Specific T cells for preventing or treating cancer and method for preparing the same

Abstract

The present invention relates to a method for preparing cancer-specific T cells based on peripheral blood or peripheral immune organs for preventing or treating cancer. Specifically, it first includes the step of separating immune cells from peripheral blood or peripheral immune organs, then the step of co-incubating with nanoparticles and / or microparticles carrying whole tumor cell antigens for a certain period to activate cancer-specific T cells, and thereafter, the step of separating the cancer-specific T cells activated by cancer antigens, amplifying them in vitro, and then re-injecting them into the body to exert an anti-cancer effect. The nanoparticles or microparticles prepared according to the present invention carry tumor antigen components on the microparticles and / or nanoparticles to activate cancer-specific T cells, and then are re-injected into patients after amplification to treat cancer or prevent recurrence or metastasis. The cancer-specific T cells separated by the screening method have high specificity and can prevent or treat cancer by killing cancer cells after amplification.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and particularly relates to specific T cells for preventing or treating cancer, a method for preparing the same, and their applications.

Background Art

[0002] Immune cells refer to cells involved in or related to immune responses, such as natural lymphocytes, various phagocytes, and lymphocytes that can recognize antigens and trigger specific immune responses (e.g., T cells, B cells, NK cells, DC cells, macrophages, granulocytes, mast cells, etc.). Autologous immune cell therapy is used to fight tumors by separating and culturing immune cells from one's own blood. By replenishing highly active immune cells, the number of immune cells in the body increases, and the original immune cells in the body are activated. As a result, the ability of immune cells to kill tumor cells, bacteria, viruses, etc. is greatly improved, and the goals of cancer prevention and anti-cancer are achieved.

[0003] In the previous application CN202011027741.0 by the invention team of the present application, a detection method capable of detecting T cells activated by nanoparticles carrying tumor components of a patient was disclosed. However, this application is a detection method rather than a treatment method, does not select or amplify activated T cells, does not require a reinjection step in the body, and in the detection process, although it is necessary to fix and stain T cells, it cannot provide corresponding active T cells. Therefore, there is no report in the prior art regarding carrying tumor antigens on nanoparticles to activate and concentrate specific types of T cells and exert a tumor treatment effect.

[0004] Cancer patients, especially those with low physical ability, have few cancer-specific T cells with low activity. Therefore, their cancer-specific T cells have problems such as being difficult to activate, slow to proliferate, having a long culture cycle, and low cell survival rate, which affect antitumor immunotherapy. The present invention further stimulates cancer-specific T cells that recognize cancer cells through tumor antigens and have the function of killing cancer cells in vitro, selects, separates, and amplifies them on a large scale, and then reinjects them into the patient. By reinjecting cancer-specific T cells, an effective method for preventing cancer occurrence, preventing cancer metastasis, and treating cancer is achieved.

[0005] The technical content such as the activation of tumor-specific T cells by nanoparticles disclosed in Chinese Patent Application (202011027741.0) is an essential component of the present invention.

Summary of the Invention

[0006] An object of the present invention is to provide a method for preparing cancer-specific T cells derived from autologous cells or allogeneic cells for preventing or treating cancer. Specifically, the method first includes the step of separating immune cells from peripheral blood or peripheral immune organs, and then co-incubating them with antigen-presenting cells, and nanoparticles and / or microparticles carrying whole tumor cell components or containing a part of whole tumor cell antigen components for a certain period to activate cancer-specific T cells. After that, it includes the step of separating the cancer-specific T cells activated by cancer antigens, amplifying them in vitro, and then reinjecting them into the body to exert an anti-cancer effect.

[0007] In a preferred technical solution of the present invention, the above preparation method specifically includes

[0008] First, separating immune cells from peripheral blood or peripheral immune organs, or selecting T cells from the immune cells,

[0009] Mixing microparticles and / or nanoparticles carrying tumor antigen components with antigen-presenting cells and T cells, and then co-incubating them to select T cells that are activated and express specific cell markers,

[0010] Incubating the T cells expressing the specific cell markers selected in step (2) with cytokines and / or antibodies to obtain amplified specific T cells,

[0011] Preferably, in step (2), the tumor antigen component may be the whole cell lysate component of tumor tissue / cancer cells, or the antigen component may be a part of the whole cell lysate component of tumor tissue / cancer cells.

[0012] The whole cell lysate component includes a water-soluble component and a water-insoluble component, and the water-insoluble component is dissolved (or solubilized) using a lysate (or solubilization solution) containing a lysing agent (or called solubilizing agent).

[0013] Preferably, in step (2), the tumor antigen component is obtained by lysing all cells of one or more cancer cells and / or tumor tissues, or is obtained by processing after lysing all cells of one or more cancer cells and / or tumor tissues, or is obtained by lysing after processing all cells of one or more cancer cells and / or tumor tissues. Preferably, at least one of the cancer cells or tumor tissues is the same as the type of the target disease, or the antigen component is composed of some components of one or more cancer cells and / or tumor tissues, and some components include protein / polypeptide components and / or mRNA components in the cell lysate,

[0014] In a preferred technical solution of the present invention, when the whole cell antigen component is a part of the whole cell lysate component, the antigen component includes protein and polypeptide components and / or mRNA components in the whole cell lysate component.

[0015] Preferably, the nanoparticles and / or microparticles carrying the tumor antigen component can be co-incubated with antigen-presenting cells and T cells simultaneously to activate cancer cell-specific T cells. First, the nanoparticles and / or microparticles carrying the tumor antigen component can be co-incubated with antigen-presenting cells to activate the antigen-presenting cells. Next, the activated antigen-presenting cells alone can be co-incubated with T cells to activate cancer cell-specific T cells. After the nanoparticles and / or microparticles carrying the tumor antigen component are co-incubated with antigen-presenting cells, the antigen-presenting cells are activated, and the antigen-presenting cells that do not require special treatment can be co-incubated with T cells to activate specific T cells. Or after the antigen-presenting cells are treated with fixation, radiation, irradiation, modification, inactivation, mineralization, etc., the antigen-presenting cells can be co-incubated with T cells to activate specific T cells.

[0016] In a preferred technical solution of the present invention, in the above step (1), immune cells of the same or different species of peripheral blood or peripheral immune system can be separated, and the cells can be directly separated and extracted without any treatment, or the cells can be separated and extracted by radiotherapy, immunotherapy, chemotherapy, particle therapy, vaccine therapy, etc.

[0017] In a preferred technical solution of the present invention, the T cells selected in the above step (1) are CD3 + T cells, CD3 + CD8 + T cells, CD3 + CD4 + Any one or a combination thereof of T cells.

[0018] In a preferred technical solution of the present invention, the selection methods in the above steps (1) and (2) are any one or a combination thereof of flow cytometry and magnetic bead method. When selecting activated cancer cell-specific T cells, one activation marker by which the T cells are activated can be used, or a combination of one or more different markers can be used as the activation marker.

[0019] In a preferred technical solution of the present invention, in step (2), the tumor antigen component can be obtained by lysing one or more cancer cells and / or all cells of the tumor tissue, or by treating the lysate of one or more cancer cells and / or all cells of the tumor tissue, or by treating one or more cancer cells and / or all cells of the tumor tissue and then lysing them. Preferably, at least one of the above cancer cells or tumor tissues is the same as the type of the target disease, or the antigen component is composed of some components of one or more cancer cells and / or tumor tissues, and some components contain protein / polypeptide components and / or mRNA components in the cell lysate.

[0020] In a preferred technical solution of the present invention, the antigen component can be (1) the whole cell lysate component of cancer cells / tumor tissues, (2) or a part of the whole cell component containing proteins and polypeptides in the whole cell lysate component of cancer cells / tumor tissues, (3) or the protein and polypeptide components and mRNA components in the whole cell component of cancer cells / tumor tissues.

[0021] In a preferred technical solution of the present invention, the tumor antigen component is the cell lysate component of the tumor tissue and / or cancer cells, and contains one or both of the water-soluble component and the water-insoluble component generated after lysing the tumor tissue and / or cancer cells. First, the water-soluble component and the water-insoluble component are collected respectively, and nanoparticles or microparticles are prepared respectively, or a lysate containing a lysing agent is directly used to directly lyse cancer cells or tumor tissues, and the whole cell component can also be dissolved to prepare nanoparticles or microparticles. The above water-insoluble component is dissolved with a lysate containing a lysing agent. Alternatively, it can be the protein and polypeptide components, or the protein and polypeptide components and mRNA components obtained after appropriate treatment of the above cell lysate components.

[0022] In a preferred technical solution of the present invention, when the antigen component is a whole cell lysate component, its preparation method is as follows: (1) First, cancer cells / tumor tissues are lysed, then water-soluble components and water-insoluble components are prepared respectively, and then the water-insoluble components are dissolved with a specific solvent containing a lysing solution and then used; (2) Cells are lysed using a lysing solution containing a solvent, and then the whole cell components lysed using the lysing solution containing a solvent are dissolved.

[0023] In a preferred technical solution of the present invention, when the antigen component is a part of the whole cell lysate component (containing proteins and polypeptide components in the whole cell components of cancer cells), the preparation method is as follows. (1) First, dissolve cancer cells / tumor tissues, then prepare water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separate and extract the protein components and polypeptide components in the water-soluble components from the water-soluble components by an appropriate method. Then, use the proteins and polypeptide components separated and extracted from the water-soluble components together with all the water-insoluble components as antigen components. (2) First, dissolve cancer cells / tumor tissues, then prepare water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separate and extract the protein components and polypeptide components in the water-soluble components from the water-insoluble components by an appropriate method. Then, use the proteins and polypeptide components separated and extracted from the water-insoluble components together with all the water-soluble components as antigen components. (3) First, dissolve cancer cells / tumor tissues, then prepare water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separately separate and extract the protein components and polypeptide components in the water-soluble components from the water-soluble components and the water-insoluble components by an appropriate method. Then, use the proteins and polypeptide components separated and extracted from the water-soluble components and the water-insoluble components as antigen components. (4) Use a lysate containing a solvent to lyse cells, then use a lysing solution containing the solvent to dissolve the lysed whole cell components. After that, separate and extract the protein and polypeptide components by an appropriate method. In the above preparation method, the step of separating and extracting total cell mRNA and using the total cell mRNA as a part of the antigen component can also be added.

[0024] Appropriate methods for separating and extracting protein and polypeptide components include, but are not limited to, salting out, heating, enzymatic hydrolysis, etc.

[0025] Separate and extract the above protein and polypeptide components and dissolve them again in a lysate containing a solvent.

[0026] In a preferred technical solution of the present invention, when incubating the above nanoparticles / microparticles alone with antigen-presenting cells, or when incubating the above nanoparticles / microparticles simultaneously with antigen-presenting cells and T cells, the concentration of the nanoparticles / microparticles is 2.5 ng / mL to 50 mg / mL, and the total incubation time is 1 to 168 hours.

[0027] In a preferred technical solution of the present invention, before and / or after lysis, after inactivating and / or denaturing, solidifying, biomineralizing, ionizing, chemically modifying, or nuclease-treating all cell components, nanoparticles or microparticles can be prepared, and they can also be directly prepared without inactivating and / or denaturing, solidifying, biomineralizing, ionizing, chemically modifying, or nuclease-treating before and / or after cell lysis.

[0028] In a preferred technical solution of the present invention, for tumor tissue cells, inactivation and / or denaturation treatment can be performed before lysis, inactivation and / or denaturation treatment can also be performed after cell lysis, or inactivation and / or denaturation treatment can be performed before and / or after cell lysis.

[0029] In a preferred technical solution of the present invention, the inactivation and / or denaturation treatment methods before and / or after cell lysis include any one or a combination of ultraviolet irradiation, high-temperature heating, radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, and freeze-drying.

[0030] In a preferred technical solution of the present invention, in the above step (2), the ratio of the number of antigen-presenting cells to T cells used is greater than 1:1, and the nanoparticles or microparticles are used to activate existing cancer-specific T cells in peripheral immune cells in vitro through the presentation of antigen-presenting cells. The above nanoparticles or microparticles are selected from nanoparticles with a particle size of 1 nm to 1000 nm or microparticles with a particle size of 1 μm to 1000 μm.

[0031] In a preferred technical solution of the present invention, the antigen-presenting cells co-incubated with T cells and nanoparticles and / or microparticles are derived from autologous, allogeneic, cell lines, stem cells or any mixture thereof, and the antigen-presenting cells co-incubated are B cells, dendritic cells, macrophages or any mixture of these three.

[0032] In a preferred technical solution of the present invention, the antigen-presenting cells are derived from autologous antigen-presenting cells, allogeneic antigen-presenting cells, antigen-presenting cell lines or antigen-presenting cells differentiated from stem cells, preferably any one of dendritic cells (DC), B cells, macrophages or a combination thereof, and more preferably, a combination of one or more antigen-presenting cells is used.

[0033] In a preferred technical solution of the present invention, the above-mentioned mixed co-incubation is selected from any one of the following three methods: (a) directly mixing these three and co-incubating for a certain period; (b) first co-incubating the microparticles and / or nanoparticles with the antigen-presenting cells for a certain period, and then adding T cells for co-incubation; (c) first co-incubating the microparticles and / or nanoparticles with the antigen-presenting cells for a certain period, selecting the incubated antigen-presenting cells, and then co-incubating the antigen-presenting cells with T cells.

[0034] Before co-incubating the cells with nanoparticles / microparticles and antigen-presenting cells, T cells alone can be statically cultured for a certain period or appropriately selected, or before co-incubating T cells with activated antigen-presenting cells, T cells alone can be statically cultured for a certain period or appropriately selected.

[0035] In a preferred technical solution of the present invention, the culture conditions for the above-mentioned mixed co-incubation are co-incubating at 30-38 °C and 1-10% CO 2 for 1-168 hours.

[0036] In a preferred technical solution of the present invention, cytokines can be added to the above-mentioned mixed co-incubation process, and the added cytokines include, but are not limited to, interleukins, tumor necrosis factors, interferons, growth factors. Preferably, the added cytokines include interleukin 7 (IL-7) and interleukin 15 (IL-15).

[0037] In a preferred technical solution of the present invention, in the above step (2), the screening method is to bind an antibody having fluorescence, magnetism or a specific ligand to a specific cell marker on the surface of T cells, and then use flow cytometry or magnetic beads, etc. to separate cells expressing the specific cell marker from the cell population.

[0038] In a preferred technical solution of the present invention, the T cells expressing the above-selected specific cell markers include any one or a combination of the following: CD69, CD137, CD25, CD134, CD80, CD86, OX40L, OX40, CD28, FAS-L, IL-2R, HLA-DR, CD127 (IL-7R), CD150, CD107A, CD83, CD166, CD39, CD178, CD212, CD229, CD100, CD107b, CD108, CD109, CD113, CD122, CD126, CD253, CD197, PD-1, TIM3, LAG-3, TIGIT, CD62L, CD70, CTLA-4 (CD152), CD27, CD26, CD30, TNFRSF9, CD74, PD-L1 (CD274), CD258, CD261, 4-1BB, CD154, ICAM-1, LFA-1, LFA-2, VLA-4, CD160, CD71, CXCR3, TNFRSF14, TNFRSF18, TNFSF4, TNFSF9, TNFSF14, CD11a, CD101, CD48, CD244, CD49a, CD95, CD44, CXCR1, CD103, CD45RO, ICOS (CD278), VTCN1, HHLA2, LGAL59, CCR7, CD357, BCL6, TCF-1, CD38, CD27, etc., but are not limited thereto.

[0039] In a preferred technical solution of the present invention, in step (3), the concentration of the above cytokine is 1 to 6000 ng / ml, preferably 5 to 200 ng / ml, more preferably 10 to 30 ng / ml.

[0040] In a preferred technical solution of the present invention, in the above step (3), the above cytokine includes, but is not limited to, interleukin, interferon, and tumor necrosis factor.

[0041] In a preferred technical solution of the present invention, the above interleukin includes, but is not limited to, interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 17 (IL-17), and interleukin 21 (IL-21).

[0042] In a preferred technical solution of the present invention, the concentration of the above antibody is 1 to 6000 ng / ml, preferably 5 to 100 ng / ml, more preferably 10 to 30 ng / ml.

[0043] In a preferred technical solution of the present invention, the above antibody includes, but is not limited to, any one or a combination thereof of αCD3 antibody, αCD28 antibody, αCD80 antibody, αCD86 antibody, and αOX40 antibody.

[0044] In a preferred technical solution of the present invention, the co-incubation time of the above nanoparticles / microparticles with the mixture of antigen-presenting cells and T cells is at least 4 hours, preferably 6 to 96 hours.

[0045] In a preferred technical solution of the present invention, the co-incubation time of the above nanoparticles / microparticles with antigen-presenting cells alone is at least 1 hour, preferably 6 to 96 hours.

[0046] In a preferred technical solution of the present invention, the co-incubation time of the above-activated antigen-presenting cells and the mixture of T cells is at least 1 hour, preferably 6 to 96 hours.

[0047] In a preferred technical solution of the present invention, the amplification culture time is at least 1 day, preferably 4 to 36 days.

[0048] In a preferred technical solution of the present invention, when the above nanoparticles / microparticles are co-incubated with antigen-presenting cells alone, the concentration of the nanoparticles / microparticles is 2.5 ng / mL to 50 mg / mL.

[0049] In a preferred technical solution of the present invention, when the above nanoparticles / microparticles are co-incubated with antigen-presenting cells and T cells, the concentration of the nanoparticles / microparticles is 2.5 ng / mL to 50 mg / mL.

[0050] In a preferred technical solution of the present invention, the content of the protein and polypeptide components in the antigen component carried by the above nanoparticles / microparticles exceeds 10 ng / mL. In a preferred technical solution of the present invention, in step (3), the specific T cells obtained after in vitro proliferation are re-injected into the body to exert an anti-cancer effect.

[0051] In a preferred technical solution of the present invention, the cancer antigen carried by the nanoparticles and / or microparticles is the whole cell component of the tumor tissue and / or cancer cells, and contains the water-soluble component and / or water-insoluble component of the tumor tissue and / or cancer cells.

[0052] In a preferred technical solution of the present invention, the method of carrying one or more components of the tumor tissue and / or cancer cells by the nanoparticles and / or microparticles to the nanoparticles or microparticles is to encapsulate the water-soluble component and water-insoluble component of the whole cell into the particle interior respectively or simultaneously, and / or carry them on the particle surface respectively or simultaneously.

[0053] In a preferred technical solution of the present invention, the original water-insoluble portion in the whole cell components derived from tumor tissue or cancer cells carried on nanoparticles and / or microparticles for activating cancer-specific T cells is changed from an insoluble state in pure water to a soluble state in an aqueous solution containing a solubilizing agent / dissolving agent or an organic solvent by an appropriate solubilization method, and the solubilizing agent / dissolving agent used is at least one selected from compounds containing the structure of Structural Formula 1, deoxycholate, lauryl sulfate, glycerol, proteolytic enzymes, albumin, lecithin, polypeptides, amino acids, glycosides, and choline.

[0054] Structural Formula 1 is as follows:

Chemical formula

[0055] , R 1 is C, N, S or O, and R 2 ~R 5 is at least one independently selected from hydrogen, alkyl, amino, carboxyl, substituted and unsubstituted guanidino.

[0056] Compounds containing Structural Formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, polyhexamethylene guanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, guanidine or other compounds containing urea, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetourea, sulfonylurea compounds (such as glybenclamide, glibencrazide, glazidone, glimepiride, etc.), thiourea compounds (such as thiouracil, imidazole, etc.), nitrosourea-based compounds containing Structural Formula 1.

[0057] In a preferred technical solution of the present invention, a target head for actively targeting antigen-presenting cells is connected to the surface of the above-mentioned nanoparticles and / or microparticles for activating cancer-specific T cells.

[0058] In a preferred technical solution of the present invention, the method in which the above-mentioned water-soluble component and / or water-insoluble component is carried on the surface of the above-mentioned cancer vaccine includes one or more of adsorption, covalent bonding, charge interaction, hydrophobic interaction, solidification in one or more steps, mineralization, and encapsulation.

[0059] In a preferred technical solution of the present invention, the particle size of the above-mentioned nanoparticles is 1 nm to 1000 nm, and the particle size of the microparticles is 1 μm to 1000 μm.

[0060] In a preferred technical solution of the present invention, the surface of the above-mentioned nanoparticles or microparticles may be electrically neutral, negatively charged, or positively charged.

[0061] In a preferred technical solution of the present invention, the preparation materials of the nano vaccine and / or micron vaccine are organic synthetic polymer materials, natural polymer materials, or inorganic materials.

[0062] In a preferred technical solution of the present invention, the above-mentioned organic synthetic polymer materials are PLGA, PLA, PGA, PEG, PCL, poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, and the natural polymer materials are lecithin, cholesterol, sodium alginate, albumin, collagen, gelatin, cell membrane components, starch, sugar, polypeptide, and the above-mentioned inorganic materials are ferric oxide, magnetite, calcium carbonate, calcium phosphate.

[0063] In a preferred technical solution of the present invention, the nanoparticles and / or microparticles for activating cancer-specific T cells can simultaneously carry one or more components of tumor tissues and / or cancer cells together with an immune adjuvant on the nanoparticles or microparticles.

[0064] In a preferred technical solution of the present invention, both the water-soluble part and the water-insoluble part can be dissolved by a solubilized aqueous solution containing a solubilizing agent or an organic solvent. The solubilizing agent is at least one of the solubilizing agents that can enhance the solubility of proteins or polypeptides in an aqueous solution, and the organic solvent is an organic solvent that can dissolve proteins or polypeptides.

[0065] In a preferred technical solution of the present invention, the original water-insoluble part is changed from an insoluble state in pure water to a soluble state in an aqueous solution or an organic solvent containing a solubilizing agent / dissolving agent by an appropriate solubilization method. The solubilizing agent / dissolving agent used is at least one selected from compounds containing the structure of Structural Formula 1, deoxycholate, lauryl sulfate, glycerol, proteolytic enzymes, albumin, lecithin, polypeptides, amino acids, glycosides, and choline. Structural Formula 1 is as follows:

Chemical Formula

[0066] R1 is C, N, S, or O, and R2 to R5 are at least one independently selected from hydrogen, alkyl, amino, carboxyl, substituted and unsubstituted guanidino.

[0067] The compounds containing Structural Formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, polyhexamethylene guanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, guanidine or other compounds containing urea, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetourea, sulfonylurea compounds (such as glibencamide, glicladide, glazidione, glimepiride, etc.), thiourea compounds (such as thiouracil, imidazole, etc.), and compounds containing Structural Formula 1 such as nitrosourea systems.

[0068] In a preferred technical solution of the present invention, the cell components carried on nanoparticles or microparticles for activating cancer-specific T cells are derived from components obtained from all cells of one or more cancer cells and / or one or more tumor tissues. To enable the nano- or micron system to contain more antigens, the water-insoluble components are carried on the delivery particles. More preferably, since the water-soluble components and the water-insoluble components are simultaneously carried on the delivery particles, the delivery particles carry all-cell component antigens.

[0069] In a preferred technical solution of the present invention, for the cell components or mixtures thereof carried on nanoparticles and / or microparticles for activating cancer-specific T cells, the mixtures include, but are not limited to, those in which the water-soluble components are mixed with each other, those in which the water-insoluble components are mixed with each other, or those in which all or part of the water-soluble components are mixed with all or part of the water-soluble components.

[0070] In a preferred technical solution of the present invention, for nanoparticles (NP) and / or microparticles (MP), cell components or a mixture thereof are carried on the inside and / or surface of the nanoparticles or microparticles. Specifically, the above carrying method is to encapsulate the water-soluble components and water-insoluble components of the cells inside the particles respectively or simultaneously, and / or carry them on the surface of the particles respectively or simultaneously. That is, encapsulating the water-soluble components inside the particles and carrying them on the surface of the particles at the same time, encapsulating the water-insoluble components inside the particles and carrying them on the surface of the particles at the same time, encapsulating the water-soluble components inside the particles and carrying the water-insoluble components on the surface of the particles, encapsulating the water-insoluble components inside the particles and carrying the water-soluble components on the surface of the particles, encapsulating the water-soluble components and water-insoluble components inside the particles, but only carrying the water-insoluble components on the surface of the particles, encapsulating the water-soluble components and water-insoluble components inside the particles, but only carrying the water-soluble components on the surface of the particles, encapsulating the water-soluble components inside the particles, but carrying the water-soluble components and water-insoluble components on the surface of the particles at the same time, encapsulating the water-insoluble components inside the particles, but carrying the water-soluble components and water-insoluble components on the surface of the particles at the same time, encapsulating the water-soluble components and water-insoluble components inside the particles at the same time, and carrying the water-soluble components and water-insoluble components on the surface of the particles at the same time, including but not limited to these.

[0071] In a preferred technical solution of the present invention, the interior and / or surface of the nanoparticles or microparticles for activating cancer-specific T cells may further contain an immune-enhancing adjuvant, and the immune-enhancing adjuvant includes, but is not limited to, at least one of immune enhancers derived from microorganisms, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicines, and other types. The immune-enhancing adjuvant includes, but is not limited to, pattern recognition receptor agonists, live tuberculosis vaccine (BCG) for tuberculosis prevention, manganese-related adjuvants, cell wall skeletons of live tuberculosis vaccine for tuberculosis prevention, methanol extraction residues of live tuberculosis vaccine for tuberculosis prevention, muramyl dipeptides of live tuberculosis vaccine for tuberculosis prevention, mycobacterium phlei, polyactin A, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virions, cholera enterotoxin, saponin and its derivatives, resiquimod, thymosin, newborn calf liver active peptide, imiquimod, polysaccharides, curcumin, immune adjuvant CpG, poly(I:C) of immune adjuvant, poly-ICLC of immune adjuvant, Corynebacterium parvum vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvant, aluminum adjuvant, calcium adjuvant, various cytokines, interleukins, interferons, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, squalene, cytokines, vegetable oils, endotoxins, liposome adjuvants, MF59, double-stranded RNA, double-stranded DNA, aluminum-related adjuvants, CAF01, ginseng, and at least one of the active ingredients of Chinese chives, but is not limited thereto. Those skilled in the art can understand that the enumeration herein is not exhaustive, and other substances that can enhance the immune response can also be used as immune-enhancing adjuvants.

[0072] In a preferred technical solution of the present invention, the immune-enhancing adjuvant is preferably a Toll-like receptor agonist.

[0073] In a preferred technical solution of the present invention, the immune-enhancing adjuvant is preferably a combination of two or more Toll-like receptor agonists.

