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

Abstract

The present invention relates to a cancer vaccine based on antigen-presenting cell membrane components and a preparation method and application thereof. The cancer vaccine includes one of the following: (1) nanovesicles prepared from activated antigen-presenting cell membrane components; (2) A second delivery particle that internally and/or externally loads the whole cell component of the cancer cell, and the surface loads the activated antigen-presenting cell membrane component. The invention realizes that the vaccine derived from dendritic cells can be loaded with broad-spectrum and diverse cancer cell antigens, and at the same time overcomes the difficulties of maintaining the activity of dendritic cells and the inability to freeze-dry long-term storage of live cell vaccines, and can prepare a broad-spectrum cancer-bearing vaccine. Cancer vaccines based on cellular epitopes for the prevention and treatment of cancer.

Description

A cancer vaccine based on antigen-presenting cell membrane components and its preparation method and application

technical field

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

Background technique

Cancer immunotherapy is one of the most important treatment methods for cancer, and cancer vaccine is one of the important methods of cancer immunotherapy. The usual mechanism of action of cancer vaccines is: the antigens loaded by cancer vaccines are phagocytosed and degraded by antigen-presenting cells, and the antigenic epitopes degraded into small peptides are combined with major histocompatibility complex (MHC) molecules and presented to the antigenic presentation cells. Present on the cell surface to activate cancer cell-specific T cells. The process of activating Naive T cells into cancer cell-specific T cells by cancer vaccines through antigen-presenting cells belongs to the initial activation of T cells, which requires dendritic cells (DC) to complete, and is completed in the lymph nodes. Once T cells are activated by antigens from Naive T cells to cancer cell-specific T cells, they can recognize antigenic polypeptides presented by various antigen-presenting cells or mutant cancer cells. In view of the core function of DCs in the primary activation of cancer cell-specific T cells, there is currently a type of cancer vaccine that uses antigen-activated DCs to infuse patients as therapeutic vaccines, such as Provenci, a prostate cancer therapeutic vaccine that has been marketed in the United States. Provenge is a DC live cell vaccine. However, DC cell vaccines also face many problems. For example, DC vaccines are subject to strict conditions during preparation, storage, transportation, and use. As live cell vaccines, DC vaccines are easily contaminated by various microorganisms, and once the cells of live cell vaccines are genetically contaminated Or the mutation is easily transmitted to other living cells in the body and so on. In addition, the antigenic epitopes of cancer cells loaded by current dendritic cell vaccines are not diverse enough, which also limits the application of dendritic cell vaccines. Therefore, the present invention aims to develop a surface-loaded antigenic epitope containing antigen-presenting cell membranes and binding to major histocompatibility complex (MHC) molecules, and the cancer cell antigenic epitopes are diverse, which can directly interact with T Cell binding activates Naive T cells, which in turn can indirectly activate Naive T cells by being phagocytosed by antigen-presenting cells in the body, which facilitates transportation and storage of cancer vaccines.

SUMMARY OF THE INVENTION

To solve the above technical problems, the present invention provides a cancer vaccine based on antigen-presenting cell membrane components.

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

(1) Nanovesicles prepared from activated antigen-presenting cell membrane components;

(2) a second delivery particle that internally and/or externally loads whole cell components of cancer cells and loads activated antigen-presenting cell membrane components on its surface;

in,

The activated antigen-presenting cell membrane components are prepared from pre-activated antigen-presenting cells, which are prepared by combining antigen-presenting cells with tumor tissue and/or cancer cell whole cell groups. A fraction of the first delivery particles were co-incubated to obtain;

The first delivery particle and the second delivery particle (hereinafter, "delivery particle" is used to refer to the first delivery particle and/or the second delivery particle, the first delivery particle and the second delivery particle may or may not be subjected to the following processes at the same time, Or one of them is nanoparticle or microparticle independently; the cell membrane component of antigen-presenting cell is the membrane component from cell membrane or extracellular vesicle membrane.

Further, the whole cell fraction of cancer cells may be a whole cell fraction containing water-soluble components and water-insoluble components obtained by lysis of cancer cells and/or tumor tissue, preferably after lysis of tumor tissue and/or cancer cells. Whole cell antigens contained in the obtained whole cell component, the whole cell antigens include water-soluble antigens and water-insoluble antigens obtained by lysis of cancer cells and/or tumor tissues, and the water-insoluble antigens are dissolved in a lysing agent and then loaded on on the delivery particles.

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

Further, the antigen presenting cells can be at least one of dendritic cells, B cells and/or macrophages, preferably, two of the above three cells can be selected, most preferably dendritic cells, B cells and macrophages.

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

Further, immune-enhancing adjuvants include but are not limited to microbial-derived immune-enhancing agents, products of human or animal immune systems, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicines and others. At least one type of; immune-enhancing adjuvants include but are not limited to pattern recognition receptor agonists, Bacille Calmette-Guérin (BCG), STING agonists, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, grass branch Bacillus, polyantibiotic A, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, miquimod, polysaccharides , Curcumin, STING agonist, Toll-like receptor agonist, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, Levamisole, polycytidylic acid, manganese adjuvant, aluminum adjuvant, calcium adjuvant, various cytokines (such as colony stimulating factor), interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, Aluminum phosphate, lanolin, squalene, cytokines, vegetable oil, endotoxin, liposome adjuvant, MF59, double-stranded RNA, double-stranded DNA, aluminum-related adjuvant, CAF01, active ingredients of ginseng, astragalus, etc.

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

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

Further, the inside and/or outside of the delivery particles are also loaded with substances that increase the escape of lysosomes.

Further, the substances that increase lysosome escape include, but are not limited to, carrier materials that increase osmotic pressure in lysosomes, carrier materials that reduce lysosomal membrane stability, and substances that have proton sponge effect.

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

Further, the inside and/or outside of the delivery particle is also loaded 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 and the like.

Further, the above-mentioned 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 includes but is not limited to Polyols, carbohydrates and amino acids.

Further, polyhydroxy compounds include, but are not limited to, one or more of glycerol, mannitol, sorbitol, inositol, thiol, polyethylene glycol, and the like.

Further, carbohydrates 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 any combination of the following:

By mass percentage,

(1) 1-5% trehalose, 1-5% mannitol and 1-5% arginine, and the rest is water;

(2) 1-5% trehalose, 1-5% mannitol and 1-5% glycine, and the rest is water;

(3) 1-5% trehalose, 1-5% mannitol and 1-5% lysine, and the rest is water;

(4) 1-5% mannitol, 1-5% sucrose and 1-5% lysine, and the rest is water;

(5) 1-5% trehalose, 1-5% mannitol and 1-5% gelatin, and the rest is water;

(6) 1-5% trehalose, 1-5% mannitol and 1-5% polyvinylpyrrolidone, and the rest is water;

(7) 1-5% trehalose, 1-5% mannitol and 1-5% albumin, and the rest is water.

Further, the particle size of the nanoparticle is 1 nm-1000 nm; the particle size of the microparticle is 1 μm-1000 μm; the surface of the nanoparticle or the microparticle is electrically neutral, negatively charged or positively charged.

Further, nanoparticles or micro-particles are prepared from organic synthetic polymer materials, natural polymer materials or inorganic materials, and can be prepared by existing preparation methods, including but not limited to common solvent evaporation methods, dialysis methods, microfluidics Control method, extrusion method, hot melt method.

Further, organic synthetic polymer materials include PLGA, PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydrides, PDON, PPDO, PMMA, polyamino acids, synthetic polypeptides, etc.; natural polymer materials include Lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, carbohydrates, polypeptides, etc.; inorganic materials include ferric oxide, ferric tetroxide, carbonate, phosphate, etc.

Further, the manner in which water-soluble antigens or water-insoluble antigens are loaded on the surface of nanoparticles or microparticles includes at least one of adsorption, covalent attachment, charge interaction, hydrophobic interaction, one-step or multi-step solidification, mineralization, and encapsulation. A sort of.

Further, the nanoparticles or microparticles may not be modified during the preparation process, and appropriate modification techniques may also be used to increase the antigen loading of the nanoparticles or microparticles. Modification techniques include, but are not limited to, biomineralization (eg, silicidation, calcification, magnesia), gelation, cross-linking, chemical modification, addition of charged species, and the like.

Further, the form in which the antigen is loaded inside the nanoparticle or the microparticle can be any manner in which the antigen can be loaded inside the nanoparticle or the microparticle, such as encapsulation.

Further, the manner in which the antigen is loaded on the surface of nanoparticles or microparticles includes, but is not limited to, adsorption, covalent attachment, charge interaction (such as adding a positively charged substance, adding a negatively charged substance), hydrophobic interaction, one-step or Multi-step curing, mineralization, wrapping, etc.

Further, the water-soluble antigens and/or water-insoluble antigens loaded on the surface of nanoparticles or microparticles are one or more layers, and when the surface is loaded with multi-layers of water-soluble antigens and/or water-insoluble antigens, the layers are In between are modifiers.

The second object of the present invention is to provide the above-mentioned preparation method of the cancer vaccine based on the antigen-presenting cell membrane component, comprising the following steps:

S1, co-incubating the antigen-presenting cells with the first delivery particles loaded with the whole cell component of the cancer cells to activate the antigen-presenting cells;

S2, preparing the cell membrane components of the antigen-presenting cells activated in S1 into nanovesicles to obtain the cancer vaccine based on the antigen-presenting cell membrane components;

Or obtaining the cell membrane fraction of the antigen-presenting cells activated in S1, and co-acting the cell membrane fraction and/or nanovesicles with the second delivery particle loaded with the cancer cell whole cell fraction to obtain the antigen-presenting-based Cancer vaccines with cell membrane components.

Further, the method of obtaining the cell membrane components of the activated antigen-presenting cells in S1 can be mechanical destruction, membrane filtration, gradient centrifugation or chemical treatment; the method of co-action is co-incubation, co-extrusion, ultrasound, stirring, homogenization. slurried or homogenized.

Further, gradient centrifugation is gradient centrifugation in which the centrifugal speed increases in turn; the pore size of the filter membrane used in membrane filtration is sequentially from large to small; the way of mechanical destruction can be ultrasonic, homogenization, homogenization, high-speed stirring, high-pressure destruction. , high shear damage, swelling; chemical treatment methods can be placed in PBS, glucose solution or saline solution containing chemical substances with low osmotic pressure, shrinkage, etc. Among them, the ultrasound is low-power ultrasound (below 500W).

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

Further, the incubation solution contains cytokines and/or antibodies during the co-incubation.

Further, the cytokine is selected from at least one of interleukin, tumor necrosis factor, interferon, growth factor and colony stimulating factor.

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), etc.

Further, antibodies include but are not limited to αCD-3 antibody, αCD-4 antibody, αCD-8 antibody, αCD-28 antibody, CD-40 antibody, CD-80 antibody, αOX-40 antibody, αOX-40L antibody, CD86 antibody Wait.

Preferably, any one of the following combinations is added during the 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, 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, a process of washing the activated antigen-presenting cells is also included, and the washing solution used in the washing process may contain protease inhibitors and/or phosphatase inhibitors.

Dendritic cell cancer vaccine is a kind of cancer vaccine. In the prior art, due to the limitation of activation mode, dendritic cells (DC) can carry relatively limited antigens, so the types of T cells activated after injection into the human body and The number of clones is small, and since DCs are live cell preparations, activated DCs used as vaccines have many defects in storage, transportation and administration. In the present invention, the antigen-presenting cells are first activated by using nano-particles and/or micro-particles loaded with cancer cell antigens, and then the antigen-presenting cells are processed by a certain method to prepare a nano-vaccine or a micro-vaccine, and the obtained vaccine is loaded with cancer cell whole Therefore, after injection into the human body, a broad spectrum of cancer cell-specific T cells can be activated, so that more and better cancer cells can be identified and killed.

T细胞作用将其激活为癌细胞特异性T细胞；此外纳米疫苗或微米疫苗还可以被抗原提呈细胞吞噬，进而通过抗原提呈细胞再间接激活癌细胞特异性T细胞。现有的疫苗只能通过抗原提呈细胞间接激活T细胞，而本发明所述纳米疫苗或微米疫苗可以通过直接激活T细胞和通过抗原提呈细胞间接激活T细胞两种方式激活T细胞。因此本发明基于上述癌症疫苗，提供了一种使用抗原提呈细胞来源的癌症疫苗直接激活T细胞的方法，将癌症疫苗与T细胞在体外共孵育以诱导激活得到癌细胞特异性T细胞。Moreover, since the surface of the nano-vaccine or micro-vaccine of the present invention is loaded with cell membrane components and the cell membrane contains broad-spectrum polypeptide antigens combined with MHC, the nano-vaccine or micro-vaccine can be used as artificial antigen-presenting cells to directly interact with

T cells activate them into cancer cell-specific T cells; in addition, nano-vaccine or micro-vaccine can also be phagocytosed by antigen-presenting cells, and then indirectly activate cancer-cell-specific T cells through antigen-presenting cells. Existing vaccines can only activate T cells indirectly through antigen-presenting cells, while the nano-vaccine or micro-vaccine of the present invention can activate T cells in two ways: direct activation of T cells and indirect activation of T cells through antigen-presenting cells. Therefore, based on the above-mentioned cancer vaccine, the present invention provides a method for directly activating T cells using antigen-presenting cell-derived cancer vaccine, and co-incubating the cancer vaccine and T cells in vitro to induce activation to obtain cancer cell-specific T cells.

The third object of the present invention is to provide the application of the above-mentioned antigen-presenting cell membrane component-based cancer vaccine in the preparation of a drug for treating or preventing cancer.

By means of the above scheme, the present invention has at least the following advantages:

(1) The present invention can integrate membrane components, loaded MHC molecules, and antigens of various antigen-presenting cells into a nano-vaccine or micro-vaccine. Therefore, a vaccine can contain DC cells, B cells or macrophages. The membrane components of various antigen-presenting cells such as cells, so a nano-vaccine particle can simultaneously have the functions and advantages of multiple antigen-presenting cells.

(2) The vaccine of the present invention can contain membrane components of various antigen-presenting cells including DC cells. The proteins on the cell membrane of antigen-presenting cells have special affinity for lymph nodes and immune cells in the lymph nodes. After entering the body, it has the characteristics of homing to the lymph nodes, and the effect of naturally targeting the lymph nodes can better activate the specific immune response of cancer cells.

(3) The present invention uses nanoparticles and/or microparticles loaded with cancer cell antigens to activate antigen-presenting cells, and then uses antigen-presenting cells to prepare a vaccine, so the prepared nanovaccine can be loaded with the prepared nanoparticles and/or microparticles With all the antigen components carried by the particles, the nanovaccine can carry a broad spectrum of cancer cell-specific antigens.

(4) The vaccine of the present invention is derived from micron-sized antigen-presenting cells, and all components are biocompatible and degradable, with good safety, and at the same time, the requirements for storage, transportation and injection of live cell vaccines are solved. Tough question and better efficacy than live cell vaccines.

The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it according to the content of the description, the following description is given with the preferred embodiments of the present invention and the detailed drawings.

Description of drawings

In order to make the content of the present invention easier to understand clearly, the present invention will be described in further detail below according to specific embodiments of the present invention and in conjunction with the accompanying drawings.

Fig. 1 is the preparation process and application schematic diagram of nano-vaccine or micro-vaccine of the present invention; Wherein, a is the schematic diagram of the collection and preparation of nano-particles or micro-particles for water-soluble antigens and water-insoluble antigens respectively; b is the use of a lysing solution containing a dissolving agent Schematic diagram of dissolving whole cell antigens of cancer cells and preparing nanoparticles or microparticles; c is the use of the above-mentioned particles prepared in a or b to activate antigen-presenting cells, and then prepare the antigen-presenting cells into nano-vaccine or micro-vaccine, and use this type of vaccine Schematic diagram of nano-vaccine or micro-vaccine to prevent or treat cancer;

代表与纳米粒/微米粒激活抗原提呈细胞过程中加入特定细胞因子共孵育后所制备的抗原提呈细胞的纳米疫苗对照组相比p＜0.05，有显著性差异；&&&表示与空白纳米粒/微米粒+游离裂解液激活的抗原提呈细胞制备的纳米疫苗对照组相比p＜0.005，有显著性差异；&&表示与空白纳米粒/微米粒+游离裂解激活的抗原提呈细胞制备的纳米疫苗对照组相比p＜0.01，有显著性差异；δδδ代表与多肽纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗相比p＜0.005，有显著性差异；δδ代表与多肽纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗组相比p＜0.01，有显著性差异；ΔΔ代表与使用被负载全细胞组分的纳米粒子激活的混合抗原提呈细胞活细胞疫苗组相比p＜0.01，有显著性差异；★代表与使用1％海藻糖+2％甘露醇+2％PVP水溶液作为冻干保护剂冷冻干燥制备的纳米疫苗或微米疫苗相比p＜0.05，有显著性差异；Ψ代表与纳米粒/微米粒激活的DC+B细胞所制备的纳米疫苗组相比p＜0.05，有显著性差异；Ω代表与纳米粒/微米粒激活的B细胞所制备的纳米疫苗组相比p＜0.05，有显著性差异；χχ代表与使用特定细胞因子组合辅助微米粒子激活的抗原提呈细胞制备的纳米疫苗或微米疫苗相比p＜0.01，有显著性差异；ω代表与只是内部负载癌细胞全细胞组分但是表面不负载被激活的抗原提呈细胞细胞膜组分的纳米疫苗或微米疫苗相比p＜0.05，有显著性差异；Μ代表与纳米粒/微米粒激活的巨噬细胞所制备的纳米疫苗组相比p＜0.05，有显著性差异；θθθ代表与不负载佐剂的纳米粒/微米粒辅助分离的T细胞组相比p＜0.005，有显著性差异；θθ代表与不负载佐剂的纳米粒/微米粒辅助分离的T细胞组相比p＜0.01，有显著性差异；ππ代表与不负载溶酶体逃逸物质的纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗组相比p＜0.01，有显著性差异；π代表与不负载溶酶体逃逸物质的纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗组相比p＜0.05，有显著性差异；ξξ代表与只负载一种CpG+Poly(I:C)混合佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗相比p＜0.01，有显著性差异；ΣΣ代表与只使用抗原提呈细胞细胞膜组分制备的纳米疫苗或微米疫苗相比p＜0.01，有显著性差异；β代表与使用2％蔗糖+2％甘露醇+1％赖氨酸水溶液作为冻干保护剂冷冻干燥制备的纳米疫苗或微米疫苗相比p＜0.05，有显著性差异；ξ代表与只负载一种CpG+Poly(I:C)混合佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗相比p＜0.05，有显著性差异；μ代表与使用两种A类CpG与toll样受体3混合佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗组相比p＜0.05，有显著性差异；ρ代表与只负载一种佐剂(两类CpG)的纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗组相比p＜0.05，有显著性差异；τ代表与纳米粒/微米粒激活抗原提呈细胞过程中不加入IL-2和IL-7共孵育所制备纳米疫苗相比p＜0.01，有显著性差异；###表示与未被任何纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗对照组相比p＜0.005，有显著性差异；##表示与未被任何纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗对照组相比p＜0.01，有显著性差异；#表示与未被任何纳米粒/微米粒激活的抗原提呈细胞制备的纳米疫苗对照组相比p＜0.05，有显著性差异。Figures 2-20 are respectively the experimental results of tumor growth rate and survival period of mice when using nano-vaccine or micro-vaccine to prevent or treat cancer in Example 1-19; a, experimental results of tumor growth rate when preventing or treating cancer (n≥ 8); b, the experimental results of mouse survival when preventing or treating cancer (n ≥ 8), each data point is the mean ± standard error (mean ± SEM); c and d are the analysis of nanometers using flow cytometry The proportion of vaccine-activated CD8 ⁺ and CD4 ⁺ cancer cell-specific T cells to total CD8 ⁺ and CD4 ⁺ T cells; e is the transmission electron microscope (TEM) scan image of the nanovaccine; the significance of the tumor growth inhibition experiment in a The difference was analyzed by ANOVA method, and the significant difference in figure b was analyzed by Kaplan-Meier and log-rank test; *** means p<0.005 compared with PBS blank control group, there is a significant difference; ** means compared with PBS blank control group Compared with the group, p<0.01, there is a significant difference;

