150 nanometers.

(4) Nano-vaccine for treating cancer

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 ⁵ And B16F10 cells. 40 μ g of nano-vaccine or PBS subcutaneously on

days

6, 10, 15 and 20 after melanoma inoculation, respectively. In the experiment, the mouse tumor volume and survival monitoring method is the same as above.

(5) Results of the experiment

As shown in fig. 16, tumors grew rapidly in the PBS control group. Compared with a control group, the growth speed of the tumor of the mouse treated by the nano vaccine prepared by the antigen presenting cell activated by the nano particles is obviously slowed down, and the survival period is obviously prolonged. Moreover, the nano-vaccine prepared by adding the antigen presenting cells activated by the nano-particles added with the lysosome escape substances is better than the nano-vaccine prepared by the antigen presenting cells activated by the nano-particles without lysosome escape substances. Moreover, the therapeutic effect of the nano-vaccine prepared by using the nano-particle activated antigen presenting cells with two kinds of CpG and Poly (I: C) as mixed adjuvants is better than that of the nano-vaccine prepared by using the nano-particle activated antigen presenting cells with only one kind of CpG and Poly (I: C) mixed adjuvants. In conclusion, the nano vaccine provided by the invention has a good treatment effect on cancer.

Example 16 Nanoprotein for the prevention of Breast cancer

This example uses 4T1 mouse triple negative breast cancer as a cancer model to illustrate how to prepare a nano-vaccine for preventing breast cancer using cancer cell whole cell antigen loaded microparticle activated antigen presenting cells. In this example, the breast cancer cells were first inactivated and denatured, then lysed, and the water-insoluble components in the cancer cells were lysed with octyl glucoside lysis. Then, PLGA is used as a microparticle framework material, CpG2007 (B-class Toll-like receptor 9 agonist), CpG2216 (A-class Toll-like receptor 9 agonist) and Poly ICLC (Toll-like receptor 3 agonist) are used as immune adjuvants, Poly-arginine and Poly-lysine are used as substances for enhancing lysosome escape, the microparticles loaded with cancer cell whole cell components are prepared, then the microparticles are used for activating antigen presenting cells, and a nano vaccine based on the activated antigen presenting cells is prepared for preventing cancer.

(1) Lysis of cancer cells

Cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells are subjected to inactivation and denaturation treatment by adopting ultraviolet rays and high-temperature heating respectively, then ultrapure water is added, freeze thawing is carried out repeatedly for 5 times, ultrasonic cracking is assisted, the cell lysate is centrifuged for 10 minutes at 5000g, the supernatant is a water-soluble component, the precipitate is dissolved by using 10% octyl glucoside to obtain a dissolved original water-insoluble component, and the water-soluble component and the water-insoluble component are mixed according to the mass ratio of 2:1 to obtain a lysate component required for preparing the micron particles.

(2) Preparation of microparticle systems

In the embodiment, a double emulsion method is adopted for preparing the micron particle system and serving as a reference micron particle, PLGA (polylactic-co-glycolic acid) serving as a micron particle skeleton material has the molecular weight of 38-54 KDa, the adopted immunologic adjuvants are CpG2007(B class), CpG2216(A class) and Poly ICLC, and the adopted lysosome escape increasing substances are polyarginine and polylysine. During preparation, the internal loaded lysate component, the adjuvant and the microparticles of the polyarginine and the polylysine are prepared by a multiple emulsion method, then 100mg of the microparticles are centrifuged at 9000g for 20 minutes, and 10mL of ultrapure water containing 4% trehalose is used for heavy suspension, and then the suspension is dried for 48 hours for later use. The average particle diameter of the micron particles is about 3.1 mu m, and the surface potential is about-7 mV; each 1mg PLGA microparticle is loaded with about 110 μ g protein or polypeptide component, each of which contains 0.01mg of CpG2007(B class), CpG2216(A class) and Poly ICLC, and each of which contains 0.02mg of polyarginine and polylysine. The preparation materials and preparation method of the control microparticle 2 are the same as the above method, but the CpG loaded on the control microparticle 2 is CpG1585 (class A) and CpG2216 (class A) of two classes A, the average particle size of the control microparticle 2 is about 3.1 μm, and the surface potential is about-7 mV; each 1mg PLGA microparticle is loaded with about 110 μ g protein or polypeptide components, 0.01mg each of CpG1585 (class A), CpG2216 (class A) and Poly ICLC, and 0.02mg each of polyarginine and polylysine.