[0074] In a preferred technical solution of the present invention, when an immunoadjuvant and cell components are co-carried on nanoparticles or microparticles, after the nanoparticles or microparticles are engulfed by antigen-presenting cells, better cancer-specific T cells.

[0075] In a preferred technical solution of the present invention, the microparticles or nanoparticles carry a substance that increases the escape of the nanoparticles and / or microparticles or the antigens carried thereon from lysosomes to the cytoplasm. The substance that increases the escape from the lysosome includes amino acids, polypeptides, sugars, lipids, inorganic salts capable of producing a proton sponge effect. Preferably, the amino acids in the substance that increases the escape from the lysosome include positively charged amino acids. Preferably, the polypeptide that increases the escape from the lysosome includes positively charged amino acids.

[0076] In a preferred technical solution of the present invention, the surface of the nanoparticles or microparticles may not be connected to a target head having an active targeting function, or may be connected to a target head having an active targeting function.

[0077] The target head of active targeting can be a normal target head such as mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, etc.

[0078] In a preferred technical solution of the present invention, the surface of the above-mentioned nanoparticles and / or microparticles is connected to a target that actively targets antigen-presenting cells.

[0079] In a preferred technical solution of the present invention, the nanoparticles or microparticles may not be modified during the preparation process, or appropriate modification techniques may be used to increase the antigen-carrying capacity of the nanoparticles or microparticles. Modification techniques include, but are not limited to, biomineralization (e.g., silicification, calcification, magnetization), gelation, crosslinking, chemical modification, addition of charged substances, etc.

[0080] In a preferred technical solution of the present invention, the form of carrying cell components or a mixture thereof inside nanoparticles or microparticles is any method capable of carrying cell components or a mixture thereof inside nanoparticles or microparticles.

[0081] In a preferred technical solution of the present invention, the methods of carrying cell components or a mixture thereof on the surface of nanoparticles or microparticles include, but are not limited to, adsorption, covalent bonding, charge interaction (for example, addition of a positively charged substance, addition of a negatively charged substance), hydrophobic interaction, solidification in one or more steps, mineralization, encapsulation, etc.

[0082] In a preferred technical solution of the present invention, the water-soluble component and / or water-insoluble component carried on the surface of nanoparticles or microparticles form one or more layers after loading. When a plurality of layers of water-soluble components and / or water-insoluble components are carried on the vaccine surface, there are modifiers between the layers.

[0083] In a preferred technical solution of the present invention, the size of the particle diameter of the above-mentioned nanoparticles is 1 nm to 1000 nm, more preferably, the size of the particle diameter is 30 nm to 1000 nm, and most preferably, the size of the particle diameter is 100 nm to 600 nm.

[0084] In a preferred technical solution of the present invention, the size of the particle diameter of the microparticles is 1 μm to 1000 μm, more preferably, the size of the particle diameter is 1 μm to 10 μm, and most preferably, the size of the particle diameter is 1 μm to 5 μm.

[0085] In a preferred technical solution of the present invention, the shape of the above-mentioned nanoparticles or microparticles includes any one of spherical, elliptical, barrel-shaped, polygonal, rod-shaped, sheet-shaped, linear, worm-shaped, square, triangular, butterfly-shaped or disc-shaped.

[0086] In a preferred technical solution of the present invention, the method in which the above-mentioned water-soluble component and / or water-insoluble component is carried on the surface of the above-mentioned cancer vaccine includes one or more of adsorption, covalent bonding, charge interaction, hydrophobic interaction, solidification in one or more steps, mineralization, and encapsulation.

[0087] In a preferred technical solution of the present invention, the preparation materials of the nano-vaccine and / or micron-vaccine are organic synthetic polymer materials, natural polymer materials or inorganic materials.

[0088] In a preferred technical solution of the present invention, the above-mentioned organic synthetic polymer material is a biocompatible or degradable polymer material, which includes any one or a combination of PLGA, PLA, PGA, PLGA-PEG, PLA-PEG, PGA-PEG, PEG, PCL, poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, synthetic lipid.

[0089] In a preferred technical solution of the present invention, the above-mentioned natural polymer material is a biocompatible or degradable polymer material, which includes any one or a combination of lecithin, cholesterol, sodium alginate, albumin, collagen, gelatin, cell membrane components, starch, sugar, polypeptide.

[0090] In a preferred technical solution of the present invention, the above-mentioned inorganic material is a non-explicit biotoxic material, which includes, but is not limited to, ferric oxide, magnetite, calcium carbonate, calcium phosphate, etc.

[0091] Another object of the present invention is to provide specific T cells prepared by the method described in the present invention.

[0092] Another object of the present invention is to provide cancer-specific T cells derived from autologous cells or allogeneic cells for preventing or treating cancer, and the above-mentioned specific T cells are CD3 + CD69 + 、CD3 +CD8 + CD69 + 、CD3 + CD4 + CD69+, CD3 + CD137 + 、CD3 + CD4 + CD137 + 、CD3 + CD8 + CD137 + 、CD3 + CD25 + 、CD3 + CD8 + CD25 + 、CD3 + CD4 + CD25 + 、、CD3 + CD134 + 、CD3 + CD8 + CD134 + 、CD3 + CD4 + CD134 + 、CD3 + IL-2R + 、CD3 + CD8 + IL-2R + 、CD3 + CD4 + IL-2R + 、CD3 + HLA-DR + 、CD3 + CD8 + HLA-DR + 、CD3 + CD4 + HLA-DR + 、CD3 + FASL + CD3 + CD8 + FASL + 、CD3 + CD4 + FASL + 、CD3 + OX40 + 、CD3 + CD8 + OX40 + 、CD3 + CD4+ OX40 + 、CD3 + TCF-1 + 、CD3 + CD8 + TCF-1 + 、CD3 + CD4 + TCF-1 + 、CD3 + PD-1 + 、CD3 + CD8 + PD-1 + 、CD3 + CD4 + PD-1 + 、CD3 + CD39 + 、CD3 + CD8 + CD39 + 、CD3 + CD4 + CD39 + 、CD3 + CD38 + 、CD3 + CD8 + CD38 + 、CD3 + CD4 + CD38 + 、CD3 + CD28 + 、CD3 + CD8 + CD28 + 、CD3 + CD4 + CD28 + 、、CD3 + CD71 + 、CD3 + CD8 + CD71 + 、CD3 + CD4 + CD71 + 、CD3 + CD44 + 、CD3 + CD8 + CD44 + 、CD3 + CD4 + CD44 + 、CD3 + CXCR3+ , CD3 + CD8 + CXCR3 + , CD3 + CD4 + CXCR3 + , CD3 + CXCR1 + , CD3 + CD8 + CXCR1 + , CD3 + CD4 + CXCR1 + , CD3 + ICAM-1 + , CD3 + CD8 + ICAM-1 + , CD3 + CD4 + ICAM-1 + , CD3 + CD70 + , CD3 + CD8 + CD70 + , CD3 + CD4 + CD70 + , CD3 + CD154 + , CD3 + CD8 + CD154 + , CD3 + CD4 + CD154 + , CD3 + CD62L + , CD3 + CD8 + CD62L + , CD3 + CD4 + CD62L + , CD3 + CD154 + , CD3 + CD8 + CD154 + , CD3 + CD4 + CD154 + , CD3 + CD160 + , CD3 + CD8 +CD160 + 、CD3 + CD4 + CD160 + 、CD3 + CD160 + 、CD3 + CD8 + CD160 + 、CD3 + CD4 + CD160 + 、CD3 + ICOS + 、CD3 + CD8 + ICOS + 、CD3 + CD4 + ICOS + 、CD3 + CD27 + 、CD3 + CD8 + CD27 + 、CD3 + CD4 + CD27 + 、CD3 + CD107A + 、CD3 + CD8 + CD107A + 、CD3 + CD4 + CD107A + including, but not limited to, any one or a combination thereof of T cells such as these, the survival rate of the specific T cells exceeds 60%, preferably exceeds 70%, more preferably exceeds 80%.

[0093] The specific T cells are obtained by co-incubating nanoparticles and / or microparticles carrying a tumor antigen component with peripheral blood mononuclear cells (PBMCs) isolated from immune cells in peripheral blood or peripheral immune organs.

[0094] In some embodiments, the PBMC cells are co-incubated with nanoparticles and / or microparticles carrying a tumor antigen component after sorting.

[0095] In some embodiments, cells co-incubated with nanoparticles and / or microparticles carrying tumor antigen components are sorted again to obtain specific T cells.

[0096] In some embodiments, the specific T cells can also undergo an in vitro amplification step, which can be performed before the aforementioned sorting step, after the aforementioned sorting step, or preferably, before the sorting step.

[0097] In some embodiments, when the above PBMC cells are co-incubated with nano / microparticles, antigen-presenting cells (APCs) are also present.

[0098] In some preferred embodiments, the specific T cells are CD3 + CD8 + CD69 + cells, and in some preferred embodiments, the specific T cells are CD3 + CD137 + cells, and in some preferred embodiments, the specific T cells are CD3 + CD8 + CD25 + cells and / or CD3 + CD8 + CD69 + cells, and in some preferred embodiments, the specific T cells are CD3 + CD69 + cells, and in some preferred embodiments, the specific T cells are CD3 + CD69 + cells, and in some preferred embodiments, the specific T cells are CD3 + CD8 + CD69 + cells, and in some preferred embodiments, the specific T cells are CD3 + CD8 + CD25 + cells and / or CD3 + CD4 + CD69 +Cells, and in some preferred embodiments, the specific T cells are CD8 + CD69 + Cells, and in some preferred embodiments, the specific T cells are CD8 + CD137 + Cells, and in some preferred embodiments, the specific T cells are CD8 + CD69 + Cells and / or CD4 + CD69 + Cells, and in some preferred embodiments, the specific T cells are CD3 + CD25 + Cells, and in some preferred embodiments, the specific T cells are CD3 + HLA-DR + And in some preferred embodiments, the specific T cells are CD3 + FASL + That's it.

[0099] Depending on the patient's needs, various types of T cells can be used alone or in combination.

[0100] An object of the present invention is to provide a method for preparing cancer-specific T cells derived from autologous cells or allogeneic cells for preventing or treating cancer, specifically including the following steps.

[0101] A step of separating mononuclear cells PBMC from immune cells in peripheral blood or peripheral immune organs, preferably, the PBMC is, by the first selection, CD3 + CD8 + T cells, CD19 + B cells, CD3 + T cells, CD8 + T cells, CD4 + T cells, B220 + B cells, CD69 - Of PBMC cells, CD25 - Of PBMC cells, CD3 + CD69 + T cells, CD11c +At least one of effector cells such as DC cells can be obtained, and before co-incubating T cells with nanoparticles / microparticles and antigen-presenting cells, T cells alone can be statically cultured for a certain period of time, or appropriately sorted, or before co-incubating T cells with activated antigen-presenting cells, T cells alone can be statically cultured for a certain period of time, or appropriately sorted.

[0102] Prepare nanoparticles and / or microparticles carrying tumor antigen components,

[0103] Preferably, in the preparation of the above tumor antigen components, first, tumor cells or tissues are separated, and the tumor cells or tissues are lysed to obtain any one or a combination of water-soluble components, water-insoluble components, and total components.

[0104] Preferably, nanoparticles and / or microparticles carrying tumor antigen components are prepared by the double emulsion method.

[0105] (3) Adding nanoparticles and / or microparticles carrying tumor antigen components to a medium and co-incubating with the PBMC cells or T cells described in (1) to obtain a cell culture, and further sorting specific T cells from the cell culture, wherein the specific T cells are CD3 + CD69 + , CD3 + CD8 + CD69 + , CD3 + CD4 + CD69+, CD3 + CD137 + , CD3 + CD4 + CD137 + , CD3 + CD8 + CD137 + , CD3 + CD25 + , CD3 + CD8 + CD25 + , CD3 + CD4 + CD25 + , CD3+ CD134 + 、CD3 + CD8 + CD134 + 、CD3 + CD4 + CD134 + 、CD3 + IL-2R + 、CD3 + CD8 + IL-2R + 、CD3 + CD4 + IL-2R + 、CD3 + HLA-DR + 、CD3 + CD8 + HLA-DR + 、CD3 + CD4 + HLA-DR + 、CD3 + FASL + CD3 + CD8 + FASL + 、CD3 + CD4 + FASL + 、CD3 + OX40 + 、CD3 + CD8 + OX40 + 、CD3 + CD4 + OX40 + 、CD3 + TCF-1 + 、CD3 + CD8 + TCF-1 + 、CD3 + CD4 + TCF-1 + 、CD3 + PD-1 + 、CD3 + CD8 + PD-1 + 、CD3 + CD4 + PD-1 + 、CD3 + CD39 + 、CD3 + CD8+ CD39 + , CD3 + CD4 + CD39 + , CD3 + CD38 + , CD3 + CD8 + CD38 + , CD3 + CD4 + CD38 + , CD3 + CD28 + , CD3 + CD8 + CD28 + , CD3 + CD4 + CD28 + , CD3 + CD71 + , CD3 + CD8 + CD71 + , CD3 + CD4 + CD71 + , CD3 + CD44 + , CD3 + CD8 + CD44 + , CD3 + CD4 + CD44 + , CD3 + CXCR3 + , CD3 + CD8 + CXCR3 + , CD3 + CD4 + CXCR3 + , CD3 + CXCR1 + , CD3 + CD8 + CXCR1 + , CD3 + CD4 + CXCR1 + , CD3 + ICAM-1 + , CD3 + CD8 + ICAM-1 + , CD3 + CD4+ ICAM-1 + and CD3 + CD70 + and CD3 + CD8 + CD70 + and CD3 + CD4 + CD70 + and CD3 + CD154 + and CD3 + CD8 + CD154 + and CD3 + CD4 + CD154 + and CD3 + CD62L + and CD3 + CD8 + CD62L + and CD3 + CD4 + CD62L + and CD3 + CD154 + and CD3 + CD8 + CD154 + and CD3 + CD4 + CD154 + and CD3 + CD160 + and CD3 + CD8 + CD160 + and CD3 + CD4 + CD160 + and CD3 + CD160 + and CD3 + CD8 + CD160 + and CD3 + CD4 + CD160 + and CD3 + ICOS + and CD3 + CD8 + ICOS + and CD3 + CD4 + ICOS + and CD3 +CD27 + , CD3 + CD8 + CD27 + , CD3 + CD4 + CD27 + , CD3 + CD107A + , CD3 + CD8 + CD107A + , CD3 + CD4 + CD107A + It includes any one or a combination thereof of specific T cells such as these, but is not limited thereto. For the surface activation marker, components of one or more surface activation markers can be used.

[0106] Preferably, in the co-incubation process, antigen-presenting cells at 10 to 50 million / ml are also added. The above antigen-presenting cells are any one or a combination thereof of B cells, DC cells, and macrophages.

[0107] Preferably, the nanoparticles and / or microparticles carrying the tumor antigen component can be co-incubated with antigen-presenting cells and T cells simultaneously to activate cancer cell-specific T cells. First, the nanoparticles and / or microparticles carrying the tumor antigen component can be co-incubated with antigen-presenting cells to activate the antigen-presenting cells, and then the activated antigen-presenting cells alone can be co-incubated with T cells to activate cancer cell-specific T cells. After co-incubating the nanoparticles and / or microparticles carrying the tumor antigen component with antigen-presenting cells, the antigen-presenting cells can be activated, and the antigen-presenting cells that do not require special treatment can be co-incubated with T cells to activate specific T cells. Or after performing treatments such as fixation, radiation, irradiation, modification, inactivation, and mineralization on the antigen-presenting cells, the antigen-presenting cells can be co-incubated with T cells to activate specific T cells.

[0108] Preferably, nanoparticles and / or microparticles carrying a tumor antigen component at 2.5 ng to 50 mg / ml are added to a culture medium, and co-incubated with PBMC cells or selected cells at 1 to 50 million / ml, at 30 to 38 °C and 1 to 5% CO 2 under the conditions for 4 to 96 hours to obtain a cell culture,

[0109] Preferably, during the co-incubation process, 10 to 500 ng / ml of interleukin is also added, and the interleukin is any one or a combination of IL-2, IL-7, IL-12, IL-15, IL-17, IL-21,

[0110] The above culture medium is any one of DMEM high glucose complete medium, RPM1640 medium, and AIMV serum-free medium,

[0111] The above co-incubation is carried out by incubating for 1 to 168 hours under the conditions of 30 to 38 °C, preferably 4 to 96 hours, more preferably 6 to 72 hours,

[0112] The above specific T cell survival rate exceeds 60%, preferably exceeds 70%, more preferably exceeds 80%, and

[0113] (4) Amplify the specific T cells obtained in (3),

[0114] Preferably, the above amplification step is as follows: add the specific T cells selected in step (2) at 1 to 50 million cells / mL to the amplification medium, and co-incubate at 30 to 38 °C and 1 to 5% CO 2 under the conditions, change the amplification medium every 2 to 3 days, and after co-incubating for 5 to 30 days, obtain amplified specific T cells,

[0115] Preferably, amplify with the amplification medium, and the above amplification medium is any one of DMEM high glucose complete medium and RPM1640 medium,

[0116] Preferably, the conditions for amplification culture are to culture at 30 to 38 °C for 4 to 72 days, preferably 5 to 30 days.

[0117] Preferably, the above amplification medium further contains 200 to 1000 U / ml of interleukin, 10 to 200 ng / ml of antibody, and / or 1 to 10 ng / mL of granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0118] Preferably, the above interleukin is any one or a combination thereof of IL-2, IL-7, IL-12, IL-15, IL-17, IL-21,

[0119] Preferably, the above antibody is any one or a combination thereof of αCD3 antibody and αCD28 antibody.

[0120] In a preferred technical solution of the present invention, in the above step (1), the above cells are selected from any part of an individual suffering from a tumor disease, preferably spleen cells and lymphocytes.

[0121] The survival rate of the above amplified specific T cells exceeds 60%, preferably exceeds 75%, and more preferably exceeds 80%.

[0122] In a preferred technical solution of the present invention, in the above step (2), the above selection method is to bind an antibody having fluorescence, magnetism or a specific ligand to a specific cell marker on the surface of T cells, and then use flow cytometry or magnetic beads, etc. to separate cells expressing the specific cell marker from the cell population.

[0123] In a preferred technical solution of the present invention, cytokines can be added to the above co-incubation process, and the added cytokines include, but are not limited to, interleukin, tumor necrosis factor, interferon, growth factor. Preferably, the added cytokines include interleukin 7 (IL-7) and interleukin 15 (IL-15).

[0124] In a preferred technical solution of the present invention, in step (3), the concentration of the above cytokine is 1 to 6000 ng / ml, preferably 5 to 100 ng / ml, more preferably 10 to 30 ng / ml.

[0125] In a preferred technical solution of the present invention, in the above step (3), the above cytokine includes, but is not limited to, interleukin, interferon, and tumor necrosis factor.

[0126] In a preferred technical solution of the present invention, the above interleukin includes, but is not limited to, interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 17 (IL-17), and interleukin 21 (IL-21).

[0127] In a preferred technical solution of the present invention, the concentration of the above antibody is 1 to 6000 ng / ml, preferably 5 to 100 ng / ml, more preferably 10 to 30 ng / ml.

[0128] In a preferred technical solution of the present invention, the above antibody includes, but is not limited to, any one or a combination thereof of αCD3 antibody, αCD28 antibody, αCD80 antibody, αCD86 antibody, and αOX40 antibody.

[0129] In a preferred technical solution of the present invention, the co-incubation time of the above nanoparticles / microparticles with the mixture of antigen-presenting cells and T cells is at least 1 hour, preferably 4 to 96 hours.

[0130] In a preferred technical solution of the present invention, the co-incubation time of the above nanoparticles / microparticles alone with antigen-presenting cells is at least 1 hour, preferably 4 to 96 hours.

[0131] In a preferred technical solution of the present invention, the co-incubation time of the above-activated antigen-presenting cells and the mixture of T cells is at least 1 hour, preferably 6 to 96 hours.

[0132] In a preferred technical solution of the present invention, the amplification culture time is at least 1 day, preferably 4 to 72 days.

[0133] In a preferred technical solution of the present invention, when the above nanoparticles / microparticles are co-incubated with antigen-presenting cells alone, the concentration of the nanoparticles / microparticles is 10 ng / mL to 5 mg / mL.

[0134] In a preferred technical solution of the present invention, when the above nanoparticles / microparticles are co-incubated with antigen-presenting cells and T cells, the concentration of the nanoparticles / microparticles is 2.5 ng / mL to 50 mg / mL.

[0135] In a preferred technical solution of the present invention, the content of the protein and polypeptide components in the antigen component carried by the above nanoparticles / microparticles exceeds 10 ng / mL.

[0136] In a preferred technical solution of the present invention, in steps (2) and (3), the above nanoparticles or microparticles are PLGA, the molecular weight is selected from 7 to 54 KDa, and the above immunoadjuvant is selected from any one or a combination of poly(I:C), polyICLC, BCG, and CpG.

[0137] In a preferred technical solution of the present invention, in steps (2) and (3), the above nanoparticles or microparticles are PLGA, the molecular weight is selected from 7 to 51 KDa, and the above immunoadjuvant is selected from any one or a combination of poly(I:C), polyICLC, BCG, and CpG.

[0138] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 24KDa to 38KDa, the immunoadjuvant used is poly(I:C), and poly(I:C) is only distributed inside the nanoparticles.

[0139] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 7Da to 17KDa, the immunoadjuvants used are poly(I:C) and CpG1018, and the mass ratio of the two immunoadjuvants is preferably 1:1.

[0140] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 7KDa to 17KDa, the immunoadjuvants used are poly(I:C), CpG2006 and CpG2216, and the mass ratio of the three immunoadjuvants is preferably 1:1:1.

[0141] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 24KDa to 38KDa, the immunoadjuvant used is poly(I:C), poly(I:C) is distributed inside the nanoparticles and also carried on the surface of the nanoparticles, and nanoparticles modified by freeze-silicification of the lysate and addition of cationic substances are carried both inside and outside.

[0142] In a preferred technical solution of the present invention, the above-mentioned microparticles use PLGA with a molecular weight of 24KDa to 38KDa, the immunoadjuvant used is poly(I:C), poly(I:C) is distributed inside the microparticles and also carried on the surface of the microparticles, and microparticles modified by freeze-silicification of the lysate and addition of cationic substances are carried both inside and outside.

[0143] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLA with a molecular weight of 20KDa, the immunoadjuvants used are poly(I:C) and CpG1018, and the mass ratio of the two immunoadjuvants is preferably 1:1.

[0144] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 24KDa to 38KDa, the immunoadjuvants used are poly(I:C) and CpG1018, and the antigen components and adjuvants are distributed on both the inside and the surface of the nanoparticles.

[0145] In a preferred technical solution of the present invention, the above-mentioned microparticles use PLGA with a molecular weight of 38KDa to 54KDa, use CpG and polyICLC as immunoadjuvants, and arginine can be added to the system to promote escape from lysosomes.

[0146] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 24KDa to 38KDa, the immunoadjuvants used are CpG and poly(I:C), and the mass ratio of the two immunoadjuvants is preferably 1:1.

[0147] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 7KDa to 17KDa, the immunoadjuvant used is BCG, and BCG is carried inside the nanoparticles.

[0148] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA and mannose-modified PLGA with a mass ratio of 4:1 and a molecular weight of 7KDa to 17KDa, the immunoadjuvants used are poly(I:C) and CpG, and the mass ratio of the two immunoadjuvants is preferably 1:1.

[0149] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 24KDa to 38KDa, the immunoadjuvants used are BCG and poly(I:C), and the mass ratio of the two immunoadjuvants is preferably 1:1.

[0150] In a preferred technical solution of the present invention, after the nanoparticles used carry whole cell antigens on the inside and surface, the nanoparticles are bio-mineralized, the molecular weight of PLGA is 7KDa to 17KDa, the immune adjuvants CpG and poly(I:C) used, and the GALA polypeptide (WEAALAEALAEALAEHLAEALAEALEALAA) that promotes escape from lysosomes are carried inside the nanoparticles, and the mass ratio of the two immune adjuvants is preferably 1:1.

[0151] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 7KDa to 17KDa, the immune adjuvants used are poly(I:C) and CpG, which promote escape from lysosomes, melittin is added to the system, and the adjuvant and melittin are encapsulated in the nanoparticles, and the mass ratio of the two immune adjuvants is preferably 1:1.

[0152] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 24KDa to 38KDa and mannan-modified PLGA, the mass ratio of PLGA and mannan-modified PLGA is 9:1, the immune adjuvants used are poly(I:C) and CpG, which promote escape from lysosomes, polyarginine and polylysine are added to the system, and the adjuvant, polyarginine and polylysine are encapsulated in the nanoparticles, and the mass ratio of the two immune adjuvants is preferably 1:1.

[0153] In a preferred technical solution of the present invention, the above-mentioned microparticles use PLGA with a molecular weight of 38KDa to 54KDa, the immune adjuvants used are CpG1018 and polyICLC, which promote escape from lysosomes, and the KALA polypeptide is added to the system, and the mass ratio of the two immune adjuvants is preferably 1:1.

[0154] In a preferred technical solution of the present invention, the microparticle skeleton material is unmodified PLA with a molecular weight of 40KDa and mannose-modified PLA, and the ratio of unmodified PLA to mannose-modified PLA is 9:1. The immune adjuvants used are CpG2395 and poly ICLC that promote escape from lysosomes, arginine and / or histidine are added to the system, and the mass ratio of the two immune adjuvants is preferably 1:1.

[0155] In a preferred technical solution of the present invention, the above nanoparticles use PLGA with a molecular weight of 7KDa to 17KDa, and the immune adjuvants used are poly(I:C) and CpG1018, which promote escape from lysosomes, and the R8 polypeptide is added to the system. The adjuvant and the R8 polypeptide are carried on the nanoparticles, and the mass ratio of the two immune adjuvants is preferably 1:1.

[0156] In a preferred technical solution of the present invention, the above nanoparticles use PLGA with a molecular weight of 7KDa to 17KDa, and poly(I:C) and CpG1018 are used as adjuvants, which promote escape from lysosomes, and NH 4 HCO 3 is added to the system, and the adjuvant and NH 4 HCO 3 are carried on the nanoparticles, and the mass ratio of the two immune adjuvants is preferably 1:1.

[0157] In a preferred technical solution of the present invention, the above nanoparticles use PLGA with a molecular weight of 20KDa to 40KDa, and the immune adjuvant used is poly(I:C).