Represents the nanovaccine control group prepared by adding specific cytokines in the process of activating antigen-presenting cells by nanoparticles/microparticles compared with the nano-vaccine control group, p<0.05, there is a significant difference; &&& means that with blank nanoparticles Compared with the control group, the nano-vaccine prepared by /microparticles + free lysate activated antigen-presenting cells has a significant difference, p<0.005; Compared with the nanovaccine control group, p<0.01, there is a significant difference; δδδ represents p<0.005 compared with the nanovaccine prepared by the antigen-presenting cells activated by polypeptide nanoparticles/microparticles; Compared with the nanovaccine group prepared by the APCs activated by particles/microparticles, p<0.01, there is a significant difference; Compared with p < 0.01, there is a significant difference; ★ means p < 0.05 compared with the nano-vaccine or micro-vaccine prepared by freeze-drying using 1% trehalose + 2% mannitol + 2% PVP aqueous solution as freeze-drying protective agent. Significant difference; Ψ represents p<0.05 compared with the nanovaccine group prepared by nanoparticle/microparticle activated DC+B cells; Ω represents the nanovaccine group prepared with nanoparticle/microparticle activated B cells Compared with the nanovaccine group, p<0.05, there is a significant difference; χχ represents a significant difference compared with p<0.01 compared with the nanovaccine or microvaccine prepared from antigen-presenting cells activated by microparticles using a specific combination of cytokines; ω Represents a significant difference compared with the nano-vaccine or micro-vaccine that only loads the whole cell components of cancer cells internally but does not load the activated antigen-presenting cell membrane components on the surface; M represents a significant difference with nanoparticles/microparticles Compared with the nanovaccine group prepared by activated macrophages, p<0.05, there is a significant difference; θθθ represents p<0.005 compared with the T cell group assisted by the separation of nanoparticles/microparticles without adjuvant, and there is a significant difference Difference; θθ represents p<0.01, significant difference compared with unadjuvanted nano/microparticle-assisted isolated T cell group; Compared with the nanovaccine group prepared by antigen-presenting cells, p<0.01, there is a significant difference; <0.05, there is a significant difference; ξξ represents that compared with the nanovaccine prepared by nanoparticle/microparticle activated antigen-presenting cells loaded with only one CpG+Poly(I:C) mixed adjuvant, p<0.01, there is a significant difference Sexual differences; ΣΣ represents p<0.01 compared with nano-vaccine or micro-vaccine prepared by using only antigen-presenting cell membrane fractions; Nanoparticles prepared by freeze-drying of aqueous acid solution as lyoprotectant Compared with vaccine or microvaccine, p<0.05, there is a significant difference; ξ represents the phase of nanovaccine prepared with nanoparticle/microparticle activated antigen-presenting cells loaded with only one CpG+Poly(I:C) mixed adjuvant Ratio p<0.05, there is a significant difference; μ represents p<0.05 compared with the nanovaccine group prepared by nanoparticles/microparticle-activated antigen-presenting cells using two kinds of A-class CpG and toll-like receptor 3 mixed adjuvants , there is a significant difference; ρ represents a significant difference compared with the nanovaccine group prepared by nanoparticle/microparticle activated antigen-presenting cells loaded with only one adjuvant (two types of CpG); p<0.05, a significant difference; τ represents Compared with the nanovaccine prepared by co-incubating with nanoparticles/microparticles without adding IL-2 and IL-7 in the process of activating antigen-presenting cells, there is a significant difference p<0.01; Compared with the nanovaccine control group prepared by the antigen-presenting cells activated by microparticles, there is a significant difference, p<0.005; Ratio p<0.01, there is a significant difference; # means p<0.05, a significant difference compared with the nanovaccine control group prepared from antigen-presenting cells not activated by any nanoparticles/microparticles.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

The cancer vaccine based on antigen-presenting cell membrane components for preventing or treating cancer according to the present invention comprises an antigen activated by nanoparticles and/or microparticles loaded with cancer cells and/or tumor tissue whole cell components Present the nanovaccine or microvaccine prepared by the cells. Nanoparticles and/or microparticles are loaded with cancer cell whole cell components or mixtures thereof. Whole cell antigens are contained in the whole cell fraction. The preparation process and application fields of nano-vaccine for preventing or treating cancer are shown in Fig. 1 .

When preparing nanoparticles or microparticles loaded with cancer cells and/or tumor tissue whole cell components for activating antigen-presenting cells, the cells or tissues can be lysed and then water-soluble antigens and water-insoluble antigens can be collected and prepared separately. Or a microparticle system; or a lysing solution containing a lysing agent can be used to directly lyse cells or tissues and lyse whole cell antigens of cancer cells to prepare a nanoparticle or microparticle system. The cancer cell whole cell antigen of the present invention can be processed before or (and) after lysis, including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, etc. Nanoparticles or microparticles are then prepared; nanoparticles can also be directly prepared before or (and) after cell lysis without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, and nuclease treatment. or micron particles. In some embodiments of the present invention, tumor tissue cells are inactivated or (and) denatured before lysis, and in actual use, inactivation or (and) denaturation can also be performed after cell lysis, or cell lysis can also be performed. Before and after lysis, inactivation or (and) denaturation treatment is performed; in some embodiments of the present invention, the inactivation or (and) denaturation treatment methods before or (and) after lysis of cells are ultraviolet irradiation and high temperature heating. In the process, treatment methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, freeze-drying and the like can also be used. Those skilled in the art can understand that in the actual application process, the skilled person can make appropriate adjustments according to specific conditions.

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

After the antigen-presenting cells are activated, low-frequency ultrasound and gradient centrifugation are used to prepare the antigen-presenting cells into nano-sized nano-vaccine or micro-vaccine. In actual preparation, other methods for preparing micron-sized live antigen-presenting cells into nano-sized or micron-sized nano-vaccine or micro-vaccine without cellular activity can also be used.

In some embodiments, the specific preparation method for preparing nano-vaccine or micro-vaccine using antigen-presenting cells activated by nanoparticles or micro-particles loaded with cancer cell whole cell antigens 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 second predetermined concentration of the raw material for preparing particles.

In some embodiments, the aqueous phase solution may contain each component in the cancer cell lysate and an immune-enhancing adjuvant; each component in the cancer cell lysate is prepared as a water-soluble antigen or soluble in urea or hydrochloric acid, respectively. Originally water-insoluble antigens in solubilizing agents such as guanidine. The concentration of the water-soluble antigen contained in the aqueous solution or the concentration of the original water-insoluble antigen, that is, the first predetermined concentration requires that the protein polypeptide concentration is greater than 1 ng/mL, which can load enough cancer cell whole cell antigens to activate relevant cells. The concentration of the immunopotentiating adjuvant in the initial aqueous phase was greater than 0.01 ng/mL.

In some embodiments, the aqueous solution contains each component in the tumor tissue lysate and an immune-enhancing adjuvant; each component in the tumor tissue lysate is water-soluble antigen or dissolved in urea or guanidine hydrochloride, respectively, during preparation The original water-insoluble antigen in the isolytic agent. The concentration of the water-soluble antigen contained in the aqueous solution or the concentration of the original water-insoluble antigen, that is, the first predetermined concentration requires the protein and polypeptide concentration to be greater than 0.01 ng/mL, which can load enough cancer cell whole cell antigens to activate relevant cells. The concentration of the immunopotentiating adjuvant in the initial aqueous phase was greater than 0.01 ng/mL.

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

In the present invention, PLGA or modified PLGA is chosen because the material is biodegradable and has been approved by the FDA for use as a drug dressing. Studies have shown that PLGA has certain immunomodulatory functions, so it is suitable as an excipient for the preparation of nanoparticles or microparticles. In practical applications, appropriate materials can be selected according to the actual situation.

In practice, the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the water phase. In the present invention, the range of the ratio of the first predetermined volume of the water phase to the second predetermined volume of the organic phase is 1:1.1-1:5000, preferably 1:10. In the specific implementation process, the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as required to adjust the size of the prepared nanoparticles or microparticles.

Preferably, when the aqueous phase solution is a lysate component solution, the concentration of proteins and polypeptides is greater than 1 ng/mL, preferably 1 mg/mL to 100 mg/mL; when the aqueous phase solution is a lysate component/immune adjuvant solution, wherein The concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL to 100 mg/mL, and the concentration of immune adjuvant is greater than 0.01 ng/mL, preferably 0.01 mg/mL to 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～ 5000 mg/mL, preferably 100 mg/mL.

In step 2, the mixed solution obtained in step 1 is subjected to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm, and the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.1 hour to 24 hours; More than 0.1 seconds, such as 2 to 200 seconds; use a high-pressure/ultra-high-pressure homogenizer or a high-shear homogenizer when using a high-pressure/ultra-high-pressure homogenizer, and the pressure is greater than 5psi, such as 20psi~100psi When the shearing homogenizer is used, the rotation speed is greater than 100rpm, such as 1000rpm to 5000rpm; the microfluidic processing flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min. Ultrasonic or stirring or homogenization treatment or microfluidic treatment for nano and/or micronization, the length of ultrasonic time or stirring speed or the pressure and time of homogenization treatment can control the size of the prepared micro-nano particles. changes in particle size.

Step 3, adding the mixture obtained after the treatment in step 2 into a third predetermined volume of an aqueous solution containing a third predetermined concentration of emulsifier and performing ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing homogenization or microfluidic control deal with. In this step, the mixture obtained in step 2 is added to the emulsifier aqueous solution and continues to be nanosized or micronized by ultrasonication or stirring. In the present invention, the ultrasonic time is greater than 0.1 seconds, such as 2-200 seconds, the stirring speed is greater than 50 rpm, such as 50-500 rpm, and the stirring time is greater than 1 minute, such as 60-6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm, and the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.5 hours to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W to 500W , the time is greater than 0.1 seconds, such as 2 to 200 seconds; high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer is used for homogenization, and the pressure is greater than 20psi, such as 20psi-100psi, When using a high shear homogenizer, the rotation speed is greater than 1000rpm, such as 1000rpm to 5000rpm; when using microfluidics, the flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min. Ultrasonic or stirring or homogenizing treatment or microfluidic treatment for nano or micronization, the length of ultrasonic time or stirring speed or homogenization treatment pressure and time can control the size of the prepared nano or micro particles, too large or too small will bring Variation in particle size.

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

In step 4, the liquid obtained after the treatment in step 3 is added to a fourth predetermined volume of the emulsifier aqueous solution of a fourth predetermined concentration, and stirred until the predetermined stirring condition is satisfied.

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

The fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size. The selection of the fourth predetermined volume is determined according to the 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 1:1.5-1:2000, preferably 1:10. In the specific implementation process, the ratio of the third predetermined volume to the fourth predetermined volume may be adjusted in order to control the size of the nano-particles or micro-particles.

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 the dichloromethane in step 1 is completed.

Step 5: After centrifuging the mixture that meets the predetermined stirring conditions in step 4 at a speed of more than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining precipitate in the fifth predetermined volume of the first. Five predetermined concentrations of the aqueous solution containing the lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).

In some embodiments of the present invention, when the pellet obtained in step 5 is resuspended in a sixth predetermined volume of PBS (or physiological saline), it does not need to be lyophilized, and the subsequent adsorption of cancer cell lysate on the surface of nanoparticles or microparticles can be carried out directly. related experiments.

In some embodiments of the present invention, when the precipitate obtained in step 5 is resuspended in an aqueous solution containing a lyoprotectant, it needs to be freeze-dried, and after freeze-drying, the subsequent adsorption of cancer cell lysates on the surface of nanoparticles or microparticles is carried out. experiment.

In the present invention, the fifth predetermined concentration of the freeze-drying protective agent in this step is 4% by mass, and the reason for this setting is to not affect the freeze-drying effect in the subsequent freeze-drying.

Step 6, after subjecting the suspension containing the freeze-drying protective agent obtained in step 5 to freeze-drying treatment, the freeze-dried substance is used for later use.

Step 7, resuspend the nanoparticle-containing suspension obtained in step 5 of the sixth predetermined volume in PBS (or physiological saline) or use the sixth predetermined volume of PBS (or physiological saline) to resuspend the suspension obtained in step 6 The freeze-dried lyophilized substance containing nanoparticles or microparticles and a lyoprotectant is used directly; or the above-mentioned sample is mixed with a seventh predetermined volume of water-soluble antigen or dissolved original water-insoluble antigen and used.

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

In some embodiments, when the volume of the resuspended nanoparticle suspension is 10 mL, the volume of the resuspended nanoparticle suspension containing the cancer cell lysate or the water-soluble antigen contained in the tumor tissue lysate or the dissolved original water-insoluble antigen is 1 mL. . In actual use, the volume and ratio of the two can be adjusted as needed.

Step 8, co-incubating the antigen-presenting cells with the above-prepared nanoparticles and/or microparticles for a certain period of time. Tumor tissue and/or cancer cells and antigen-presenting cells from which nanoparticles and/or microparticles are prepared can be derived from autologous or allogeneic sources.

Step 9, collect the antigen-presenting cells after co-incubation, which can be washed or not washed and then mechanically destroyed or added with chemical substances to destroy the cell structure but still preserve the cell membrane components, the method of mechanical destruction or chemical addition used Including but not limited to sonication below 500W, high pressure homogenization, agitation, swelling, shrinking, high shear failure, small pore size extrusion failure, extrusion, addition of low osmotic pressure PBS or glucose solution containing chemicals Or one or more of the salt solutions.

Step 10, prepared by centrifuging a sample mechanically disrupted or adding a specific chemical substance, and/or filtering through a filter membrane with a specific pore size, extruding, and/or co-acting with nanoparticles or microparticles loaded with whole cell components Nano-vaccine or micro-vaccine. Means of mechanical disruption or addition of specific chemical substances to the co-action of the sample with the whole-cell component-loaded nanoparticles or microparticles include, but are not limited to, co-incubation, sonication, extrusion, mechanical stirring, high pressure homogenization, extrusion, etc. The manner in which membrane components are loaded onto the surface of nanoparticles or microparticles.

In other embodiments, the specific preparation method for preparing the antigen-loaded nanoparticles or microparticles is as follows:

Steps 1 to 4 are the same as above.

Step 5: After centrifuging the mixture that meets the predetermined stirring conditions in step 4 at a speed of more than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining precipitate in the fifth predetermined volume of the first. five predetermined concentrations of a solution containing water-soluble and/or water-insoluble antigens in cancer cell whole cell antigens, or resuspend the remaining pellet in a fifth predetermined volume of a fifth predetermined concentration of cancer cell whole cell-containing antigens A solution of water-soluble and/or water-insoluble antigen in antigen mixed with adjuvant.

Step 6: After centrifuging the mixture that meets the predetermined stirring conditions in step 5 at a speed of more than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining precipitate in a sixth predetermined volume of solidification. The treatment reagent or the mineralization treatment reagent, after acting for a certain period of time, is centrifuged and washed, and then the seventh predetermined submission containing the positively charged or negatively charged substance is added and acted for a certain period of time.

In some embodiments of the present invention, after the pellet obtained in step 6 is resuspended in the seventh predetermined volume of charged substance, lyophilization may not be required, and subsequent experiments related to the loading of cancer cells/tissue lysates on the surface of nanoparticles or microparticles can be performed directly.

In some embodiments of the present invention, the precipitate obtained in step 6 is resuspended in an aqueous solution containing a drying protective agent, and then subjected to room temperature vacuum drying or freeze-vacuum drying, and after drying, subsequent adsorption of cancer cell lysate on the surface of nanoparticles or microparticles is carried out. related experiments.

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

In step 7, after drying the suspension containing the drying protective agent obtained in step 6, the dried substance is used for later use.

Step 8, resuspend the nanoparticle-containing suspension obtained in step 6 of the eighth predetermined volume in PBS (or physiological saline) or use the eighth predetermined volume of PBS (or physiological saline) to resuspend the suspension obtained in step 7 The dried substance containing nanoparticles or microparticles and drying protective agent is used directly; or mixed with the ninth predetermined volume of water-soluble antigen or water-insoluble antigen for use.

In the present invention, the modification and antigen loading steps of steps 5 to 8 can be repeated multiple times to increase the antigen loading. Moreover, when adding positively charged or negatively charged substances, substances with the same charge can be added multiple times, or substances with different charges can be added alternately.

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

Step 9, co-incubating the antigen-presenting cells with the above-prepared nanoparticles and/or microparticles for a certain period of time. Tumor tissue and/or cancer cells and antigen-presenting cells from which nanoparticles and/or microparticles are prepared can be derived from autologous or allogeneic sources.

Step 10: Collect the antigen-presenting cells after co-incubation, which can be washed or not washed and then mechanically destroyed or added with chemical substances to destroy the cell structure but still preserve the cell membrane components, the method of mechanical destruction or chemical addition used Including but not limited to sonication below 500W, high pressure homogenization, stirring, extrusion, swelling, shrinking, high shear failure, small pore size extrusion failure, addition of low osmotic pressure PBS or glucose solution containing chemicals Or one or more of the salt solutions.

Step 11, centrifuging the mechanically damaged or added sample with specific chemical substances, and/or filtering through a filter membrane with a specific pore size, and/or co-acting with nanoparticles or microparticles loaded with whole cell components to prepare a nanovaccine or a nanovaccine. Micron vaccine. Means of mechanical disruption or addition of specific chemical substances to the co-action of the sample with the whole-cell component-loaded nanoparticles or microparticles include, but are not limited to, co-incubation, sonication, mechanical stirring, extrusion, high pressure homogenization, extrusion, etc. The manner in which membrane components are loaded onto the surface of nanoparticles or microparticles.