(3) Preparation of antigen-presenting cells

This example uses the DC2.4 cell line as an antigen presenting cell.

(4) Activation of antigen presenting cells

Cancer cell Whole cell fraction-loaded microparticles (1000. mu.g) were incubated with DC2.4(1000 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), and CD80 antibody (10 ng/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The post-incubation DCs were collected by centrifugation at 400g for 5 minutes, followed by washing the cells twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by 2 minutes of low power (20W) sonication at 4 ℃. And centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, filtering and extruding the supernatant through a 0.45-micrometer membrane, centrifuging at 12000g for 90 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine with the average particle size of 250 nanometers.

(6) Nano-vaccine cancer prevention

Female BALB/c of 6-8 weeks is selected as a model mouse to prepare a breast cancer tumor-bearing mouse. Mice were subcutaneously vaccinated with 50 μ g of nano-vaccine or PBS on days-35, 21 and 7, respectively, before they were vaccinated with cancer cells. Each mouse was inoculated subcutaneously with 1X 10 injection on day 0 ⁶ 4T1 cells, the size of the tumor volume of the mice was recorded every 3 days from day 3, the mice were tumorigenicThe detection method of the long and life cycle is the same as the above.

(7) Results of the experiment

As shown in fig. 17, the tumor growth rate of the mice treated with the nano-vaccine prepared from the antigen-presenting cells activated by the microparticles was significantly slowed and the survival time of the mice was significantly prolonged, compared to the control group. Furthermore, the use of a class B Toll-like receptor 9 agonist in combination with a class A Toll-like receptor 9 agonist and a Toll-like receptor 3 agonist as a mixed adjuvant for deactivating antigen-presenting cells is more effective than the use of two class A Toll-like receptor 9 agonists in combination with a Toll-like receptor 3 agonist as a mixed adjuvant for deactivating antigen-presenting cells. Therefore, the nano vaccine based on the activated antigen presenting cells has a prevention effect on breast cancer.

Example 17 Nanoprotein for prevention of Breast cancer

This example uses 4T1 mouse triple negative breast cancer as a cancer model to illustrate how nanoparticles activate antigen presenting cells and then prepare a nano-vaccine for preventing cancer.

(1) Lysis of cancer cells

Cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells are respectively inactivated and denatured by ultraviolet rays and high-temperature heating, and then 8M urea aqueous solution (containing 500mM sodium chloride) is used for cracking the cancer cells and dissolving a lysate component, namely the antigen component for preparing the micron particle system.

(2) Preparation of microparticle systems

In this example, the system for preparing microparticles and the reference microparticles were prepared by a multiple emulsion method, wherein the matrix material of the microparticles was unmodified PLA and mannose-modified PLA, the molecular weights were both 40KDa, and the ratio of unmodified PLA to mannose-modified PLA was 4: 1. The adopted immune adjuvants are CpG2006, CpG2216 and Poly ICLC, and the adopted lysosome escape increasing substances are arginine and histidine. The preparation method comprises the steps of firstly preparing the micro-particles internally loaded with the lysate component, the adjuvant, the arginine and the histidine by a multiple emulsion method, then centrifuging 100mg of the micro-particles at 9000g for 20 minutes, using 10mL of ultrapure water containing 4% trehalose for resuspension, and drying for 48 hours for later use. The average particle size of the micron particle system is about 2.1 mu m, and the surface potential of the micron particle system is about-7 mV; each 1mg PLGA microparticle loaded about 100 μ g protein or polypeptide component, 0.01mg each of CpG2006, CpG2216 and Poly ICLC, and 0.05mg each of arginine and histidine. The control micrometer particles 2 were prepared using the same materials and methods as the micrometer particles described in this example, having a particle size of about 2.1 μm and a surface potential of about-7 mV, but loaded with arginine and histidine and an equal amount of the cell lysate fraction, but without any adjuvant.

(3) Preparation of antigen-presenting cells

(4) Activation of antigen presenting cells

Microparticles (1000. mu.g) were incubated with BMDCs (500 ten thousand) and B cells (500 ten thousand) in 15mL high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), IFN- γ (500U/mL), and CD80 antibody (10ng/mL) and CD40 antibody (20 mg/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated DC and B cells were harvested by centrifugation at 400g for 5 minutes, followed by washing twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by 2 minutes of low power (20W) sonication at 4 ℃. And centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting the supernatant, centrifuging at 16000g for 60 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, resuspending the precipitate in PBS, and filtering and extruding through a 0.22 mu m membrane to obtain the nano vaccine with the particle size of 150 nanometers.