[0158] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLA (molecular weight 30-40 kDa) and mannan-PEG2000-PLA (PLA with a molecular weight of 30-40 kDa), and the mass ratio of PLA (molecular weight 30-40 kDa) to mannan-PEG2000-PLA (PLA with a molecular weight of 30-40 kDa) is 9:1. The immunoadjuvants used are CpG2006 (class B), CpG2216 (class A) and poly ICLC, and the mass ratio of the three immunoadjuvants is preferably 1:1:1.

[0159] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 10 kDa to 20 kDa, and the immunoadjuvants used are poly(I:C), CpG7909 and CpG2395, and the mass ratio of the three immunoadjuvants is preferably 1:1:1.

[0160] In a preferred technical solution of the present invention, the above-mentioned nanoparticles use PLGA with a molecular weight of 10 kDa to 20 kDa, and the immunoadjuvants carried are poly(I:C) and CpG7909, and the mass ratio of the two immunoadjuvants is preferably 1:1.

[0161] In a preferred technical solution of the present invention, the above-mentioned microparticles use PLGA with a molecular weight of 38 kDa to 54 kDa, and the immunoadjuvants used are CpG1018 and poly ICLC, which promote escape from lysosomes, and the KALA polypeptide is added to the system, and the mass ratio of the two immunoadjuvants is preferably 1:1.

[0162] In a preferred technical solution of the present invention, the tumor antigen component contains a protein / polypeptide component in the cell lysate of tumor tissue and / or cancer cells, and its preparation method is as follows. (1) First, prepare a cell lysate of tumor tissue / cancer cells, then prepare the water-soluble component and water-insoluble component in the cell lysate, and then prepare the protein / polypeptide components in the water-soluble component and water-insoluble component respectively by methods such as salting out, heating, and enzymatic hydrolysis. Subsequently, carry them as antigen components on nano- or microparticles. (2) Alternatively, a solubilization solution containing a solubilizing agent can be directly used to directly lyse cells or tissues to dissolve all cell components. Next, the protein / polypeptide components therein are prepared respectively by methods such as salting out, heating, and enzymatic hydrolysis, and they are carried as antigen components on nano- or microparticles. The above-mentioned water-insoluble component and the precipitate obtained by treatments such as salting out, heating, and enzymatic hydrolysis are dissolved with a lysate containing a solubilizing agent.

[0163] In a preferred technical solution of the present invention, the preparation method of whole cells is as follows. (1) First, lyse cancer cells / tumor tissue, then prepare the water-soluble component and water-insoluble component respectively. Subsequently, dissolve the water-insoluble component with a specific solubilizing agent containing a lysate and then use it. (2) Use a lysate containing a solubilizing agent to lyse cells, and then use a lysate containing a solubilizing agent to dissolve all cell components lysed.

[0164] In a preferred technical solution of the present invention, a method for preparing a part of the whole cell components containing all cell proteins and polypeptide components in the whole cell components of cancer cells is as follows. (1) First, dissolve cancer cells / tumor tissues, then prepare water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separate and extract the protein components and polypeptide components in the water-soluble components from the water-soluble components by an appropriate method. Then, use the proteins and polypeptide components separated and extracted from the water-soluble components together with all the water-insoluble components as antigen components. (2) First, dissolve cancer cells / tumor tissues, then prepare water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separate and extract the protein components and polypeptide components in the water-soluble components from the water-insoluble components by an appropriate method. Then, use the proteins and polypeptide components separated and extracted from the water-insoluble components together with all the water-soluble components as antigen components. (3) First, decompose cancer cells / tumor tissues, then prepare water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separate and extract the protein components and polypeptide components in the water-soluble components from the water-soluble components and the water-insoluble components by an appropriate method respectively. Then, use the proteins and polypeptide components separated and extracted from the water-soluble components and the water-insoluble components as antigen components. (4) Use a lysate containing a solvent to lyse cells. Then, use a lysing solution containing the solvent to dissolve the lysed whole cell components. After that, separate and extract the protein and polypeptide components by an appropriate method.

[0165] Appropriate methods for separating and extracting protein and polypeptide components include, but are not limited to, salting out, heating, enzymatic hydrolysis, etc.

[0166] Separate and extract the above-mentioned protein and polypeptide components and dissolve them again in a lysate containing a solvent.

[0167] In a preferred technical solution of the present invention, in step (2), in the preparation of the above tumor antigen component, first, tumor cells or tissues are separated, and the tumor cells or tissues are lysed to obtain any one or a combination of a water-soluble component, a water-insoluble component, and a total component.

[0168] In a preferred technical solution of the present invention, in step (2), the above tumor antigen component is appropriately treated by methods such as salting out, heating, and enzymatic hydrolysis.

[0169] In a preferred technical solution of the present invention, both the water-soluble part and the water-insoluble part can be dissolved by a solubilized aqueous solution containing a solubilizing agent or an organic solvent.

[0170] In a preferred technical solution of the present invention, the above solubilizing agent is at least one of the solubilizing agents that can enhance the solubility of proteins or polypeptides in an aqueous solution, and the organic solvent is an organic solvent that dissolves proteins or polypeptides.

[0171] In a preferred technical solution of the present invention, the above water-insoluble part is changed from a state insoluble in pure water to a state soluble in an aqueous solution or an organic solvent containing a solubilizing agent by an appropriate solubilization method, and the solubilizing agent used is at least one selected from compounds containing the structure of Structural Formula 1, deoxycholate, lauryl sulfate, glycerol, proteolytic enzymes, albumin, lecithin, polypeptides, amino acids, glycosides, and choline. Structural Formula 1 is as follows:

Chemical formula

[0172] R1 is C, N, S, or O, and R2 to R5 are at least one independently selected from hydrogen, alkyl, amino, carboxyl, substituted and unsubstituted guanidino.

[0173] Compounds containing the structure of Structural Formula 1 include metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, other compounds containing guanidine such as guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoylurea, acetourea, sulfonylurea compounds (such as glibenclamide, glibencarpid, glazidone, glimepiride, etc.), thiourea compounds (such as thiouracil, imidazole, etc.), nitrosourea systems, etc., but are not limited thereto.

[0174] Containing the structure of Structural Formula 1 such as urea and guanidine hydrochloride, the inventor has found that substances having the structure of Structural Formula 1 can be used as a solubilizer in a dissolution solution for dissolving water-insoluble components in cells or tumor tissues. Therefore, in addition to general compounds containing guanidine such as urea, guanidine hydrochloride, and metformin, other compounds containing this structure also have the ability to act as a solubilizer for dissolving water-insoluble components.

[0175] In a preferred technical solution of the present invention, when an immunoadjuvant and cell components are co-loaded on nanoparticles or microparticles, after the nanoparticles or microparticles are engulfed by antigen-presenting cells, better cancer-specific T cells.

[0176] In a preferred technical solution of the present invention, the preparation method of the above-mentioned water-soluble components is as follows. Cut the tumor tissue or cancer cells finely, grind them, filter to obtain a single-cell suspension, add water and repeat freezing and thawing 1 to 5 times, dissolve with ultrasonic waves, and centrifuge the lysate at a rotation speed of 5000 to 10000 g for 5 to 10 minutes. Then, obtain the supernatant as the water-soluble component and the precipitate part as the water-insoluble component.

[0177] In a preferred technical solution of the present invention, the antigen component is a soluble component obtained by adding a solubilizing agent to a water-insoluble component, and the solubilizing agent is any one of urea, sodium deoxycholate, guanidine hydrochloride, N-octyl-β-D-glucopyranoside, arginine, glycerin, semicarbazide hydrochloride, and agmatine sulfate, or a combination thereof.

[0178] In a preferred technical solution of the present invention, the antigen component is a soluble component obtained by adding an aqueous urea solution to a water-insoluble component, and is mixed with the water-soluble component at a ratio of 3 to 1:1 to obtain a mixture.

[0179] In a preferred technical solution of the present invention, the antigen component is a mixture of the water-soluble component of tumor cells and the water-soluble component of cancer cells at a ratio of 1:1, or a mixture of the soluble component obtained by adding a solubilizing agent to the water-insoluble component of tumor cells and the soluble component obtained by adding a solubilizing agent to the water-insoluble component of cancer cells at a ratio of 1:1.

[0180] In a preferred technical solution of the present invention, for tumor tissues or cancer cells, any one of inactivation and denaturation treatment by ultraviolet high-temperature heating, and nuclease inactivation, or a combination thereof, is performed in advance.

[0181] In a preferred technical solution of the present invention, the antigen component is a component obtained by salting out and heat-precipitating a water-soluble component.

[0182] In a preferred technical solution of the present invention, the antigen component is the precipitate portion after dissolving and centrifuging tumor tissues, that is, a soluble component obtained by adding a solubilizing agent to the precipitate obtained by adding a solubilizing agent to the water-insoluble component and dissolving it twice, and the solubilizing agent is any one of Tween 80 and guanidine sulfate, or a combination thereof.

[0183] Another object of the present invention is to provide a method for preparing a pharmaceutical composition containing the specific T cells described in the present invention, and the preparation method includes a step of adding natural immune system enhancing substances such as albumin, NK cells, neutrophils, γδ T cells, and NK T cells to the cancer-specific T cells before reinjecting the cancer-specific T cells into a patient.

[0184] In a preferred technical solution of the present invention, the cell concentration of the above specific T cells is (0.01~100)×10 7 cells / ml, preferably (0.1~8)×10 7 cells / ml.

[0185] In a preferred technical solution of the present invention, the above pharmaceutical composition further contains any one or a combination of hydroxyethyl starch, sugar, and salt.

[0186] Another object of the present invention is the application of the specific T cells of the present invention in the preparation of drugs for treating or preventing cancer.

[0187] In a preferred technical solution of the present invention, the above specific T cells are administered multiple times before cancer occurs, after cancer occurs, or after surgical removal of tumor tissue.

[0188] Another object of the present invention is the application of the specific T cells of the present invention in the preparation of drugs for preventing cancer recurrence or metastasis.

[0189] Another object of the present invention is to provide the application of the specific T cells of the present invention in the preparation of anti-tumor immunotherapy products.

[0190] In a preferred technical solution of the present invention, the above-mentioned tumor is selected from solid tumors, hematological tumors, and lymphomas. Blood malignancies such as lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, leukemia, brain tumors, head and neck cancers, gliomas, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumors, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancer, metastatic cancer, circulating tumor cells, including any one or a combination thereof, but not limited thereto.

[0191] Preferably, the above-mentioned tumor is selected from any one or a combination thereof of melanoma, colon cancer, triple-negative breast cancer, pancreatic cancer, metastatic cancer, liver cancer, colon cancer, lymphoma, esophageal cancer, and non-small cell lung cancer.

[0192] In a preferred technical solution of the present invention, the above-mentioned immunotherapy is selected from any one or a combination thereof of immunotherapy in anti-tumor treatment and immunotherapy after radical surgery.

[0193] In a preferred technical solution of the present invention, the above-mentioned immunotherapy is selected from immunotherapy after radical resection of primary hepatocellular carcinoma.

[0194] In a preferred technical solution of the present invention, the above-mentioned drug is used for adult or pediatric patients.

[0195] Another object of the present invention is to provide the application of the specific T cells of the present invention in immunotherapy.

[0196] In a preferred technical solution of the present invention, the administration method of the above immunotherapy is one or a combination of intravenous injection, subcutaneous injection, intratumoral injection, intraperitoneal injection, intramuscular injection, and intradermal injection.

[0197] Another object of the present invention is to provide the application of the specific T cells of the present invention in combination with any one of radiotherapy, chemotherapy, targeted therapy, surgical treatment, or immunotherapy for anti-tumor or tumor immunotherapy.

[0198] Another object of the present invention is to provide the application of specific T cells in the preparation of drugs for enhancing anti-viral ability.

[0199] Another object of the present invention is to provide the application of specific T cells in the preparation of drugs for enhancing the treatment of autoimmune diseases.

[0200] In a preferred technical solution of the present invention, the autoimmune disease is selected from any one of systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, skin diseases, or a complication thereof.

[0201] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; and the rest is weight / weight percentage.

[0202] Compared with the prior art, the present invention has the following beneficial effects.

[0203] 1. The nanoparticles or microparticles prepared by the present invention carry tumor antigen components on the microparticles and / or nanoparticles in order to activate cancer cell-specific T cells in a broad-spectrum, and utilize the activated characteristics of cancer cell-specific T cells. After screening and amplification, it is reinjected into the patient for the treatment, prevention of recurrence or metastasis of cancer. The obtained cancer-specific T cells have a broad spectrum, high specificity, and can prevent or treat cancer by killing cancer cells after amplification.

[0204] 2. The present invention screens antigen components and nanoparticles obtained by a specific method, scientifically screens the conditions for screening and amplification, and the obtained T cells have high survival rate, excellent cell stability, high immune cell targeting and anti-tumor activity. When used for the treatment of tumor patients, it can prevent tumor recurrence, extend the tumor growth rate, prevent cancer metastasis, and extend the survival period.

[0205] 3. The method of the present invention has the advantages of being easy to operate, having controllable quality, and being suitable for industrial production.

Brief Description of the Drawings

[0206]

Figure 1

Figure 2

Figure 30

[0207] In the above drawings, unless otherwise specified, *** indicates a significant difference with p < 0.005 compared to the PBS blank control group, ** indicates a significant difference with p < 0.001 compared to the PBS blank control group, * indicates a significant difference with p < 0.001 compared to the PBS blank control group, τττ indicates a significant difference with p < 0.005 compared to the cancer-specific T cell group further sorted without nanoparticle stimulation, ■■■ indicates a significant difference with p < 0.005 compared to the cell control group obtained by the auxiliary sorting of blank nanoparticles + free cell lysate containing an immune adjuvant, λ indicates a significant difference with p < 0.05 compared to the cancer-specific T cell group sorted without adding IL-7 during the co-incubation process, ε indicates a significant difference with p < 0.05 compared to the cancer-specific T cell group co-incubated, sorted, and amplified with only one type of antigen-presenting cell, ε indicates a significant difference with p < 0.01 compared to the cancer-specific T cell group co-incubated, sorted, and amplified with only one type of antigen-presenting cell, η indicates a significant difference with p < 0.05 compared to the cancer-specific T cell group separated and amplified by nanoparticles / microparticles carrying an adjuvant, $ indicates a significant difference with p < 0.05 compared to the cancer-specific T cell group obtained by the auxiliary sorting of polypeptide nanoparticles or microparticles, $$ indicates a significant difference with p < 0.01 compared to the cancer-specific T cell group obtained by the auxiliary sorting of polypeptide nanoparticles or microparticles, δ indicates a significant difference with p < 0.05 compared to the cancer-specific T cell group obtained by the auxiliary sorting of nanoparticles without using an adjuvant, σ indicates CD8 obtained by the auxiliary sorting of nanoparticle 1 + indicates a significant difference with p < 0.05 compared to the cancer-specific T cell group, and Δ indicates CD8 obtained by the auxiliary sorting of nanoparticle 2 + cancer-specific T cell + CD4 + indicates a significant difference with p < 0.05 compared to the cancer-specific T cell group. # indicates a significant difference with p < 0.05, ## indicates a significant difference with p < 0.01, and indicates a significant difference with p < 0.005. ns indicates no significant difference.

Mode for Carrying Out the Invention

[0208] In the following examples, first, an antigen component is prepared. The antigen component can be (1) the whole cell lysate of cancer cells, (2) or a part of the whole cell component containing proteins and polypeptides in the whole cell component of cancer cells, (3) the protein, polypeptide component, and mRNA component in the whole cell component of cancer cells / tumor tissues.

[0209] The method for preparing whole cells is as follows. (1) First, cancer cells / tumor tissues are lysed. Next, a water-soluble component and a water-insoluble component are prepared respectively. Then, the water-insoluble component is dissolved with a specific solvent containing a lysate and then used. (2) Cells are lysed using a lysate containing a solvent. Next, the whole cell component lysed using the lysate containing a solvent is dissolved.

[0210] A method for preparing a part of the whole cell components containing the whole cell proteins and polypeptide components in the whole cell components of cancer cells is as follows. (1) First, dissolve the cancer cells / tumor tissues, then prepare the water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separate and extract the protein components and polypeptide components in the water-soluble components from the water-soluble components by an appropriate method. Then, use the proteins and polypeptide components separated and extracted from the water-soluble components together with all the water-insoluble components as antigen components. (2) First, dissolve the cancer cells / tumor tissues, then prepare the water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separate and extract the protein components and polypeptide components in the water-soluble components from the water-insoluble components by an appropriate method. Then, use the proteins and polypeptide components separated and extracted from the water-insoluble components together with all the water-soluble components as antigen components. (3) First, dissolve the cancer cells / tumor tissues, then prepare the water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separately separate and extract the protein components and polypeptide components in the water-soluble components from the water-soluble components and the water-insoluble components by an appropriate method. Then, use the proteins and polypeptide components separated and extracted from the water-soluble components and the water-insoluble components as antigen components. (4) Use a lysate containing a solvent to lyse the cells, then use a lysing solution containing the solvent to dissolve the lysed whole cell components. After that, separate and extract the protein and polypeptide components by an appropriate method. In the above preparation method, a step of separating and extracting the whole cell mRNA and using the whole cell mRNA as a part of the antigen components can also be added.

[0211] Appropriate methods for separating and extracting protein and polypeptide components include, but are not limited to, salting out, heating, enzymatic hydrolysis, etc.

[0212] Separate and extract the above-mentioned protein and polypeptide components, and dissolve them again in a lysate containing a solvent.

[0213] The method for preparing protein, polypeptide, and mRNA components in the total cell components of cancer cells / tumor tissues is as follows. (1) First, dissolve the cancer cells / tumor tissues, then prepare the water-soluble components and water-insoluble components respectively. After that, dissolve the water-insoluble components with a specific solvent containing a lysate and then use them. Subsequently, separately isolate and extract the protein, polypeptide, and mRNA components in the water-soluble components and / or water-insoluble components. Then, after mixing the protein, polypeptide, and mRNA components, use them as antigen components.

[0214] The above solvent is at least one selected from compounds containing the structure of Structural Formula 1, deoxycholate, lauryl sulfate, glycerol, protease, albumin, lecithin, polypeptide, amino acid, glycoside, and choline. Structural Formula 1 is as follows:

Chemical formula

[0215] R1 is C, N, S, or O, and R2 to R5 are at least one independently selected from hydrogen, alkyl, amino, carboxyl, substituted and unsubstituted guanidino.

[0216] Compounds containing Structural Formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, polyhexamethylene guanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, guanidine, or other compounds containing urea, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoylurea, acetourea, sulfonylurea compounds (such as glibenclamide, glibencarpid, glazidone, glimepiride, etc.), thiourea compounds (such as thiouracil, imidazole, etc.), and compounds containing Structural Formula 1 such as nitrosoureas.

[0217] Next, the antigen component is supported on nanoparticles or microparticles. The nanoparticles or microparticles carrying the cell component can be obtained by any preparation method for supporting the antigen component on nano / microparticles, including but not limited to any one of methods such as the solvent evaporation method, dialysis method, microfluidic method, extrusion method, and thermal melting method.

[0218] The antigen component can be supported inside the nanoparticles / microparticles, on the surface of the nanoparticles / microparticles, or on both the inside and the surface of the nanoparticles / microparticles.

[0219] In the examples of the present invention, the solvent evaporation method is used to illustrate a specific implementation, but in actual applications, other feasible preparation methods can also be used.

[0220] The preparation method of the above-mentioned nanoparticles or microparticles includes the following steps.

[0221] A first predetermined volume of an aqueous phase solution containing a first predetermined concentration is added to a second predetermined volume of an organic phase containing a medical polymer material with a second predetermined concentration.

[0222] The mixed solution obtained in Step 1 is subjected to ultrasonic treatment for 2 seconds or more, or stirring or homogenization treatment or microfluidic treatment for 1 minute or more.

[0223] The mixture obtained after the treatment in Step 2 is added to a third predetermined volume of an aqueous solution containing an emulsifier with a third predetermined concentration, and ultrasonic treatment for 2 seconds or more, or stirring or homogenization treatment or microfluidic treatment for 1 minute or more is performed.

[0224] The liquid obtained after the treatment in Step 3 is added to a fourth predetermined volume of an aqueous solution containing an emulsifier with a fourth predetermined concentration, and stirring is performed until predetermined stirring conditions are satisfied.

[0225] After the treatment in step 4, the mixed solution that satisfies the predetermined stirring conditions is centrifuged at a rotational speed exceeding 100 RPM for 1 minute or more, the supernatant is removed, and the remaining precipitate is resuspended in an aqueous solution of a fifth predetermined volume containing a cryoprotectant of a fifth predetermined concentration, or in a sixth predetermined volume of PBS (or physiological saline).

[0226] After freeze-drying the suspension containing the cryoprotectant obtained in step 5, a freeze-dried substance is obtained and prepared for use.

[0227] A sixth predetermined volume of suspension containing nanoparticles resuspended in the PBS (or physiological saline) obtained in step 5, or the freeze-dried nanoparticles or microparticles obtained in step 6 and the freeze-dried substance containing the cryoprotectant are resuspended using a sixth predetermined volume of PBS (or physiological saline) and used directly, or the above sample is used after mixing with a seventh predetermined volume of a water-soluble component or the solubilized original water-insoluble component.

[0228] In the method for preparing the above nanoparticles or microparticles, the aqueous phase solution can contain each component in the cancer cell lysate and an immunopotentiating adjuvant such as poly(I:C), BCG, manganese adjuvant, calcium adjuvant or CpG. Each component in the cancer cell lysate is respectively the original water-insoluble component dissolved in a water-soluble component, urea or guanidine hydrochloride during preparation. The concentration of the water-soluble component derived from cancer cells contained in the above aqueous phase solution, or the concentration of the original water-insoluble component derived from cancer cells dissolved in urea or guanidine hydrochloride, that is, at the first predetermined concentration, it is necessary that the concentration of proteins and polypeptides exceeds 0.01 ng / mL and can carry cancer antigens sufficient to activate the relevant immune response. The concentration of the immunopotentiating adjuvant in the initial aqueous phase exceeds 0.01 ng / mL.

[0229] The aqueous solution can contain each component in the tumor lysate and an immunopotentiating adjuvant such as poly(I:C), BCG, manganese adjuvant, calcium adjuvant or CpG. Each component in the tumor lysate is, respectively, the original water-insoluble component dissolved in a water-soluble component, urea or guanidine hydrochloride during preparation. The concentration of the water-soluble component derived from the tumor tissue contained in the above aqueous solution, or the concentration of the original water-insoluble component derived from the tumor tissue dissolved in urea or guanidine hydrochloride, that is, at the first predetermined concentration, the concentration of proteins and polypeptides exceeds 0.01 ng / mL, and it is necessary to be able to carry a cancer antigen sufficient to activate the related immune response. The concentration of the immunopotentiating adjuvant in the initial aqueous phase exceeds 0.01 ng / mL.

[0230] Dissolve the medical polymer material in an organic solvent to obtain a second predetermined volume of organic phase containing the medical polymer material at a second predetermined concentration. In some embodiments, the medical polymer material is PLGA, and the organic solvent is selected as dichloromethane. Also, in some embodiments, the range of the second predetermined concentration of the medical polymer material is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0231] Actually, the second predetermined volume of the organic phase is set according to the ratio to the first predetermined volume of the aqueous phase. In the present invention, the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1:1.1 to 1:5000, preferably 1:10. During 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 needed to adjust the size of the prepared nanoparticles or microparticles.

[0232] Preferably, when the aqueous solution is a solution of the dissolved component, the concentration of the protein and polypeptide exceeds 1 ng / mL, preferably 1 mg / mL to 100 mg / mL. When the aqueous solution is a solution of the dissolved component / immune adjuvant, the concentration of the protein and polypeptide exceeds 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of the immune adjuvant exceeds 0.01 ng / mL, preferably 0.01 mg / mL to 20 mg / mL. In the organic phase solution of the polymer material, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane, and the concentration of the polymer material is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL. The first emulsifier solution is preferably an aqueous solution of polyvinyl alcohol having a concentration of 10 mg / mL to 50 mg / mL, preferably 20 mg / mL. The second emulsifier solution is preferably an aqueous solution of polyvinyl alcohol having a concentration of 1 mg / mL to 20 mg / mL, preferably 5 mg / mL. The dispersion is PBS buffer, physiological saline, pure water, etc.

[0233] Step 2: Perform ultrasonic treatment on the mixed solution obtained in Step 1 for 2 seconds or more, or stirring or homogenization treatment or microfluidic treatment for 1 minute or more. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed exceeds 50 rpm, and the stirring time exceeds 1 minute. For example, the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.1 hour to 24 hours. In the case of ultrasonic treatment, the ultrasonic output exceeds 5 W, and the time exceeds 0.1 second. For example, it is 2 to 200 seconds. In the case of homogenization treatment, when using a high-pressure / ultra-high-pressure homogenizer, the pressure exceeds 5 psi. For example, it is 20 psi to 100 psi. When using a high-shear homogenizer, the rotation speed exceeds 100 rpm. For example, it is 1000 rpm to 5000 rpm. When using microfluidic treatment, the flow rate exceeds 0.01 mL / min. For example, it is 0.1 mL / min to 100 mL / min. Nanonization and / or micronization are performed by ultrasonic or stirring or homogenization treatment or microfluidic treatment. The length of the ultrasonic treatment time, or the stirring speed, or the pressure and time of homogenization can control the size of the prepared microparticles and nanoparticles. If it is too large or too small, the particle size will change.