Example 1DC-derived nanovaccine for the prevention of melanoma

This example uses mouse melanoma as a cancer model to illustrate how to use DC-derived nanovaccine for the prevention of melanoma. In this example, the B16F10 melanoma tumor tissue was lysed to prepare water-soluble antigens and water-insoluble antigens of the tumor tissue. Then, the organic polymer material PLGA was used as the nanoparticle skeleton material, and Polyinosinic-polycytidylic acid (poly(I:C )) using solvent volatilization method for immune adjuvant to prepare nanoparticle system loaded with water-soluble antigens and water-insoluble antigens of tumor tissue, then using nanoparticles to activate DC cells, and then using DC cells to prepare nano-vaccine for cancer prevention and treatment .

(1) Lysis of tumor tissue and collection of components

1.5×10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse, the mice were sacrificed when the tumors grew to a volume of about 1000 mm ³ , and tumor tissues were excised. The tumor tissue was cut into pieces and ground, and an appropriate amount of ultrapure water was added through a cell strainer and freeze-thawed 5 times, accompanied by ultrasound to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was taken to be the water-soluble antigen soluble in pure water; adding 8M urea to the obtained precipitate to dissolve the precipitated part to make it insoluble in pure water. The water-insoluble antigen was converted to soluble in 8 M urea in water. Mixing the water-soluble antigen and the water-insoluble antigen in a mass ratio of 1:1 is the source of the antigen raw material for preparing the nanoparticles.

(2) Preparation of nanoparticle system

In this example, the nanoparticles were prepared by the double emulsion method in the solvent evaporation method. The used nanoparticle preparation material PLGA has a molecular weight of 24KDa-38KDa, and the used immune adjuvant is poly(I:C) and poly(I:C) is only contained in the nanoparticle. The preparation method was as described above. In the preparation process, the whole cell lysate component and adjuvant were first loaded into the nanoparticles by double emulsion method. After the cell lysate component and the adjuvant were loaded inside, 100 mg of nanoparticles were centrifuged at 10,000 g for 20 minutes. , and resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h. The average particle size of the nanoparticles is about 250nm, and the surface potential of the nanoparticles is about -3mV; each 1mg PLGA nanoparticles is loaded with about 100μg protein or polypeptide components, and the poly(I:C) immune adjuvant used per 1mg PLGA nanoparticles is 0.02mg. The blank nanometer preparation material and preparation method are the same, the particle size is about 240nm, and the same amount of adjuvant is used to replace the whole cell component.

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

This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare BMDCs. First, a 6-8-week-old C57 mouse was sacrificed by cervical dislocation. The tibia and femur of the hind leg were surgically removed and placed in PBS. The muscle tissue around the bone was removed with scissors and forceps. Cut off both ends of the bone with scissors, and then use a syringe to extract the PBS solution. The needles are inserted into the bone marrow cavity from both ends of the bone, and the bone marrow is repeatedly washed into the Petri dish. The bone marrow solution was collected, centrifuged at 400 g for 3 min, and then 1 mL of erythrocyte lysate was added. 3 mL of RPMI 1640 (10% FBS) medium was added to terminate the lysis, centrifuged at 400 g for 3 min, and the supernatant was discarded. The cells were placed in a 10 mm petri dish and cultured in RPMI 1640 (10% FBS) medium with the addition of recombinant mouse GM-CSF (20 ng/mL) at 37 degrees, 5% CO ₂ for 7 days. On day 3, the flasks were shaken gently and supplemented with the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng/mL). On the 6th day, the medium was subjected to half volume exchange. On the 7th day, a small amount of suspension and semi-adherent cells were collected, and by flow cytometry, when the ratio of CD86 ⁺ CD80 ⁺ cells in CD11c ⁺ cells was between 15-20%, the induced cultured BMDCs could be used for the next step. One step experiment.

(4) Activation of antigen presenting cells

Nanoparticles (500 μg) or blank nanoparticles (500 μg) + free lysate loaded with whole cell fractions of cancer cells derived from tumor tissue were incubated with BMDC (10 million) in 15 mL of RPMI1640 complete medium for 48 hours (37°C). , 5% CO ₂ ); the incubation system contains cytokine combination 1: granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL) mL), IL-12 (1000U/mL), or with cytokine components 2: GM-CSF, (1000U/mL), IL-4 (100U/mL), tumor necrosis factor alpha (TNF-α, 200U/mL) ).

(5) Preparation of DC-derived nanovaccine

The incubated DCs were collected by centrifugation at 400g for 5 minutes, then the cells were washed twice with saline, resuspended in saline and sonicated at 7.5W for 20 minutes. The samples were then centrifuged at 2000g for 20 minutes and the supernatant was collected, the supernatant was centrifuged at 7000g for 20 minutes and the supernatant was collected and then centrifuged at 15000g for 120 minutes. The supernatant was discarded to collect the pellet and the pellet was placed in PBS The nano-vaccine was obtained after resuspending, and the particle size of the nano-vaccine was 120 nm.

(6) Nano vaccines for cancer prevention

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. Each mouse was subcutaneously injected with 500 μg of nanovaccine on day -42, day -35, day -28, day -21, day -14 and day -7, respectively, before the mice were inoculated with cancer cells. On day 0, each mouse was inoculated subcutaneously with ^1.5 x 105 B16F10 cells on the lower right side of the back. Mouse tumor growth rate and mouse survival were monitored. In the experiment, the size of mouse tumor volume was recorded every 3 days starting from day 3. The tumor volume was calculated using the formula v=0.52×a×b ² , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm ³ , the mouse is regarded as dead and the mouse is euthanized.

(7) Experimental results

As shown in Figure 2, mice in the PBS control group had fast tumor growth and short survival. Mice treated with nanovaccine prepared from blank nanoparticles + free lysate-activated DC had slower tumor growth. Mice treated with the nanovaccine prepared from nanoparticle-activated DCs loaded with whole-cell lysates of cancer cells had significantly slower tumor growth and longest survival than the above two groups. Among them, the effect of adding cytokine combination 1 during the activation of DC cells was better than that of cytokine combination 2. In conclusion, the nano-vaccine of the present invention has a good preventive effect on melanoma.

Example 2 DC-based cancer vaccines for melanoma prevention

This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticles to assist in activating DCs, and then prepare DCs into cancer vaccines for the prevention of melanoma. In this example, the B16F10 melanoma tumor tissue was split to prepare water-soluble antigens and water-insoluble antigens of the tumor tissue, then, PLGA was used as the nanoparticle skeleton material, and poly(I: C), CpG2216 and CpG2395 are mixed immune adjuvants to prepare nanoparticle systems loaded with water-soluble antigens and non-water-soluble antigens of tumor tissue by solvent evaporation method, and then use nanoparticles to activate DCs, and DCs are prepared into sources after appropriate treatment In vivo prevention of melanoma by injection of nanovaccine on DC.

(1) Lysis of tumor tissue and collection of components

1.5×10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse, the mice were sacrificed when the tumors grew to a volume of about 1000 mm ³ , and tumor tissues were excised. The tumor tissue was cut into pieces and ground, and an appropriate amount of pure water was added through a cell strainer and freeze-thawed for 5 times. Ultrasound was also used to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at 5000g for 5 minutes, and the supernatant was taken to be the water-soluble antigen soluble in pure water; adding 8M urea to the obtained precipitate to dissolve the precipitated part to make it insoluble in pure water. The water-insoluble antigen was converted to soluble in 8 M urea in water. The above is the source of antigen raw materials for the preparation of nanoparticle systems.

(2) Preparation of nanoparticle system

In this example, the nanovaccine and the blank nanoparticle as a control were prepared by solvent evaporation method. During preparation, the nanovaccine loaded with the water-soluble antigen in the cancer cell whole cell antigen and the nanoparticle loaded with the water-insoluble antigen in the cancer cell whole cell antigen are prepared separately, and then used together when used. The used nanoparticle preparation material PLGA has a molecular weight of 7Da-17KDa, and the used immune adjuvants are poly(I:C), CpG2216 and CpG2395, and the adjuvants are encapsulated inside the nanoparticles. The preparation method was as described above. In the preparation process, the antigen and adjuvant were first loaded into the nanoparticles by the double emulsion method. After the antigen was loaded inside, 100 mg of the nanoparticles were centrifuged at 10,000 g for 20 minutes, and 10 mL of 4% trehalose was used. Resuspend in ultrapure water and freeze-dry for 48 h. The average particle size of the nanoparticles is about 280 nm; each 1 mg of PLGA nanoparticles is loaded with about 100 μg of protein and polypeptide components, and each 1 mg of PLGA nanoparticles uses 0.025 mg of poly(I:C), CpG2216 and CpG2395 immune adjuvants. In this example, four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were loaded with equal mass. The nanoparticles were used as control nanoparticles. The size of the control nanoparticles was about 260 nm, loaded with 100 μg of polypeptide components, and loaded with an equal amount of adjuvant. The particle size of the blank nanoparticles is about 250 nm, and only the same amount of immune adjuvant is loaded without any antigenic components.

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

This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare BMDCs. First, a 6-8-week-old C57 mouse was sacrificed by cervical dislocation. The tibia and femur of the hind leg were surgically removed and placed in PBS. The muscle tissue around the bone was removed with scissors and forceps. Cut off both ends of the bone with scissors, and then use a syringe to extract the PBS solution. The needles are inserted into the bone marrow cavity from both ends of the bone, and the bone marrow is repeatedly washed into the Petri dish. The bone marrow solution was collected, centrifuged at 400 g for 3 min, and then 1 mL of erythrocyte lysate was added. 3 mL of RPMI 1640 (10% FBS) medium was added to terminate the lysis, centrifuged at 400 g for 3 min, and the supernatant was discarded. The cells were placed in a 10 mm petri dish and cultured in RPMI 1640 (10% FBS) medium with the addition of recombinant mouse GM-CSF (20 ng/mL) at 37 degrees, 5% CO ₂ for 7 days. On day 3, the flasks were shaken gently and supplemented with the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng/mL). On the 6th day, the medium was subjected to half volume exchange. On the 7th day, a small amount of suspension and semi-adherent cells were collected, and by flow cytometry, when the ratio of CD86 ⁺ CD80 ⁺ cells in CD11c ⁺ cells was between 15-20%, the induced cultured BMDCs could be used for the next step. One step experiment.

(4) Activation of DC

Nanoparticles (500μg, 250μg of nanoparticles loaded with water-soluble components, 250μg of nanoparticles loaded with non-water-soluble components) or polypeptide nanoparticles (500μg) or blank will be loaded with whole cell components of cancer cells derived from tumor tissue. Nanoparticles (500μg) + free lysate and BMDC (10 million) were co-incubated in 15mL RPMI1640 complete medium for 48 hours (37°C, 5% CO ₂ ), and the incubation system contained granulocyte-macrophage colony-stimulating factor ( GM-CSF, 2000 U/mL), IL-2 (500 U/mL), IL-7 (1000 U/mL), IL-12 (500 U/mL) and CD86 antibody (20 ng/mL).

(5) Preparation of DC-derived nanovaccine

Incubated DCs were collected by centrifugation at 400 g for 5 min, then cells were washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, cells were resuspended in PBS water at 4°C low power (22.5W) Sonicate for 1 minute. The samples were then centrifuged at 3000g for 15 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 8000g for 15 minutes, and then after 90 minutes at 16000g. The supernatant was discarded to collect the pellet and the pellet was placed in PBS After resuspension, a membrane filter was used to filter the sample to obtain a nano-vaccine, and the particle size of the nano-vaccine was 120 nm.

(6) Nano vaccines for cancer prevention

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice, on the -35th, -28th, -21st, -14th and -7 days each mouse was vaccinated with 500 μg of nanovaccine. On day 0, ^1.5 x 105 B16F10 cells were inoculated subcutaneously in the lower right back of each recipient mouse. Mouse tumor growth rate and mouse survival were monitored. In the experiment, the size of mouse tumor volume was recorded every 3 days starting from day 3. The tumor volume was calculated using the formula v=0.52×a×b ² , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm ³ , the mouse is regarded as dead and the mouse is euthanized.

(7) Analysis of nano-vaccine activation of cancer cell-specific T cells

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. Each mouse was injected subcutaneously with 100 μg of nanovaccine or PBS on day 0, day 7, day 14, day 28 and day 42, respectively. The mice were sacrificed on day 45, and the spleens of the mice were harvested to prepare a single-cell suspension of spleen cells, and B cells and T cells were separated from the mouse spleen cells by magnetic bead sorting. 100 μg of nanoparticles loaded with whole cell components, 5 million B cells and 1 million T cells were co-incubated in 5 mL of RPMI1640 complete medium for 48 hours (37° C., 5% CO ₂ ). The incubated cells were then collected and labeled with live dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody and IFN-γ antibody, and the proportion of IFN-γ ⁺ T cells in T cells was analyzed by flow cytometry. The cancer cell whole cell antigens loaded by nanoparticles can be degraded into antigenic epitopes after being phagocytosed by antigen-presenting cells B cells and presented on the surface of antigen-presenting cells. Specific T cells that can recognize cancer cell whole cell antigens are It can be activated and secrete killer cytokines after recognizing whole cell antigen epitopes of cancer cells. IFN-γ is the most important cytokine secreted after antigen-specific T cells are activated and activated after recognizing antigen. IFN-γ ⁺ T cells analyzed by flow cytometry are cancer cell-specific T cells that can recognize and kill cancer cells.

(8) Experimental results

As shown in a and b in Figure 3, both the PBS control group and the blank nanoparticle control group had fast tumor growth and short survival time. Compared with the above two groups of control groups, the tumor growth rate in mice treated with nanovaccine prepared by nanoparticle-activated DC was significantly slower, and some mice tumors disappeared and recovered. Moreover, the effect of nanovaccine prepared from DCs activated by nanoparticles loaded with whole cell antigens of cancer cells is better than that prepared by DCs activated by nanoparticles loaded with four antigen polypeptides. This indicates the types of antigenic epitopes processed and presented by DCs activated by nanoparticles loaded with four neoantigen polypeptides, and thus the cancer cell-specific T antigen-presenting cells that can be activated by the prepared nanovaccine. T cells contained in cancer vaccines With fewer clones, fewer cancer cells can be identified and killed. However, the DCs activated by nanoparticles loaded with whole cell antigens of cancer cells can process and present a broader spectrum of cancer cell antigens, so the number of T cell clones that can be activated by the prepared nanovaccine is also broader, and can recognize and kill cancer cells. The more cancer cells are killed, the better the effect of treating or preventing cancer.

As shown in c and d in Figure 3, the CD8+IFN-γ+T cells and CD4+IFN-γ+T cells that can be activated by the nanovaccine prepared from the DC cells activated by nanoparticles loaded with the whole cell components accounted for the proportion of CD8+ IFN-γ+ T cells. The ratios of T cells and CD4+T cells were significantly higher than those activated by the nanovaccine prepared by polypeptide-loaded nanoparticles and blank nanoparticles+DC cells activated by free lysate. It can be seen that the nano-vaccine prepared from DC cells activated by nanoparticles loaded with whole cell components according to the present invention can better activate cancer cell-specific T cells with the ability to recognize cancer cells and kill cancer cells.

Example 3

This example uses mouse melanoma as a cancer model to illustrate how to use nanovaccine to treat cancer. In this example, B16F10 melanoma tumor tissue and cancer cells were firstly 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 The water-soluble antigen mixture and the water-insoluble antigen mixture were mixed in a mass ratio of 1:1. Then, nanoparticles loaded with lysate components were prepared with PLGA as nanoparticle skeleton material and Poly(I:C) and CpG2006 as adjuvants, and then the nanoparticles were co-incubated with T antigen presenting cells in vitro for a certain period of time, and then used Activated antigen-presenting cells to prepare nanovaccine for cancer treatment.

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

When collecting tumor tissue, firstly inoculate 1.5×10 ⁵ B16F10 cells subcutaneously on the back of each C57BL/ ⁶ mouse. When the tumor grows to a volume of about 1000 mm, the mice are sacrificed and the tumor tissue is excised. The tumor tissue is cut into pieces. Grind, add an appropriate amount of pure water through a cell strainer and freeze and thaw for 5 times, and sonicate to destroy the lysed samples; when collecting the cultured B16F10 cancer cell line, first centrifuge to remove the medium, then wash twice with PBS and centrifuge Cancer cells were collected, resuspended in ultrapure water, freeze-thawed 3 times, and lysed cancer cells with ultrasonic damage. After the tumor tissue or cancer cells are lysed, the lysate is centrifuged at 5000g for 5 minutes, and the supernatant is taken to be the water-soluble antigen soluble in pure water; 8M urea is added to the obtained precipitate to dissolve the precipitated part. Water-insoluble antigens that were insoluble in pure water were converted to be soluble in 8M aqueous urea. The water-soluble antigen of tumor tissue and the water-soluble antigen of cancer cells are mixed in a mass ratio of 1:1; the water-insoluble antigen of tumor tissue and the water-insoluble antigen of cancer cells are mixed in a mass ratio of 1:1. Mixing the water-soluble antigen mixture and the water-insoluble antigen mixture in a mass ratio of 1:1 is the source of antigen raw materials for preparing nanoparticles.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by the double emulsion method. The used nanoparticle preparation material PLGA has a molecular weight of 7KDa-17KDa, and the used immunoadjuvant is poly(I:C) and CpG2006, and the adjuvant is encapsulated in the nanoparticle. The preparation method was as described above. In the preparation process, the lysate components and adjuvants were first loaded into the nanoparticles by the double emulsion method, and then 100 mg of the nanoparticles were centrifuged at 10,000 g for 20 minutes, and 10 mL of ultrapure 4% trehalose was used. After resuspending in water, it was freeze-dried for 48 hours; before use, it was resuspended in 9 mL of PBS, then 1 mL of lysate fraction (protein concentration 80 mg/mL) was added and treated at room temperature for 10 min to obtain a nanoparticle system loaded with lysate inside and outside. The average particle size of the nanoparticles is about 270nm, the surface potential is about -5mV, each 1mg PLGA nanoparticle is loaded with about 130μg protein or polypeptide components, and each 1mg PLGA nanoparticle is loaded with poly(I:C) and CpG2006 immune adjuvant 0.02 each. mg.