(6) Cancer cell-specific T cells for prevention of cancer

Selecting female BALB/c of 6-8 weeks as a model mouse to prepare a breast cancer tumor-bearing mouse. Each mouse was inoculated with cancer cells on days-35, 21 and 7 before the mice were inoculated with cancer cells50 μ g of nano-vaccine or PBS was inoculated. Each mouse was also inoculated subcutaneously 1X 10 on day 0 ⁶ The 4T1 cells, mouse tumor volume and survival monitoring methods were as above.

(7) Results of the experiment

As shown in fig. 18, the tumor growth rate of the mice treated with the nano-vaccine prepared using the micron-sized activated antigen-presenting cells was significantly slowed and the survival time of the mice was significantly prolonged, compared to the control group. Moreover, the effect of the nano-vaccine prepared from the antigen presenting cells activated by the micro-particles containing the substance for increasing the lysosome escape function and the mixed adjuvant is better than that of the nano-vaccine prepared from the antigen presenting cells activated by the micro-particles containing only the substance for increasing the lysosome escape function and no mixed adjuvant. Therefore, the antigen presenting cell-based nano vaccine has a prevention effect on breast cancer, and the mixed adjuvant is beneficial to the activation of the antigen presenting cell and the activity of the subsequent nano vaccine.

Example 18 Nanoprotein for treatment of cancer

This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticle-activated antigen-presenting cells to prepare a nano-vaccine for the treatment of melanoma. In this example, tumor tissue and cancer cells are first lysed to prepare water-soluble components, then PLGA is used as a scaffold material, Poly (I: C) and CpG1018 are used as immunoadjuvants, R8 (rrrrrrrrrrrrrrrrrr) polypeptide is used as a material for dissolving lysosome escape capacity to prepare nanoparticles loaded with water-soluble components, and then nano-vaccines are prepared using antigen-presenting cells activated by nanoparticles to treat cancer.

(1) Lysis of tumor tissue and cancer cells and collection of fractions

Tumor tissue was collected by subcutaneous dorsal vaccination of 1.5X 10 mice per C57BL/6 mouse ⁵ B16F10 cells, which were about 1000mm in tumor length to volume ³ Killing mouse and picking tumor tissue, cutting tumor tissue into pieces, grinding, adding appropriate amount of pure water through cell filter screen, repeatedly freezing and thawing for 5 times (with ultrasound) to destroy the sample obtained by lysis, adding nuclease for 10min, heating at 95 deg.C for 10min to inactivate nuclei(ii) an acid enzyme; when the cultured B16F10 cancer cell line is collected, the culture medium is removed by centrifugation, then the cancer cells are washed twice by PBS and collected by centrifugation, the cancer cells are resuspended in ultrapure water, freeze-thawing is repeated for 3 times, the cancer cells are cracked by ultrasonic damage, and then nuclease is added into a sample for action for 10 minutes and then heated for 5 minutes at 95 ℃ to inactivate the nuclease. After the tumor tissue or cancer cell is treated by enzyme action, the lysate is centrifuged for 5 minutes at 5000g of rotation speed, and the supernatant is taken as a water-soluble component which can be dissolved in pure water. The water-soluble components of the tumor tissue and the water-soluble components of the cancer cells are mixed according to the mass ratio of 1:1, and the mixture is the source of the antigen raw material for preparing the nanoparticle system.

(2) Preparation of nanoparticle systems

In this example, the nanoparticles were prepared by a multiple emulsion method. The molecular weight of PLGA used as a material for preparing the nano particles is 7-17 KDa, the adopted immunologic adjuvant is poly (I: C) and CpG1018, the R8 polypeptide is a substance for increasing lysosome escape, and the adjuvant and the R8 polypeptide are loaded in the nano particles. As mentioned above, in the preparation process, firstly, the lysate component, the adjuvant and the R8 polypeptide are loaded inside the nanoparticles by a multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 12000g for 25 minutes, and are re-suspended by using 10mL of ultrapure water containing 4% trehalose, and then are frozen and dried for 48 hours for later use. The average particle diameter of the nano particles is about 250nm, and the surface potential of the nano particles is about-5 mV; each 1mg PLGA nano particle is loaded with about 120 mug protein or polypeptide component, each 1mg PLGA nano particle is loaded with 0.03mg of poly (I: C) and CpG1018 immunologic adjuvant, and each 1mg PLGA nano particle is loaded with 0.03mg of R8 polypeptide.