[0234] Step 3: Add the mixture obtained after the treatment in Step 2 to an aqueous solution with a third predetermined volume containing an emulsifier at a third predetermined concentration, and perform ultrasonic treatment for 2 seconds or more, or stirring for 1 minute or more, or homogenization treatment, or microfluidic treatment. In this step, the mixture obtained in Step 2 is added to the aqueous emulsifier solution, and ultrasonic treatment or stirring is continued to perform nanosizing or microparticulation. This step is executed for nanosizing or microparticulation. The length of the ultrasonic treatment time or the stirring speed and time can control the size of the microparticles and nanoparticles to be prepared. If it is too large or too small, the particle size will change. Therefore, it is necessary to select an appropriate ultrasonic treatment time. In the present invention, the ultrasonic treatment time exceeds 0.1 second, for example, it is 2 to 200 seconds, the stirring speed exceeds 50 rpm, for example, it is 50 rpm to 500 rpm, and the stirring time exceeds 1 minute, for example, it is 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed exceeds 50 rpm, the stirring time exceeds 1 minute. For example, the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.5 hour to 5 hours. In the case of ultrasonic treatment, the ultrasonic output is 50 W to 500 W, and the time exceeds 0.1 second, for example, it is 2 to 200 seconds. In the case of homogenization treatment, when using a high-pressure / ultra-high-pressure homogenizer, the pressure exceeds 20 psi, for example, it is 20 psi to 100 psi. When using a high-shear homogenizer, the rotation speed exceeds 1000 rpm, for example, it is 1000 rpm to 5000 rpm. When using microfluidic treatment, the flow rate exceeds 0.01 mL / min, for example, it is 0.1 mL / min to 100 mL / min. Nanosizing or microparticulation is performed by ultrasonic or stirring or homogenization treatment or microfluidic treatment. The length of the ultrasonic treatment time, or the stirring speed, or the pressure and time of homogenization can control the size of the nanoparticles and microparticles to be prepared. If it is too large or too small, the particle size will change.

[0235] In the present invention, 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 the 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 1:1.1 to 1:1000, and preferably can be 2:5. In order to control the size of nanoparticles or microparticles during a specific implementation, the ratio between the second predetermined volume and the third predetermined volume can be adjusted. Similarly, the ultrasonic treatment time or stirring time, the volume and concentration of the aqueous emulsifier solution in this step are all based on obtaining nanoparticles or microparticles of an appropriate size.

[0236] Step 4: Add the liquid obtained after the treatment in Step 3 to an aqueous solution with a fourth predetermined volume containing an emulsifier with a fourth predetermined concentration, and stir until the predetermined stirring conditions are satisfied.

[0237] In this step, the aqueous emulsifier solution is still PVA.

[0238] The fourth predetermined concentration is 5 mg / mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of an appropriate size. The selection of the fourth predetermined volume is determined based on the ratio between the third predetermined volume and the fourth predetermined volume. In the present invention, the range of the ratio between the third predetermined volume and the third predetermined volume is set to 1:1.5 to 1:2000, and preferably is 1:10. In order to control the size of nanoparticles or microparticles during a specific implementation, the ratio between the third predetermined volume and the fourth predetermined volume can be adjusted.

[0239] In the present invention, the predetermined stirring conditions in this step are until the volatilization of the organic solvent is completed, that is, until the volatilization of dichloromethane in Step 1 is completed.

[0240] Step 5: After the treatment in Step 4, the mixed solution that satisfies the predetermined stirring conditions is centrifuged at a rotation speed exceeding 100 RPM for 1 minute or more, the supernatant is removed, and the remaining precipitate is resuspended in an aqueous solution of a fifth predetermined volume containing a cryoprotectant at a fifth predetermined concentration, or in a sixth predetermined volume of PBS (or physiological saline).

[0241] In some embodiments of the present invention, when the precipitate obtained in Step 5 is resuspended in a sixth predetermined volume of PBS (or physiological saline), it is not necessary to perform lyophilization, and subsequent experiments regarding the adsorption of cancer cell lysates onto the surface of the nanoparticles or microparticles can be directly carried out.

[0242] In some embodiments of the present invention, when the precipitate obtained in Step 5 is resuspended in an aqueous solution containing a cryoprotectant, lyophilization is required. After lyophilization, subsequent experiments regarding the adsorption of cancer cell lysates onto the surface of the nanoparticles or microparticles are performed.

[0243] In the present invention, the cryoprotectant is trehalose.

[0244] In the present invention, the fifth predetermined concentration of the cryoprotectant in this step is set to 4% by mass percentage because it does not affect the lyophilization effect during subsequent lyophilization.

[0245] Step 6: After the suspension containing the cryoprotectant obtained in Step 5 is lyophilized, a lyophilized substance is obtained and prepared for use.

[0246] Step 7: The sixth predetermined volume of suspension containing nanoparticles obtained in Step 5 and resuspended in PBS (or physiological saline), or the lyophilized nanoparticles or microparticles obtained in Step 6 containing the cryoprotectant and the lyophilized substance are resuspended using the sixth predetermined volume of PBS (or physiological saline), and used directly, or the above sample is used after being mixed with a seventh predetermined volume of a water-soluble component or the solubilized original water-insoluble component.

[0247] 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.

[0248] In some embodiments, when the volume of the resuspended nanoparticle suspension is 10 mL, the volume of the water-soluble component in the lysate containing cancer cells or the lysate containing tumor tissue, or the solubilized original water-insoluble component is 1 mL. In actual use, the two volumes and ratios can be adjusted as needed.

[0249] In a preferred technical solution of the present invention, the method for preparing nanoparticles or microparticles by the double emulsion method includes the following steps.

[0250] Add a first predetermined volume of aqueous phase solution containing a first predetermined concentration to a second predetermined volume of organic phase containing a medical polymer material of a second predetermined concentration.

[0251] Perform ultrasonic treatment on the mixed solution obtained in step 1 for 2 seconds or more, or stirring or homogenization treatment or microfluidic treatment for 1 minute or more.

[0252] Add the mixture obtained after the treatment in step 2 to a third predetermined volume of aqueous solution containing an emulsifier of a third predetermined concentration, and perform ultrasonic treatment for 2 seconds or more or stirring or homogenization treatment or microfluidic treatment for 1 minute or more.

[0253] Add the liquid obtained after the treatment in step 3 to a fourth predetermined volume of aqueous solution containing an emulsifier of a fourth predetermined concentration, stir until a predetermined stirring condition is satisfied, or directly perform the next treatment without stirring.

[0254] After the treatment of step 4, the mixed solution satisfying the predetermined stirring conditions is centrifuged at a rotational speed exceeding 100 RPM for 1 minute or more, the supernatant is removed, and the remaining precipitate is resuspended in a fifth predetermined volume of a solution containing water-soluble and / or water-insoluble components in the whole cell components at a fifth predetermined concentration, or the remaining precipitate is resuspended in a fifth predetermined volume of a solution containing a mixture of water-soluble and / or water-insoluble components and an adjuvant in the whole cell components at a fifth predetermined concentration.

[0255] After the treatment of step 5, the mixed solution satisfying the predetermined stirring conditions is centrifuged at a rotational speed exceeding 100 RPM for 1 minute or more, the supernatant is removed, and the remaining precipitate is resuspended in a sixth predetermined volume of a solidification treatment reagent or a mineralization treatment reagent, allowed to act for a certain period, then centrifuged and washed, and then a seventh predetermined volume of a positively charged substance or a negatively charged substance is added and allowed to act for a certain period.

[0256] After drying the suspension containing the cryoprotectant obtained in step 6, a dried substance is obtained and prepared for use.

[0257] Resuspend in the PBS (or physiological saline) obtained in step 6 to obtain an eighth predetermined volume of a suspension containing nanoparticles, or use an eighth predetermined volume of PBS (or physiological saline) to resuspend the dried nanoparticles or microparticles and the dried substance containing the cryoprotectant obtained in step 7 and use directly, or mix with a ninth predetermined volume of water-soluble components or water-insoluble components and use.

[0258] In some embodiments, the aqueous solution can contain each component in the cancer cell lysate and an immune enhancer adjuvant such as poly(I:C), manganese adjuvant, calcium adjuvant, BCG or CpG. Each component in the cancer cell lysate is, respectively, the original water-insoluble component dissolved in a water-soluble component or urea or guanidine hydrochloride during preparation. The concentration of the water-soluble component derived from cancer cells contained in the above aqueous solution, or the concentration of the original water-insoluble component derived from cancer cells dissolved in urea or guanidine hydrochloride, that is, at the first predetermined concentration, the concentration of proteins and polypeptides exceeds 0.01 ng / mL, and it is necessary to be able to carry a cancer antigen sufficient to activate the related immune response. The concentration of the immune enhancer adjuvant in the initial aqueous phase exceeds 0.01 ng / mL.

[0259] In some embodiments, the aqueous solution can contain each component in the tumor tissue lysate and an immune enhancer adjuvant such as poly(I:C), manganese adjuvant, calcium adjuvant, BCG or CpG. Each component in the tumor tissue lysate is, respectively, the original water-insoluble component dissolved in a water-soluble component or urea or guanidine hydrochloride during preparation. The concentration of the water-soluble component derived from tumor tissue contained in the above aqueous solution, or the concentration of the original water-insoluble component derived from tumor tissue dissolved in urea or guanidine hydrochloride, that is, at the first predetermined concentration, the concentration of proteins and polypeptides exceeds 0.01 ng / mL, and it is necessary to be able to carry a cancer antigen sufficient to activate the related immune response. The concentration of the immune enhancer adjuvant in the initial aqueous phase exceeds 0.01 ng / mL.

[0260] In the present invention, a medical polymer material is dissolved in an organic solvent to obtain a second predetermined volume of an organic phase containing the medical polymer material at a second predetermined concentration. In some embodiments, the medical polymer material is PLGA, and the organic solvent is dichloromethane. Also, in some embodiments, the range of the second predetermined concentration of the medical polymer material is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0261] In the present invention, PLGA or modified PLGA is selected because the material is a biodegradable material and its use as a pharmaceutical dressing has been approved by the FDA. Research has shown that PLGA has specific immunomodulatory functions and is thus suitable as an excipient in the preparation of nanoparticles or microparticles.

[0262] Actually, the second predetermined volume of the organic phase is set according to the ratio to the first predetermined volume of the aqueous phase. In the present invention, the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1:1.1 to 1:5000, preferably 1:10. During 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 needed to adjust the size of the prepared nanoparticles or microparticles.

[0263] Preferably, when the aqueous phase solution is the lysate component solution, the concentration of the protein and polypeptide exceeds 1 ng / mL, preferably 1 mg / mL to 100 mg / mL. When the aqueous phase solution is the lysate component / immune adjuvant solution, the concentration of the protein and polypeptide exceeds 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of the immune adjuvant exceeds 0.01 ng / mL, preferably 0.01 mg / mL to 20 mg / mL. In the organic phase solution of the polymer material, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane, and the concentration of the polymer material is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL. The first emulsifier solution is preferably an aqueous polyvinyl alcohol solution with a concentration of 10 mg / mL to 50 mg / mL, preferably 20 mg / mL. The second emulsifier solution is preferably an aqueous polyvinyl alcohol solution with a concentration of 1 mg / mL to 20 mg / mL, preferably 5 mg / mL. The dispersion is PBS buffer, physiological saline, pure water, etc.

[0264] Step 2: Perform ultrasonic treatment for 2 seconds or more, or stirring or homogenization treatment or microfluidic treatment for 1 minute or more on the mixed solution obtained in Step 1. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed exceeds 50 rpm, and the stirring time exceeds 1 minute. For example, the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.1 hour to 24 hours. In the case of ultrasonic treatment, the ultrasonic output exceeds 5 W, and the time exceeds 0.1 second. For example, it is 2 to 200 seconds. In the case of homogenization treatment, when using a high-pressure / ultra-high-pressure homogenizer, the pressure exceeds 5 psi. For example, it is 20 psi to 100 psi. When using a high-shear homogenizer, the rotation speed exceeds 100 rpm. For example, it is 1000 rpm to 5000 rpm. When using microfluidic treatment, the flow rate exceeds 0.01 mL / min. For example, it is 0.1 mL / min to 100 mL / min. Nanonization and / or micronization are performed by ultrasonic or stirring or homogenization treatment or microfluidic treatment. The length of the ultrasonic treatment time, or the stirring speed, or the pressure and time of homogenization can control the size of the prepared microparticles and nanoparticles. If it is too large or too small, the particle size will change.

[0265] Step 3: Add the mixture obtained after the treatment in Step 2 to an aqueous solution with a third predetermined volume containing an emulsifier at a third predetermined concentration, and perform ultrasonic treatment for 2 seconds or more, or stirring for 1 minute or more, or homogenization treatment, or microfluidic treatment. In this step, the mixture obtained in Step 2 is added to the aqueous emulsifier solution, and ultrasonic treatment or stirring is continued to perform nanosizing or microparticulation. This step is executed for nanosizing or microparticulation. The length of the ultrasonic treatment time or the speed and time of stirring can control the sizes of the microparticles and nanoparticles to be prepared. If it is too large or too small, the particle size will change. Therefore, it is necessary to select an appropriate ultrasonic treatment time. In the present invention, the ultrasonic treatment time exceeds 0.1 second, for example, it is 2 to 200 seconds, the stirring speed exceeds 50 rpm, for example, it is 50 rpm to 500 rpm, and the stirring time exceeds 1 minute, for example, it is 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed exceeds 50 rpm, the stirring time exceeds 1 minute. For example, the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.5 hour to 5 hours. In the case of ultrasonic treatment, the ultrasonic output is 50 W to 500 W, the time exceeds 0.1 second, for example, it is 2 to 200 seconds. In the case of homogenization treatment, when using a high-pressure / ultra-high-pressure homogenizer, the pressure exceeds 20 psi, for example, it is 20 psi to 100 psi. When using a high-shear homogenizer, the rotation speed exceeds 100 rpm, for example, it is 1000 rpm to 5000 rpm. When using microfluidic treatment, the flow rate exceeds 0.01 mL / min, for example, it is 0.1 mL / min to 1000 mL / min. Nanosizing or microparticulation is performed by ultrasonic treatment, or stirring, or homogenization treatment, or microfluidic treatment. The length of the ultrasonic treatment time, or the stirring speed, or the pressure and time of homogenization can control the sizes of the nanoparticles and microparticles to be prepared. If it is too large or too small, the particle size will change.

[0266] In the present invention, 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 the 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 1:1.1 to 1:1000, and preferably can be 2:5. In order to control the size of nanoparticles or microparticles during a specific implementation, the ratio between the second predetermined volume and the third predetermined volume can be adjusted. Similarly, the ultrasonic treatment time or stirring time, the volume and concentration of the aqueous emulsifier solution in this step are all based on obtaining nanoparticles or microparticles of an appropriate size.

[0267] Step 4: Add the liquid obtained after the treatment in Step 3 to an aqueous solution with a fourth predetermined volume containing an emulsifier at a fourth predetermined concentration, and stir until the predetermined stirring conditions are met, or the next treatment can be directly performed without stirring.

[0268] In this step, the aqueous emulsifier solution is still PVA.

[0269] The fourth predetermined concentration is 5 mg / mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of an appropriate size. The selection of the fourth predetermined volume is determined based on the ratio between the third predetermined volume and the fourth predetermined volume. In the present invention, the range of the ratio between the third predetermined volume and the fourth predetermined volume is set to 1:1.5 to 1:2000, and preferably is 1:10. In order to control the size of nanoparticles or microparticles during a specific implementation, the ratio between the third predetermined volume and the fourth predetermined volume can be adjusted.

[0270] In the present invention, the predetermined stirring conditions in this step are that the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in Step 1 is completed. The subsequent experiment can also be performed without stirring.

[0271] Step 5: After the treatment in Step 4, centrifuge the mixed solution that meets the predetermined stirring conditions at a rotation speed exceeding 100 RPM for 1 minute or more, remove the supernatant, and resuspend the remaining precipitate in a fifth predetermined volume of a solution containing water-soluble and / or water-insoluble components in the whole cell components at a fifth predetermined concentration, or resuspend the remaining precipitate in a fifth predetermined volume of a solution containing a mixture of water-soluble and / or water-insoluble components in the whole cell components at a fifth predetermined concentration and an adjuvant.

[0272] Step 6: After the treatment in Step 5, centrifuge the mixed solution that meets the predetermined stirring conditions at a rotation speed exceeding 100 RPM for 1 minute or more, remove the supernatant, and resuspend the remaining precipitate in a sixth predetermined volume of a solidification treatment reagent or a mineralization treatment reagent, allow it to act for a certain period, then centrifuge and wash, and then add a seventh predetermined volume of a positively charged substance or a negatively charged substance and allow it to act for a certain period.

[0273] In some embodiments of the present invention, after resuspending the precipitate obtained in Step 6 in a seventh predetermined volume of the charged substance, it is not necessary to perform freeze-drying, and subsequent experiments regarding the loading of cancer cell / tissue lysates onto the surface of the nanoparticles or microparticles can be directly carried out.

[0274] In some embodiments of the present invention, after resuspending the precipitate obtained in Step 6 in an aqueous solution containing a cryoprotectant, perform room temperature vacuum drying or freeze vacuum drying. After drying, conduct subsequent experiments regarding the adsorption of cancer cell lysates onto the surface of the nanoparticles or microparticles.

[0275] In the present invention, the cryoprotectant is selected from trehalose or a mixture of mannitol and sucrose. In the present invention, the fifth predetermined concentration of the cryoprotectant in this step is set to 4% by mass percentage because it does not affect the drying effect during subsequent drying.

[0276] Step 7: After drying the suspension containing the cryoprotectant obtained in Step 6, obtain a dried substance for use.

[0277] Step 8: Resuspend in the PBS (or physiological saline) obtained in Step 6 to obtain an eighth predetermined volume of suspension containing nanoparticles, or use the eighth predetermined volume of PBS (or physiological saline) to resuspend the dried nanoparticles or microparticles obtained in Step 7 and the dried substance containing the cryoprotectant, and use it directly, or mix it with a ninth predetermined volume of water-soluble component or water-insoluble component and then use it.

[0278] In the present invention, the modification steps of Steps 5 to 8 and the antigen loading step can be repeated multiple times to improve the antigen loading amount. Also, when adding a positively or negatively charged substance, substances with the same charge can be added multiple times, or substances with different charges can be added alternately.

[0279] In some embodiments, when the volume of the resuspended nanoparticle suspension is 10 mL, the volume of the water-soluble component in the lysate containing cancer cells or the lysate containing tumor tissue, or the original water-insoluble component, is 0.1 to 100 mL. In actual use, the two volumes and ratios can be adjusted as needed.

[0280] In the present invention, the water-soluble component or the original water-insoluble component containing the cancer cell lysate or tumor tissue lysate used contains poly(I:C), manganese adjuvant, live vaccine for tuberculosis prevention (BCG), or CpG, and the concentration of poly(I:C), calcium adjuvant, BCG, or CpG exceeds 0.01 ng / mL.

[0281] Furthermore, during the preparation of the nanoparticles or microparticles described in the present invention, when activating cancer-specific T cells in vitro, nanoparticles and / or microparticles carrying only the water-soluble component and nanoparticles and / or microparticles carrying only the water-insoluble component can be used simultaneously, or nanoparticles and / or microparticles carrying only the water-soluble component can be used, nanoparticles and / or microparticles carrying only the water-insoluble component can be used, or nanoparticles and / or microparticles carrying both the water-soluble component and the water-insoluble component can be used.

[0282] After preparing nanoparticles or microparticles carrying tumor antigens by any method, the nanoparticles or microparticles are co-incubated with antigen-presenting cells and T cells simultaneously to activate cancer cell-specific T cells. Alternatively, nanoparticles and / or microparticles carrying tumor antigen components are first co-incubated with antigen-presenting cells to activate the antigen-presenting cells, and then the activated antigen-presenting cells alone are co-incubated with T cells to activate cancer cell-specific T cells.

[0283] Before co-incubating T cells with nanoparticles / microparticles and antigen-presenting cells, T cells alone can be cultured in a quiescent state for a certain period, or appropriately sorted. Alternatively, before co-incubating T cells with activated antigen-presenting cells, T cells alone can be cultured in a quiescent state for a certain period, or appropriately sorted.

[0284] After nanoparticles and / or microparticles carrying tumor antigen components are co-incubated with antigen-presenting cells, the antigen-presenting cells are activated, and the antigen-presenting cells that do not require special treatment can be co-incubated with T cells to activate specific T cells. Alternatively, after performing treatments such as fixation, radiation, irradiation, modification, inactivation, and mineralization on the antigen-presenting cells, the antigen-presenting cells can be co-incubated with T cells to activate specific T cells.

[0285] After cancer cell-specific T cells are activated, cell separation methods such as flow cytometry or magnetic bead sorting are used to select the activated cancer cell-specific T cells from the incubated cells. Next, after amplifying the cancer cell-specific T cells in vitro for a certain period, the amplified cancer cell-specific T cells are used for cancer prevention or treatment.

[0286] When selecting activated cancer cell-specific T cells, either one activation marker by which the T cells are activated or a combination of one or more different markers can be used as the activation marker.

[0287] Molecules that can be used as surface markers include, but are not limited to, any one or any combination of CD69, CD137, CD25, CD134, CD80, CD86, OX40L, OX40, CD28, FAS-L, IL-2R, HLA-DR, CD127 (IL-7R), CD150, CD107A, CD83, CD166, CD39, CD178, CD212, CD229, CD100, CD107b, CD108, CD109, CD113, CD122, CD126, CD253, CD197, PD-1, TIM3, LAG-3, TIGIT, CD62L, CD70, CTLA-4 (CD152), CD27, CD26, CD30, TNFRSF9, CD74, PD-L1 (CD274), CD258, CD261, 4-1BB, CD154, ICAM-1, LFA-1, LFA-2, VLA-4, CD160, CD71, CXCR3, TNFRSF14, TNFRSF18, TNFSF4, TNFSF9, TNFSF14, CD11a, CD101, CD48, CD244, CD49a, CD95, CD44, CXCR1, CD103, CD45RO, ICOS (CD278), VTCN1, HLA2, LGAL59, CCR7, CD357, BCL6, TCF-1, CD38, CD27, etc.

[0288] Hereinafter, the present invention will be further described with reference to the accompanying drawings and specific examples so that those skilled in the art can better understand and implement the present invention, but these examples are not intended to limit the present invention.

Examples

[0289] Example 1, Separation and Amplification of T Cells for Melanoma Prevention In this example, mouse melanoma is used as a cancer model to explain how to use nanoparticles to separate and amplify peripheral cancer cell-specific T cells for cancer prevention. In this example, the tumor tissue of B16F10 melanoma is lysed to prepare the water-soluble component and water-insoluble component of the tumor tissue. Next, the organic polymer material PLGA is used as the nanoparticle skeleton material, and polyinosinic acid-polycytidylic acid (poly(I:C)) is used as an immunoadjuvant. The water-soluble component and water-insoluble component of the tumor tissue are prepared by the solvent evaporation method. Subsequently, the nanoparticles assist in the separation of cancer cell-specific T cells in the organ, amplify the separated cancer cell-specific T cells, and then reinject them into the body to prevent melanoma. In this example, immune cells in the peripheral spleen cells of mice are used, and in actual applications, peripheral blood or peripheral lymph node cells can be directly used.

[0290] (1) Preparation of antigen component 1.5×10 5 B16F10 cells are subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor volume reaches about 1000 mm 3 , the mouse is sacrificed and the tumor tissue is removed. After cutting the tumor tissue into small pieces, it is ground and passed through a cell filter to prepare a single-cell suspension of the tumor tissue (containing cancer cells). Next, an appropriate amount of pure water is added to the single-cell suspension of the tumor tissue, and freezing and thawing are repeated 5 times. In some cases, ultrasonic waves may also be used in combination to disrupt the lysed cells. After lysing the cells, the lysate is centrifuged at a rotation speed of 5000 g for 5 minutes, and the supernatant is taken as the water-soluble component. 8 M urea aqueous solution (containing 500 mM sodium chloride) is added to the obtained precipitate to dissolve the precipitate, whereby the water-insoluble component insoluble in pure water can be solubilized in 8 M urea aqueous solution. The above is the antigen component for preparing nanoparticles.

[0291] (2) Preparation of nanoparticles In this example, nanoparticles are prepared by the double emulsion method in the solvent evaporation method. During the preparation, nanoparticles carrying water-soluble components in the whole cell components and nanoparticles carrying water-insoluble components in the whole cell components are prepared separately and then used in combination during use. The molecular weight of PLGA, which is the material for preparing the nanoparticles used, is 24KDa to 38KDa, and the immunoadjuvant used is poly(I:C). The preparation method is as described above. In the preparation process, first, the double emulsion method is used to carry the antigen component and the adjuvant in the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 10,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then freeze-dried for 48 hours. The average particle size of the nanoparticles is about 280 nm, the surface potential of the nanoparticles is about -3 mV, about 100 μg of protein or polypeptide component is carried per 1 mg of the PLGA nanoparticles, and 0.02 mg of poly(I:C) is carried per 1 mg of the PLGA nanoparticles. The materials and methods for preparing the blank nanoparticles are the same, and the particle size is about 260 nm. When preparing the blank nanoparticles, pure water containing the same amount of poly(I:C) or 8 M urea is used respectively to replace the corresponding water-soluble component and water-insoluble component.

[0292] (3) Selection and amplification of cancer-specific T cells 0.5×10 5 Individual B16F10 cells are subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor reaches about 1000 mm 3 , the mouse is sacrificed, the mouse spleen is removed, a single cell suspension of mouse spleen cells is prepared, and erythrocyte lysis treatment is performed to remove erythrocytes from the single cell suspension. First, the above cells are sequentially co-incubated with a T cell magnetic bead selection reagent (CD3+ magnetic bead selection reagent, CD8 + magnetic bead selection reagent) or a B cell magnetic bead selection reagent, and then a magnetic bead selection device is used to select CD3 + CD8 + T cells and CD19 + B cells from mouse spleen cells.

[0293] In the one-step selection control group, CD3 selected above + CD8 + T cells (10,000 cells, cell viability 60%) were incubated with IL-2 (500 U / mL), IL-7 (200 U / mL), IL-15 (200 U / mL), and αCD3 / αCD28 (10 ng / mL) in 10 mL of RPMI1640 complete medium at 37 °C (5% CO 2 ) for 12 days (changing the medium containing the above cytokines and antibodies every 3 days) and co-incubated to amplify the obtained CD8 + T cells (cell viability 60%).

[0294] In the two-step selection method, 1 million selected CD8 + T cells and 10 million B cells were co-incubated with nanoparticles carrying the whole component antigen of tumor tissue (250 μg of nanoparticles carrying water-soluble components + 250 μg of nanoparticles carrying water-insoluble components) or blank nanoparticles (500 μg) + the same amount of free cell lysate in 10 mL of DMEM high-glucose complete medium for 96 hours. Next, the incubated cells were sequentially co-incubated with CD3 + T cell magnetic bead selection reagent, CD8 + T cell magnetic bead selection reagent, and CD69 magnetic bead selection reagent, and selected accordingly. Using the magnetic bead selection method, CD3 in the incubated T cells, which are cancer-specific T cells activated by cancer antigens + CD8 + CD69 + T cells (cell viability 60%) were selected. 10,000 cancer-specific T cells selected above were co-incubated with IL-2 (500 U / mL), IL-7 (200 U / mL), IL-15 (200 U / mL), αCD3 / αCD28 (10 ng / mL) in RPMI1640 complete medium at 37 °C (5% CO 2 ) for 12 days (changing the medium containing the above cytokines and antibodies every 3 days) and co-incubated to amplify the selected cancer cell-specific T cells (cell viability 60%).