(3) Isolation of B cells

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

(4) Activation of antigen presenting cells

Nanoparticles (500 μg) or polypeptide nanoparticles (500 μg) loaded with whole cell components of cancer cells were co-incubated with DC2.4 (10 million) and B cells (10 million) in 15 mL of complete RPMI1640 medium for 48 hours (37°C, 5% CO ₂ ), the incubation system contains granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL) ), IL-12 (200 U/mL), albumin (50 ng/mL), and CD80 antibody (10 ng/mL). After incubation, the incubated cells were centrifuged at 400g for 5 minutes, the supernatant was discarded, and then washed twice with PBS to obtain activated antigen-presenting cells. The activated mixed antigen-presenting cells can be used as antigen-presenting cells. Cellular vaccines are used.

(5) Preparation of antigen-presenting cell-derived nanovaccine

DC and B cells (20 million, 10 million each of DC and B cells) after incubation in step (4) were collected by centrifugation at 400g for 5 minutes, then washed three times with PBS, and the mixed cells were resuspended in PBS water. Sonicate at low power (10W) for 30 minutes. Then the sample was centrifuged at 500g for 5 minutes and the supernatant was collected. The supernatant was filtered through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 0.45 μm and 0.22 μm in turn. The obtained filtrate samples were collected and prepared with step (2). The nanoparticles (20 mg) were co-extruded through a 0.45 μm filter after co-incubation for 15 min, and the supernatant was discarded after centrifugation at 13,000 g for 25 min. mannitol + 1% arginine) was resuspended and then freeze-dried to obtain a nano-vaccine with the whole cell components of cancer cells loaded inside and the cell membrane components of mixed antigen-presenting cells loaded on the surface. The particle size of the nano-vaccine was 280 nanometers.

(6) Nano vaccines for cancer treatment

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. ^1.5 x 105 B16F10 cells were inoculated subcutaneously in the lower right back of each mouse on day 0. Activated by subcutaneous injection of 100 μg nanovaccine per mouse or subcutaneous injection of 10 million steps (4) on days 4, 7, 10, 15, 20, and 25 after melanoma inoculation, respectively of mixed antigen-presenting cells (5 million DC + 5 million B cells) or injected subcutaneously with 100 μL of PBS. In the experiment, the size of mouse tumor volume was recorded every 3 days starting from day 3. The tumor volume was calculated using the formula v=0.52×a×b ² , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm ³ , the mouse is regarded as dead and the mouse is euthanized.

(7) Analysis of nano-vaccine activation of cancer cell-specific T cells

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. On day 0, mice were inoculated with 2×10 ⁵ B16F10 cells, and on day 7, day 12, and day 17, each mouse was subcutaneously injected with 2 mg of the cancer cell-loaded whole cell fraction prepared in step (2). Nanoparticles. The mice were sacrificed on the 20th day, and the spleens of the mice were harvested to prepare a single-cell suspension of spleen cells, and B cells and T cells were separated from the mouse spleen cells by magnetic bead sorting. Co-incubate 100 μg of the nanovaccine prepared in step (5) and 1 million T cells in 5 mL of RPMI1640 complete medium for 48 hours (37°C, 5% CO ₂ ); or 10 million mixed antigens activated in step (4) Presenter cells and 1 million T cells were co-incubated in 5 mL of RPMI1640 complete medium for 48 hours (37°C, 5% CO ₂ ). The incubated cells were then collected and labeled with live dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody and IFN-γ antibody, and the proportion of IFN-γ ⁺ T cells in T cells was analyzed by flow cytometry. Specific T cells that can recognize cancer cell antigens are activated and secrete killer cytokines after recognizing cancer cell antigen epitopes. IFN-γ is the most important cytokine secreted after antigen-specific T cells are activated and activated after recognizing antigen. IFN-γ ⁺ T cells analyzed by flow cytometry are cancer cell-specific T cells that can recognize and kill cancer cells.

(8) Experimental results

As shown in a and b in Figure 4, the tumor growth rate of the mice in the PBS control group was very fast, while the antigen-presenting cell vaccine activated by nanoparticles or the nanovaccine prepared in step (6) could significantly slow down the tumor in the mice Grow and prolong tumor survival in mice and cure some mice. Moreover, the nanovaccine prepared by the nanoparticle-activated mixed antigen-presenting cells loaded with the whole-cell component is more effective than the nanoparticle-activated live antigen-presenting cell vaccine. In conclusion, the antigen-presenting cell-based cancer vaccine of the present invention has an excellent therapeutic effect on cancer.

As shown in c and d in Figure 4, the CD8 ⁺ IFN-γ ⁺ T cells and CD4 ⁺ IFN-γ ⁺ T cells that can be activated by the nanovaccine prepared in step (6) accounted for the majority of CD8 ⁺ T cells and CD4 ⁺ T cells The ratio was significantly higher than that which could be activated by nanoparticle-activated live mixed antigen-presenting cells. It can be seen that, compared with live antigen-presenting cells activated by nanoparticles, the nanovaccine of the present invention can better activate cancer cell-specific T cells with the ability to recognize and kill cancer cells.

Example 4 Nano-vaccine for the prevention of melanoma lung metastasis

This example uses a mouse melanoma lung model to illustrate how to use antigen-presenting cell-derived nanovaccine to prevent cancer metastasis. In this example, B16F10 melanoma tumor tissue was firstly lysed to prepare water-soluble antigens and water-insoluble antigens of tumor tissue; then, a nanoparticle system loaded with water-soluble antigens and water-insoluble antigens of tumor tissue was prepared. In this example, the methods of silicidation and addition of charged substances were used to increase the antigen loading, and only one round of mineralization was performed. In this example, the antigen-presenting cells are activated by using nanoparticles first, and then the nano-vaccine is prepared by using the antigen-presenting cells to prevent cancer metastasis.

(1) Lysis of tumor tissue and collection of components

1.5×10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse, the mice were sacrificed when the tumors grew to a volume of about 1000 mm ³ , and tumor tissues were excised. The tumor tissue was cut into pieces and ground, added collagenase and incubated in RPMI1640 medium for 30min, then added an appropriate amount of pure water through the cell strainer and repeated freezing and thawing 5 times, accompanied by ultrasound to destroy the lysed cells. After the cells are lysed, the lysate is centrifuged at 5000g for 5 minutes and the supernatant is taken as the water-soluble antigen soluble in pure water; add 10% sodium deoxycholate to the obtained precipitate to dissolve the precipitate. Convert the water-insoluble antigen insoluble in pure water to be soluble in 10% sodium deoxycholate aqueous solution, and mix the water-soluble antigen and the water-insoluble antigen in a mass ratio of 2:1, which is the source of antigen raw materials for preparing particles.

(2) Preparation of nanoparticles

The nanoparticles in this example and the blank nanoparticles as a control were prepared by solvent evaporation method, and appropriate modification was carried out. In the preparation process of nanoparticles, two modification methods, low temperature silicidation technology and addition of charged substances were used to improve the antigen loading capacity. . The used nanoparticle preparation material PLGA has a molecular weight of 24KDa-38KDa, and the used immune adjuvant is poly(I:C). The preparation method was as described above. In the preparation process, the antigen and adjuvant were first loaded inside the nanoparticles by double emulsion method. After loading the antigen (lysed components) inside, 100 mg of nanoparticles were centrifuged at 10,000 g for 20 minutes, and then 7 mL of PBS was used. Nanoparticles were resuspended and mixed with 3 mL of PBS containing cell lysate (60 mg/mL), followed by centrifugation at 10,000 g for 20 min, followed by 10 mL of silicate solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate, and 1.0 mM orthosilicate). HCl, pH 3.0) was resuspended and fixed at room temperature for 10 min, then fixed at -80 °C for 24 h, centrifuged with ultrapure water, washed with 3 mL of protamine (5 mg/mL) and polylysine (10 mg/mL) ) in PBS for 10min, then centrifuged at 10000g for 20min, washed with 10mL PBS solution containing lysate (50mg/mL) and acted for 10min, then centrifuged at 10000g for 20min and used 10mL supernatant containing 4% trehalose. The particles were resuspended in pure water and then freeze-dried for 48 hours; before the particles were used, they were resuspended in 7 mL of PBS, then 3 mL of adjuvanted cancer tissue lysate fraction (protein concentration 50 mg/mL) was added and allowed to act for 10 min at room temperature to obtain both internal and external load lysis. Cryosilicidation of substances and modified nanoparticle systems with addition of cationic species. The average particle size of the nanoparticle is about 350nm, and the surface potential of the nanoparticle is about -3mV; each 1mg PLGA nanoparticle is loaded with about 300μg protein or polypeptide components, and each 1mg PLGA nanoparticle is used inside and outside the poly(I:C) immune adjuvant. A total of about 0.02mg and half inside and outside.

The control nanoparticles replaced the loaded cancer cell whole cell antigen with four kinds of melanoma antigen polypeptides with equal mass, and others were the same as the nanoparticles loaded with cancer cell whole cell antigen. The control nanoparticles used 0.02 mg of poly(I:C) per 1 mg of PLGA nanoparticles, the average particle size was about 350 nm, and the surface potential of the nanoparticles was about -3 mV. The four peptide neoantigens loaded were B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL).

(3) Preparation of dendritic cells

This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare bone marrow-derived dendritic cells (BMDC). First, a 6-8-week-old C57 mouse was sacrificed by cervical dislocation. The tibia and femur of the hind leg were surgically removed and placed in PBS. The muscle tissue around the bone was removed with scissors and forceps. Cut off both ends of the bone with scissors, and then use a syringe to extract the PBS solution. The needles are inserted into the bone marrow cavity from both ends of the bone, and the bone marrow is repeatedly washed into the Petri dish. The bone marrow solution was collected, centrifuged at 400 g for 3 min, and then 1 mL of erythrocyte lysate was added. 3 mL of RPMI 1640 (10% FBS) medium was added to terminate the lysis, centrifuged at 400 g for 3 min, and the supernatant was discarded. The cells were placed in a 10 mm petri dish and cultured in RPMI 1640 (10% FBS) medium with the addition of recombinant mouse GM-CSF (20 ng/mL) at 37 degrees, 5% CO ₂ for 7 days. On day 3, the flasks were shaken gently and supplemented with the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng/mL). On the 6th day, the medium was subjected to half volume exchange. On the 7th day, a small amount of suspension and semi-adherent cells were collected, and by flow cytometry, when the ratio of CD86 ⁺ CD80 ⁺ cells in CD11c ⁺ cells was between 15-20%, the induced cultured BMDCs could be used for the next step. One step experiment.

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

The C57 mice were anesthetized and killed by dislocation. The mice were sterilized with 75% ethanol, and then a small incision was cut on the back of the mice with scissors. skin. Use scissors to remove the hindlimb along the greater trochanter at the base of the thigh of the mouse, remove the muscle tissue and place it in a petri dish containing 75% ethanol for 5 min, then replace the petri dish with a new 75% ethanol and move it into a clean bench. Transfer the ethanol-soaked leg bones into cold PBS to wash off the ethanol on the surface of the tibia and femur. This process can be repeated 3 times. Separate the cleaned femur and tibia, and cut both ends of the femur and tibia with scissors. Use a 1 mL syringe to suck out the cold induction medium to blow out the bone marrow from the femur and tibia. Repeat the blowing 3 times until the inside of the leg bone is no longer visible. to a clear red. The medium containing bone marrow cells was repeatedly pipetted with a 5mL pipette to disperse the cell clumps, then the cells were sieved using a 70μm cell strainer, transferred to a 15mL centrifuge tube, centrifuged at 1500rpm/min for 5min, the supernatant was discarded, and red blood cells were added for lysis. The solution was resuspended for 5 min and then centrifuged at 1500 rpm/min for 5 min. The supernatant was discarded and resuspended in cold prepared bone marrow macrophage induction medium (DMEM high glucose medium containing 15% L929 medium) and plated. Cells were cultured overnight to remove other fast-adhering miscellaneous cells such as fibroblasts and the like. Collect non-adherent cells and seed them into dishes or cell culture plates according to the experimental design. Macrophage colony-stimulating factor (M-CSF) was stimulated at a concentration of 40 ng/mL to differentiate myeloid cells into mononuclear macrophages. After 8 days of culture, the morphological changes of macrophages were observed under a light microscope. After 8 days, the cells were digested and collected. After incubation with anti-mouse F4/80 antibody and anti-mouse CD11b antibody for 30 min at 4°C in the dark, flow cytometry was used to identify the proportion of successfully induced macrophages.

(5) Activation of antigen presenting cells

Nanoparticles (500 μg) or polypeptide nanoparticles (500 μg) loaded with cancer cell whole cell components were co-incubated with prepared BMDC (10 million) and BMDM cells (10 million) in 15 mL high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ), the incubation system contains granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL) , IL-12 (200U/mL) and CD40 antibody (10ng/mL).

(6) Preparation of antigen-presenting cell-derived nanovaccine

The incubated BMDCs and BMDMs were collected by centrifugation at 400 g for 5 min, then the cells were washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, and the cells were resuspended in PBS water at low power (22.5°C) at 4°C. W) Sonication for 1 minute. The samples were then centrifuged at 3000g for 15 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 8000g for 15 minutes, and then after 90 minutes at 16000g. The supernatant was discarded to collect the pellet and the pellet was placed in PBS After resuspension the samples were extrusion filtered using a 0.22 μm membrane filter. Then the sample was freeze-dried in mixed lyophilized protective agent aqueous solution 1 (1% trehalose + 2% mannitol + 2% gelatin) for 48 hours to obtain nanovaccine 1, and the particle size of nanovaccine 1 was 150 nm; Mix the freeze-drying protective agent aqueous solution 2 (1% trehalose + 2% mannitol + 2% PVP) and freeze-dry for 48 hours to obtain the nano-vaccine 2, and the particle size of the nano-vaccine 2 is 150 nm.

(7) Nano-vaccine prepared by antigen-presenting cells for the prevention of cancer metastasis

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. Each mouse was injected subcutaneously with 120 μg of nanovaccine on days -35, -28, -21, -14 and -7 days before cancer modeling in mice. On the 0th day, each mouse was inoculated with 0.5×10 ⁵ B16F10 cells by intravenous injection, and the mice were sacrificed on the 14th day, and the number of melanoma tumor foci in the lungs of the mice was observed and recorded.

(8) Experimental results

As shown in Figure 5, the control mice had more and larger cancer foci, while the nanovaccine-treated mice had fewer cancers. Moreover, the nanovaccine prepared by nanoparticle-activated DCs and macrophages loaded with cancer cell whole cell antigens is more effective in preventing cancer lung metastasis than the nanovaccine prepared by nanoparticle-activated DCs and macrophages loaded with four antigen polypeptides . This indicates that the nanovaccine prepared by the antigen-presenting cells activated by nanoparticles loaded with cancer cell whole cell antigens can activate a wider spectrum and variety of cancer cell-specific T cells, and thus the more cancer cells that can be recognized and killed. The more, the better the effect of preventing cancer metastasis. Moreover, the effect of freeze-drying the vaccine prepared by using the mixed lyophilized protective agent 1 is better than that of the vaccine prepared by using the mixed lyophilized protective agent 2.

Example 5 Micron-particle-activated antigen-presenting cells to prepare micro-vaccine for cancer prevention

In this example, the whole cell fraction of B16F10 melanoma cancer cells was firstly lysed with 6M guanidine hydrochloride. Whole cell antigens are contained in the whole cell fraction. Then, using PLGA as the microparticle skeleton material, poly(I:C), CpGM362 and CpGBW006 as immune adjuvants to prepare microparticles loaded with cancer cell whole cell antigens, and use the microparticles to activate antigen-presenting cells, and then use Antigen-presenting cells prepare micro-vaccine for cancer prevention.

(1) Lysis of cancer cells

The cultured B16F10 melanoma cancer cell line was collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, then the cancer cells were resuspended and lysed with 6M guanidine hydrochloride, and the cancer cell whole cell antigen was lysed and dissolved in 6M Guanidine hydrochloride is the source of antigen raw material for the preparation of microparticle system.

(2) Preparation of microparticle system

In this example, the microparticles were prepared by the double emulsion method. The molecular weight of the microparticle preparation material PLGA was 38KDa-54KDa, and the immunoadjuvants used were poly(I:C), CpGM362 and CpG BW006. The preparation method was as described above. In the preparation process, the whole-cell antigen and adjuvant of cancer cells were first loaded into the microparticles by double emulsion method, and then 100 mg of microparticles were centrifuged at 10,000 g for 15 minutes, and then resuspended with 10 mL of 4% trehalose aqueous solution. The microparticles were freeze-dried for 48 hours and used for later use. The average particle size of the microparticles is about 2.50μm, and the surface potential of the microparticles is about -2mV; each 1mg PLGA microparticle is loaded with about 100μg protein or polypeptide components, and each 1mg PLGA microparticle is loaded with poly(I:C), CpGM362 and CpG BW006 Each 0.02 mg of immune adjuvant. The microparticles were defined as the control microvaccine 3 in the cancer prevention experiment in the following step (6).

(3) Preparation of antigen presenting cells

In this example, BMDC and B cells were mixed with antigen presenting cells. The preparation method of BMDC is the same as that in Example 1. After the mice were sacrificed, the mouse spleen was harvested, and the spleen was minced and passed through a cell mesh to prepare a single-cell suspension of spleen cells, and then CD19 ⁺ B cells were separated from the single-cell suspension of spleen cells by magnetic bead sorting. . Mixing BMDC and B cells according to the quantity ratio of 1:1 is the mixed antigen-presenting cells.

(4) Activation of antigen presenting cells

Microparticles (500 μg) loaded with cancer cell whole cell fractions were co-incubated with mixed antigen-presenting cells (10 million) in 20 mL of high glucose DMEM complete medium for 72 hours (37° C., 5% CO ₂ ). The incubation system contains cytokine components 1: IL-2 (500U/mL), IL-7 (200U/mL), IL-15 (200U/mL), and the activated antigen-presenting cells are activated mixed antigens Presenting cells 1; or the incubation system contains control cytokine combination 2: IL-4 (500U/mL), IL-6 (200U/mL), IL-10 (200U/mL), activated antigen-presenting cells 2 for activated mixed antigen presenting cells.

(5) Preparation of antigen-presenting cell-derived micro-vaccine

The incubated pooled antigen-presenting cells were collected by centrifugation at 400 g for 5 min, then washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, resuspended in PBS water at low power at 4°C (22.5W) sonicated for 2 minutes. The samples were then centrifuged at 3000g for 15 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 8000g for 15 minutes, and then after 90 minutes at 16000g. The supernatant was discarded to collect the pellet and the pellet was placed in PBS After resuspension, the mixture was filtered and extruded through a 0.22 μm membrane to obtain the cell membrane fraction of the mixed antigen-presenting cells. Mix 10 mL of the mixed antigen-presenting cell membrane fraction (4 mg) with 100 mg of the microparticles prepared in step (2), then sonicate at 4°C with low power (22.5W) for 1 minute, and then use a homogenizer to stir for 5 minutes, and then a total of Incubate for 10 minutes, then centrifuge at 12,000 g for 10 minutes, discard the supernatant, and resuspend the pellet in 10 mL of lyoprotectant (2% trehalose + 2% mannitol + 1% arginine in water) to freeze dry 48 Backup after hours. Among them, the micro-vaccine prepared from the activated mixed antigen-presenting cells 1 is the micro-vaccine 1 with a particle size of 2.51 μm; the micro-vaccine prepared from the activated mixed antigen-presenting cells 2 is the micro-vaccine 2 with a particle size of 2.51 μm μm.