(3) Preparation of antigen-presenting cells

(4) Activation of antigen presenting cells

Nanoparticles (1000. mu.g) were incubated with BMDCs (500 ten thousand) and B cells (500 ten thousand) in 15mL high-glucose DMEM complete medium for 24 hours (37 ℃, 5% CO) ₂ ) (ii) a Alternatively, BMDCs (500 million) and B cells (500 million) were co-incubated in 15mL high-glucose DMEM complete medium for 24 hours (37)℃，5％CO ₂ ). In both cases the incubation system contained GM-CSF (2000U/mL), IL-2(500U/mL), IL-7(200U/mL), IL-12(200U/mL), and CD80 antibody (10ng/mL) and CD40 antibody (20 mg/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated DC and B cells were harvested by centrifugation at 400g for 5 minutes, followed by washing twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by 2 minutes of low power (20W) sonication at 4 ℃. And then centrifuging the sample at 4000g for 5 minutes and collecting supernatant, centrifuging the supernatant at 6000g for 10 minutes, collecting supernatant, filtering and extruding the supernatant through a 0.22 mu m membrane, centrifuging at 17000g for 40 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the antigen presenting cell membrane component. Mixing 10mL of antigen presenting cell membrane components (10mg) with 50mg of the nanoparticles prepared in the step (2), incubating at 4 ℃ for 15 minutes, filtering and extruding through a 0.45-micron membrane, centrifuging at 17000g for 40 minutes, collecting, discarding supernatant, collecting precipitate, resuspending the precipitate in an aqueous solution containing a freeze-drying protective agent (2% trehalose + 2% mannitol + lysine), and freeze-drying for 48 hours to obtain the nano vaccine. The nano vaccine prepared by loading the cell membrane component of the activated mixed antigen presenting cell on the surface of the nano particle is nano vaccine 1, and the particle size is 260 nm; the nano vaccine prepared by loading the inactivated mixed antigen presenting cell membrane components on the surface of the nano particle is nano vaccine 2, and the particle size is 260 nm.

(6) Nano vaccine for treating cancer

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. Each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 ⁵ And B16F10 cells. 50 μ g of nano-vaccine or PBS was injected subcutaneously on

days

4, 7, 10, 15 and 20 after melanoma inoculation, respectively. Mouse tumor growth and survival monitoring methods are as above.

(7) Results of the experiment

As shown in fig. 19, the tumor growth rate of the nano-vaccine treated mice was significantly slowed and the survival time of the mice was significantly prolonged compared to the control group. Moreover, the effect of the 1 nanometer vaccine prepared by using the antigen presenting cells activated by the nanoparticles is obviously better than that of the 2 nanometer vaccine prepared by using the antigen presenting cells not activated by the nanoparticles. Thus, the nanoparticle activation of the present invention can improve the efficacy of antigen presenting cell based nano-vaccines.

EXAMPLE 19 Nanoprotein for treatment of Colon cancer

This example uses mouse colon cancer as a cancer model to illustrate how to prepare a nano-vaccine for treating colon cancer using nanoparticle-activated antigen-presenting cells loaded with cancer cell whole-cell antigen derived from colon cancer tumor tissue. In this example, 8M urea solution was used to lyse colon cancer tumor tissue and to dissolve the lysed components, and then PLGA was used as the scaffold, Poly (I: C), CpG2336 and CpG2006 were used as adjuvants, NH 2006 was used ₄ HCO ₃ To increase lysosomal escape material, nanoparticles are prepared, and then nano-vaccines are prepared using nanoparticle-activated antigen-presenting cells for cancer treatment.

(1) Lysis of tumor tissue and Collection of fractions

Tumor tissue was collected by subcutaneous dorsal vaccination of 2X 10 mice per C57BL/6 mouse ⁶ MC38 colon cancer cells, which grow to a volume of about 1000mm in tumor ³ The mice are sacrificed and the tumor tissue is picked up, the tumor tissue is cut into pieces and ground, 8M urea aqueous solution is added through a cell filter screen to understand the tumor tissue and the whole cell components after the lysis are dissolved. The above is the source of the antigen raw material for preparing the nano particles.