[0295] CD8 amplified by the one-step selection method of the control group +500,000 T cells, or 500,000 cancer-specific T cells amplified by a two-step selection method, were co-incubated with 2 million B cells and nanoparticles carrying whole cell components (30 μg of nanoparticles carrying water-soluble components + 30 μg of nanoparticles carrying water-insoluble components) in 3 mL of DMEM high-glucose complete medium for 48 hours. Next, the incubated cells were collected and labeled using CD3 antibody, CD8 antibody, and IFN-γ antibody equipped with different fluorescent probes. Then, the CD8 + CD8 against T cells + IFN-γ + ratio in the cells amplified after one-step selection and two-step selection was analyzed using flow cytometry. The cancer cell antigen carried by the nanoparticles can be degraded into antigen epitopes after being engulfed by antigen-presenting cells (B cells), can be presented on the surface of antigen-presenting cells, and specific T cells capable of recognizing the cancer cell antigen are activated after recognizing the cancer cell antigen epitope and can secrete killer cytokines. IFN-γ is the most important cytokine secreted by antigen-specific T cells activated after recognizing an antigen. However, since it is a secreted cytokine, it is necessary to fix the cells by adding 4% paraformaldehyde, then disrupt the membrane with a membrane-disrupting agent, and then stain the inside of the cells using an antibody (the cells after analysis are dead cells), and the cells fixed with paraformaldehyde are four cells. The CD8 + IFN-γ + T cells (cell viability 0%) are cancer-specific T cells that can specifically recognize cancer cell antigen epitopes.

[0296] (4) Cancer-specific T cells for cancer prevention To prepare melanoma tumor-bearing mice, female C57BL / 6 mice aged 6 - 8 weeks were selected as model mice. One day before transplanting cells into the mice, to remove the recipient mice's immune cells, cyclophosphamide was intraperitoneally injected into the recipient mice at a dose of 100 mg / kg. Next, 2 million cancer cell-specific T cells prepared by the two-step screening method in step (3), or 2 million CD3 + T cells amplified by the one-step screening method were intravenously injected into the recipient mice. The next day, 1.5×10 5 B16F10 cells were subcutaneously inoculated under the lower right back of each recipient mouse. The tumor growth rate and survival period of the mice were monitored. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52×a×b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. According to the ethics of animal experiments, in the experiment of the mouse survival period, when the tumor volume exceeded 2000 mm 3 , the mice were considered dead and euthanized.

[0297] (5) Experimental results As shown in a and b of Figure 2, the mice in the PBS control group had the fastest tumor growth rate and the shortest survival period. The tumor growth rate and survival period of the mice in the T cell treatment groups obtained by the one-step screening method and the T cell treatment group obtained by blank nanoparticle-assisted screening and amplification were improved compared with the PBS control group. Compared with the above three groups, the mice treated with cancer cell-specific T cells obtained by the assisted screening and amplification of nanoparticles carrying all cell components of cancer cells had the slowest tumor growth rate and the longest survival period, indicating that the cell line treated by the screening and amplification described in the present invention has a good preventive effect against cancer.

[0298] As shown in c of FIG. 2, the cancer cell-specific T cells obtained by the two-step selection method were co-incubated with nanoparticles carrying all cell components of antigen-presenting cells and cancer cells, and then recognized almost 100% of cancer cell antigens and could be activated by cancer antigens. However, in the T cells obtained by amplification of the one-step selection method, after co-incubation, CD8 + IFN-γ + T cells accounted for less than 5% of CD8 + T cells. As can be seen from this, the selection method described in the present invention can effectively enrich cancer cell-specific T cells having a killing ability.

[0299] In this example, surface markers are used as markers for T cell activation, but in actual applications, combinations of one or more different markers can also be used as activation markers.

[0300] Example 2. Isolation and amplification of peripheral cancer cell-specific T cells for the prevention of melanoma In this example, the tumor tissue of B16F10 melanoma was lysed to prepare water-soluble components and water-insoluble components of the tumor tissue. Next, an organic polymer material was used as a nanoparticle skeleton material, and poly(I:C) and CpG1018 were used as immunoadjuvants to prepare water-soluble components and water-insoluble components of the tumor tissue by the solvent evaporation method. Subsequently, nanoparticles were used to select and amplify peripheral cancer cell-specific T cells. In this example, immune cells in the peripheral spleen cells of mice were used, and in actual applications, peripheral blood or peripheral lymph node cells can be used.

[0301] (1) Preparation of antigen components 1.5×10 5 Individual B16F10 cells were subcutaneously inoculated into the back of each C57BL / 6 mouse, and when the tumor reached about 1000 mm 3When this occurs, the mouse is sacrificed and the tumor tissue is excised. After cutting the tumor tissue into small pieces, it is ground, passed through a cell filter to prepare a single cell suspension, and then an appropriate amount of pure water is added. Freezing and thawing are repeated 5 times, and sometimes ultrasonic waves are also used in combination to disrupt the lysed cells. After lysing the cells, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as the water-soluble component soluble in pure water. 8 M urea is added to the obtained precipitate portion to dissolve the precipitate portion, whereby the water-insoluble component insoluble in pure water can be solubilized in an 8 M urea aqueous solution. The above is the antigen component for preparing the nanoparticles.

[0302] (2) Preparation of Nanoparticles In this example, nanoparticles and blank nanoparticles as a control are prepared by the solvent evaporation method. During preparation, nanoparticles carrying the water-soluble components in the whole cell components and nanoparticles carrying the water-insoluble components in the whole cell components are prepared separately and then used in combination during use. The molecular weight of PLGA, which is the preparation material of the nanoparticles used, is 7 KDa to 17 KDa, and the immunoadjuvants used are poly(I:C) and CpG1018. The preparation method is as described above. In the preparation process, first, the double emulsion method is used to carry the antigen component and the adjuvant in the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 10000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then freeze-dried for 48 hours. The average particle size of the nanoparticles is about 280 nm, the surface potential of the nanoparticles is about -3 mV, about 100 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.02 mg of poly(I:C) and CpG1018 are carried respectively. The preparation materials and preparation methods of the blank nanoparticles are the same, the particle size is about 260 nm, and the same amount of adjuvant is carried in the blank nanoparticles, but no arbitrary antigen component is carried.

[0303] (3) Isolation and Amplification of Cancer-Specific T Cells 1.5×10 5Individual B16F10 cells were subcutaneously inoculated into the back of each C57BL / 6 mouse. On the 4th, 7th, 10th, 15th, 20th, 25th, and 30th days, 1 mg of PLGA nanoparticles carrying water-soluble components and 1 mg of PLGA nanoparticles carrying water-insoluble components were subcutaneously injected into the mice respectively. On the 34th day, the mice were sacrificed, the spleens of the mice were collected, a single cell suspension of mouse spleen cells was prepared, and the above cells were co-incubated with a T cell magnetic bead sorting reagent and a B cell magnetic bead sorting reagent respectively, and sequentially sorted. CD3 + T cells and CD19 + B cells were sequentially sorted.

[0304] In the one-step sorting control group, 1 million CD3 + T cells were incubated with IL-2 (500 U / mL), IL-7 (200 U / mL), IL-15 (200 U / mL), and αCD3 / αCD28 (10 ng / mL each) in 10 mL of RPMI1640 complete medium at 37 °C (5% CO 2 ) for 14 days (changing the medium containing the above cytokines and antibodies every 2 days) and co-incubated to amplify the obtained T cells (cell viability 65%).

[0305] In the two-step sorting method, 5 million CD3 + T cells and 10 million B cells were co-incubated with nanoparticles carrying whole tumor tissue component antigens (250 μg of nanoparticles carrying water-soluble components + 250 μg of nanoparticles carrying water-insoluble components) or blank nanoparticles (500 μg) + the same amount of free antigen component in 10 mL of RPMI1640 complete medium for 48 hours. Then, the incubated cells were co-incubated with the reagent in the magnetic bead sorting method, and using flow cytometry, CD3 among the incubated T cells, which are cancer-specific T cells (cell viability 65%) activated by cancer antigens + CD137 +Select T cells. The cancer cell-specific T cells (1 million) selected above are incubated with IL-2 (500 U / mL), IL-7 (200 U / mL), IL-15 (200 U / mL), αCD3 / αCD28 (10 ng / mL each) and 10 mL of RPMI1640 complete medium (37 °C, 5% CO 2 ) for 14 days (replace the medium containing the above cytokines and antibodies every 2 days) to co-incubate and amplify the selected cancer cell-specific T cells (cell viability 65%).

[0306] T cells (500,000) amplified by the one-step selection method in the control group, or cancer cell-specific T cells (500,000) amplified by the two-step selection method are co-incubated with B cells (2 million) and nanoparticles (60 μg) carrying whole cell components in 3 mL of RPMI1640 complete medium for 48 hours. Next, the incubated cells are collected, and CD3 antibodies, CD8 antibodies and IFN-γ antibodies with different fluorescent probes are used simultaneously to label the incubated cells. Then, using flow cytometry, CD3 in the cells amplified after one-step selection and two-step selection + CD3 against T cells + IFN-γ + The ratio of T cells is analyzed. CD3 obtained by the analysis of flow cytometry + IFN-γ + T cells (cell viability 0%) are cancer cell-specific T cells that can specifically recognize cancer cell antigens.

[0307] (4) Cancer cell-specific T cells for cancer prevention To prepare melanoma tumor-bearing mice, 6- to 8-week-old female C57BL / 6 mice are selected as model mice. One day before adoptively transferring cells to the mice, to remove the immune cells of the recipient mice, cyclophosphamide is intraperitoneally injected into the recipient mice at a dose of 100 mg / kg. Next, 2 million cancer cell-specific T cells obtained by the two-step selection and amplification in step (3), or 2 million T cells amplified after one-step selection are intravenously injected into the recipient mice. The next day, 1.5×10 5Inject individual B16F10 cells subcutaneously into the lower right back of each recipient mouse. The method for monitoring the tumor growth rate and survival period of the mouse is the same as described above.

[0308] (5) Experimental results As shown in FIGS. 3a and 3b, the mice in the PBS control group had the fastest tumor growth rate and the shortest survival period. The tumor growth rate and survival period of the mice in the T cell treatment group obtained by the one-step selection method and the T cell treatment group obtained by blank nanoparticle-assisted selection were improved compared with the PBS control group. Compared with the above three groups, the mice treated with cancer cell-specific T cells obtained by the selection and amplification of nanoparticles carrying the whole cell antigen component had the slowest tumor growth rate and the longest survival period, indicating that the cell line treated by the selection and amplification described in the present invention has a good preventive effect against cancer.

[0309] As shown in FIG. 3c, the cancer cell-specific T cells obtained by the two-step selection method, after being co-incubated with antigen-presenting cells and nanoparticles carrying the whole cell components of cancer cells, recognized almost 100% of the cancer cell antigens and could be activated by the cancer cell antigens. However, for the T cells obtained by the amplification of the one-step selection method, after co-incubation, the CD3 + IFN-γ + T cells only accounted for less than 5% of the CD3 + T cells. As can be seen from this, the selection method described in the present invention can effectively enrich cancer cell-specific T cells with killing ability.

[0310] Example 3. Cancer cell-specific T cells after separation and amplification for melanoma treatment In this example, first, the tumor tissue and cancer cells of B16F10 melanoma are lysed to prepare a water-soluble component mixture (mass ratio 1:1) and a water-insoluble component mixture (mass ratio 1:1) of the tumor tissue and cancer cells. Next, PLGA is used as a nanoparticle skeleton material, and poly(I:C), CpG2006, and CpG2216 are used as immunoadjuvants to prepare a nanoparticle system carrying the water-soluble component mixture and the water-insoluble component mixture. Then, the nanoparticles are co-incubated with T cells and antigen-presenting cells in vitro to activate existing cancer cell-specific T cells. After the cancer cell-specific T cells are activated, specific molecules are highly expressed and can be sorted using flow cytometry. Next, after amplification, it is used for cancer treatment.

[0311] (1) Preparation of antigen components When collecting tumor tissue, first, 1.5×10 5 B16F10 cells are subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor volume reaches about 1000 mm 3 , the mouse is sacrificed, the tumor tissue is removed, the tumor tissue is cut into small pieces and then ground, an appropriate amount of pure water is added after passing through a cell filter, and freezing and thawing are repeated 5 times. In some cases, ultrasonic waves may also be used in combination to break and dissolve the obtained sample. When collecting the cultured B16F10 cancer cell line, first, the medium is removed by centrifugation, then washed twice with PBS, the cancer cells are collected by centrifugation, the cancer cells are resuspended in ultrapure water, and freezing and thawing are repeated 3 times. In some cases, ultrasonic waves may also be used in combination to break and dissolve the cancer cells. After lysing the tumor tissue and cancer cell line, the lysate is centrifuged at a rotation speed of 5000 g for 5 minutes, the supernatant is taken as the water-soluble component soluble in pure water, 8 M urea is added to the obtained precipitate part to dissolve the precipitate part, whereby the water-insoluble component insoluble in pure water can be solubilized in an 8 M urea aqueous solution. The water-soluble component of the tumor tissue and the water-soluble component of the cancer cell line are mixed at a mass ratio of 1:1, and the water-insoluble component of the tumor tissue and the water-insoluble component of the cancer cell line are mixed at a mass ratio of 1:1. The above is the antigen component for preparing nanoparticles.

[0312] (2) Preparation of nanoparticles In this example, nanoparticles are prepared by the double emulsion method. The molecular weight of PLGA, which is the material for preparing the nanoparticles, is 7 KDa to 17 KDa, and the immunoadjuvants used are poly(I:C), CpG2006, and CpG2216. The preparation method is as described above. First, an antigen component and an adjuvant are loaded into the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 12,000 g for 25 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. Before use, it is resuspended in 9 mL of PBS, and then 1 mL of the cell lysate component (protein concentration 80 mg / mL) is added and allowed to act at room temperature for 10 minutes to obtain nanoparticles carrying the antigen component both inside and outside. The average particle size of the nanoparticles is about 280 nm, the surface potential of the nanoparticles is about -5 mV, about 130 μg of protein or polypeptide component is carried per 1 mg of the PLGA nanoparticles, and 0.02 mg of poly(I:C), CpG2006, and CpG2216 immunoadjuvants are each carried per 1 mg of the PLGA nanoparticles. The preparation materials and preparation methods of the blank nanoparticles are the same, the particle size is about 260 nm, and the same amount of adjuvant is carried in the blank nanoparticles, but the cancer cell lysate is not carried.

[0313] (3) Isolation and amplification of cancer-specific T cells 1.5×10 5 B16F10 cells are subcutaneously inoculated into the back of each C57BL / 6 mouse, and on the 4th, 7th, 10th, 15th, and 20th days, 2 mg of PLGA nanoparticles (carrying the antigen component and the adjuvant) are subcutaneously injected into the mice respectively. On the 24th day, the mice are sacrificed, the peripheral blood of the mice is collected, and peripheral blood mononuclear cells (PBMC) are separated from the peripheral blood of the mice using gradient centrifugation. The PBMC are co-incubated with CD3 antibody and CD19 antibody labeled with different fluorescent probes, and then CD3 + T cells and CD19 + B cells are sorted.

[0314] The method for preparing mouse bone marrow-derived macrophages (BMDMs) is a conventional preparation method. The method for preparing BMDMs is as follows. Anesthetize C57 mice and kill them by dislocation. Disinfect the mice with 75% ethanol. Next, use scissors to make a small opening on the back of the mouse, and directly tear the mouse's skin by hand up to the calf joint of the mouse, and remove the ankle joint and skin of the mouse. Use scissors to remove the hind limbs along the greater trochanter at the base of the mouse's thigh, remove the muscle tissue, then place it in a culture dish containing 75% ethanol and soak for 5 minutes, replace it with a new culture dish of 75% ethanol, and transfer it to a clean bench. Transfer the ethanol-soaked foot bones to cold PBS, wash the ethanol adhering to the surface of the tibia and femur, and this process can be repeated 3 times. Separate the washed femur and tibia, use scissors to cut both ends of the femur and tibia respectively, use a 1 mL syringe to aspirate cold induction medium, blow the bone marrow out of the femur and tibia, and repeat the purge 3 times until no obvious redness can be seen in the foot bones. Repeatedly spray the medium containing bone marrow cells using a 5 mL pipette to disperse the cell mass. Next, use a 70 μm cell strainer to filter the cells, transfer them to a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add red blood cell lysate and resuspend for 5 minutes, then centrifuge at 1500 rpm for 5 minutes, discard the supernatant, resuspend with cold-prepared bone marrow macrophage induction medium (DMEM high glucose medium containing 15% L929 medium), and inoculate on a cell count plate. Culture the cells overnight to remove other miscellaneous cells that adhere immediately, such as fibroblasts. Collect the non-adherent cells and inoculate them into dishes or cell culture plates according to the experimental plan. Macrophage colony-stimulating factor (M-CSF) stimulates the differentiation of bone marrow cells into mononuclear macrophages at a concentration of 40 ng / mL. After culturing for 8 days, observe the morphological changes of macrophages under an optical microscope. After 8 days, digest and collect the cells, use anti-mouse F4 / 80 antibody and anti-mouse CD11b antibody, incubate in the dark at 4°C for 30 minutes, and then only use flow cytometry to identify the proportion of macrophages that have been successfully induced.

[0315] 5 million selected CD3 + T cells, 5 million B cells, 5 million BMDMs, and nanoparticles (500 μg) carrying the whole-component antigen of tumor tissue were co-incubated in 10 mL of RPMI 1640 complete medium for 48 hours, or 1 million selected CD3 + T cells, 10 million mouse BMDMs, and nanoparticles (500 μg) carrying the whole-component antigen of tumor tissue were co-incubated in 10 mL of RPMI 1640 complete medium for 48 hours, or 1 million selected CD3 + T cells, 5 million B cells, 5 million mouse BMDMs, IL-7 (10 ng / mL), and nanoparticles (500 μg) carrying the whole-component antigen of tumor tissue were co-incubated in 10 mL of RPMI 1640 complete medium for 48 hours. Next, using flow cytometry, CD3 + CD137 + T cells (cell viability 70%) are selected. 1 million cancer-specific T cells selected above are co-incubated with IL-2 (1000 U / mL) and IL-7 (1000 U / mL) in 10 mL of RPMI 1640 complete medium (37 °C, 5% CO 2 ) for 14 days (changing the medium every 2 days) to amplify the selected cancer cell-specific T cells.

[0316] (4) Cancer cell-specific T cells for cancer treatment To prepare melanoma tumor-bearing mice, female C57BL / 6 at 6 - 8 weeks old are selected as model mice. On day 0, 1.5×10 5 cells of B16F10 are subcutaneously inoculated into the lower right of the back of each mouse. On the 4th, 7th, 10th, 15th, 20th, and 25th days after melanoma inoculation, 2 million cancer-specific T cells are intravenously injected respectively. The monitoring methods for the tumor volume and survival period of the mice are the same as above.

[0317] (5) Experimental results As shown in Fig. 4, the mice in the PBS control group had the fastest tumor growth rate and the shortest survival period. The tumor growth rate and survival period of the mice in some treatment groups after amplification of the selected T cells were significantly improved compared with those of the PBS control group. The anti-cancer effect after amplification of cancer-specific T cells obtained by a two-step selection method using B cells and BMDM as mixed antigen-presenting cells was higher than that after amplification of cancer-specific T cells obtained by a two-step selection method using BMDM as antigen-presenting cells. As can be seen, when selecting cancer-specific T cells, it is more effective to use B cells and BMDM as mixed antigen-presenting cells than to use BMDM antigen-presenting cells alone. In addition, cancer-specific T cells selected by adding IL-7 during the co-incubation process of nanoparticles, antigen-presenting cells, and T cells were more effective than cancer cell-specific T cells selected without adding IL-7 during the incubation process.

[0318] Example 4, Cell Line for Preventing Lung Metastasis of Melanoma In this example, a mouse melanoma lung model was used to illustrate how to prevent cancer metastasis using a cell line. In this example, first, B16F10 melanoma tumor tissue was lysed to prepare the water-soluble and water-insoluble components of the tumor tissue, and then nanoparticles carrying the water-soluble and water-insoluble components of the tumor tissue were prepared. In this example, a series of mineralization treatments were performed. In this example, first, dendritic cells (DC) were activated in vitro using nanoparticles, and then the dendritic cells and cancer-specific T cells were co-incubated and activated to assist in the selection of cancer cell-specific T cells. In actual applications, antigen-presenting cells can be used alive, or antigen-presenting cells inactivated by paraformaldehyde fixation treatment, radiation inactivation treatment, etc. can also be used.

[0319] (1) Preparation of Antigen Components 1.5×10 5 individual B16-F10 cells were subcutaneously inoculated into the back of each C57BL / 6 mouse, and when the tumor reached about 1000 mm 3When this occurs, the mouse is sacrificed and the tumor tissue is excised. After mincing the tumor tissue, it is ground, collagenase (1 mg / mL) is added, and it is incubated in RPMI 1640 complete medium for 30 minutes. Next, a single cell suspension is prepared by passing it through a cell filter, an appropriate amount of pure water is added, freezing and thawing are repeated 5 times, and in some cases, ultrasonic waves are also used in combination to disrupt the lysed cells. After lysing the cells, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as a water-soluble component soluble in pure water. To the obtained precipitate portion, 10% sodium deoxycholate (containing 0.8 M arginine) is added to dissolve the precipitate portion, whereby the water-insoluble component insoluble in pure water can be solubilized in a 10% aqueous sodium deoxycholate solution. The water-soluble component and the water-insoluble component are mixed at a mass ratio of 3:1 to obtain an antigen component.

[0320] (2) Preparation of nanoparticles In this example, nanoparticles are prepared by the double emulsion method in the solvent evaporation method, and the double emulsion method is appropriately modified and improved. The molecular weight of PLGA, which is the material for preparing the nanoparticles used, is 24KDa to 38KDa. The immunoadjuvant used is poly(I:C), and poly(I:C) is distributed inside the nanoparticles but supported on the surface of the nanoparticles. The preparation method is as described above. During the preparation process, first, by the double emulsion method, a dissolved component and an adjuvant are supported inside the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 10,000 g for 20 minutes. Subsequently, the nanoparticles are resuspended in 7 mL of PBS and mixed with 3 mL of a PBS solution containing cell lysate (60 mg / mL). Then, it is centrifuged at 10,000 g for 20 minutes. Then, it is resuspended in 10 mL of a silicate solution (containing 150 mM of NaCl, 80 mM of tetramethyl orthosilicate, and 1.0 mM of HCl, pH 3.0), fixed at room temperature for 10 minutes, and then fixed at -80°C for 24 hours. It is centrifuged and washed using ultrapure water, resuspended in PBS (3 mL) containing protamine (5 mg / mL) and polylysine (10 mg / mL), allowed to act for 10 minutes, then centrifuged at 10,000 g and washed for 20 minutes, resuspended in a PBS solution (10 mL) containing cell lysate (50 mg / mL), allowed to act for 10 minutes, subsequently centrifuged at 10,000 g for 20 minutes, resuspended in ultrapure water (10 mL) containing 4% trehalose, and then freeze-dried for 48 hours. Before use, the particles are resuspended in 7 mL of PBS, and then a cancer tissue cell lysate component (3 mL) (protein concentration 50 mg / mL) containing an adjuvant is added and allowed to act at room temperature for 10 minutes. Nanoparticles modified by freeze-silicification of the lysate and addition of cationic substances both inside and outside are supported. The average particle size of the nanoparticles is about 350 nm, the surface potential of the nanoparticles is about -3 mV, about 300 μg of protein or polypeptide component is supported per 1 mg of PLGA nanoparticles, 0.02 mg of poly(I:C) immunoadjuvant is supported per 1 mg of PLGA nanoparticles, and both the inside and outside are supported equally.

[0321] The preparation process of unmodified nanoparticles is basically the same as that of modified nanoparticles, except that it does not go through the steps of low-temperature silicification treatment and addition of charged substances. In the preparation process, first, antigens are loaded into the nanoparticles by the double emulsion method. After loading the antigen (dissolved component) inside, it is centrifuged at 10,000 g for 20 minutes. Next, it is resuspended in ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. Before use, the particles are resuspended in 7 mL of PBS. Then, a cancer tissue cell lysate (3 mL) containing an adjuvant (protein concentration 50 mg / mL) is added and allowed to act at room temperature for 10 minutes to obtain nanoparticles with the lysate loaded inside and outside. The average particle size of the nanoparticles is about 320 nm, the surface potential of the nanoparticles is about -5 mV, about 150 μg of protein or polypeptide component is loaded per 1 mg of PLGA nanoparticles, 0.02 mg of poly(I:C) is loaded per 1 mg of PLGA nanoparticles, and the inside and outside are each loaded half.

[0322] The preparation materials and methods of blank nanoparticles are the same, the particle size is about 300 nm, the same amount of adjuvant is loaded on the blank nanoparticles, but no arbitrary antigen component is loaded.

[0323] (3) Preparation of dendritic cells In this example, taking the preparation of dendritic cells from mouse bone marrow cells as an example, how to prepare bone marrow-derived dendritic cells (BMDC) will be described. First, one 6- to 8-week-old C57 mouse is killed by cervical dislocation. After surgically removing the tibia and femur of the hindlimb and placing them in PBS, the muscle tissue around the bone is completely removed using scissors and forceps. The two ends of the bone are cut with scissors, and then a syringe is used to collect the PBS solution. The needle is inserted into the bone marrow cavity from both ends of the bone respectively, and the bone marrow is repeatedly flushed into the culture dish. The bone marrow solution is collected and centrifuged at 400 g for 3 minutes. Then, 1 mL of red blood cell lysate is added to lyse the red blood cells. 3 mL of RPMI1640 (10% FBS) medium is added to stop the lysis, and it is centrifuged at 400 g for 3 minutes, and the supernatant is discarded. The cells are placed in a 10 mm culture dish and cultured in RPMI1640 (10% FBS) medium. At the same time, recombinant mouse GM-CSF (20 ng / mL) is added, and cultured at 37 °C in 5% CO 2 for 7 days. On the 3rd day, the culture bottle is gently shaken, and the same amount of RPMI1640 (10% FBS) medium containing GM-CSF (20 ng / mL) is added. On the 6th day, half of the medium is replaced. On the 7th day, a small amount of floating cells and semi-adherent cells are collected and detected by flow cytometry. The proportion of CD86 + in CD11c + CD80 + cells is 15 - 20%, and the induced and cultured BMDC can be used in the next experiment.