(6) Micron vaccine for cancer prevention

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. Each mouse was vaccinated with 100 μg of the micro-vaccine (Micro-vaccine 1, Or micro-vaccine 2 or micro-particles prepared in step (2) are defined as micro-vaccine 3) or PBS. Each mouse was inoculated subcutaneously with 1.5×10 ⁵ B16F10 cells on day 0, and the size of mouse tumor volume was recorded every 3 days starting from day 3. The tumor volume was calculated using the formula v=0.52×a×b ² , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm ³ , the mouse is regarded as dead and the mouse is euthanized.

(7) Experimental results

As shown in Figure 6, the tumors of the mice in the PBS control group grew rapidly, and the mice died quickly. However, the tumor growth rate of mice treated with the micro-vaccine was significantly slower and the survival time was significantly prolonged. Moreover, the effect of the micro-vaccine 1 prepared by using the mixed antigen-presenting cells 1 activated by the cytokine combination 1 was significantly better than that of the micro-vaccine 2 prepared by using the mixed antigen-presenting cells 2 activated by the cytokine component 2. Moreover, the effect of micro-vaccine 2 is also significantly better than that of micro-vaccine 3, which only loads the whole cell components of cancer cells inside and does not load any antigen-presenting cell membrane components on the surface. It can be seen that loading the cell membrane components of the antigen-presenting cells on the surface can significantly improve the efficacy of the micro-vaccine; moreover, adding specific cytokines when the micro-particles activate the antigen-presenting cells can improve the micro-vaccine prepared by the activated antigen-presenting cells. curative effect.

Example 6 Nano-vaccine for cancer prevention

In this example, B16F10 melanoma tumor tissue was firstly lysed with 8M urea, and the tumor tissue lysate fraction was dissolved. Then, using PLGA as nanoparticle skeleton material, using Poly(I:C) and CpG2395 as immune adjuvants to prepare nanoparticles loaded with cancer cell whole cell antigens, using nanoparticles to activate antigen-presenting cells and then using antigen-presenting cells to prepare Nanovaccine for cancer prevention.

(1) Collection and lysis of tumor tissue

1.5×10 ⁵ B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse, the mice were sacrificed when the tumors grew to a volume of about 1000 mm ³ , and tumor tissues were excised. The tumor tissue was cut into pieces and ground, and an appropriate amount of 8M urea was added to lyse the cells through a cell strainer, and the cell lysate was dissolved. The above is the source of antigen raw materials for the preparation of nanoparticle systems.

(2) Preparation of nanoparticle system

The nanoparticles in this example and the blank nanoparticles as a control were prepared by solvent evaporation method. The adopted nanoparticle preparation material PLGA has a molecular weight of 7KDa-17KDa, the adopted immunoadjuvant is poly(I:C) and CpG2395, and the lysate components and adjuvant are encapsulated in the nanoparticle. The preparation method was as described above. In the preparation process, the lysate components and adjuvants were first loaded into the nanoparticles by double emulsion method, and then 100 mg of nanoparticles were centrifuged at 12,000 g for 20 minutes, and 10 mL of ultrapure 4% trehalose was used. After resuspension in water, freeze-dry for 48 hours, and obtain freeze-dried powder for later use. The average particle size of the nanoparticle is about 270nm, and the surface potential of the nanoparticle is about -3mV; each 1mg PLGA nanoparticle is loaded with about 110μg protein or polypeptide components, and the poly(I:C) and CpG2395 used in each 1mg PLGA nanoparticle are immune Adjuvants were each 0.02 mg.

The preparation materials and preparation methods of the control nanoparticles are the same, each 1mg PLGA nanoparticles is loaded with 0.02mg of poly(I:C) and CpG2395, the particle size is about 270nm, the surface potential of the nanoparticles is about -3mV, and each 1mg PLGA nanoparticles is loaded with about 110μg peptide components. The four peptide neoantigens loaded were B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL).

(3) Preparation of DC and B cells

After C57BL/6 was sacrificed, the lymph nodes of the mice were harvested to prepare a single-cell suspension of mouse lymph nodes, and then CD11c ⁺ DC and CD19 ⁺ B cells were sorted from the single-cell suspension of lymph node cells by flow cytometry.

(4) Activation of antigen presenting cells

Nanoparticles (1 mg) or polypeptide nanoparticles (1 mg) loaded with whole cell components of cancer cells were co-incubated with DC (20 million) and B cells (20 million) in 20 mL of high glucose DMEM complete medium for 72 hours (37 °C, 5% CO ₂ ), or co-incubate nanoparticles (500 μg) loaded with cancer cell whole cell fractions with DCs (10 million) in 20 mL of high glucose DMEM complete medium for 72 hours (37 ° C, 5% CO _{2 )} . ); the incubation system contains granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL) ) and CD86 antibody (10ng/mL).

(5) Preparation of antigen-presenting cell-derived nanovaccine

Incubated DC and B cells (10 million DC cells + 10 million B cells) or only DC cells (20 million) were collected by centrifugation at 400 g for 5 min, followed by 4°C phosphate buffer containing protease inhibitors The cells were washed twice with the solution (PBS), and the cells were resuspended in PBS water and disrupted with a homogenizer at 2000 rpm for 25 minutes at 4°C. Then the sample was centrifuged at 3000g for 15 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 8000g for 15 minutes, and then the obtained supernatant was compared with the corresponding activated antigen-presenting cells prepared in step 3. Nanoparticles (30mg) were mixed and extruded using 0.45μm membrane filtration after 50W ultrasound for 3 minutes, and then the supernatant was discarded after centrifugation at 12000g for 30 minutes to collect the precipitate, and the precipitate was resuspended in PBS to obtain a nanovaccine. The particle size of the nanovaccine is 310 nm.

(6) Antigen-presenting cell-derived nanovaccine for cancer prevention

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. Each mouse was injected subcutaneously with 40 μg of nanovaccine or PBS on days -42, -28 and -14 days before inoculation with cancer cells. On day 0, 1.5×10 ⁵ B16F10 cells were subcutaneously injected into each mouse, and the methods for monitoring the tumor volume and survival period of the mice were the same as those in Example 5.

(7) Experimental results

As shown in Figure 7, the tumors of the control mice grew rapidly, while the tumors of the mice treated with the nanovaccine significantly slowed down or disappeared. Moreover, the preventive effect of the nanovaccine prepared from DCs and B cells activated by nanoparticles loaded with cancer cell whole cell antigens was better than that prepared by nanoparticles activated DCs and B cells loaded with four antigen polypeptides. Moreover, the nanovaccine prepared by the mixture of DCs and B cells activated by nanoparticles loaded with cancer cell whole cell antigens is more effective than the nanovaccine prepared by DCs activated by nanoparticles loaded with cancer cell whole cell antigens, indicating that a variety of nanoparticles activated The nano-vaccine prepared by antigen-presenting cells is more effective. This may be because some of the components in the activated B cells can help enhance the activation of cancer cell-specific T cells by the nanovaccine after being included in the nanovaccine prepared by antigen-presenting cells.

Example 7 Nano-vaccine prepared by antigen-presenting cells is used to treat colon cancer

This example uses MC38 mouse colon cancer as a cancer model to illustrate how to use the nanovaccine prepared from antigen-presenting cells to treat colon cancer. First, colon cancer tumor tissue and lung cancer cancer cells were lysed to prepare a mixture of water-soluble antigens (mass ratio 1:1) and water-insoluble antigens (mass ratio 1:1). Mass ratio 1:1 mix. Then, nanoparticles were prepared with PLA as nanoparticle skeleton material, CpGSL03 and Bacille Calmette-Guérin (BCG) as immune adjuvants, and the nanoparticles were used to activate antigen-presenting cells in vitro to prepare a nanovaccine to treat colon cancer.

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

2×10 ⁶ MC38 cells were inoculated subcutaneously on the back of each C57BL/6 mouse. The mice were sacrificed when the tumors grew to a volume of about 1000 mm ³ , respectively, and the tumor tissue was excised. The tumor tissue was cut into pieces and ground, and an appropriate amount of pure water was added through a cell strainer and freeze-thawed for 5 times. Ultrasound was also used to destroy the lysed cells. After the cells are lysed, the lysate is centrifuged for 5 minutes at a rotating speed greater than 5000g and the supernatant is taken as the water-soluble antigen soluble in pure water; 5% sodium dodecylbenzenesulfonate ( The SDS) aqueous solution dissolves the precipitated part to convert the water-insoluble antigen that is insoluble in pure water into soluble in the aqueous solution.

The cultured LLC lung cancer cell line was collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, then the cells were resuspended in ultrapure water and freeze-thawed 5 times, and sonicated to destroy the lysed cells . After the cells were lysed, the lysate was centrifuged at 3000g for 6 minutes, and the supernatant was taken to be the water-soluble antigen soluble in pure water; adding 5% SDS aqueous solution to the obtained precipitate to dissolve the precipitate, the insoluble antigen was Water-insoluble antigens in pure water are converted to soluble in aqueous SDS.

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

(2) Cleavage of BCG and collection of components

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

(3) Preparation of nanoparticles

In this example, the nanoparticles were prepared by solvent evaporation method. The molecular weight of the nanoparticle preparation material PLA is 20KDa, and the used immune adjuvants are CpGSL03 and BCG, and the adjuvants are distributed both inside and on the surface of the nanoparticles. The preparation method was as described above. In the preparation process, the lysate mixture and adjuvant were first loaded inside the nanoparticles by double emulsion method, and then 100 mg of nanoparticles were centrifuged at 10,000 g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose was used. Freeze-dried for 48h after resuspension. Before use, 20 mg of nanoparticles were resuspended in 0.9 mL of PBS and incubated with 0.1 mL of the sample containing the lysate mixture (80 mg/mL) and adjuvant for 5 minutes at room temperature before use. The average particle size of the nanoparticles is about 280nm, and the surface potential of the nanoparticles is about -3mV; each 1 mg of PLGA nanoparticles is loaded with about 140 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles contains 0.04 mg of CpGSL03 and 0.04 mg of BCG immune adjuvant.

(3) Preparation of antigen presenting cells

Peripheral blood of mice was collected after sacrifice of C57BL/6, peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood, and CD11c ⁺ DC and CD19 ⁺ B cells were sorted from PBMC using flow cytometry. In this example, both BMDC and BMDM were used as antigen-presenting cells. The preparation method of BMDC is the same as that of Example 2, and the preparation method of BMDM is the same as that of Example 4.

(4) Activation of antigen presenting cells

Nanoparticles (1000 μg) loaded with whole cell fractions of cancer cells were mixed with peripheral blood-derived DC (5 million), BMDC (5 million), BMDM (5 million) and B cells (5 million) in 20 mL RPMI1640 Co-incubated for 48 hours in complete medium (37°C, 5% CO ₂ ), or nanoparticles loaded with whole cell fractions of cancer cells (500 μg) with peripheral blood-derived DCs (10 million) and BMDCs (10 million) Incubate in RPMI1640 complete medium for 72 hours (37°C, 5% CO ₂ ); the incubation system contains granulocyte-macrophage colony-stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL) , IL-7 (200U/mL), IL-12 (200U/mL) and CD86 antibody (10ng/mL).

(5) Preparation of antigen-presenting cell-derived nanovaccine

Peripheral blood-derived DCs, B cells, and BMDMs after incubation were collected by centrifugation at 400 g for 5 min (DC 5 million + B cells 5 million + BMDM 5 million) or only DC and B cells (DC 7.5 million + B cells 750 10,000 cells), cells were then washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, resuspended in PBS water and treated in a high pressure homogenizer (5000 bar) for 5 minutes. Then the sample was centrifuged at 2000g for 15 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 8000g for 15 minutes, and the supernatant was co-incubated with the corresponding nanoparticles (50mg) prepared in step 3 at room temperature After 6 hours, it was filtered and extruded using a 0.45 μm membrane, and then centrifuged at 13,000 g for 20 minutes to collect and discard the supernatant to collect the precipitate. After resuspending the precipitate in PBS, the nanovaccine was obtained. The particle size of the nanovaccine was 320 nm.

(6) Nano vaccines for cancer treatment

Select 6-8 week old female C57BL/6 as model mice to prepare colon cancer tumor-bearing mice. Each mouse was inoculated subcutaneously with 2×10 ⁶ MC38 cells on day 0, and 50 μg of nanovaccine was subcutaneously injected into each mouse on days 4, 7, 10, 15 and 20, respectively. The size of mouse tumor volume was recorded every 3 days starting from day 3. The tumor volume was calculated using the formula v=0.52×a×b ² , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm ³ , the mouse is regarded as dead and the mouse is euthanized.

(7) Experimental results

As shown in Figure 8, the tumors of the control mice grew rapidly, while the tumor growth rate of the mice treated with the nanovaccine was significantly slowed down or the tumors disappeared. Moreover, the nanovaccine prepared by the mixture of nanoparticle-activated DCs, B cells and macrophages is more effective than the nanovaccine prepared by nanoparticle-activated DC and B cells, indicating that a variety of nanoparticle vaccines prepared by nanoparticle-activated antigen-presenting cells Vaccines work better. In conclusion, the nano-vaccine prepared by the antigen-presenting cells of the present invention has a good therapeutic effect on colon cancer.

Example 8 Nano-vaccine prepared by antigen-presenting cells is used to treat 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 tissue, and then use antigen-presenting cells to prepare nanovaccine and use the nanovaccine Treatment of melanoma. In this example, B16F10 melanoma tumor tissue and LLC lung cancer tumor tissue were firstly lysed to prepare a water-soluble antigen mixture (mass ratio 3:1) and a water-insoluble antigen mixture (3:1) of tumor tissue. Using PLGA as the nanoparticle skeleton material and Poly ICLC and CpG2395 as immune adjuvants, the nanoparticles loaded with the above mixture were prepared, and then the nanoparticles were used to activate antigen-presenting cells, and the antigen-presenting cells were used to prepare nano-vaccine for cancer treatment.

(1) Lysis of tumor tissue and collection of components

1.5×10 ⁵ B16F10 cells or 2×10 ⁶ LLC lung cancer cells were subcutaneously inoculated on the back of each C57BL/6 mouse, and the mice were sacrificed when the tumor grew to a volume of about 1000 mm ³ , respectively, and the tumor tissue was excised. The methods of tumor lysis and fraction collection were the same as in Example 1. The water-soluble antigens from melanoma tumor tissue and from lung cancer tumor tissue and the original water-insoluble antigens dissolved in 8M urea were mixed in a ratio of 3:1 to obtain the respective mixtures. Then, the water-soluble antigen mixture and the water-insoluble antigen mixture are mixed in a mass ratio of 1:1 to prepare the antigen source of the nanoparticles.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by the double emulsion method. The used nanoparticle preparation material PLGA has a molecular weight of 24KDa-38KDa, and the used immune adjuvants are Poly ICLC and CpG2395. The preparation method is as described above. First, lysate components and adjuvants were loaded into PLGA nanoparticles, then 100 mg of nanoparticles were centrifuged at 10,000 g for 25 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours before use. The average particle size of the nanoparticles is about 300nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with about 80 μg of protein and polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.04 mg of poly ICLC and 0.04 mg of CpG2395.

(3) Preparation of antigen presenting cells

DCs are mixed DCs derived from peripheral blood and BMDCs. The preparation method of BMDC is the same as above. After killing C57BL/6 mice, peripheral blood was collected, peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood, and CD11c ⁺ DCs were sorted from PBMCs using flow cytometry. The quantity ratio is 1:1 mixed, that is, the mixed DC used in this example.

(4) Activation of antigen presenting cells

Nanoparticles (800 μg) loaded with cancer cell whole cell components and mixed DCs (10 million) were co-incubated in 15 mL of high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ), and the incubation system contained granulocytes - Macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL) and CD86 antibody (10ng/mL) ).

(5) Preparation of antigen-presenting cell-derived nanovaccine

Incubated DCs (10 million) were collected by centrifugation at 400 g for 5 min, then cells were washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, and cells were resuspended in PBS water at 4°C low Power (20 W) was sonicated for 1 minute and then treated with a homogenizer at 1000 rpm for 3 minutes. Then the sample was centrifuged at 3000g for 10 minutes and the supernatant was collected. The supernatant was centrifuged at 8000g for 5 minutes and the supernatant was collected. The supernatant was mixed with the nanoparticles (50mg) prepared in step (2) and DSPE-PEG - CD32 monoclonal antibody (50μg) was mixed, sonicated at 100W for 2 minutes, filtered through a 0.45μm membrane, and then centrifuged at 15,000g for 30 minutes to collect and discard the supernatant to collect the precipitate, and resuspend the precipitate in PBS to obtain a nanovaccine 1. The particle size of the nano-vaccine is 280 nanometers.

Incubated DCs (10 million) were collected by centrifugation at 400 g for 5 min, then cells were washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, and cells were resuspended in PBS water at 4°C low Power (20 W) was sonicated for 1 minute and then treated with a homogenizer at 1000 rpm for 3 minutes. The samples were then centrifuged at 3000g for 10 minutes and the supernatant was collected. The supernatant was centrifuged at 8000g for 5 minutes and the supernatant was collected. The supernatant was mixed with DSPE-PEG-CD32 mAb (50μg) and then sonicated at 100W. 2 minutes, and then centrifuged at 18,000 g for 90 minutes to collect and discard the supernatant to collect the precipitate. After resuspending the precipitate in PBS, nanovaccine 2 was obtained, and the particle size of the nanovaccine was 150 nm.

(6) Nano vaccines for cancer treatment

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. Each mouse was inoculated subcutaneously with 1.5×10 ⁵ B16F10 cells on day 0, and mice were injected subcutaneously with 100 μg of nanovaccine on days 4, 7, 10, 15, and 20, respectively. Mouse tumor volume and survival were monitored as above.