(2) Preparation of nanoparticle systems

In this example, the nanoparticles were prepared by a multiple emulsion method. The preparation material of the nano particle 1, PLGA, has the molecular weight of 7-17 KDa, takes Poly (I: C) and CpG as adjuvant and NH ₄ HCO ₃ To increase lysosomal escape material, adjuvant and NH ₄ HCO ₃ Loaded within the nanoparticles; the preparation method is as described above, in the preparation process, firstly, the lysate component and the adjuvant are loaded in the nano particles, and then 100mg of nano particles are centrifuged for 20 minutes at 10000gAfter being resuspended by using 10mL of ultrapure water containing 4% trehalose, the mixture is frozen and dried for 48h for standby; the average particle diameter of the nano particles is about 260nm, and the surface potential is about-7 mV; about 90 mug of protein and polypeptide components are loaded on each 1mg of PLGA nano particles, and 0.02mg of each of poly (I: C), CpG2336 and CpG2006 immunoadjuvants loaded on each 1mg of PLGA nano particles are loaded with NH ₄ HCO ₃ 0.01 mg. The preparation material and the preparation method of the nano-particle 2 are the same as those of the nano-particle 1, the particle diameter is about 260nm, the surface potential is about minus 7mV, about 90 mug of protein and polypeptide components are loaded on each 1mg of PLGA nano-particle, and NH is loaded on each 1mg of PLGA nano-particle ₄ HCO ₃ 0.01mg, loading CpG2336 and CpG2006, 0.03mg each.

(3) Preparation of antigen-presenting cells

(4) Activation of antigen presenting cells

Nanoparticle 1 (500. mu.g) or nanoparticle 2 (500. mu.g) loaded with cancer cell whole cell fraction was incubated with BMDCs (250 ten thousand) and B cells (250 ten thousand) in 10mL of high-glucose DMEM complete medium for 69 hours (37 ℃, 5% CO) ₂ ) Incubation systems containing GM-CSF (500U/mL), IL-2(300U/mL), IL-7(200U/mL), IL-12(500U/mL), and CD80 antibody (10ng/mL) and CD40 antibody (20 mg/mL); alternatively, cancer cell whole cell fraction-loaded nanoparticle 1 (500. mu.g) was incubated with BMDCs (250 ten thousand) and B cells (250 ten thousand) in 10mL high-glucose DMEM complete medium for 69 hours (37 ℃, 5% CO2) in an incubation system containing GM-CSF (500U/mL), IL-12(1000U/mL) and CD80 antibody (10 ng/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated DC and B cells were harvested by centrifugation at 400g for 5 minutes, followed by washing twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by 2 minutes of low power (20W) sonication at 4 ℃. And centrifuging the sample at 3000g for 30 minutes, discarding the precipitate, only collecting the supernatant, centrifuging the supernatant at 8000g for 15 minutes, discarding the precipitate, collecting the supernatant, filtering and extruding the supernatant through a 0.22 mu m membrane, centrifuging at 18000g for 90 minutes, collecting the supernatant, discarding the precipitate, collecting the precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine with the particle size of 120 nanometers.

(6) Cancer cell specific T cells for the treatment of cancer

Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare colon cancer mice. Each mouse was inoculated subcutaneously at day 0 in the lower right back of 2X 10 mice ⁶ MC38 cells. Each mouse was subcutaneously injected with 50 μ g of the nano-vaccine on

days

6, 9, 12, 15, 20 and 25 after inoculation with colon cancer cells. Mouse tumor growth and survival monitoring methods are as above.