[0324] (4) Isolation and amplification of cancer cell-specific T cells 1.5×10 5 B16F10 cells are subcutaneously inoculated into the back of each C57BL / 6 mouse. On the 4th, 7th, 10th, 15th, 20th, and 25th days, 1 mg of PLGA nanoparticles (100 μL) are subcutaneously injected into the mouse respectively. In the blank nanoparticle + free cell lysate control group, the same dose is injected at the corresponding time as a control. On the 24th day, the mouse is killed, the spleen is collected, a single cell suspension of mouse spleen cells is prepared, and the mouse spleen cells are co-incubated with CD3 antibody. Then, by flow cytometry, CD3 in the single cell suspension of spleen cells +Sort T cells. Mix 1 million BMDCs prepared in step 3 with 40 μg of nanoparticles loaded with whole tumor tissue antigen, and co-incubate them in 3 mL of RPMI 1640 complete medium (at 37 °C, 5% CO 2 ) for 48 hours. Next, centrifuge at 400 g for 5 minutes to collect the BMDCs, and co-incubate the sorted CD3 + T cells with the BMDCs for 24 hours. Then, using flow cytometry, select CD3 + CD8 + CD69 + T cells (cell viability 75%) and CD3 + CD4 + CD25 + T cells (cell viability 75%). Incubate 500,000 cancer cell-specific T cells selected above with IL-2 (200 U / mL), IL-7 (200 U / mL), IL-15 (200 U / mL), αCD3 / αCD28 and 10 mL of RPMI 1640 complete medium for 14 days (changing the medium every 2 days) to amplify the selected cancer cell-specific T cells (cell viability 75%).

[0325] (5) Allogeneic cell line for preventing cancer metastasis To prepare melanoma tumor-bearing mice, select 6- to 8-week-old female C57BL / 6 as model mice. One day before transplanting cells into the mice, intraperitoneally inject cyclophosphamide into the recipient mice at a dose of 100 mg / kg to remove the immune cells of the recipient mice. On day 0, intravenously inject 2 million cancer cell-specific T cells (1 million CD3 + CD8 + CD69 + T cells and 1 million CD3 + CD4 + CD25 + T cells) (100 μL) into the mice. At the same time, on day 1, intravenously inject 0.5×10 5 cells of B16F10 into each mouse. On day 14, sacrifice the mice and observe and record the number of melanoma cancer nests in the lungs of the mice.

[0326] (6) Experimental results As shown in Figure 5, many cancer nests are seen in the mice of the PBS control group, but there are almost no cancer nests in the mice treated with T cells. Compared with the control group, the mice treated with cancer cell-specific T cells obtained by assisting the selection and amplification of modified or unmodified nanoparticles have significantly fewer cancer nests. This indicates that both T cells selected and amplified by modified or unmodified nanoparticles can effectively prevent cancer metastasis.

[0327] Example 5, cancer cell-specific T cells selected and amplified with the assistance of microparticles for cancer prevention In this example, during the preparation of microparticles, silanization is performed twice. After selecting and amplifying cancer cell-specific T cells obtained by microparticles carrying antigens, they are injected into mice to prevent cancer.

[0328] (1) Preparation of antigen component After collecting the cultured B16F10 melanoma cancer cell line, it is centrifuged at 350 g for 5 minutes. Then, the supernatant is discarded, and it is washed twice with PBS. After that, the cancer cells are resuspended and lysed with 6 M guanidine hydrochloride, and the lysed whole cell components are dissolved to obtain the antigen component for preparing the microparticle system.

[0329] (2) Preparation of microparticles In this example, microparticles 1 are prepared by the double emulsion method, and the double emulsion method is appropriately modified and improved. The molecular weight of PLGA, which is the material for preparing the microparticles used, is 38 KDa to 54 KDa, and the immunoadjuvant used is poly(I:C). Poly(I:C) is distributed within the microparticles but is carried on the surface of the nanoparticles. The preparation method is as described above. During the preparation process, first, by the double emulsion method, whole cell components are carried within the microparticles and lysate components are carried inside. Then, 100 mg of the microparticles are centrifuged at 10,000 g for 15 minutes. Subsequently, the microparticles are resuspended in 7 mL of PBS and mixed with a PBS solution (3 mL) containing cell lysate (50 mg / mL). Then, it is centrifuged at 10,000 g for 20 minutes. Next, it is resuspended in 10 mL of silicate solution (containing 120 mM of NaCl, 100 mM of tetramethyl orthosilicate, and 1.0 mM of HCl, pH 3.0), fixed at room temperature for 12 hours, centrifuged and washed with ultrapure water. Then, it is resuspended in PBS (3 mL) containing polyaspartic acid (10 mg / mL), allowed to act for 10 minutes, then centrifuged at 10,000 g and washed for 15 minutes, resuspended in a PBS solution (10 mL) containing cell lysate (50 mg / mL), allowed to act for 10 minutes, and then centrifuged at 10,000 g for 20 minutes. Next, 10 mL of silicate solution (containing 150 mM of NaCl, 80 mM of tetramethyl orthosilicate, and 1.0 mM of HCl, pH 3.0) is used, fixed at room temperature for 12 hours, centrifuged and washed with ultrapure water. Then, it is resuspended in PBS (3 mL) containing protein (5 mg / mL) and polyarginine (10 mg / mL), allowed to act for 10 minutes, then centrifuged at 10,000 g and washed for 15 minutes, resuspended in a PBS solution (10 mL) containing cell lysate (50 mg / mL), allowed to act for 10 minutes, and then centrifuged at 10,000 g for 20 minutes. After that, it is resuspended in ultrapure water (10 mL) containing 4% trehalose and freeze-dried for 48 hours. Before use, the particles are resuspended in 7 mL of PBS, and then a cancer cell lysate component (3 mL) containing an adjuvant (protein concentration 50 mg / mL) is added and allowed to act at room temperature for 10 minutes. Microparticles modified by two freeze-silicification of the lysate both inside and outside and the addition of cationic and anionic substances are carried.The average particle size of the microparticles is about 1.1 μm, the surface potential of the microparticles is about -2 mV, about 340 μg of protein or polypeptide component is carried per 1 mg of PLGA microparticles, 0.02 mg of CpG is carried per 1 mg of PLGA microparticles, and half of the interior and exterior are each carried.

[0330] The preparation materials and preparation method of blank microparticles 2 are the same, the particle size is about 1.1 μm, the surface potential is about -3 mV, the same amount of adjuvant is carried on the blank microparticles, but no antigen component is carried.

[0331] (3) Preparation of dendritic cells In this example, taking the preparation of dendritic cells from mouse bone marrow cells as an example, how to prepare bone marrow-derived dendritic cells (BMDC) will be described. First, one 6-8 week-old C57 mouse is killed by cervical dislocation. After surgically removing the tibia and femur of the hind limb and placing them in PBS, the muscle tissue around the bone is completely removed with scissors and forceps. The two ends of the bone are cut with scissors, then a syringe is used to collect the PBS solution, and the needle is inserted into the bone marrow cavity from both ends of the bone respectively, and the bone marrow is repeatedly flushed into the culture dish. The bone marrow solution is collected, centrifuged at 400 g for 3 minutes, then 1 mL of red blood cell lysate is added to lyse the red blood cells. 3 mL of RPMI1640 (10% FBS) medium is added to stop the lysis, centrifuged at 400 g for 3 minutes, and the supernatant is discarded. The cells are placed in a 10 mm culture dish and cultured in RPMI1640 (10% FBS) medium. At the same time, recombinant mouse GM-CSF (20 ng / mL) is added, and cultured at 37 °C, 5% CO 2 for 7 days. On the 3rd day, the culture bottle is gently shaken, and the same amount of RPMI1640 (10% FBS) medium containing GM-CSF (20 ng / mL) is added. On the 6th day, half of the medium is replaced. On the 7th day, a small amount of floating cells and semi-adherent cells are collected and detected by flow cytometry for CD11c + CD86 in cells + CD80 + When the proportion of cells is 15-20%, the induced and cultured BMDC can be used in the next experiment.

[0332] (4) Isolation and Amplification of Cancer Cell-Specific T Cells 1.5×10 5 Inject 1.5×10 individual B16F10 cells subcutaneously into the back of each C57BL / 6 mouse. On the 4th, 7th, 10th, 15th, 20th, and 25th days, inject 1 mg of PLGA microparticles (100 μL) subcutaneously into the mice respectively. On the 30th day, sacrifice the mice, collect the spleens, and prepare a single-cell suspension of mouse spleen cells. First, use the magnetic bead sorting method to select CD3 + T cells in the single-cell suspension of spleen cells. After mixing the selected T cells (20 million), the BMDC prepared in step 3 (20 million), and 20 μg of microparticle 1 (or blank microparticle 2 + the same amount of free cell lysate), incubate them together for 24 hours in 10 mL of RPMI complete medium (containing 5% FBS), and then, by the magnetic bead sorting method, select CD3 + CD69 + T cells (cell survival rate 75%) that are cancer-specific T cells activated by cancer antigens. Incubate 1 million cancer-specific T cells selected above together with IL-2 (2000 U / mL) and αCD3 / αCD28 antibody (20 ng / mL) in 10 mL of DMEM high-glucose complete medium (37 °C, 5% CO 2 ) for 7 days (change the medium every 2 days) to amplify the selected cancer cell-specific T cells (cell survival rate 75%).

[0333] (5) Amplified Cancer Cell-Specific T Cells for Cancer Prevention To prepare melanoma tumor-bearing mice, select 6- to 8-week-old female C57BL / 6 as model mice. One day before adoptively transferring cells to the mice, inject cyclophosphamide intraperitoneally into the recipient mice at a dose of 100 mg / kg to remove the immune cells of the recipient mice. On day 0, inject 2 million cancer-specific T cells (100 μL) intravenously into the mice. At the same time, on day 0, subcutaneously inoculate 1.5×10 5 individual B16F10 cells into each mouse. The method for monitoring the tumor volume and survival period of the mice is the same as above.

[0334] (6) Experimental Results As shown in Fig. 6, the tumors of the mice in the PBS control group had a fast growth rate and a short survival period, while the tumors of the mice treated with T cells had a significantly slower growth rate and a significantly extended survival period. This indicates that T cells sorted and amplified by microparticles can effectively prevent cancer.

[0335] Example 6, Cancer-specific T cells for cancer prevention In this example, first, 8M urea was used to lyse B16F10 melanoma tumor tissue and dissolve the tumor tissue lysate components. Next, using PLA as the nanoparticle backbone material and poly(I:C) and CpG1018 as immunoadjuvants, nanoparticles carrying whole cell antigens were prepared, and the nanoparticles assist in the sorting and amplification of cell-specific T cells.

[0336] (1) Preparation of antigen components 1.5×10 5 individual B16F10 cells were subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor reached about 1000 mm 3 , the mouse was sacrificed and the tumor tissue was removed. After cutting the tumor tissue into small pieces, it was ground, passed through a cell filter to obtain a single cell suspension, and an appropriate amount of 8M urea aqueous solution was added to lyse the cells and dissolve the cell lysate. The above is the antigen component for preparing nanoparticles.

[0337] (2) Preparation of nanoparticles In this example, nanoparticles 1 are prepared by the solvent evaporation method. The molecular weight of PLGA, which is the material for preparing nanoparticles 1, is 20 KDa, and the immunoadjuvants used are poly(I:C) and CpG1018. The preparation method is as described above. First, the double emulsion method is used to carry the antigen component and the adjuvant in the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 12,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours to obtain the lyophilized product for use. The average particle size of the nanoparticles 1 is about 250 nm, the surface potential is about -3 mV, about 110 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.02 mg of poly(I:C) and CpG1018 are each carried.

[0338] The preparation materials and preparation method of nanoparticles 2 are the same as above. The particle size is about 250 nm, the surface potential is about -3 mV, about 110 μg of protein or polypeptide component and 0.04 mg of poly(I:C) are carried per 1 mg of PLGA nanoparticles.

[0339] (3) Isolation and amplification of cancer cell-specific T cells 1.5×10 5 Individual B16F10 cells are subcutaneously inoculated into the back of each C57BL / 6 mouse. On the 4th, 7th, 10th, 15th, 20th, and 25th days, 1 mg of PLA nanoparticles 1 (100 μL) are subcutaneously injected into the mice respectively. On the 29th day, the mice are sacrificed and the spleens are collected to prepare a single cell suspension of mouse spleen cells. The CD3 + T cells in the spleen cells of the mice are sorted using the magnetic bead sorting method. Next, the sorted T cells (10 million) are combined with allogeneic B cells (15 million) and 40 mg of nanoparticles (nanoparticles 1 or 2) and 20 mL of RPMI1640 complete medium (37 °C, 5% CO 2Incubate together for 48 hours. Next, use CD3 antibody and CD69 antibody labeled with different fluorescent probes to label the incubated cells, and then use flow cytometry to detect CD3 in the incubated cells, which are cancer cell-specific T cells activated by cancer cell antigens. + CD69 + Sort T cells (cell viability 75%). Incubate 1 million cancer cell-specific T cells sorted above together with IL-2 (1000 U / mL) and αCD3 / αCD28 antibody (20 ng / mL) in 10 mL of high-glucose DMEM complete medium for 11 days (change the medium every 2 days) to amplify the sorted cancer cell-specific T cells (cell viability 75%).

[0340] (4) T cells for cancer prevention To prepare melanoma tumor-bearing mice, select 6- to 8-week-old female C57BL / 6 as model mice. One day before transplanting the cancer cell-specific T cells of the mice, intraperitoneally inject cyclophosphamide into the recipient mice at a dose of 100 mg / kg to remove the immune cells of the recipient mice. On day 0, subcutaneously inject 2 million amplified cancer-specific T cells (100 μL) into the mice. At the same time, on day 0, subcutaneously inoculate 1.5×10 5 B16F10 cells into each mouse, and the monitoring methods for the tumor volume and survival period of the mice are the same as above.

[0341] (5) Experimental results As shown in Figure 6, the mice in the control group had the fastest tumor growth rate and the shortest survival period. Both the cancer cell-specific T cells sorted and amplified using nanoparticle 1 and nanoparticle 2 could slow down the tumor growth rate and extend the survival period of the mice.

[0342] Example 7, Cancer cell-specific T cells after sorting and amplification for colon cancer treatment First, dissolve colon cancer tumor tissue and lung cancer cell lines to prepare a water-soluble component mixture (mass ratio 1:1) and a water-insoluble component mixture (mass ratio 1:1), and also mix the water-soluble component mixture and the water-insoluble component mixture at a mass ratio of 1:1. Next, using the organic polymer material PLGA as a nanoparticle skeleton material and CpG1018 and poly(I:C) as immunoadjuvants, prepare nanoparticles, and use the nanoparticles to select and amplify cancer-specific T cells for colon treatment.

[0343] (1) Preparation of antigen components 2×10 6 MC38 cells were subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor reached approximately 1000 mm 3 , the mouse was sacrificed and the tumor tissue was removed. After cutting the tumor tissue into small pieces, it was ground, passed through a cell filter to prepare a single-cell suspension, then an appropriate amount of pure water was added, and freezing and thawing were repeated 5 times. In some cases, ultrasonic waves were also used in combination to disrupt the lysed cells. After lysing the cells, the lysate was centrifuged at a rotational speed of more than 5000 g for 5 minutes, and the supernatant was taken as the water-soluble component soluble in pure water. 10% N-octyl-β-D-glucopyranoside aqueous solution was added to the obtained precipitate portion to dissolve the precipitate portion, whereby the water-insoluble component insoluble in pure water can be solubilized in 10% N-octyl-β-D-glucopyranoside aqueous solution. The cultured LLC lung cancer cell line was collected, centrifuged at 350 g for 5 minutes, then the supernatant was discarded, washed twice with PBS, and then the cells were resuspended in ultrapure water, and freezing and thawing were repeated 5 times. In some cases, ultrasonic waves were also used in combination to disrupt the lysed cells. After lysing the cells, the lysate was centrifuged at a rotational speed of 3000 g for 6 minutes, and the supernatant was taken as the water-soluble component soluble in pure water. 10% N-octyl-β-D-glucopyranoside aqueous solution was added to the obtained precipitate portion to dissolve the precipitate portion, whereby the water-insoluble component insoluble in pure water can be solubilized in 10% N-octyl-β-D-glucopyranoside aqueous solution.

[0344] Mix the water-soluble components from colon cancer tumor tissues and lung cancer cells in a mass ratio of 1:1, and also mix the water-insoluble components dissolved in 10% N-octyl-β-D-glucopyranoside in a mass ratio of 1:1. Next, mix the water-soluble component mixture and the water-insoluble component mixture in a mass ratio of 1:1, and this mixture is the antigen component for preparing nanoparticles.

[0345] (2) Preparation of nanoparticles In this example, nanoparticles are prepared by the double emulsion method. The molecular weight of PLGA, which is the material for preparing nanoparticles, is 24KDa - 38KDa, and the immunoadjuvants used are poly(I:C) and CpG1018. The antigen component and the adjuvant are distributed on both the inside and the surface of the nanoparticles. The preparation method is as described above. In the preparation process, first use the double emulsion method to carry the antigen component and the adjuvant inside the nanoparticles, then centrifuge 100 mg of the nanoparticles at 10,000 g for 20 minutes, resuspend them using ultrapure water (10 mL) containing 4% trehalose, and then lyophilize for 48 hours. Before use, resuspend 20 mg of the nanoparticles in 0.9 mL of PBS, mix them with a sample (0.1 mL) containing the antigen component (80 mg / mL) and the adjuvant at room temperature, and incubate for 5 minutes to be ready for use. The average particle size of the nanoparticles is about 280 nm, the surface potential is about -3 mV, about 100 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and the carried CpG1018 and immunoadjuvant are each 0.02 mg.

[0346] The preparation materials and preparation method of nanoparticles 2 are the same. The particle size is about 280 nm, the surface potential is -3 mV, about 100 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles 2, and the carried CpG1018 is 0.04 mg.

[0347] (3) Selection and amplification of cancer cell-specific T cells 1.5×10 5Individual B16F10 cells are subcutaneously inoculated into the back of each C57BL / 6 mouse, and on the 4th, 7th, 10th, 15th, 20th, and 25th days, 2 mg of PLGA nanoparticles (100 μL) are subcutaneously injected into the mice respectively. On the 30th day, the mice are sacrificed, and the spleens of the mice in each group are collected respectively. A single cell suspension of mouse spleen cells is prepared, and by flow cytometry, CD3 + CD8 + T cells and CD19 + B cells are sorted. 3 million CD8 + T cells, 6 million CD19 + B cells and nanoparticles (50 μg) are co-incubated in 2 mL of RPMI complete medium for 96 hours (37 °C, 5% CO 2 ), and then, using flow cytometry, CD3 + CD8 + CD69 + T cells (cell viability 70%), which are cancer cell-specific T cells activated by cancer cell antigens in the incubated cells, are sorted. 200,000 cancer cell-specific T cells sorted above are co-incubated with IL-2 (2000 U / mL), IL-7 (500 U / mL), IL-15 (500 U / mL), and αCD3 / αCD28 antibody (20 ng / mL) in 10 mL of high-glucose DMEM complete medium (37 °C, 5% CO 2 ) for 7 days (changing the medium every 2 days) to amplify the sorted cancer cell-specific T cells (cell viability 70%).

[0348] (4) Cancer cell-specific T cells for cancer treatment To prepare colon cancer tumor-bearing mice, female C57BL / 6 at 6 - 8 weeks old are selected as model mice. On the 0th day, 2×10 6 individual MC38 cells are subcutaneously inoculated into each mouse, and on the 4th, 7th, 10th, 15th, and 20th days, 100,000 cancer cell-specific T cells (100 μL) are injected into the mice respectively. The method for monitoring the tumor growth and survival period of the mice is the same as above.

[0349] (5) Experimental results As shown in Fig. 7, the mice in the control group had a fast tumor growth rate, while the cancer cell-specific T cells obtained by sorting and amplifying nanoparticles 1 and 2 could effectively slow down the tumor growth rate and extend the survival period of the mice.

[0350] Example 8. T cells after sorting and amplification for breast cancer prevention (1) Preparation of antigen component The cultured 4T1 cells were centrifuged at 400 g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells were inactivated and denatured by ultraviolet light and heat treatment respectively, and then an appropriate amount of 8 M urea was used to lyse the breast cancer cells. The lysate was dissolved to obtain an antigen component for preparing particles.

[0351] (2) Preparation of microparticles In this example, microparticles were prepared by the double emulsion method. The molecular weight of PLGA, the skeletal material of microparticle 1, was 38 KDa - 54 KDa. The immunoadjuvants used were CpG and poly ICLC, and the substance for increasing immune escape from lysosomes was arginine. During the preparation, first, microparticles carrying antigen components, adjuvants and arginine inside were prepared by using the double emulsion method. Then, 100 mg of the microparticles were centrifuged at 9000 g for 20 minutes, resuspended in ultrapure water (10 mL) containing 4% trehalose, and then dried for 48 hours before use. The average particle size of the microparticle system was about 2.1 μm, the surface potential of the microparticles was about -5 mV, about 110 μg of protein or polypeptide components were carried per 1 mg of PLGA microparticles, 0.01 mg of CpG and poly ICLC were carried respectively, and 0.05 mg of arginine was carried. The preparation materials and methods of blank microparticle 2 were the same, with a particle size of about 2.0 μm. The same amounts of CpG, poly ICLC and arginine were carried on the blank microparticles, but no arbitrary antigen components were carried.

[0352] (3) Isolation and amplification of cancer cell-specific T cells Six- to eight-week-old female BALB / c mice are selected and on days 0, 4, 7, 14, 21, and 28, microparticles 1 (100 μL) containing 2 mg of PLGA are each subcutaneously injected. On day 32, the mice are sacrificed, peripheral blood is collected, and then peripheral blood mononuclear cells (PBMCs) are isolated from the peripheral blood. First, in the first step, flow cytometry is used to separate CD3 + CD8 + T cells and B220 + B cells from PBMCs, and 200,000 sorted CD8 + T cells, 300,000 B cells, IL-7 (10 ng / mL), and 40 μg of microparticles (microparticles 1 or microparticles 2 + the same amount of free lysate) are co-incubated in 2 mL of RPMI 1640 complete medium for 96 hours. Next, using flow cytometry, CD3 + CD8 + CD69 + T cells (cell viability 85%) that are cancer cell-specific T cells capable of recognizing cancer cell antigens are further sorted. The cancer cell-specific T cells sorted in the above two steps are co-incubated with IL-2 (2000 U / mL), IL-7 (200 U / mL), IL-15 (200 U / mL), and αCD3 / αCD28 antibody for 7 days to amplify the sorted cancer cell-specific T cells (cell viability 85%).

[0353] (4) T cells sorted and amplified for cancer prevention To prepare breast cancer tumor-bearing mice, six- to eight-week-old female BALB / c mice are selected as model mice. One day before adoptively transferring cells into the mice, to remove the recipient mice's immune cells, cyclophosphamide is intraperitoneally injected into the recipient mice at a dose of 100 mg / kg. On day 0, 2 million amplified cancer cell-specific CD8 + T cells are intravenously injected into the mice. At the same time, on day 0, 4 × 10 5 cells of 4T1 cells are subcutaneously inoculated into each mouse, and the method for monitoring the growth and survival period of the mice's tumors is the same as above.

[0354] (5) Experimental results As shown in Figure 9, compared with the control group, the tumor growth rate of the cancer cell-specific T cell treatment group was significantly slower, and the survival period of the mice was significantly extended. In addition, when the nanoparticles carrying all cell components assisted in the selection and amplification of the obtained cancer cell-specific T cells, the preventive effect was superior to that when the blank nanoparticles + free cell lysate assisted in the selection and amplification of the obtained cancer cell-specific T cells. As can be seen from this, the cancer cell-specific T cells selected and amplified in the two steps described in the present invention have an excellent preventive effect against breast cancer.

[0355] Example 9, Cancer Cell-Specific T Cells for Preventing Cancer Metastasis In this example, a mouse lung metastasis cancer model of mouse melanoma is used to illustrate the prevention of cancer metastasis using the selected and amplified cancer cell-specific T cells. In actual applications, specific dosage forms, adjuvants, incubation times, incubation concentrations, administration times, administration frequencies, and administration schedules can be adjusted according to the situation. In this example, mouse melanoma tumor tissues and cancer cell lines were dissolved in 8M urea and then melted. Next, the tumor tissue lysate components and cancer cell line lysate components were loaded onto nanoparticles at a mass ratio of 1:2. In addition, the particles were used to activate cancer-specific T cells and assist in the selection of cancer-specific T cells. The obtained T cells, after amplification, prevent cancer metastasis in the mouse body. In this example, four types of polypeptide neoantigens

[0356] B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM),

[0357] B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD),

[0358] Nanoparticles carrying B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were used as control nanoparticles, and the effectiveness of cancer-specific T cells assisted in selection and amplification by nanoparticles carrying whole cell antigens and nanoparticles carrying several types of polypeptide neoantigens in preventing cancer lung metastasis was analyzed.

[0359] (1) Preparation of antigen component After collecting mouse B16F10 melanoma tumor tissues and cultured B16F10 cancer cell lines, 8M urea was used to lyse and dissolve the whole cell components of the tumor tissues and cancer cells. Next, the tumor tissue components and cancer cell line lysate components were mixed and melted at a mass ratio of 1:2 to obtain the antigen component.

[0360] (2) Preparation of nanoparticles In this example, nanoparticles 1 were prepared by the solvent evaporation method. The PLGA molecular weight of the preparation material of nanoparticles 1 used was 24KDa - 38KDa, the immunoadjuvants used were CpG7909 and poly(I:C), and the substance used to increase escape from lysosomes was the KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA). The preparation method was as described above. In the preparation process, first, the double emulsion method was used to carry the antigen component, adjuvant, and KALA polypeptide inside the nanoparticles. After carrying the antigen component and adjuvant inside, 100 mg of the nanoparticles were centrifuged at 10,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then freeze-dried for 48 hours for use. The average particle size of the nanoparticles 1 was about 470 nm. Each 1 mg of PLGA nanoparticles 1 carried about 10 μg of the protein and polypeptide components of the lysate, contained 0.02 mg of CpG7909 and poly(I:C) respectively, and contained 0.04 mg of the KALA polypeptide. The preparation method of the control nanoparticles 2 carrying four types of antigen polypeptides was the same as above. The particle size of the control nanoparticles 2 was about 460 nm, and each 1 mg of PLGA nanoparticles carried about 10 μg of the antigen polypeptide, the same amount of adjuvant, and the KALA polypeptide.