(7) Experimental results

As shown in Figure 9, the tumors of the mice in the PBS control group grew rapidly. Compared with the control group, the tumor growth rate of the mice treated with nanovaccine 1 and nanovaccine 2 prepared by nanoparticle-activated DCs was significantly slower. And some mice tumors disappeared and recovered. Moreover, the effect of nanovaccine 1, which is loaded with whole cell components of cancer cells and surface-loaded with DC cell membrane components, is significantly better than that of nanovaccine 2 prepared only from DC cell membrane components. The therapeutic effect, and the internal loading of whole cell components of cancer cells can make the nano-vaccine more effective. In the nanovaccine of this example, CD32 monoclonal antibody is used as the active target. In practical applications, any target with a target such as mannose, CD3 antibody, CD56 antibody, mannan, CD205 monoclonal antibody, and CD19 monoclonal antibody can also be used. Targeting the ability to target cells.

Example 9 micron vaccine for breast cancer prevention

This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use 8M urea to dissolve 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 are used to prevent breast cancer.

(1) Lysis of cancer cells

The cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells were inactivated and denatured by ultraviolet rays and high temperature heating respectively, and then the breast cancer cells were lysed with an appropriate amount of 8M urea, and the lysate was dissolved as the raw material source for preparing the particle system.

(2) Preparation of microparticle system

In this example, the microparticles were prepared by the double emulsion method, the molecular weight of the microparticle skeleton material PLGA was 38KDa-54KDa, and the immunoadjuvants used were CpG2395 and Poly(I:C). During preparation, the microparticles loaded with lysate components and adjuvants were prepared by the double emulsion method, and then 100 mg of microparticles were centrifuged at 9000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use. The average particle size of the microparticle system is about 2.18 μm, and the surface potential is about -6mV; every 1 mg of PLGA microparticles is loaded with about 110 μg of protein or polypeptide components, and each of CpG2395 and Poly(I:C) is loaded with 0.03 mg.

(3) Preparation of BMDC and B cells

The preparation of BMDC is the same as that of Example 2. The preparation of B cells was the same as in Example 3.

(4) Activation of antigen presenting cells

Microparticles (1000 μg) loaded with cancer cell whole cell fractions were co-incubated with BMDC (10 million) in 15 mL high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ); Microparticles (1000 μg) of cell fractions were incubated with B cells (10 million) in 15 mL high glucose DMEM complete medium for 72 hours (37°C, 5% CO ₂ ); the incubation system contained granulocyte-macrophage colonies Stimulatory factor (GM-CSF, 2000 U/mL), IL-2 (500 U/mL), IL-7 (200 U/mL), IL-12 (200 U/mL) and CD86 antibody (10 ng/mL).

(5) Preparation of antigen-presenting cell-derived micro-vaccine

Incubated DCs (10 million) or B cells (10 million) were collected by centrifugation at 400 g for 5 min, then resuspended by washing the cells twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors Low power (10 W) sonication at 4°C for 10 minutes after PBS water. Then the sample was filtered through membranes with pore sizes of 10 μm, 5 μm, 2 μm, 1 μm, 0.45 μm and 0.22 μm in turn to collect the filtrate. The filtrate was mixed with the microparticles (40 mg) prepared in step 2, and then sonicated at 50 W for 1 minute for a total of After incubation for 10 minutes, the supernatant was discarded after centrifugation at 9000g for 15 minutes to collect the pellet, and the pellet was resuspended in PBS to obtain a micro-vaccine with a particle size of 2.20 μm.

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

Female BALB/c aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. On the -35th, -28th, -21st, -14th and -7th days before the mice were inoculated with cancer cells, each mouse in the vaccine group was inoculated with 80 μg of micron vaccine; the PBS control group was inoculated with 100 μL of PBS. Each mouse was inoculated with 1×10 ⁶ 4T1 cells by subcutaneous injection on day 0, and the size of mouse tumor volume was recorded every 3 days from day 3 onwards. The mouse tumor monitoring method is the same as above.

(7) Experimental results

As shown in Figure 10, compared with the PBS control group, the tumor growth rate of the mice treated with the micro-vaccine prepared from the microparticle-activated antigen-presenting cells was significantly slower and the survival period was significantly prolonged. Moreover, the micro-vaccine prepared by microparticle-activated DC was more effective than the micro-vaccine prepared by microparticle-activated B cells. It can be seen that the micron vaccine of the present invention has a preventive effect on breast cancer.

Example 10 Nano-vaccine for the prevention of cancer metastasis

(1) Lysis of tumor tissue and cancer cells

After collecting mouse B16F10 melanoma tumor tissue and cultured cancer cells, 8M urea was used to lyse and dissolve the cancer cell whole cell fractions from the tumor tissue and cancer cells, and then the tumor tissue fraction and cancer cell fraction were in a mass ratio of 1:2 Miscible.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by solvent evaporation method. The molecular weight of the nanoparticle preparation material PLGA was 24KDa-38KDa, and poly ICLC and CpG1018 were used as immune adjuvants. The preparation method was as described above. In the preparation process, the cell components and adjuvants were first loaded inside the nanoparticles by double emulsion method, and then 100 mg of nanoparticles were centrifuged at 10,000 g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose was used. After resuspension, freeze-dried for 48h before use. The average particle size of the nanoparticles is about 250 nm; each 1 mg of PLGA nanoparticles is loaded with about 90 μg of protein and polypeptide components, and loaded with 0.03 mg each of polyICLC and CpG1018.

(3) Preparation of DC

This example uses a mixed DC of BMDC and peripheral blood DC. The preparation method of BMDC is the same as that in Example 2. The preparation method of peripheral blood DC is to collect the peripheral blood of the mice after killing the mice, then separate the mouse PBMCs, and then use flow cytometry to separate the CD11c ⁺ DCs from the mouse PBMCs. 1:1 mix.

(4) Activation of antigen presenting cells

Nanoparticles (1000μg) and DCs (10 million) loaded with cancer cell whole cell components were co-incubated in 15mL high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ); the incubation system contained granulocytes- Macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and CD86 antibody (10ng/mL) .

(4) Preparation of antigen-presenting cell-derived nanovaccine

After incubation, peripheral blood-derived DCs and BMDCs were collected by centrifugation at 400 g for 5 min, then the mixed cells were washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, and the cells were resuspended in PBS water at 4 °C and 20W sonication for 5 min. The samples were then centrifuged at 3000g for 5 minutes and the supernatant was collected. The supernatant was centrifuged at 8000g for 15 minutes and the precipitate was discarded and the supernatant was collected. After centrifugation at 18000g for 90 minutes, the supernatant was filtered with a 0.22 μm filter and the supernatant was discarded. Then, 10 mL of the cell membrane fraction (5 mg) was mixed with 100 mg of the nanoparticles prepared in step (2), incubated at room temperature for 15 minutes, and then passed through 0.45 Filter and extrude with a μm filter membrane, and then centrifuge at 14,000 g for 25 minutes to obtain a nano-vaccine with surface-loaded antigen-presenting cell membrane components. The above-mentioned nano-vaccine was resuspended with 10 mL of lyophilized protective agent 1 (2% trehalose+2% mannitol+1% arginine aqueous solution) and then freeze-dried for 48 hours. The obtained nano-vaccine was Nano-vaccine 1, and the particle size was 260 nm; The above nanovaccine was resuspended with 10 mL of lyophilized protective agent 2 (2% trehalose + 2% mannitol + 1% glycine) and then freeze-dried for 48 hours to obtain nanovaccine 2 with a particle size of 260 nm. Nanovaccine 1 and Nanovaccine 2 were stored at room temperature for 360 days before use.

The newly lyophilized nanovaccine 1 and the nanovaccine 1 stored at room temperature for 360 days were analyzed for the particle size of the nanoparticles respectively. The results showed that after 360 days of storage at room temperature, the particle size of the nanovaccine 1 was 260 nm, indicating that the nanovaccine 1 has good stability. . In addition, the newly prepared nanovaccine 1 and the nanovaccine 1 stored at room temperature for 360 days were used to analyze the efficacy of preventing cancer metastasis.

(5) Nano-vaccine for the prevention of cancer metastasis

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. Each mouse was vaccinated with 100 μg of nanovaccine (freshly prepared on days -42, -35, -28, -21, -14 and -7 days before inoculation with cancer cells) Nanovaccine 1, or Nanovaccine 1 after storage at room temperature for 360 days, or Nanovaccine 2) after storage at room temperature for 360 days, or PBS. On the 0th day, each mouse was inoculated with 0.5×10 ⁵ B16F10 cells by intravenous injection, and the mice were sacrificed on the 14th day, and the number of melanoma tumor foci in the lungs of the mice was observed and recorded.

(6) Experimental results

As shown in Figure 11, the nanovaccine prepared from nanoparticle-activated DCs can effectively prevent cancer metastasis. Moreover, after being placed at room temperature for 360 days, the properties of the nanovaccine are stable and the effect is consistent. Moreover, the effect of nanovaccine 1 prepared by using 2% trehalose+2% mannitol+1% arginine as lyoprotectant is better than using 2% trehalose+2% mannitol+1% glycine as lyoprotectant The prepared nanovaccine 2; the nanovaccine 1 after being stored at room temperature for 360 days and the newly prepared nanovaccine 1 have no significant difference in efficacy, which shows that the nanovaccine of the present invention can maintain long-term stability, and the freeze-drying process may destroy the vaccine Its structure makes the vaccine unstable and reduces its activity. The specific freeze-drying protective agent of the present invention can improve the therapeutic effect of the prepared nano-vaccine when it undergoes freeze-drying and long-term storage.

Example 11 Nano-vaccine for the treatment of pancreatic cancer

In this example, mice Pan02 pancreatic cancer tumor tissue and MC38 colon cancer tumor tissue split fractions were loaded on nanoparticles at a ratio of 3:1, and the nanoparticle-activated antigen-presenting cells were used to prepare nanovaccine for treatment Pancreatic cancer. In the experiment, mouse pancreatic cancer and colon cancer tumor tissues were obtained and lysed to prepare water-soluble antigens and original water-insoluble antigens dissolved in 6M guanidine hydrochloride. When preparing the particles, PLGA is used as the nanoparticle skeleton material, and BCG is used as the adjuvant to prepare the nanoparticles, and then the nanoparticle is used to activate the antigen-presenting cells to prepare the nanovaccine.

(1) Lysis of tumor tissue and collection of components

Each C57BL/6 mouse was inoculated subcutaneously with 2 x 10 ⁶ MC38 colon cancer cells or 1 x 10 ⁶ Pan02 pancreatic cancer cells in the armpit, when the inoculated tumors in each mouse grew to a volume ^of approximately 1000 mm, respectively Mice were sacrificed and tumor tissues were excised. After the tumor tissue was mechanically disrupted, it was digested in RPMI1640 complete medium containing 5 mg/mL type IV collagenase, 5 mg/mL type I collagenase, 1.5 mg/mL hyaluronidase and 1.5 mg/mL DNase for 20 min at 37°C , collect the cell solution and filter through 300 mesh sterile nylon mesh to remove small debris. Then, pure tumor cells were obtained by negative sorting with CD45 magnetic beads. The obtained tumor cells were repeatedly frozen and thawed in ultrapure water for 3 times, and the tumor cells were lysed by ultrasonic, and then centrifuged at 3000g for 5 minutes. It is a water-insoluble component. The water-soluble component is a 3:1 mixture of the water-soluble component of pancreatic cancer tumor tissue and the water-soluble component of colon cancer tumor tissue; the water-insoluble component is the water-insoluble component of pancreatic cancer tumor tissue and the water-insoluble component of colon cancer tumor tissue. A 3:1 mixture of sexual components. The water-soluble component mixture and the water-insoluble component mixture are mixed in a mass ratio of 1:1. The method of lysing and dissolving BCG is the same as that of tumor tissue, and the water-soluble component and the water-insoluble component are mixed in a mass ratio of 1:1. The whole-cell water-soluble fraction contains whole-cell water-soluble antigens, and the whole-cell water-insoluble fraction contains whole-cell water-insoluble antigens.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by the double emulsion method. The used nanoparticle preparation material PLGA has a molecular weight of 7KDa-17KDa, the used immune adjuvant is BCG, and BCG is encapsulated in the nanoparticle. The preparation method was as described above. In the preparation process, the lysate components and adjuvants were first loaded into the nanoparticles by double emulsion method, and then 100 mg of nanoparticles were centrifuged at 12,000 g for 20 minutes, and 10 mL of ultrapure 4% trehalose was used. After resuspension in water, freeze-dry for 48 hours, and obtain freeze-dried powder for later use. 20 mg of nanoparticles were dissolved in 0.9 mL of PBS prior to nanoparticle injection, mixed with 0.1 mL of sample containing lysate (80 mg/mL) and used after 10 min at room temperature. The average particle size of the nanoparticles is about 260 nm, and the surface potential of the nanoparticles is about -4mV; each 1 mg of PLGA nanoparticles is loaded with about 130 μg of protein and polypeptide components, and 0.08 mg of BCG immune adjuvant is used per 1 mg of PLGA nanoparticles.

(3) Preparation of antigen presenting cells

In this example, BMDC or BMDM were used as antigen-presenting cells. The preparation method of BMDC is the same as that of Example 2, and the preparation method of BMDM is the same as that of Example 4.

(4) Activation of antigen presenting cells

Nanoparticles (1000 μg) loaded with cancer cell whole cell fractions were co-incubated with BMDC (10 million) or with BMDM (10 million) in 15 mL high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ) ; The incubation system contains GM-CSF (2000U/mL), M-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN-γ (500 U/mL) and CD80 antibody (10 ng/mL).

(4) Preparation of antigen-presenting cell-derived nanovaccine

Incubated BMDCs or BMDMs were collected by centrifugation at 400 g for 5 min, then washed three times by centrifugation at 1200 rpm for 3 min in 30 mM pH 7.0 Tris-HCl buffer containing 0.0759 M sucrose and 0.225 M mannitol, and then treated with phosphatase inhibitor and protease inhibitor Antigen presenting cells were mechanically disrupted using a homogenizer at 2000 rpm for 10 minutes in the presence of the agent, followed by sonication at 20W for 1 minute. Cell membranes obtained after centrifugation were washed with a solution of 10 mM Tris-HCl pH 7.5 and 1 mM EDTA. Then, the samples were filtered through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 2 μm, and 0.45 μm in turn. The filtrate was centrifuged at 12,000 g for 25 minutes, and the supernatant was discarded to collect the precipitate. The precipitate was placed in physiological saline containing 4% mannitol. The nano-vaccine is obtained after being resuspended and then freeze-dried, and the particle size of the nano-vaccine is 260 nm.

(5) Nano-vaccine for cancer treatment

The 6-8 week old female C57BL/6 was selected as model mice to prepare pancreatic cancer tumor-bearing mice. Each mouse was inoculated subcutaneously with ¹ x 106 Pan02 cells on day 0, and mice were injected subcutaneously with 400 μg of Nanovaccine or PBS. The size of mouse tumor volume was recorded every 3 days starting from day 3. The tumor volume calculation method and the mouse survival monitoring method are the same as above.

(6) Experimental results

As shown in Figure 12, compared with the PBS control group, the nanovaccine prepared by nanoparticle-activated DCs or macrophages can effectively treat pancreatic cancer, and the nanovaccine prepared by using nanoparticle-activated DCs is more effective.

Example 12 Nano-vaccine for cancer prevention

This example uses mannose as the target to illustrate how to use antigen-presenting cells activated by active targeting nanoparticles to prepare nano-vaccine for cancer prevention. In actual application, the specific dosage form, adjuvant, administration time, administration frequency, and administration schedule can be adjusted according to the situation. The nanoparticle system can be taken up into dendritic cells through mannose receptors on the surface of DCs, and then activated DCs to prepare nanovaccine for cancer prevention.

(1) Lysis of cancer cells

After collecting the cultured B16F10 cancer cells, 8M urea was used to lyse and lyse the cancer cell whole cell fractions derived from the cancer cells.

(2) Preparation of nanoparticle system

In this example, the nanoparticle system was prepared by the double emulsion method. The used nanoparticle preparation materials are PLGA and mannose-modified PLGA, both of which have a molecular weight of 7KDa-17KDa. The mass ratio of the two is 4:1 when used together to prepare nanoparticles with target heads. The immunoadjuvants used were Poly(I:C) and CpG SL03. The preparation method was as described previously. The lysate components and adjuvants were co-loaded inside the nanoparticles by the double emulsion method, and then 100 mg of the nanoparticles were centrifuged at 10,000 g for 20 minutes and resuspended in 10 mL of ultrapure water containing 4% trehalose. Freeze-dried for 48h for later use. The average particle size of the nanoparticles with the target head is about 270 nm, and each 1 mg of PLGA nanoparticles is loaded with about 80 μg of protein and polypeptide components, including 0.04 mg each of Poly(I:C) and CpGSL03. The particle size of the control nanoparticles without adjuvant but with mannose target is also about 270nm. The same amount of cell components are used in preparation but without any immune adjuvant. Each 1mg PLGA nanoparticles is loaded with about 80μg protein and peptide group. point. The particle size of blank nanoparticles with mannose targets is about 250 nm, and the same amount of adjuvant is used for preparation, but no cell lysis components are loaded.

(3) Preparation of antigen presenting cells

This example used BMDC and DC2.4 as antigen presenting cells. The preparation method of BMDC is the same as that in Example 2.

(4) Activation of antigen presenting cells

The nanoparticles loaded with whole cell components of cancer cells (1000μg) or blank nanoparticles nanoparticles (1000μg) + free lysate were mixed with BMDC (5 million) and DC2.4 cells (5 million) in 15mL of high glucose DMEM. The medium was co-incubated for 48 hours (37°C, 5% CO ₂ ); the incubation system contained GM-CSF (2000U/mL), M-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200 U/mL), IL-12 (200 U/mL), IFN-γ (500 U/mL) and CD80 antibody (10 ng/mL).

(5) Preparation of antigen-presenting cell-derived nanovaccine

Incubated DCs were collected by centrifugation at 400 g for 5 min, cells were washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, cells were resuspended in PBS water and sonicated at 4°C at low power (20W) 2 minutes. Then the sample was centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 5000g for 10 minutes and the supernatant was collected. The supernatant was filtered through a 0.45 μm membrane and then used an ultrafiltration membrane (molecular weight cut-off 50KDa) to supernatant. Filtration, centrifugal filtration and concentration, the filtered and concentrated samples were centrifuged at 17,000g for 120 minutes, collected, discarded the supernatant, collected the precipitate, and resuspended the precipitate in PBS to obtain a nano-vaccine with a particle size of 120 nm.

(6) Nano vaccines for cancer prevention

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice, which were given on the 35th, 28th, 21st, 14th and 7th days before the mice were inoculated with cancer cells, respectively. Each mouse was inoculated with 400 μg of nanovaccine or PBS, and 1.5×10 ⁵ B16F10 cells were subcutaneously inoculated into the lower right back of each recipient mouse on day 0. Mouse tumor growth rate and mouse survival were monitored. Tumor growth and survival monitoring methods are the same as above.