(7) Results of the experiment

As shown in fig. 20, the tumor growth rate of the mice treated with the nano-vaccine prepared from the antigen-presenting cells activated by the nanoparticles was significantly slowed and the survival time of the mice was significantly prolonged, compared to the control group. Moreover, the nano-vaccine prepared by using the nano-particle activated antigen presenting cell simultaneously loaded with the lysate component, the mixed adjuvant and the lysosome escaping substance is obviously better than the nano-vaccine prepared by using the nano-particle activated antigen presenting cell simultaneously loaded with the lysate component, the two CpG adjuvants and the lysosome escaping substance. Moreover, the effect of adding IL-7, IL-2 and antibody for co-incubation in the process of activating antigen presenting cells by the nanoparticles is better than that of not adding the two interleukins and the antibodies for co-incubation. Therefore, the nano vaccine prepared based on the antigen presenting cells activated by the nanoparticles has excellent treatment effect on cancer, and the use of the mixed adjuvant and the addition of the antibody, IL-7 and IL-2 during the activation of the antigen presenting cells have enhancement effect on the finally prepared nano vaccine.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A cancer vaccine based on antigen presenting cell membrane components, wherein the cancer vaccine based on antigen presenting cell membrane components comprises one of:

wherein the content of the first and second substances,

the activated antigen-presenting cell membrane fraction is prepared from pre-activated antigen-presenting cells obtained by co-incubating antigen-presenting cells with first delivery particles loaded with cancer cell whole cell fraction;

the first delivery particle and the second delivery particle are each independently a nanoparticle or a microparticle.

2. A cancer vaccine based on antigen presenting cell membrane components according to claim 1, characterized in that: the antigen presenting cell is at least one of a dendritic cell, a B cell and a macrophage.

3. A cancer vaccine based on antigen presenting cell membrane components according to claim 1, characterized in that: the cancer cell whole cell component is a whole cell component which is obtained by cracking cancer cells and/or tumor tissues and contains a water-soluble component and a water-insoluble component, and the water-insoluble antigen is dissolved by a dissolving agent and then loaded on the first delivery particle or the second delivery particle; wherein the dissolving agent is at least one selected from urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, inorganic salt, Triton, Tween, amino acid, glucoside and choline.

4. A cancer vaccine based on antigen presenting cell membrane components according to claim 1, characterized in that: the first delivery particle or the second delivery particle is loaded with a substance that increases lysosomal escape.

5. A cancer vaccine based on antigen presenting cell membrane components according to claim 1, characterized in that: the cancer vaccine based on the antigen presenting cell membrane component is a freeze-dried preparation, and the freeze-dried preparation is prepared by freeze drying the cancer vaccine based on the antigen presenting cell membrane component; wherein the freeze-drying protective agent is any one of the following (1) to (7):

(1) trehalose, mannitol and arginine;

(2) trehalose, mannitol and glycine;

(3) trehalose, mannitol and lysine;

(4) mannitol, sucrose and lysine;

(5) trehalose, mannitol and gelatin;

(6) trehalose, mannitol, and polyvinylpyrrolidone;

(7) trehalose, mannitol and albumin.

6. A method for preparing a cancer vaccine based on antigen presenting cell membrane components, comprising the steps of:

or obtaining cell membrane components of the activated antigen-presenting cells in S1, and enabling the cell membrane components and/or the nano vesicles to act together with second delivery particles loaded with cancer cell whole cell components to obtain the cancer vaccine based on the antigen-presenting cell membrane components.

7. The method of claim 6, wherein: when in co-action or co-incubation, the system contains cell factors, proteins and/or antibodies; the cell factor is at least one of interleukin, tumor necrosis factor, interferon, growth factor and colony stimulating factor; the antibody is selected from at least one of an alpha CD-3 antibody, an alpha CD-4 antibody, an alpha CD-8 antibody, an alpha CD-28 antibody, a CD40 antibody, a CD80 antibody, an alpha OX-40L antibody, and a CD86 antibody.

8. The process according to claim 7, wherein the system comprises any one of the following combinations (1) to (11):

(5) interleukin 2, interleukin 7, and interleukin 15;

(6) interleukin 4, interleukin 6, and interleukin 10;

(10) granulocyte-macrophage colony stimulating factor, interleukin 2, interleukin 7, interleukin 12, gamma interferon, and CD80 antibody;

9. Use of the antigen presenting cell membrane fraction based cancer vaccine according to any one of claims 1 to 5 or the cancer vaccine prepared by the preparation method according to any one of claims 6 to 8 for the preparation of a medicament for the treatment or prevention of cancer.

10. A method of inducing T cells in vitro to cancer cell-specific T cells, comprising: co-incubating a cancer vaccine with T cells in vitro to induce the cancer cell-specific T cells, wherein the cancer vaccine is the cancer vaccine based on the antigen presenting cell membrane component according to any one of claims 1 to 5 or the cancer vaccine prepared by the preparation method according to any one of claims 6 to 8.