[0361] (3) Selection and amplification of cancer cell-specific T cells Female C57BL / 6 mice at 6 - 8 weeks of age are selected and subcutaneously injected with 2 mg of PLGA nanoparticles (200 μL) on day 0, day 4, day 7, day 14, day 21, and day 28 respectively. On day 32, the mice are sacrificed, peripheral blood is collected, and then peripheral blood mononuclear cells (PBMCs) are isolated from the peripheral blood. First, CD3 + T cells and CD19 + B cells are sorted. Next, 7 million T cells and 7 million B cells sorted in the first step are co - incubated with 4 mg of nanoparticle 1 or nanoparticle 2 and 10 mL of RPMI1640 complete medium for 96 hours. Then, using flow cytometry, CD3 + CD8 + CD69 + T cells and CD3 + CD4 + CD69 + T cells in the incubated cells, which are cancer - cell - specific T cells (cell viability 80%) that can recognize cancer cell antigens, are sorted. 1 million CD3 + CD8 + CD69 + T cells or 1 million CD3 + CD4 + CD69 + T cells are co - incubated with IL - 2 (1000 U / mL), IL - 12 (1000 U / mL), and αCD3 / αCD28 antibody (10 ng / mL) and 10 mL of RPMI1640 complete medium for 14 days to amplify cancer - cell - specific T cells (cell viability 80%).

[0362] (4) Cancer - cell - specific T cells for cancer metastasis prevention To prepare melanoma - tumor - bearing mice, female C57BL / 6 at 6 - 8 weeks of age are selected as model mice. One day before adoptively transferring cells into the mice, to remove the recipient mice's immune cells, cyclophosphamide is intraperitoneally injected into the recipient mice at a dose of 100 mg / kg. On day 0, 1.5 million cancer - cell - specific CD8 + T cells and 0.5 million cancer - cell - specific CD4 + T cells are intravenously injected into the mice. At the same time, on day 1, 0.5×105 Inject the individual B16F10 cells intravenously for inoculation, sacrifice the mice on the 14th day, and observe and record the number of melanoma cancer nests in the lungs of the mice.

[0363] (5) Experimental results As shown in Figure 10, the cancer cell-specific T cells obtained by the assistance of nanoparticle 1 and nanoparticle 2 in sorting can effectively prevent cancer metastasis after proliferation. Also, compared with nanoparticle 2 carrying four types of neoantigen polypeptides, nanoparticle 1 carrying whole cell components was more excellent in assisting the sorting and amplification of cancer cell-specific T cells.

[0364] Example 10, T cells after sorting and amplification for pancreatic cancer prevention In this example, the lysed components of mouse Pan02 pancreatic cancer tumor tissue and MC38 colon cancer tumor tissue are loaded onto nanoparticles at a ratio of 3:1. The nanoparticles assist in the separation of cancer cell-specific T cells in the peripheral blood of mice and then are amplified for treating pancreatic cancer. In the experiment, first, mouse pancreatic cancer and colon cancer tumor tissues are obtained and lysed to prepare the water-soluble components and the original water-insoluble components dissolved in 6M guanidine hydrochloride. During particle preparation, the water-soluble components are a 3:1 mixture of the water-soluble components of pancreatic cancer tumor tissue and the water-soluble components of colon cancer tumor tissue, and the water-insoluble components are a 3:1 mixture of the water-insoluble components of pancreatic cancer tumor tissue and the water-insoluble components of colon cancer tumor tissue. Using PLGA as the nanoparticle skeleton material and BCG as the adjuvant, nanoparticles are prepared, and the nanoparticles assist in the sorting and amplification of cancer cell-specific T cells.

[0365] (1) Preparation of antigen components Inject 2×10 6 individual MC38 colon cancer cells or 1×10 6 individual Pan02 pancreatic cancer cells subcutaneously under the armpit of each C57BL / 6 mouse, and the tumors inoculated into each mouse are about 1000 mm 3When this occurs, the mouse is sacrificed and the tumor tissue is excised. The lysis method and the method for collecting each component are the same as in Example 1, except that 6 M guanidine hydrochloride is used instead of 8 M urea to dissolve the water-insoluble components, and the tumor tissue lysate component is the antigen component. The BCG lysis method is the same as the tumor tissue lysis method.

[0366] (2) Preparation of nanoparticles In this example, nanoparticles are prepared by the double emulsion method. The molecular weight of PLGA, which is the nanoparticle preparation material used for Nanoparticle 1, is 7 KDa to 17 KDa, the immunoadjuvant used is the BCG lysate component, and the preparation method is as described above. First, the antigen component and the adjuvant are loaded into the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 12,000 g for 20 minutes, resuspended in ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours to obtain a lyophilized powder. For use, before injecting Nanoparticle 1, 20 mg of the nanoparticles are dissolved in 0.9 mL of PBS, mixed with a sample (0.1 mL) containing the antigen component (80 mg / mL) and GM-CSF (50 ng / mL), allowed to act at room temperature for 10 minutes, and then used. The average particle size of Nanoparticle 1 is about 160 nm, and the surface potential is about -4 mV. About 130 μg of protein or polypeptide component is loaded per 1 mg of PLGA Nanoparticle 1, and 0.02 mg of BCG is loaded per 1 mg of PLGA Nanoparticle 1. The preparation material and the preparation method of Nanoparticle 2 are the same as those of Nanoparticle 1. Its particle size is about 160 nm, and the surface potential is about -4 mV. About 130 μg of protein or polypeptide component in the tumor tissue lysate is loaded per 1 mg of PLGA Nanoparticle 2, but no arbitrary adjuvant is contained.

[0367] (3) Preparation of cancer cell-specific T cells Female C57BL / 6 mice aged 6 - 8 weeks are selected and 2 mg of PLGA nanoparticles (200 μL) are subcutaneously injected on days 0, 4, 7, 14, 21, and 28 respectively. On day 32, the mice are sacrificed and peripheral blood is collected. Next, peripheral blood mononuclear cells (PBMC) are separated from the peripheral blood. First, using the magnetic bead sorting method, CD3 is separated from PBMC+ T cells and CD19 + Sort B cells, and 9 million CD3 T cells sorted in the first step + and 12 million CD19 B cells + Incubate the B cells with 50 μg of nanoparticles (nanoparticle 1 or nanoparticle 2) for 6 hours. Next, use flow cytometry to detect CD3, a cancer cell-specific T cell + CD69 + Sort the T cells (cell viability 75%). 1 million CD3 CD69 T cells sorted in the above two steps + CD69 + Incubate the T cells with IL-2 (1000 U / mL) and granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng / mL) in 15 mL of high-glucose DMEM complete medium (37 °C, 5% CO 2 2) for 14 days (change the medium every 2 days) to amplify the selected cancer cell-specific T cells.

[0368] (4) Cancer cell-specific T cells for pancreatic cancer treatment To prepare pancreatic cancer tumor-bearing mice, select 6- to 8-week-old female C57BL / 6 as model mice. At the same time, on day 0, inoculate each mouse subcutaneously with 1×10 6 cells of Pan02 pancreatic cancer cells. On days 6, 9, 12, 17, and 23, intravenously inject 2 million amplified cancer cell-specific T cells into the mice respectively. The method for monitoring and recording the tumor volume of the mice is the same as above.

[0369] (5) Experimental results As shown in Figure 11, the cancer cell-specific T cells obtained by selection and amplification with nanoparticle 1 and nanoparticle 2 used in the present invention can effectively treat pancreatic cancer.

[0370] Example 11, T cells after selection and amplification for cancer treatment In this example, we will explain how to select and amplify cancer cell-specific T cells by using mannose as a targeting head for active targeting. In actual applications, it can be adjusted according to the situation. The nanoparticle system can be taken up by dendritic cells through the mannose receptor on the surface of dendritic cells, and the antigen carried by the particles can activate cancer cell-specific T cells after being presented by dendritic cells. In actual applications, nanoparticles or microparticles can also be modified using targeting heads such as mannan, anti-CD32 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-B220 antibody, anti-CD11c antibody, etc. that can actively target antigen-presenting cells.

[0371] (1) Preparation of antigen component After collecting the cultured B16F10 cancer cells, the cancer cells are lysed using an 8M urea aqueous solution, and then the whole cell components of the lysed cancer cells are dissolved using an 8M urea aqueous solution to obtain the antigen component.

[0372] (2) Preparation of nanoparticles In this example, nanoparticle 1 is prepared by the double emulsion method. The materials used for preparing the nanoparticles are PLGA and mannose-modified PLGA. When preparing nanoparticles with a targeting head, when the two are used in combination, the mass ratio is 4:1 and the molecular weight is 7KDa - 17KDa. The immunoadjuvants used are poly(I:C) and CpG7909. The preparation method is as described above. First, the antigen component and the adjuvant are co-loaded into the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 12000 g for 25 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours for use. The average particle size of nanoparticle 1 is about 120 nmV, about 80 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.02 mg of poly(I:C) and CpG7909 are each contained.

[0373] (3) Preparation of dendritic cells (DC) In this example, bone marrow-derived dendritic cells (BMDCs) are used as antigen-presenting cells. The preparation method is the same as above.

[0374] (4) Activation of dendritic cells Mouse BMDCs are inoculated into a cell culture plate, 5 mL of RPMI1640 (10% FBS) medium is added per 100,000 BMDC cells, and then 30 μg of nanoparticle 1 is added, followed by co-incubation with BMDCs for 48 hours (37 °C, 5% CO 2 ), and then, after collecting the BMDCs, they are centrifuged at 300 g for 5 minutes, washed twice with phosphate-buffered saline (PBS), and then resuspended in PBS for use.

[0375] (5) Preparation of cancer cell-specific T cells Six- to eight-week-old female C57BL / 6 mice are selected and subcutaneously injected with 2 mg of nanoparticle 1 (200 μL) on days 0, 4, 7, 14, 21, and 28, respectively. On day 32, the mice are sacrificed, peripheral blood is collected, and then peripheral blood mononuclear cells (PBMCs) are separated from the peripheral blood. Using magnetic bead sorting, CD3 + CD8 + T cells are sorted. Two million sorted CD8 + T cells are co-incubated with 5 million BMDCs prepared in step (4), 40 μg of nanoparticle 1, 10 ng / mL of IL-7, and 4 mL of DMEM high-glucose complete medium for 18 hours. Next, using flow cytometry, the incubated CD8 + T cells, which are cancer cell-specific T cells capable of recognizing cancer antigens, are sorted for CD8 + CD69 + T cells (cell viability 75%). Two hundred thousand CD8 + CD69 + T cells sorted in the above two steps are co-incubated with IL-2 (1000 U / mL) and IL-7 (1000 U / mL) for 10 days to amplify cancer cell-specific T cells (cell viability 75%).

[0376] (6) Cancer cell-specific T cells for cancer prevention To prepare melanoma tumor-bearing mice, female C57BL / 6 mice aged 6 - 8 weeks were selected as model mice. One day before transplanting cells into the mice, to remove the immune cells of the recipient mice, cyclophosphamide was intraperitoneally injected into the recipient mice at a dose of 100 mg / kg. Next, 2 million cancer-specific T cells prepared in step (5) were subcutaneously injected into the recipient mice. The next day, 1.5×10 5 cells of B16F10 were subcutaneously inoculated at the lower right of the back of each recipient mouse. The method for monitoring the tumor growth rate and survival period of the mice is the same as above.

[0377] (7) Experimental results As shown in Figure 12, compared with the PBS control group, the tumor growth rate of the mice treated with T cells was significantly slower.

[0378] Example 12, Cancer cell-specific T cells after screening and amplification for liver cancer prevention In this example, first, Hepa1-6 liver cancer cells were lysed. Using PLGA as the nanoparticle skeleton material, poly(I:C) and live vaccine against bacterial tuberculosis (BCG) as immune adjuvants, nanoparticles carrying the whole cell antigen of liver cancer cells were prepared using the solvent evaporation method. Next, the particles were mixed with B cells and T cells, and after screening and amplification, cancer cell-specific T cells were obtained.

[0379] (1) Preparation of antigen components After collecting the cultured Hepa1-6 liver cancer cells, they were washed twice with PBS, treated with heat and ultraviolet irradiation, and then 8M urea was used to dissolve and lyse all cell components of the cancer cells to obtain the antigen components. The lysis method of BCG is the same as above, and the BCG lysate is used as an adjuvant.

[0380] (2) Preparation of nanoparticles In this example, nanoparticles were prepared by the solvent evaporation method. The molecular weight of PLGA, which is the material for preparing the nanoparticles used, was 24 KDa to 38 KDa, and the immune adjuvants used were BCG and poly(I:C). The preparation method was as described above. First, antigen components and adjuvants were loaded into the nanoparticles. Next, 100 mg of the nanoparticles were centrifuged at 10,000 g for 25 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours for use. The particle size of the nanoparticles was about 270 nm, about 100 μg of protein or polypeptide components were loaded per 1 mg of the PLGA nanoparticles, and 0.02 mg of BCG and poly(I:C) were each loaded.

[0381] (3) Preparation of cancer cell-specific T cells Female C57BL / 6 mice at 6 to 8 weeks of age were selected and 2 mg of PLGA nanoparticles (200 μL) were subcutaneously injected on days 0, 4, 7, 14, 21, and 28, respectively. On day 32, the mice were sacrificed and peripheral blood was collected. Next, peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood, and CD8 + T cells were selected from the PBMCs using the magnetic bead sorting method. 2 million selected CD8 + T cells, nanoparticles (500 μg), 8 million BAF3 mouse B cell lines, and IL-7 (10 ng / mL) were co-incubated in 2 mL of RPMI1640 complete medium for 48 hours. Next, using flow cytometry, CD8 + CD8 + CD69 + T cells (cell viability 80%) in the incubated CD8 + CD69 + T cells, which are cancer-specific T cells that can recognize cancer antigens, were selected. The selected CD8 + CD69 + T cells were co-incubated with IL-2 (1000 U / mL), IL-7 (500 U / mL), IL-15 (500 U / mL), and αCD3 / αCD28 antibodies for 7 days to amplify the selected cancer cell-specific T cells (cell viability 80%).

[0382] (4) Cancer cell-specific T cells for cancer prevention To prepare liver cancer tumor-bearing mice, female C57BL / 6 mice aged 6 - 8 weeks are selected as model mice. One day before transplanting cells into the mice, to remove the immune cells of the recipient mice, cyclophosphamide is intraperitoneally injected into the recipient mice at a dose of 100 mg / kg. On day 0, 2 million amplified cancer cell-specific T cells are injected into the mice. At the same time, on day 0, 1.0×10 6 cells of Hepa1-6 liver cancer cells are subcutaneously injected into the back of each mouse, and the tumor growth and the survival period of the mice are recorded by the same method as above.

[0383] (5) Experimental results As shown in Figure 13, compared with the control group, the mice treated with cancer cell-specific T cells have a significantly slower tumor growth rate.

[0384] Example 13, Cancer cell-specific T cells selected and amplified with the assistance of calcified nanoparticles for cancer prevention In this example, calcified nanoparticles are used to select cancer cell-specific T cells. In actual applications, other biomineralization techniques, modified particles such as crosslinking and gelation can also be used. In actual applications, specific dosage forms, adjuvants, administration times, administration frequencies, administration schedules, etc. can all be adjusted according to the situation. In this example, mouse melanoma tumor tissues and cancer cell lines are dissolved with 8M urea and then melted. Next, the tumor tissue lysate components and cancer cell line lysate components are loaded onto nanoparticle 1 at a mass ratio of 1:1. In this example, four types of polypeptide neoantigens

[0385] B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM),

[0386] B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD),

[0387] B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and

[0388] Use nanoparticles carrying TRP2:180-188 (SVYDFFVWL) as control nanoparticles 2.

[0389] (1) Preparation of antigen components After collecting mouse B16F10 melanoma tumor tissues and cultured B16F10 cancer cell lines, use 8M urea to lyse and dissolve all cell components of the tumor tissues and cancer cell lines. Next, mix and fuse the tumor tissue components and cancer cell line components at a mass ratio of 1:1 to obtain antigen components.

[0390] (2) Preparation of nanoparticles In this example, after carrying whole cell antigen on the inside and surface of the nanoparticles, the nanoparticles are bio-limeified. In this example, nanoparticles 1 are prepared by the solvent evaporation method. The PLGA molecular weight of the preparation material of nanoparticles 1 is 7KDa - 17KDa, the immunoadjuvants are CpG2006 and poly(I:C), and the substance to enhance escape from lysosomes is GALA polypeptide (WEAALAEALAEALAEHLAEALAEALEALAA). The preparation method is as follows. First, carry antigen components, adjuvants and GALA polypeptide into the nanoparticles. Next, centrifuge 100 mg of PLGA nanoparticles at 13000 g for 20 minutes, resuspend with 18 mL of PBS, then add the antigen component (60 mg / mL) (2 mL) dissolved in 8M urea, allow to act at room temperature for 10 minutes, then centrifuge at 12000 g for 20 minutes, and collect the precipitate. Next, resuspend the 100 mg of PLGA nanoparticles in 20 mL of DMEM medium, add 200 μL of CaCl 2 (1 mM), and react at 37 °C for 2 hours. Subsequently, centrifuge at 10000 g for 20 minutes, collect the precipitate, resuspend with ultrapure water, and then centrifuge and wash twice. The average particle size of the nanoparticles is about 290 nm. For every 1 mg of PLGA nanoparticles, about 230 μg of protein and polypeptide components in the antigen component are carried, 0.02 mg of CpG and poly(I:C) are carried respectively, and 0.001 mg of GALA polypeptide is carried.

[0391] The preparation materials and preparation method of the control nanoparticles 2 carrying several types of antigen polypeptides are the same as above. The particle size of the control nanoparticles is about 290 nm, and about 230 μg of antigen polypeptide, the same amount of adjuvant and GALA polypeptide are carried per 1 mg of PLGA nanoparticles.

[0392] (3) Preparation of cancer-specific T cells Select 6-8 week-old female C57BL / 6 mice. On day 0, subcutaneously inoculate 1.5×10 5 cells of B16F10 into the back of the mice. Next, on days 10, 13, 17, 21 and 28, intraperitoneally inject the mice with a dose of 10 mg / kg of PD-1 antibody respectively. Kill the mice on day 30, collect peripheral blood, and then isolate peripheral blood mononuclear cells (PBMC) from the peripheral blood. Using flow cytometry, first select CD8 + T cells and B cells from PBMC. The selected CD8 + T cells (5 million cells), nanoparticles (500 μg), B cells (20 million cells) and IL-7 (10 ng / mL) are co-incubated in 20 mL of RPMI1640 complete medium for 48 hours. Next, using flow cytometry, select the CD8 + CD137 + T cells (cell viability 75%) in the incubated CD8 + T cells that can recognize cancer antigens. 500,000 CD8 + CD137 + T cells selected above are co-incubated with IL-2 (1000 U / mL), IL-7 (500 U / mL), IL-15 (500 U / mL) and αCD3 antibody (10 ng / mL) for 14 days to amplify cancer cell-specific T cells (cell viability 75%).

[0393] (4) Cancer cell-specific T cells for cancer prevention Six- to eight-week-old female C57BL / 6 mice were selected. One day before transplanting cells into the mice, to remove the immune cells of the recipient mice, cyclophosphamide was intraperitoneally injected into the recipient mice at a dose of 100 mg / kg. On day 0, 1 million amplified cancer cell-specific T cells were injected into the mice. At the same time, on day 0, 1.5×10 5 B16F10 melanoma cells were subcutaneously injected into the back of each mouse, and the tumor growth and the survival period of the mice were monitored in the same manner as described above.

[0394] (5) Experimental results As shown in Figure 14, compared with the control group, the cancer cell-specific T cells activated by calcified nanoparticles and assisted in sorting can extend the survival period of mice and effectively prevent cancer. In addition, the effect of the nanoparticles carrying the whole cell components was superior to that of the nanoparticles carrying four types of neoantigen polypeptides.

[0395] Example 14, Cancer cell-specific T cells for melanoma treatment In this example, first, the tumor tissue of B16F10 melanoma was lysed to prepare the water-soluble and water-insoluble components of the tumor tissue. Then, using PLGA as the nanoparticle backbone material, poly(I:C) and CpG2395 as immune adjuvants, and melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-amide) as the component to promote the escape from lysosomes, nanoparticles were prepared by the solvent evaporation method.

[0396] (1) Preparation of antigen components When collecting tumor tissue, first, 1.5×10 5 B16F10 cells were subcutaneously inoculated into the back of each C57BL / 6 mouse, and when the tumor reached about 1000 mm 3When this occurs, the mouse is sacrificed, the tumor tissue is excised, the excised tumor tissue is minced and then ground, passed through a cell filter to prepare a single cell suspension, an appropriate amount of pure water is added, and freezing and thawing are repeated 5 times. In some cases, ultrasonic waves may also be used in combination to disrupt and dissolve the obtained sample. After dissolving the tumor tissue, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as a water-soluble component soluble in pure water. An aqueous sodium chloride solution containing 2.0 M arginine and 10% sodium deoxycholate is added to the obtained precipitate portion to dissolve the precipitate portion, whereby water-insoluble components insoluble in pure water can be solubilized in the aqueous solution. The above is the antigen component for preparing the nanoparticle system.

[0397] (2) Preparation of Nanoparticles In this example, nanoparticles are prepared by the double emulsion method. During preparation, nanoparticles carrying water-soluble components and nanoparticles carrying water-insoluble components are prepared separately and used in combination during application. The molecular weight of PLGA, which is the material for preparing the nanoparticles used, is 7 KDa to 17 KDa. The immunoadjuvants used are poly(I:C) and CpG2395, and the substance escaping from lysosomes is melittin. The preparation method is as described above. First, antigen components, adjuvants, and melittin are carried inside the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 10000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. Before use, it is resuspended in 9 mL of PBS, and then 1 mL of the antigen component (protein concentration 80 mg / mL) is added and allowed to act at room temperature for 10 minutes to obtain nanoparticles with the lysate carried inside and outside. The average particle size of the nanoparticles is about 290 nm. Each 1 mg of the PLGA nanoparticles carries about 140 μg of protein and polypeptide components in the antigen component, 0.02 mg of each of the poly(I:C) and CpG2395 immunoadjuvants, and 0.05 mg of melittin.

[0398] (3) Preparation of Cancer Cell-Specific T Cells Female C57BL / 6 mice aged 6 - 8 weeks are selected. On day 0, 1.5×105 Inject individual B16F10 subcutaneously, and then, on the 7th, 10th, 15th, 20th, and 25th days, inject 1 mg of nanoparticles carrying the water-soluble component and 1 mg of nanoparticles carrying the water-insoluble component subcutaneously into the mice respectively. On the 30th day, kill the mice, collect the PBMC of the mice, and use flow cytometry to obtain CD8 + T cells, CD4 + T cells and B cells are sorted. The CD8 + T cells (1 million), CD4 + T cells (1 million), nanoparticles (nanoparticles carrying the water-soluble component and nanoparticles carrying the water-insoluble component (200 μg each)), B cells (3 million), and IL-15 (50 ng / mL) are co-incubated in 2 mL of RPMI1640 complete medium for 72 hours, or the CD8 + T cells (1 million), CD4 + T cells (1 million), nanoparticles (nanoparticles carrying the water-soluble component and nanoparticles carrying the water-insoluble component (200 μg each)), B cells (3 million) are co-incubated in 2 mL of RPMI1640 complete medium for 72 hours, or the CD8 + T cells (1 million), CD4 + T cells (1 million), nanoparticles (nanoparticles carrying the water-soluble component and nanoparticles carrying the water-insoluble component (200 μg each)), B cells (3 million), and Flt3L (50 ng / mL) are co-incubated in 2 mL of RPMI1640 complete medium for 72 hours. Next, using flow cytometry, the CD8 + CD8 + CD69 + T cells (cell viability 80%) and CD4 + CD4 + CD69 + T cells (cell viability 80%) are sorted. 100,000 CD8 + CD69 + T cells or 100,000 CD4 + CD69 +T cells were co-incubated with IL-2 (1000 U / mL), IL-7 (500 U / mL), IL-15 (500 U / mL) and αCD3 antibody (10 ng / mL) in 20 mL of RPMI1640 complete medium (37 °C, 5% CO 2 ) for 11 days (changing the medium every 2 days) to amplify cancer cell-specific T cells.

[0399] (4) Amplified cancer cell-specific T cells for cancer treatment To prepare melanoma tumor-bearing mice, female C57BL / 6 at 6 - 8 weeks of age were selected as model mice. On day 0, 1.5×10 5 cells of B16F10 were subcutaneously inoculated into the lower right back of each mouse. On days 4, 7, 10, 15 and 20 after melanoma inoculation, 800,000 cancer cell-specific CD8 + T cells and 200,000 cancer cell-specific CD4 + T cells were intravenously injected respectively. In the experiment, the monitoring methods of the tumor volume and survival period of mice were the same as above.

[0400] (5) Experimental results As shown in Figure 15, the sorted and amplified cancer cell-specific T cells in the present invention have a good therapeutic effect on melanoma treatment.

[0401] Example 15, Cancer cell-specific T cells for melanoma treatment In this example, first, the tumor tissue of B16F10 melanoma was lysed, and then, using PLGA as the nanoparticle backbone material, poly(I:C) and CpG7909 as immune adjuvants, and polyarginine and polylysine as components to promote escape from lysosomes, nanoparticles were prepared.

[0402] (1) Preparation of antigen components When collecting tumor tissue, first, 1.5×10 5 cells of B16F10 were subcutaneously inoculated into the back of each C57BL / 6 mouse, and when the tumor reached about 1000 mm 3When this occurs, the mouse is sacrificed, the tumor tissue is excised, the tumor tissue is agitated and pulverized using a tissue homogenizer, then, after adding an appropriate amount of ultrapure water, freezing and thawing are repeated to lyse the cells, subsequently, nuclease is added and allowed to act for 5 minutes, and then, it is allowed to act at 95 °C for 10 minutes to inactivate the nuclease, and a whole cell antigen component (mainly proteins and polypeptides) from which the nucleic acid components of all cells are removed is obtained. Then, centrifugation is performed at 8000 g for 3 minutes, the supernatant portion is a water-soluble component, and for the precipitate portion, a water-insoluble component is dissolved using 0.1 M metformin hydrochloride and 0.1 M arginine aqueous solution. The water-soluble component and the water-insoluble component dissolved in the solution are mixed and melted at a mass ratio of 1:1 to obtain an antigen component for preparing nanoparticles.