(5) Experimental results

As shown in Figure 13, compared with the PBS control group and the nanovaccine control group prepared from actively targeted blank nanoparticles + free lysate-activated DCs, the nanovaccine treatment of nanoparticle-activated DCs loaded with whole cell components of mice had significantly slower tumor growth. With or without adjuvant, nanovaccine prepared from nanoparticle-activated DCs can effectively prevent cancer, but with adjuvant, the effect is better. And this shows that the nano-vaccine of the present invention can effectively prevent cancer.

Example 13 Nano-vaccine for the prevention of liver cancer

In this example, Hepa1-6 liver cancer cells were firstly lysed, PLGA was used as nanoparticle skeleton material, and Poly(I:C) and BCG were used as immune adjuvants to prepare nanoparticles loaded with whole cell components of cancer cells derived from liver cancer cells , and then prepare nano-vaccine with DC and B cells activated by the particle for the prevention of liver cancer.

(1) Lysis of cancer cells and collection of components

The cultured Hepa 1-6 hepatoma cells were collected and washed twice with PBS. The hepatoma cells were treated with heating and ultraviolet irradiation, and then 8M urea aqueous solution (containing 200mM sodium chloride) was used to lyse and lyse the whole cell group of cancer cells derived from cancer cells. point. Use 8M urea aqueous solution (containing 200mM sodium chloride) to dissolve BCG and dissolve the cleavage components as an adjuvant.

(3) Preparation of nanoparticle system

In this example, the nanoparticle system is prepared by solvent evaporation method, the molecular weight of the nanoparticle preparation material PLGA is 24KDa-38KDa, the adopted immune adjuvant is BCG and Poly(I:C), and the adjuvant is encapsulated in the nanoparticle Inside. The preparation method was as described above. In the preparation process, the whole cell components of cancer cells and adjuvants were first loaded into the nanoparticles by the double emulsion method, and then 100 mg of the nanoparticles were centrifuged at 10,000 g for 20 minutes, and 10 mL of 4% trehalose was used. Resuspend in ultrapure water and freeze-dry for 48 h before use. The average particle size of the nanoparticles is about 285 nm; each 1 mg of PLGA nanoparticles is loaded with about 100 μg of protein and polypeptide components, and each of BCG and Poly(I:C) is 0.04 mg. The average particle size of the control nanoparticles was about 285 nm, and each 1 mg of PLGA nanoparticles loaded about 100 μg of protein and polypeptide components without any adjuvant.

(3) Preparation of antigen presenting cells

This example used BMDC and B as antigen presenting cells. The preparation method of BMDC is the same as that in Example 2. B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.

(4) Activation of antigen presenting cells

Nanoparticles (1000 μg) loaded with cancer cell whole cell fractions were co-incubated with BMDC (5 million) and B cells (5 million) in 15 mL high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ) ; The incubation system contains GM-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) and CD40 antibody (20 mg/mL).

(5) Preparation of antigen-presenting cell-derived nanovaccine

After incubation, DCs and B cells (5 million DC + 5 million B cells) were collected by centrifugation at 400 g for 5 min, and then washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors. After resuspension in PBS water, low power (20W) sonication was performed at 4°C for 2 minutes. Then the sample was centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 5000g for 10 minutes and the supernatant was collected. The supernatant was filtered through a 0.45 μm membrane and then used an ultrafiltration membrane (molecular weight cut-off 50KDa) to supernatant. Filtration and centrifugal filtration and concentration, the filtered and concentrated sample was mixed with the nanoparticles (10 mg) prepared in step 2, treated with a high pressure homogenizer (10000 bar) for 3 minutes, filtered through a 0.45 μm filter membrane, and then centrifuged at 13000 g for 30 After 10 minutes, discard the supernatant to collect the precipitate, resuspend the precipitate in a mixed lyophilized protective agent aqueous solution (2% trehalose + 2% mannitol + 1% albumin), and then freeze-dry for 48 hours to obtain a nano-vaccine, Its particle size is 300 nanometers.

(4) Prevention of liver cancer

The 6-8 week old female C57BL/6 was selected as the model mice to prepare liver cancer tumor-bearing mice. The mice were injected with 80 μg of nanovaccine or PBS on days -42, -35, -28, -21 and -7 days before inoculation of cancer cells. At the same time, 1.0×10 ⁶ Hepa1-6 hepatoma cells were subcutaneously injected into each mouse on day 0, and the tumor growth and mouse survival were recorded in the same manner as above.

(5) Experimental results

As shown in Figure 14, compared with the PBS control group, the tumor growth rate of the mice treated with the nanovaccine prepared from the nanoparticle co-activated antigen-presenting cells was significantly slower. Moreover, nanovaccine prepared by nanoparticle-activated antigen-presenting cells can effectively prevent cancer with or without adjuvant, but the effect with adjuvant is better. This shows that the nanovaccine of the present invention can effectively prevent cancer.

Example 14 Nano-vaccine prepared by antigen-presenting cells for cancer prevention

(1) Lysis of tumor tissue and cancer cells

After collecting mouse B16F10 melanoma tumor tissue and cultured cancer cells, 10% sodium deoxycholate aqueous solution (containing 8M arginine) was used to lyse and dissolve the whole cell components of cancer cells derived from tumor tissue and cancer cells, and then tumor tissue The components and the cancer cell components were miscible at a mass ratio of 1:1.

(2) Preparation of nanoparticles

In this example, the nanoparticles are prepared by solvent evaporation method, the molecular weight of the nanoparticle preparation material PLGA is 7KDa-17KDa, the immune adjuvants used are CpG2006, CpG1018 and Poly(I:C), and the lysosome escape substances are Arginine, immune adjuvant and arginine were all loaded inside the nanoparticles. The preparation method was as described above. Antigen, arginine and adjuvant were loaded inside the nanoparticles by double emulsion method, and then 100 mg of PLGA nanoparticles were centrifuged at 13000 g for 20 min, the supernatant was discarded, and the precipitate was collected, and the precipitate was resuspended in 4 . % trehalose, freeze-dried for 48 hours before use. The average particle size of the nanoparticles is about 240 nm; each 1 mg of PLGA nanoparticles is loaded with about 80 μg of protein or polypeptide components, 0.03 mg of CpG2006, CpG1018 and Poly(I:C), and 0.02 mg of arginine.

(3) Preparation of antigen presenting cells

This example used BMDC and B as antigen presenting cells. The preparation method of BMDC is the same as that in Example 2. B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.

(4) Activation of antigen presenting cells

Nanoparticles (1000 μg) loaded with cancer cell whole cell fractions were co-incubated with BMDC (5 million) and B cells (5 million) in 15 mL high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ) ; The incubation system contains GM-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) and CD40 antibody (20 mg/mL).

(5) Preparation of antigen-presenting cell-derived nanovaccine

Incubated DCs and B cells were collected by centrifugation at 400 g for 5 min, then washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, resuspended in PBS water at 4°C low power ( 10W) sonicated for 10 minutes. Then, the samples were filtered through 2 μm, 1 μm, 0.45 μm and 0.22 μm membranes in turn, and the filtrate was collected, and then the filtrate was collected after centrifugation at 15,000 g for 30 minutes. components. Then 10 mL of antigen-presenting cell membrane fraction (5 mg) was mixed with 100 mg of the nanoparticles prepared in step (2) and incubated for 10 minutes at room temperature after sonication at low power (20 W) for 2 minutes, and then filtered and squeezed through a 0.45 μm membrane. After centrifugation at 15000g for 20 minutes, the supernatant was discarded, and the precipitate was resuspended in a 4% trehalose aqueous solution, and then freeze-dried for 48 hours to obtain nano-vaccine 2, and the particle size of nano-vaccine 2 was 250 nm; or The precipitate was resuspended in a mixed lyophilized protective agent aqueous solution (2% sucrose + 2% mannitol + 1% lysine), and then freeze-dried for 48 hours to obtain nanovaccine 1 with a particle size of 250 nm. The freeze-dried nanovaccine 1 and nanovaccine 2 were used after being placed at room temperature for 180 days, and 1 mg of the nanovaccine was resuspended in 1 mL of PBS during use.

(4) Nano-vaccine cancer prevention

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice, on the -35th, -28th, -21st, -14th and -7 days Each mouse was vaccinated with 100 μg of Nanovaccine 1 or 100 μg of Nanovaccine 2 or PBS, which had been left at room temperature for more than 180 days, respectively. On day 0, ^1.5 x 105 B16F10 cells were inoculated subcutaneously in the lower right back of each recipient mouse. The method for monitoring tumor growth and survival in mice is the same as above.

(5) Experimental results

As shown in Figure 15, compared with the PBS control group, both nanovaccine 1 and nanovaccine 2 prepared by nanoparticle-activated antigen-presenting cells can significantly prolong the survival period of mice and effectively prevent cancer. Moreover, the effect of the nanovaccine 1 prepared by the freeze-drying of the mixed freeze-drying protective agent (2% sucrose + 2% mannitol + 1% lysine) was better than that prepared by the freeze-drying of the 4% trehalose freeze-drying protectant. Nanovaccine 2.

Example 15 Nano-vaccine for the treatment of melanoma

This example uses mouse melanoma as a cancer model to illustrate how to use nanovaccine to treat melanoma.

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

When collecting tumor tissue, firstly inoculate 1.5×10 ⁵ B16F10 cells subcutaneously on the back of each C57BL/ ⁶ mouse. When the tumor grows to a volume of about 1000 mm, the mice are sacrificed and the tumor tissue is excised. The tumor tissue is cut into pieces. Grind, pass through a cell strainer to prepare a single cell suspension, add ultrapure water, freeze and thaw repeatedly and lyse the above cells with ultrasonication, then add nuclease for 5 minutes, and then inactivate nuclease at 95°C for 10 minutes. Then, centrifuge at 8000g for 3 minutes, and the supernatant part is water-soluble antigen; the precipitate part is dissolved in water-insoluble antigen with 10% sodium deoxycholate aqueous solution. The water-soluble antigen and the water-insoluble antigen after dissolving sodium deoxycholate are mixed in a mass ratio of 1:1, which is the whole cell antigen raw material source for preparing the nanoparticle system.

(2) Preparation of nanoparticle system

In this example, the nanoparticles are prepared by the double emulsion method, and have the ability to target dendritic cells. The used nanoparticle preparation materials are PLGA and mannan-modified PLGA, both of which have molecular weights of 24KDa-38KDa, and the mass ratio of unmodified PLGA and mannan-modified PLGA is 9:1. The adopted immune adjuvants are poly(I:C), CpG1018 and CpG2216, and the substances that increase lysosomal immune escape are KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA), and the adjuvant and KALA polypeptide are encapsulated in nanoparticles. The preparation method was as described above. In the preparation process, the lysate components, adjuvant, and KALA polypeptide were first loaded into the nanoparticles by the double emulsion method. And use 10mL of ultrapure water containing 4% trehalose to resuspend and freeze-dry for 48h. The average particle size of the nanoparticles is about 250nm, and the surface potential is about -5mV; each 1mg PLGA nanoparticles is loaded with about 100μg protein or polypeptide components, and the poly(I:C), CpG1018 and CpG2216 loaded per 1mg PLGA nanoparticles are immune Each adjuvant is 0.02mg, and the load of KALA polypeptide is 0.05mg. The preparation material and method of nanoparticle 2 are the same, the particle size is about 250nm, the surface potential is about -5mV, the KALA polypeptide is not loaded, and the same amount of adjuvant and cell lysis components are loaded. The preparation material and preparation method of nanoparticle 3 are the same, about 250nm, and the surface potential is about -5mV; each 1mg PLGA nanoparticle is loaded with about 100μg protein and polypeptide components, and each 1mg PLGA nanoparticle is loaded with poly(I:C) 0.02mg, load CpG1018 is 0.04mg, load KALA polypeptide 0.05mg.

(3) Preparation of antigen presenting cells

This example used BMDC and B as antigen presenting cells. The preparation method of BMDC is the same as that in Example 2. B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.

(4) Activation of antigen presenting cells

Nanoparticles (1000 μg) loaded with cancer cell whole cell fractions were co-incubated with BMDC (5 million) and B cells (5 million) in 15 mL high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ) ; The incubation system contains GM-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) and CD40 antibody (20 mg/mL).

(5) Preparation of antigen-presenting cell-derived nanovaccine

Incubated DCs and B cells were collected by centrifugation at 400 g for 5 min, then washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, resuspended in PBS water at 4°C low power ( 20W) sonicated for 2 minutes. The samples were then centrifuged at 3000g for 15 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 8000g for 15 minutes, the supernatant was extruded by filtration through a 0.22 μm membrane, and then collected after centrifugation at 16000g for 60 minutes The supernatant was discarded to collect the precipitate, and the precipitate was resuspended in PBS to obtain a nano-vaccine. The particle size of the nano-vaccine was 150 nm.

(4) Nano-vaccine for the treatment of cancer

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. ^1.5 x 105 B16F10 cells were inoculated subcutaneously in the lower right back of each mouse on day 0. 40 μg of nanovaccine or PBS was subcutaneously administered on days 6, 10, 15 and 20 after melanoma inoculation, respectively. In the experiment, the mouse tumor volume and survival monitoring methods were the same as above.

(5) Experimental results

As shown in Figure 16, tumors in the PBS control group all grew rapidly. Compared with the control group, the tumor growth rate of the mice treated with the nanovaccine prepared from the nanoparticle-activated antigen-presenting cells was significantly slower, and the survival period was significantly prolonged. Moreover, the nanovaccine prepared by the nanoparticle-activated antigen-presenting cells with the addition of substances that increase lysosomal escape is better than the nanovaccine prepared by the antigen-presenting cells without the addition of the nanoparticle-activated lysosomal escape. Moreover, the nanovaccine prepared from nanoparticle-activated antigen-presenting cells using two CpG and Poly(I:C) adjuvants as mixed adjuvants was better than the one using only one CpG and Poly(I:C) mixed adjuvant. Nanovaccine prepared from nanoparticle-activated antigen-presenting cells. To sum up, the nano-vaccine of the present invention has a good therapeutic effect on cancer.

Example 16 Nano-vaccine for breast cancer prevention

This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use antigen-presenting cells activated by microparticles loaded with cancer cell whole cell antigens to prepare nano-vaccine for breast cancer prevention. In this example, breast cancer cells were first inactivated and denatured, and then the cells were lysed, and the water-insoluble components in the cancer cells were lysed and lysed with octyl glucoside. Then, PLGA was used as the microparticle framework material, and CpG2007 (Class B Toll-like receptor 9 agonist), CpG2216 (Class A Toll-like receptor 9 agonist), and Poly ICLC (Toll-like receptor 3 agonist) were used as Immune adjuvant, using polyarginine and polylysine as substances to enhance lysosomal escape, prepare microparticles loaded with whole cell components of cancer cells, and then use the microparticles to activate antigen-presenting cells, and prepare microparticles based on A nanovaccine of activated antigen-presenting cells prevents cancer.

(1) Lysis of cancer cells

The cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells were inactivated and denatured by ultraviolet and high temperature heating respectively, and then ultrapure water was added and freeze-thawed 5 times, supplemented by ultrasonic lysis, and the cell lysate was centrifuged at 5000g for 10 minutes, and the supernatant was water-soluble. It is the original water-insoluble component after dissolving the precipitate with 10% octyl glucoside, and the water-soluble component and the water-insoluble component are mixed in a mass ratio of 2:1, that is, the preparation Lysate components required for microparticles.

(2) Preparation of microparticle system

In this example, the microparticle system was prepared and used as a control microparticle by the double emulsion method. The molecular weight of the microparticle skeleton material PLGA was 38KDa-54KDa, and the immune adjuvants used were CpG2007 (type B), CpG2216 (type A) and PolyICLC, The lysosomal escape enhancing substances employed were polyarginine and polylysine. During preparation, the lysate component, adjuvant and microparticles loaded with polyarginine and polylysine were prepared by the double emulsion method, and then 100 mg of microparticles were centrifuged at 9000g for 20 minutes, and 10 mL of 4% trehalose was used. Resuspend in ultrapure water and dry for 48h before use. The average particle size of the microparticles is about 3.1μm, and the surface potential is about -7mV; each 1mg PLGA microparticle is loaded with about 110μg protein or polypeptide components, including CpG2007 (type B), CpG 2216 (type A) and Poly ICLC each 0.01 mg, containing 0.02 mg each of polyarginine and polylysine. The preparation material and preparation method of the control microparticles 2 are the same as the above methods, but the CpGs loaded by the control microparticles 2 are CpG1585 (type A) and CpG2216 (type A) of two types of A, and the average particle size of the control microparticles 2 is 3.1 About μm, the surface potential is about -7mV; each 1mg PLGA microparticles load about 110μg protein or polypeptide components, including CpG1585 (type A), CpG2216 (type A) and Poly ICLC each 0.01mg, containing polyarginine and poly Lysine 0.02mg each.

(3) Preparation of antigen presenting cells

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

(4) Activation of antigen presenting cells

Microparticles (1000 μg) loaded with whole cell components of cancer cells were co-incubated with DC2.4 (10 million) in 15 mL high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ); the incubation system contained GM - CSF (2000 U/mL), IL-2 (500 U/mL), IL-7 (200 U/mL), IL-12 (200 U/mL) and CD80 antibody (10 ng/mL).

(5) Preparation of antigen-presenting cell-derived nanovaccine

Incubated DCs were collected by centrifugation at 400 g for 5 min, cells were washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, cells were resuspended in PBS water and sonicated at 4°C at low power (20W) 2 minutes. The samples were then centrifuged at 3000g for 15 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 8000g for 15 minutes, the supernatant was extruded by filtration through a 0.45 μm membrane, and then collected after centrifugation at 12000g for 90 minutes The supernatant was discarded to collect the precipitate, and the precipitate was resuspended in PBS to obtain a nano-vaccine. The average particle size of the nano-vaccine was 250 nm.

(6) Nano-vaccine cancer prevention

Female BALB/c aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. Each mouse was subcutaneously inoculated with 50 μg nanovaccine or PBS on days -35, -21 and -7 days before inoculation with cancer cells. On day 0, 1×10 ⁶ 4T1 cells were subcutaneously injected into each mouse, and the tumor volume of mice was recorded every 3 days from day 3. The methods for detecting tumor growth and survival were the same as above.

(7) Experimental results

As shown in Figure 17, compared with the control group, the tumor growth rate of the mice treated with the nanovaccine prepared from the antigen-presenting cells activated by microparticles was significantly slower and the survival time of the mice was significantly prolonged. Moreover, 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 to activate antigen-presenting cells was more effective than Antigen-presenting cells were deactivated using two class A Toll-like receptor 9 agonists in combination with a Toll-like receptor 3 agonist as a mixed adjuvant. It can be seen that the nanovaccine based on the activated antigen-presenting cells of the present invention has a preventive effect on breast cancer.

Example 17 Nano-vaccine for breast cancer prevention

This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to prepare nano-vaccine for cancer prevention after microparticles activate antigen-presenting cells.

(1) Lysis of cancer cells

The cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells were inactivated and denatured by ultraviolet rays and high temperature heating respectively, and then 8M urea aqueous solution (containing 500mM sodium chloride) was used to lyse the cancer cells and dissolve the lysate components, which are the antigen components of the microparticle system.

(2) Preparation of microparticle system

In this example, the microparticle system was prepared and used as a control microparticle by the double emulsion method. The framework materials of the microparticles were unmodified PLA and mannose-modified PLA, and the molecular weights were both 40KDa. The unmodified PLA and mannose-modified PLA had The ratio is 4:1. The used immune adjuvants were CpG2006, CpG2216 and Poly ICLC, and the used lysosomal escape-increasing substances were arginine and histidine. During the preparation, the double emulsion method was used to prepare microparticles loaded with lysate components, adjuvants, arginine and histidine. Then, 100 mg of microparticles were centrifuged at 9000g for 20 minutes, and 10 mL of ultra-high-density aliquot containing 4% trehalose was used. Resuspend in pure water and dry for 48 hours before use. The average particle size of the microparticle system is about 2.1μm, and the surface potential of the microparticle system is about -7mV; each 1mg PLGA microparticle is loaded with about 100μg protein or polypeptide components, including 0.01mg each of CpG2006, CpG2216 and Poly ICLC, and arginine. 0.05 mg each of acid and histidine. The preparation material and preparation method of the control microparticle 2 are the same as the microparticles described in this example, the particle size is about 2.1 μm, the surface potential is about -7mV, and only arginine and histidine and the same amount of cell lysate are loaded. without any adjuvant.

(3) Preparation of antigen presenting cells

This example used BMDC and B as antigen presenting cells. The preparation method of BMDC is the same as that in Example 2. B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.

(4) Activation of antigen presenting cells

Microparticles (1000 μg) were co-incubated with BMDC (5 million) and B cells (5 million) in 15 mL high glucose DMEM complete medium for 48 hours (37°C, 5% CO ₂ ); 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 nanovaccine

Incubated DCs and B cells were collected by centrifugation at 400 g for 5 min, then washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, resuspended in PBS water at 4°C low power ( 20W) sonicated for 2 minutes. The samples were then centrifuged at 3000g for 15 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 8000g for 15 minutes, and then after 60 minutes at 16000g. The supernatant was discarded to collect the pellet and the pellet was placed in PBS After resuspension, it is filtered and extruded through a 0.22 μm membrane to obtain a nano-vaccine, and the particle size of the nano-vaccine is 150 nm.

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

Female BALB/c aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. Each mouse was inoculated with 50 μg of nanovaccine or PBS on days -35, -21 and -7 days before inoculation of cancer cells. At the same time, 1×10 ⁶ 4T1 cells were subcutaneously injected into each mouse on the 0th day, and the methods for monitoring the tumor volume and survival period of the mice were the same as above.

(7) Experimental results

As shown in Figure 18, compared with the control group, the mice treated with the nanovaccine prepared by using microparticle-activated antigen-presenting cells had significantly slower tumor growth and significantly longer survival. Moreover, the nano-vaccine prepared by microparticle-activated antigen-presenting cells containing substances that increase lysosomal escape function and mixed adjuvants is better than that of microparticles activated by substances containing only lysosomal escape functions without mixed adjuvants. Nanovaccine prepared from antigen-presenting cells. It can be seen that the nano-vaccine based on antigen-presenting cells of the present invention has a preventive effect on breast cancer, and the use of mixed adjuvants is helpful for the activation of antigen-presenting cells and the subsequent activity of the nano-vaccine.

Example 18 Nano-vaccine for the 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 nanovaccine for the treatment of melanoma. In this example, tumor tissue and cancer cells were firstly lysed to prepare water-soluble components, then, PLGA was used as the skeleton material, Poly(I:C) and CpG1018 were used as immune adjuvants, and R8(RRRRRRRR) polypeptide was used as the lysing agent. Substances with the ability to escape from enzymes, prepare nanoparticles loaded with water-soluble components, and then use nanoparticle-activated antigen-presenting cells to prepare nanovaccine to treat cancer.

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

When collecting tumor tissue, firstly inoculate 1.5×10 ⁵ B16F10 cells subcutaneously on the back of each C57BL/ ⁶ mouse. When the tumor grows to a volume of about 1000 mm, the mice are sacrificed and the tumor tissue is excised. The tumor tissue is cut into pieces. Grind, add an appropriate amount of pure water through the cell strainer and freeze and thaw 5 times (accompanied by ultrasound) to destroy the lysed samples, add nuclease for 10 minutes, then heat at 95 °C for 10 minutes to inactivate the nuclease; collect the cultured B16F10 For cancer cell lines, the culture medium was first removed by centrifugation, then washed twice with PBS, and the cancer cells were collected by centrifugation. The cancer cells were resuspended in ultrapure water, freeze-thawed three times, and lysed cancer cells with ultrasonic damage. Nuclease was added to the sample for 10 minutes and then heated at 95°C for 5 minutes to inactivate the nuclease. After the tumor tissue or cancer cells were enzymatically treated, the lysate was centrifuged at 5,000 g for 5 minutes, and the supernatant was collected as a water-soluble component soluble in pure water. The water-soluble components of tumor tissue and the water-soluble components of cancer cells are mixed in a mass ratio of 1:1, which is the source of antigen raw materials for preparing the nanoparticle system.

(2) Preparation of nanoparticle system

In this example, the nanoparticles were prepared by the double emulsion method. The molecular weight of the nanoparticle preparation material PLGA is 7KDa-17KDa, the immunoadjuvant used is poly(I:C) and CpG1018, the R8 polypeptide is a substance that increases lysosome escape, and the adjuvant and R8 polypeptide are loaded on the nanoparticle. within the particles. The preparation method was as described above. In the preparation process, the lysate components, adjuvant and R8 polypeptide were first loaded into the nanoparticles by the double emulsion method, and then 100 mg of the nanoparticles were centrifuged at 12,000 g for 25 minutes, and 10 mL of 4% seaweed was used. The sugar was resuspended in ultrapure water and freeze-dried for 48 h for use. The average particle size of the nanoparticle is about 250nm, and the surface potential of the nanoparticle is about -5mV; each 1mg PLGA nanoparticle is loaded with about 120μg protein or polypeptide components, and each 1mg PLGA nanoparticle is loaded with poly(I:C) and CpG1018 immune Each adjuvant is 0.03 mg, and the R8 polypeptide is loaded with 0.03 mg.

(3) Preparation of antigen presenting cells

This example used BMDC and B as antigen presenting cells. The preparation method of BMDC is the same as that in Example 2. B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.

(4) Activation of antigen presenting cells

Nanoparticles (1000 μg) were co-incubated with BMDC (5 million) and B cells (5 million) in 15 mL high glucose DMEM complete medium for 24 hours (37°C, 5% CO ₂ ); or BMDC (5 million) ) and B cells (5 million) were co-incubated in 15 mL of high glucose DMEM complete medium for 24 hours (37°C, 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 (20mg/mL).

(5) Preparation of antigen-presenting cell-derived nanovaccine

Incubated DCs and B cells were collected by centrifugation at 400 g for 5 min, then washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, resuspended in PBS water at 4°C low power ( 20W) sonicated for 2 minutes. The samples were then centrifuged at 4000g for 5 minutes and the supernatant was collected, the supernatant was collected after centrifugation at 6000g for 10 minutes, the supernatant was extruded by filtration through a 0.22 μm membrane, and then collected after centrifugation at 17000g for 40 minutes Discard the supernatant to collect the pellet, and resuspend the pellet in PBS to obtain the cell membrane fraction of antigen-presenting cells. 10 mL of antigen-presenting cell membrane fraction (10 mg) was mixed with 50 mg of the nanoparticles prepared in step (2), then incubated at 4°C for 15 minutes, filtered and extruded through a 0.45 μm membrane, and then collected and discarded after centrifugation at 17,000 g for 40 minutes. The supernatant was removed to collect the precipitate, the precipitate was resuspended in an aqueous solution containing a lyoprotectant (2% trehalose + 2% mannitol + lysine), and the nanovaccine was obtained after lyophilization for 48 hours. The nanovaccine prepared by using the cell membrane components of the activated mixed antigen-presenting cells to be loaded on the surface of the nanoparticles is Nanovaccine 1 with a particle size of 260 nm; the cell membrane components of the non-activated mixed antigen-presenting cells are loaded on the surface of the nanoparticles The prepared nanovaccine is Nanovaccine 2 with a particle size of 260 nm.

(6) Nano-vaccine for cancer treatment

Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. ^1.5 x 105 B16F10 cells were inoculated subcutaneously in the lower right back of each mouse on day 0. 50 μg of nanovaccine or PBS was subcutaneously injected on the 4th, 7th, 10th, 15th and 20th days after melanoma inoculation, respectively. The method for monitoring tumor growth and survival in mice is the same as above.

(7) Experimental results

As shown in Figure 19, compared with the control group, the tumor growth rate of the nanovaccine-treated mice was significantly slower and the survival time of the mice was significantly prolonged. Moreover, the effect of nanovaccine 1 prepared by using nanoparticle-activated antigen-presenting cells was significantly better than that of nanovaccine 2 prepared by antigen-presenting cells that were not activated by nanoparticles. It can be seen that the nanoparticle activation of the present invention can improve the efficacy of the antigen-presenting cell-based nanovaccine.

Example 19 Nano-vaccine for the treatment of colon cancer

This example uses mouse colon cancer as a cancer model to illustrate how to prepare a nanovaccine to treat colon cancer using nanoparticle-activated antigen-presenting cells loaded with cancer cell whole cell antigens derived from colon cancer tumor tissue. In this example, 8M urea aqueous solution was used to lyse colon cancer tumor tissue and the lysis components were firstly dissolved. Then, PLGA was used as the framework material, Poly(I:C), CpG2336 and CpG2006 were used as adjuvants, and NH ₄ HCO ₃ was used as adjuvant. Increase lysosomal escape substances, prepare nanoparticles, and then use nanoparticle-activated antigen-presenting cells to prepare nanovaccine for cancer treatment.

(1) Lysis of tumor tissue and collection of components

When collecting tumor tissue, 2 × 10 ⁶ MC38 colon cancer cells were subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of about 1000 mm, the mice ^were sacrificed and the tumor tissue was excised. After the blocks were ground, 8M aqueous urea solution was added through a cell strainer to dissociate the tumor tissue and lyse the whole cell fraction after lysis. The above is the source of antigenic raw materials for the preparation of nanoparticles.

(2) Preparation of nanoparticle system

In this example, the nanoparticles were prepared by the double emulsion method. The preparation material of nanoparticle 1 PLGA molecular weight is 7KDa-17KDa, with Poly(I:C) and CpG as adjuvant, with NH ₄ HCO ₃ as the substance to increase lysosomal escape, and the adjuvant and NH ₄ HCO ₃ are loaded on the nanoparticle In-particle; the preparation method was as described above. During the preparation process, lysate components and adjuvants were first loaded inside the nanoparticles, and then 100 mg of nanoparticles were centrifuged at 10,000 g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose was used. After resuspension, freeze-drying is used for 48h; the average particle size of the nanoparticle is about 260nm, and the surface potential is about -7mV; every 1mg PLGA nanoparticle is loaded with about 90 μg of protein and polypeptide components, and the poly(I) loaded by every 1mg PLGA nanoparticle : C), CpG2336 and CpG2006 immunoadjuvant 0.02 mg each, loaded with NH ₄ HCO ₃ 0.01 mg. The preparation material and preparation method of nanoparticle 2 are the same as nanoparticle 1, the particle size is about 260nm, the surface potential is about -7mV, each 1mg PLGA nanoparticle is loaded with about 90μg protein and polypeptide components, and each 1mg PLGA nanoparticle is loaded with NH ₄ HCO ₃ 0.01 mg, loaded with 0.03 mg of CpG2336 and CpG2006 each.

(3) Preparation of antigen presenting cells

This example used BMDC and B as antigen presenting cells. The preparation method of BMDC is the same as that in Example 2. B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.

(4) Activation of antigen presenting cells

Nanoparticles 1 (500 μg) or nanoparticle 2 (500 μg) loaded with cancer cell whole cell fractions were co-incubated with BMDC (2.5 million) and B cells (2.5 million) in 10 mL of high-glucose DMEM complete medium for 69 hours ( 37°C, 5% CO ₂ ), the incubation system contains GM-CSF (500U/mL), IL-2 (300U/mL), IL-7 (200U/mL), IL-12 (500U/mL) and CD80 Antibody (10ng/mL) and CD40 antibody (20mg/mL); or nanoparticle 1 (500μg) loaded with whole cell fraction of cancer cells with BMDC (2.5 million) and B cells (2.5 million) in 10 mL of high glucose DMEM complete medium was incubated for 69 hours (37°C, 5% CO2) containing GM-CSF (500U/mL), IL-12 (1000U/mL) and CD80 antibody (10ng/mL).

(5) Preparation of antigen-presenting cell-derived nanovaccine

Incubated DCs and B cells were collected by centrifugation at 400 g for 5 min, then washed twice with 4°C phosphate buffered saline (PBS) containing protease inhibitors, resuspended in PBS water at 4°C low power ( 20W) sonicated for 2 minutes. The sample was then centrifuged at 3000g for 30 minutes to discard the precipitate and only the supernatant was collected. The supernatant was centrifuged at 8000g for 15 minutes and the precipitate was discarded to collect the supernatant. The supernatant was filtered and extruded through a 0.22 μm membrane, and then After centrifugation at 18000g for 90 minutes, the supernatant was discarded and the precipitate was collected, and the precipitate was resuspended in PBS to obtain a nano-vaccine, and the particle size of the nano-vaccine was 120 nm.

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

Colon cancer mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. On day 0, each mouse was inoculated subcutaneously with ² x 106 MC38 cells on the lower right side of the back. Each mouse was subcutaneously injected with 50 μg of the nanovaccine on days 6, 9, 12, 15, 20 and 25 after colon cancer inoculation. The method for monitoring tumor growth and survival in mice is the same as above.

(7) Experimental results

As shown in Figure 20, compared with the control group, the tumor growth rate of the mice treated with the nanovaccine prepared by the nanoparticle-activated antigen-presenting cells was significantly slower and the survival time of the mice was significantly prolonged. Moreover, nanovaccine prepared using nanoparticle-activated antigen-presenting cells loaded with lysate fraction, mixed adjuvant, and lysosomal escape material was significantly better than that prepared with lysate fraction, two CpG adjuvants, and lysosomal escape materials. Nanovaccine prepared from antigen-presenting cells activated by nanoparticles of enzymatic escape substances. Moreover, the co-incubation effect of adding IL-7, IL-2 and antibody in the process of activating antigen-presenting cells by nanoparticles is better than that without adding the two interleukins and antibody. It can be seen that the nanovaccine prepared based on the nanoparticle-activated antigen-presenting cells of the present invention has an excellent therapeutic effect on cancer, and the use of mixed adjuvants and the addition of antibodies and IL-7, IL to activate the antigen-presenting cells -2 has an enhanced effect on the final prepared nano-vaccine.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, other different forms of changes or modifications can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. However, the obvious changes or changes derived from this are still within the protection scope of the present invention.

Claims (10)

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 the following: (1) Nanovesicles prepared from activated antigen-presenting cell membrane components; (2) a second delivery particle that internally and/or externally loads whole cell components of cancer cells and loads activated antigen-presenting cell membrane components on its surface; in, The activated antigen-presenting cell membrane components are prepared from pre-activated antigen-presenting cells, and the pre-activated antigen-presenting cells pass through the antigen-presenting cells and the first delivery particles loaded with the cancer cell whole cell components co-incubated; The first delivery particle and the second delivery particle are each independently a nanoparticle or a microparticle. 2 . The cancer vaccine based on antigen-presenting cell membrane components according to claim 1 , wherein the antigen-presenting cells are at least one of dendritic cells, B cells and macrophages. 3 . 3. The cancer vaccine based on antigen-presenting cell membrane components according to claim 1, wherein the cancer cell whole cell components are obtained by lysis of cancer cells and/or tumor tissues and contain water-soluble components and The whole cell component of the water-insoluble component, the water-insoluble antigen is dissolved on the first delivery particle or the second delivery particle after being dissolved by a dissolving agent; wherein, the dissolving agent is selected from urea, guanidine hydrochloride, deoxychol at least one of acid salts, lauryl sulfate, glycerol, protein degrading enzymes, albumin, lecithin, inorganic salts, Triton, Tween, amino acids, glycosides, and choline. 4 . The cancer vaccine based on antigen-presenting cell membrane components according to claim 1 , wherein the first delivery particle or the second delivery particle is loaded with a substance that increases lysosome escape. 5 . 5. The cancer vaccine based on antigen-presenting cell membrane components according to claim 1, wherein the cancer vaccine based on antigen-presenting cell membrane components is a freeze-dried preparation, and the freeze-dried preparation is prepared from the antigen-presenting cell membrane components. The cancer vaccine in the form of cell membrane components is prepared by freeze-drying; 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 following steps: S1, co-incubating the antigen-presenting cells with the first delivery particles loaded with the whole cell component of the cancer cells to activate the antigen-presenting cells; S2, preparing the cell membrane components of the antigen-presenting cells activated in S1 into nanovesicles to obtain the cancer vaccine based on the antigen-presenting cell membrane components; Or obtaining the cell membrane fraction of the antigen-presenting cells activated in S1, and co-acting the cell membrane fraction and/or nanovesicles with the second delivery particle loaded with the cancer cell whole cell fraction to obtain the antigen-presenting-based Cancer vaccines with cell membrane components. 7. The preparation method according to claim 6, characterized in that: during co-action or co-incubation, the system contains cytokines, proteins and/or antibodies; the cytokines are selected from interleukin, tumor necrosis factor, interferon , at least one of growth factor and colony stimulating factor; the antibody is selected from αCD-3 antibody, αCD-4 antibody, αCD-8 antibody, αCD-28 antibody, CD40 antibody, CD80 antibody, αOX-40 antibody, αOX - at least one of 40L antibody and CD86 antibody. 8. The preparation method according to claim 7, wherein the system contains any combination of the following (1) to (11): (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, 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. 9. The cancer vaccine based on the antigen-presenting cell membrane component according to any one of claims 1-5 or the cancer vaccine prepared by the preparation method according to any one of claims 6-8 is prepared for the treatment or prevention of cancer application in medicine. 10. A method for inducing T cells into cancer cell-specific T cells in vitro, wherein the cancer vaccine is co-incubated 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 of any one of claims 1-5 or the cancer vaccine prepared by the preparation method of any one of claims 6-8.

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