[0403] (2) Preparation of nanoparticles In this example, nanoparticle 1 is prepared by the double emulsion method, which has the ability to target dendritic cells. The materials used for preparing nanoparticle 1 are PLA with a molecular weight of 20 KDa to 30 KDa and mannan-modified PLA, and during use, the mass ratio of unmodified PLA to mannan-modified PLA is 9:1. The immunoadjuvants used are poly(I:C) and CpG7909, and the substances that increase immune escape from lysosomes are polyarginine and polylysine. The preparation method is as described above. First, a cell lysate component, an adjuvant, polyarginine, and polylysine are loaded into the nanoparticles, and after the above components are loaded inside, 100 mg of the nanoparticles are centrifuged at 10000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then freeze-dried for 48 hours. The average particle size of the nanoparticles is 360 nm, about 100 μg of protein and polypeptide components in the antigen component are loaded per 1 mg of PLGA nanoparticles, 0.02 mg of poly(I:C) and CpG7909 immunoadjuvants are each loaded, and 0.01 mg of polyarginine and polylysine are each loaded.

[0404] (3) Preparation of cancer cell-specific T cells Female C57BL / 6 mice at 6 - 8 weeks of age are selected. On day 0, 1.5×10 5Subcutaneously inoculate individual B16F10, and then subcutaneously inject 2 mg of nanoparticle 1 into the mice on the 7th, 10th, 15th, 20th, and 25th days, respectively. On the 30th day, sacrifice the mice, collect the PBMC of the mice, and use flow cytometry to obtain CD3 from the PBMC + T cells and B cells are sorted. The sorted CD3 + T cells (2 million cells), nanoparticle 1 (40 μg), B cells (3 million cells), and IL-15 (10 ng / mL) are co-incubated in 2 mL of RPMI1640 complete medium for 6 hours. Next, using flow cytometry, CD3 in the incubated T cells, which are cancer-specific T cells capable of recognizing cancer antigens + CD69 + T cells (cell survival rate 85%) are sorted. 300,000 CD3 + CD69 + T cells are co-incubated with IL-2 (1000 U / mL), IL-7 (1000 U / mL), IL-15 (1000 U / mL), and αCD3 antibody (20 ng / mL) in 2 mL of RPMI1640 complete medium for 14 days to amplify cancer cell-specific T cells.

[0405] (4) T cells for cancer treatment To prepare melanoma tumor-bearing mice, female C57BL / 6 at 6 - 8 weeks of age are selected as model mice. On the 0th day, subcutaneously inoculate 1.5×10 5 individual B16F10 cells under the lower right of the back of each mouse. On the 4th, 7th, 10th, 15th, and 20th days after melanoma inoculation, inject 800,000 amplified CD3 + T cells intravenously, respectively. The monitoring methods for the tumor volume and survival period of the mice are the same as above.

[0406] (5) Experimental results As shown in Figure 16, the tumors in the PBS control group grow very rapidly, but the tumor growth rate of the mice treated with cancer cell-specific T cells sorted and amplified with nanoparticles is significantly slower.

[0407] Example 16, Sorted and Amplified T Cells for Breast Cancer Prevention In this example, first, breast cancer cells are inactivated and denatured, and then the cells are lysed. N-octyl-β-D-glucopyranoside is used to dissolve and solubilize the water-insoluble components in the cancer cells. Next, using PLGA as the microparticle skeleton material and CpG1018 and poly ICLC as immunoadjuvants, microparticles carrying whole cell antigens are prepared.

[0408] (1) Preparation of antigen components The cultured 4T1 cells are centrifuged at 400 g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells are inactivated and denatured by ultraviolet light and high-temperature heating respectively. Next, ultrapure water is added, and freezing and thawing are repeated 5 times. Ultrasonic waves are applied to lyse the cancer cells. The cell lysate is centrifuged at 5000 g for 10 minutes. The supernatant is used as the water-soluble component, and the precipitate is dissolved with 10% N-octyl-β-D-glucopyranoside. After dissolution, the original water-insoluble component is obtained. When the water-soluble component and the water-insoluble component are mixed at a mass ratio of 2:1, antigen component 1 required for preparing microparticles is obtained.

[0409] (2) Preparation of microparticles In this example, microparticle 1 is prepared by the double emulsion method. The molecular weight of PLGA, which is the skeleton material of microparticle 1, is 38 KDa to 54 KDa, and the immunoadjuvants used are CpG1018 and poly ICLC. During the preparation, first, microparticles carrying antigen component 1 and adjuvants inside are prepared by the double emulsion method. Next, 100 mg of the microparticles are centrifuged at 9000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then dried for 48 hours for use. The average particle size of the microparticle system is about 5.0 μm. About 410 μg of the protein or polypeptide component of cancer cells is carried per 1 mg of PLGA microparticles, and 0.01 mg of CpG1018 and poly ICLC are carried respectively.

[0410] (3) Preparation of dendritic cells In this example, BMDC is used as the antigen-presenting cell. The preparation method is as described above.

[0411] (4) Activation of dendritic cells 5 million BMDCs, 2 mg of microparticles, and IL-15 (20 ng / mL) were co-incubated in 5 mL of RPMI 1640 (10% FBS) medium for 8 hours. Then, after collecting the BMDCs, the activated dendritic cells were irradiated with radiation to inactivate the dendritic cells, and the inactivated dendritic cells were used to activate T cells.

[0412] (5) Preparation of cancer cell-specific T cells Female BALB / c mice aged 6 - 8 weeks were selected and 2 mg of microparticle 1 (100 μL) was subcutaneously injected into each mouse on days 0, 4, 7, 14, 21, and 28. On day 32, the mice were sacrificed, and the PBMCs of the mice were collected. Using flow cytometry, CD3 + T cells were sorted from the PBMCs. The sorted CD3 + T cells (2 million cells), the inactivated BMDCs (3 million cells) prepared in step 4, and IL-7 (10 ng / mL) were co-incubated in 10 mL of RPMI 1640 complete medium for 18 hours. Next, using flow cytometry, CD3 + CD69 + T cells (cell viability 85%) in the incubated T cells, which are cancer cell-specific T cells capable of recognizing cancer antigens, were sorted. 300,000 CD3 + CD69 + T cells selected above were co-incubated with IL-2 (1000 U / mL), IL-7 (1000 U / mL), IL-15 (1000 U / mL), and αCD3 antibody (10 ng / mL) in 10 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 14 days to amplify cancer cell-specific T cells (cell viability 85%).

[0413] (6) Cancer cell-specific T cells for cancer prevention To prepare breast cancer tumor-bearing mice, female BALB / c mice aged 6 - 8 weeks are selected as model mice. One day before transplanting cells into the mice, to remove the recipient mice's immune cells, cyclophosphamide is intraperitoneally injected into the recipient mice at a dose of 100 mg / kg. On day 0, 1.2 million amplified CD3 + T cells (100 μL) are subcutaneously injected into the mice. At the same time, on day 0, 1×10 6 cells of 4T1 are subcutaneously inoculated into each mouse, and the size of the mouse tumor volume is recorded every 3 days starting from day 3.

[0414] (7) Experimental results As shown in Figure 17, compared with the control group, the tumor growth rate of the cancer cell-specific T cell treatment group sorted and amplified with microparticles is significantly slower, and the survival period of the mice is significantly extended. As can be seen from this, the cancer cell-specific T cells described in the present invention have a preventive effect against breast cancer.

[0415] Example 17, T cells sorted and amplified with microparticles for breast cancer prevention In this example, first, breast cancer cells are lysed using an 8M urea solution to dissolve the lysis components. Next, using PLA as the microparticle skeleton material and CpG2395 and poly ICLC as adjuvants, microparticles are prepared.

[0416] (1) Preparation of antigen components The cultured 4T1 cells are centrifuged at 400 g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells are inactivated and denatured by ultraviolet light and high-temperature heating respectively, and then the cancer cells are lysed using an 8M urea aqueous solution to dissolve the lysate components and obtain the antigen components for preparing microparticles.

[0417] (2) Preparation of microparticles In this example, microparticles 1 are prepared by the double emulsion method. The molecular weight of PLA, which is the skeletal material of microparticles 1, is 40 KDa, and CpG2395 and poly ICLC are immune adjuvants. During the preparation, first, antigen components and adjuvants are carried inside, and then 100 mg of the microparticles are centrifuged at 9000 g for 20 minutes, resuspended in ultrapure water (10 mL) containing 4% trehalose, dried for 48 hours, and then prepared for use. The average particle size of the microparticles is about 2.5 μm, about 600 μg of protein or polypeptide components are carried per 1 mg of PLGA microparticles, and 0.02 mg of CpG2395 and poly ICLC are carried respectively.

[0418] (3) Preparation of cancer cell-specific T cells Female C57BL / 6 mice aged 6 - 8 weeks are selected, and on days 0, 4, 7, 14, 21, and 28, microparticles 1 (100 μL) containing 2 mg of PLGA are subcutaneously injected respectively. On day 32, the mice are sacrificed, PBMC in the peripheral blood of the mice are collected, and using flow cytometry, CD3 + T cells and CD19 + B cells are sorted. The sorted CD3 + T cells (8 million), microparticles 1 (500 μg), B cells (9 million), IL-7 (10 ng / mL), and IL-15 (10 ng / mL) are co-incubated in 5 mL of RPMI1640 complete medium for 24 hours. Next, using flow cytometry, CD3 + CD69 + T cells are cancer-specific T cells that can recognize cancer antigens, and the incubated CD3 + CD3 in T cells + CD69 + T cells (cell survival rate 80%) are sorted. 500,000 CD3 + CD69 + T cells sorted above are cultured with IL-2 (1000 U / mL), IL-7 (1000 U / mL), IL-15 (1000 U / mL), and αCD3 antibody (10 ng / mL) in 10 mL of DMEM complete medium (37 °C, 5% CO 2) Incubate together for 10 days to amplify cancer cell-specific T cells (cell survival rate 80%), and the resulting cells are T cell 1.

[0419] Alternatively, the selected CD3 + T cells (2 million cells), microparticles 1 (50 μg), B cells (6 million cells), IL-7 (10 ng / mL), and IL-15 (10 ng / mL) are incubated together in 5 mL of RPMI 1640 complete medium for 24 hours. Next, the T cells therein are separated without any selection and amplification, and the resulting cells are T cell 2 (cell survival rate 80%).

[0420] (4) Cancer cell-specific T cells for cancer prevention To prepare breast cancer tumor-bearing mice, female BALB / c mice aged 6 - 8 weeks are selected as model mice. One day before adoptively transferring cells into the mice, to remove the recipient mice's immune cells, cyclophosphamide is intraperitoneally injected into the recipient mice at a dose of 100 mg / kg. On day 0, the sorted and amplified T cell 1 and T cell 2 (1 million cells) are subcutaneously injected. At the same time, on day 0, 1×10 6 4T1 cells are subcutaneously inoculated into each mouse, and the monitoring methods for the tumor volume and survival period of the mice are the same as above.

[0421] (5) Experimental results As shown in Figure 18, compared with the control group, the tumor growth rate in the T cell treatment group was significantly slower, the survival period was significantly extended, and the effect of T cell 1 was significantly superior to that of T cell 2.

[0422] Example 18, Cancer cell-specific T cells for melanoma treatment (1) Preparation of antigen components When collecting tumor tissues, first, 1.5×10 5 B16F10 cells are subcutaneously inoculated on the back of each C57BL / 6 mouse, and when the tumors reach approximately 1000 mm 3When this occurs, kill the mouse, extract the tumor tissue, finely cut the tumor tissue and then grind it, pass it through a cell filter, add an appropriate amount of pure water, repeat freezing and thawing 5 times (ultrasonic waves may be added), break and dissolve the obtained sample, add nuclease and let it act for 10 minutes, then heat at 95 °C for 10 minutes to inactivate the nuclease. When collecting the cultured B16F10 cancer cell line, first remove the medium by centrifugation, wash twice with PBS, centrifuge and collect the cancer cells, resuspend the cancer cells in ultrapure water, repeat freezing and thawing 3 times, use ultrasonic waves in combination to break and dissolve the cancer cells, then add nuclease to the sample and let it act for 10 minutes, and then heat at 95 °C for 5 minutes to inactivate the nuclease. After treating the tumor tissue or cancer cell line with an enzyme, centrifuge the cell lysate at a rotational speed of 5000 g for 5 minutes, take the supernatant as a water-soluble component soluble in pure water, add 50% glycerin to the obtained precipitate part, dissolve and solubilize the precipitate part. Mix the water-soluble components of the tumor tissue and the cancer cell line at a mass ratio of 1:1, and mix the water-insoluble components of the tumor tissue and the cancer cell line at a mass ratio of 1:1. The above is the antigen component for preparing the nanoparticles.

[0423] (2) Preparation of Nanoparticles In this example, nanoparticles 1 are prepared by the double emulsion method. During the preparation, nanoparticles carrying a water-soluble component mixture and nanoparticles carrying a water-insoluble component mixture are prepared separately and used in combination during application. The molecular weight of PLGA, which is the material for preparing the nanoparticles used, is 7KDa to 17KDa. The immunoadjuvants used are poly(I:C) and CpG1018, and the R8 polypeptide (RRRRRRRR) is a substance that increases the escape from lysosomes. The preparation method is as described above. First, cell lysate components, an adjuvant, and the R8 polypeptide are carried in the nanoparticles by the double emulsion method. Next, 100 mg of the nanoparticles are centrifuged at 12,000 g for 25 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, then lyophilized for 48 hours. Before use, it is resuspended in 9 mL of PBS, and then 1 mL of cell lysate components (protein / polypeptide concentration 80 mg / mL) is added and allowed to act at room temperature for 10 minutes to obtain nanoparticles with the lysate carried both inside and outside. The average particle size of the nanoparticles is about 290 nm. Each 1 mg of the PLGA nanoparticles carries about 140 μg of protein and polypeptide components in the antigen component, 0.02 mg of each of the poly(I:C) and CpG1018 immunoadjuvants, and 0.1 mg of the polypeptide.

[0424] (3) Preparation of cancer-specific T cells Female C57BL / 6 mice aged 6 - 8 weeks are selected and, on days 0, 4, 7, 14, 21, and 28, 1 mg of PLGA nanoparticles carrying water-soluble components and 1 mg of PLGA nanoparticles carrying water-insoluble components (100 μL) are each subcutaneously injected. On day 32, the mice are sacrificed, and the PBMC of the mice are collected. Using flow cytometry, CD8 + T cells and CD4 + T cells are sorted. The sorted CD8 + T cells (2 million) and CD4 +T cells (1 million), nanoparticles (50 μg, where 25 μg are nanoparticles carrying water-soluble components and 25 μg are nanoparticles carrying water-insoluble components), DC2.4 cell line (300,000), and IL-7 (10 ng / mL) were co-incubated in 2 mL of RPMI1640 complete medium for 96 hours. Next, using flow cytometry, the incubated CD8, which is a cancer-specific T cell capable of recognizing cancer antigens + CD8 in T cells + CD69 + T cells (cell viability 80%) and CD4 + CD4 in T cells + CD69 + Select T cells (cell viability 80%). 200,000 CD8 + CD69 + Cancer cell-specific T cells or 100,000 CD4 + CD69 + The cancer cell-specific T cells were co-incubated with IL-2 (1000 U / mL), IL-7 (1000 U / mL), IL-21 (1000 U / mL), and αCD3 antibody (10 ng / mL) in 10 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 14 days to amplify cancer cell-specific T cells (cell viability 80%).

[0425] Alternatively, 200,000 CD8 + T cells or 100,000 CD4 + T cells (without further selection) were directly co-incubated with IL-2 (1000 U / mL), IL-7 (1000 U / mL), IL-21 (1000 U / mL), and αCD3 antibody (10 ng / mL) in 10 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 14 days to amplify cancer cell-specific T cells.

[0426] (4) T cells treat cancer Select 6- to 8-week-old female C57BL / 6 as model mice. On day 0, 1.5×10 5Inject 1 × 10⁶ B16F10 cells subcutaneously into the lower right back of each mouse. On the 4th, 7th, 10th, 15th, and 20th days after melanoma inoculation, 800,000 amplified cancer-specific CD8 + T cells and 400,000 cancer-specific CD4 + T cells (after two-step sorting) are each injected intravenously, or on the above days, 800,000 CD8 + T cells and 400,000 CD4 + T cells only (without nanoparticle sorting) are injected. The method for monitoring the tumor growth and survival period of the mice is the same as above.

[0427] (5) Experimental results As shown in Figure 19, compared with the control group, the tumor growth rate of the T cell treatment group is significantly slower, and the survival period is significantly extended. In addition, the cancer-specific T cells (after two-step sorting) obtained by sorting and amplifying the nanoparticles carrying the lysate components were superior to the T cells directly amplified without nanoparticle sorting.

[0428] Example 19, Cancer cell-specific T cells for colon cancer treatment (1) Preparation of antigen components When collecting tumor tissue, first, inoculate 2 × 10 6 ⁶ MC38 colon cancer cells subcutaneously into the back of each C57BL / 6 mouse. When the tumor volume reaches about 1000 mm 3 ³, sacrifice the mouse, extract the tumor tissue, finely cut the tumor tissue and then grind it, pass it through a cell filter, add an 8M urea aqueous solution to dissolve the tumor tissue, and dissolve the dissolved components. The above is the antigen component for preparing nanoparticles.

[0429] (2) Preparation of nanoparticles In this example, nanoparticles are prepared by the double emulsion method. The molecular weight of PLGA, the preparation material of nanoparticle 1, is 7 kDa to 17 kDa. Using poly(I:C) and CpG1018 as adjuvants, NH 4 HCO 3It is used as a substance that increases the escape from lysosomes. The preparation method is as described above. During the preparation process, first, cell lysate components, an adjuvant, and NH 4 HCO 3 are carried in the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 10,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. For use, the average particle size of the nanoparticles is about 260 nm, about 90 μg of protein and polypeptide components are carried per 1 mg of PLGA nanoparticles, 0.02 mg of poly(I:C) and CpG1018 are each carried, and NH 4 HCO 3 is carried at 0.01 mg. The preparation materials and method of nanoparticle 2 are the same as those of nanoparticle 1, but no adjuvant is added, only the substance that increases the escape from lysosomes is added, the particle size is about 260 nm, the surface potential is about -7 mV, about 90 μg of protein and peptide components are carried per 1 mg of PLGA nanoparticles, and 0.01 mg of NH 4 HCO 3 is carried per 1 mg of PLGA nanoparticles, but no adjuvant is carried.

[0430] (3) Preparation of cancer-specific T cells Six- to eight-week-old female C57BL / 6 mice are selected and, on days 0, 4, 7, 14, 21, and 28, 100 μL of nanoparticle 1 containing 2 mg of PLGA is subcutaneously injected respectively. On day 32, the mice are sacrificed, and the PBMC of the mice are collected. Using flow cytometry, CD8 + T cells, CD4 + T cells, and B cells are sorted. The sorted CD8 + T cells (2 million), CD4 + T cells (1 million), nanoparticles (50 μg), B cells (3 million), and IL-7 (10 ng / mL) are co-incubated in 2 mL of RPMI1640 complete medium for 48 hours. Next, using flow cytometry, the CD8 + T cells in the incubated CD8+ CD69 + T cells (cell viability 80%) and CD4 + CD4 in T cells + CD69 + Select T cells (cell viability 80%). 200,000 CD8 selected as above + CD69 + T cells or 200,000 CD4 + CD69 + T cells are co-incubated with IL-2 (1000 U / mL), IL-7 (1000 U / mL), IL-15 (1000 U / mL) and αCD3 antibody (10 ng / mL) in 10 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 14 days to amplify cancer cell-specific T cells (cell viability 80%).

[0431] (4) T cells for cancer treatment To prepare colon cancer mice, female C57BL / 6 at 6 - 8 weeks of age are selected as model mice. On day 0, 2×10 6 individual MC38 cells are subcutaneously inoculated into the lower right of the back of each mouse. On days 6, 9, 12, 15, 20 and 25 after inoculation with colon cancer cells, 1 million CD8 + cancer cell-specific T cells and 500,000 CD4 + cancer-specific T cells are respectively intravenously injected, or on the above days, 1.5 million CD8 + cancer-specific T cells are injected. The method for monitoring the tumor growth and survival period of mice is the same as above.

[0432] (5) Experimental results As shown in Figure 20, compared with the control group, the tumor growth rate of the cancer-specific T cell treatment group selected and amplified with nanoparticles is significantly slower, and the survival period is significantly extended. Also, simultaneously, the CD8 + T cells and CD4 + T cells obtained by nanoparticles only assisting in selection and amplification were superior to the CD8 + T cells obtained only by selection and amplification with nanoparticles.

[0433] Example 20, Cancer cell-specific T cells for melanoma treatment (1) Preparation of antigen component 1.5×10 5 Individual B16F10 cells were subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor reached approximately 1000 mm 3 , the mouse was sacrificed and the tumor tissue was excised. After mincing the tumor tissue, it was ground, passed through a cell filter, an appropriate amount of ultrapure water was added, freezing and thawing were repeated 5 times, and ultrasound may also be used in combination to disrupt the lysed cells. After lysis, the lysate was centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant was taken as the water-soluble component soluble in pure water. To the obtained precipitate, an aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate was added to dissolve the precipitate, whereby the water-insoluble component insoluble in pure water can be solubilized in the aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate. An aqueous solution of saturated ammonium sulfate was added dropwise to the water-soluble component in the cell lysate. After precipitation was complete, the obtained sample was centrifuged at 3000 g for 5 minutes. The precipitate was dissolved in an aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate. For use, the supernatant was heated at 100°C for 5 minutes, the obtained sample was centrifuged at 3000 g for 5 minutes, the supernatant was discarded, the precipitate was dissolved in an aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate. Next, after combining the precipitate after salting out dissolved in the aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate and the precipitate after heating dissolved in the aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate, it was used as part of the water-soluble component. The water-insoluble component in the cell lysate dissolved in the 2M semicarbazide hydrochloride aqueous solution and the components of the precipitate after salting out and heating in the water-soluble component dissolved in the 2M semicarbazide hydrochloride and 0.2M agmatine sulfate were mixed at a mass ratio of 1:1 to obtain antigen component 1 for preparing nanoparticle 1.

[0434] 1.5×10 5 Individual B16F10 cells were subcutaneously inoculated into the back of each C57BL / 6 mouse. When the volume of the tumor reached approximately 1000 mm 3When this occurs, the mouse is sacrificed and the tumor tissue is excised. After mincing the tumor tissue, it is ground, passed through a cell filter, and an appropriate amount of ultrapure water is added. Freezing and thawing are repeated 5 times, and sometimes ultrasonic waves are also used in combination to disrupt the lysed cells. After lysis, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as a water-soluble component soluble in pure water. To the obtained precipitate, an aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate is added to dissolve the precipitate, whereby the water-insoluble component insoluble in pure water can be solubilized in the aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate. An aqueous saturated ammonium sulfate solution is added dropwise to the water-soluble component in the cell lysate. After precipitation is complete, the obtained sample is centrifuged at 3000 g for 5 minutes, and the precipitate is dissolved in an aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate and used as part of the water-soluble component. The water-insoluble component in the cell lysate dissolved in the above aqueous solution of 2M semicarbazide hydrochloride and 0.2M agmatine sulfate and the components of the precipitate after salting out in the water-soluble component dissolved in 2M semicarbazide hydrochloride and the above 0.2M agmatine sulfate are mixed at a mass ratio of 1:1 to obtain antigen component 2 for preparing nanoparticle 2.

[0435] 1.5×10 5 1.5×10⁶ B16F10 cells are subcutaneously inoculated into the back of each C57BL / 6 mouse, and when the tumor reaches approximately 1000 mm 3When this occurs, the mouse is sacrificed and the tumor tissue is excised. After cutting the tumor tissue into small pieces, it is ground, passed through a cell filter, and an appropriate amount of ultrapure water is added. Freezing and thawing are repeated 5 times, and sometimes ultrasonic waves are also used in combination to disrupt the lysed cells. After lysis, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as the water-soluble components soluble in pure water. To the obtained precipitate, an aqueous solution of 2M semicarbazide hydrochloride and 0.2M arginine sulfate is added to dissolve the precipitate, whereby the water-insoluble components insoluble in pure water can be solubilized in the aqueous solution of 2M semicarbazide hydrochloride and 0.2M arginine sulfate. The water-soluble components in the cell lysate are heated at 100 °C for 5 minutes, and then the obtained sample is centrifuged at 3000 g for 5 minutes. After discarding the supernatant, the precipitate is dissolved in an aqueous solution of 2M semicarbazide hydrochloride and 0.2M arginine sulfate to obtain a part of the water-soluble components. The water-insoluble components in the cell lysate dissolved in the above-mentioned aqueous solution of 2M semicarbazide hydrochloride and 0.2M arginine sulfate, and the components of the precipitate after heating in the water-soluble components dissolved in the above-mentioned 2M semicarbazide hydrochloride and 0.2M arginine sulfate are mixed at a mass ratio of 1:1 to obtain the antigen component 3 for preparing nanoparticle 3.

[0436] (2) Preparation of Nanoparticles In this example, nanoparticle 1 (Nanoparticle 1) is prepared by the double emulsion method in the solvent evaporation method. The molecular weight of PLGA, which is the material for preparing the antigen delivery nanoparticles used, is 20 KDa to 40 KDa, and the immunoadjuvant used is poly(I:C). The preparation method is as described above. First, the double emulsion method is used to carry the antigen component 1 and the adjuvant inside the nanoparticles. Next, 300 mg of the nanoparticles are centrifuged at 14000 g for 30 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then freeze-dried for 48 hours. The average particle size of the nanoparticle 1 is about 100 nm, about 250 μg of protein or polypeptide component is carried per 1 mg of the PLGA nanoparticles, and 0.01 mg of poly(I:C) is carried per 1 mg of the PLGA nanoparticles.

[0437] In this example, the preparation method and materials of Nanoparticle 2 are the same as those of Nanoparticle 1. The preparation method is as described above. First, the double emulsion method is used to carry the antigen component 2 and the adjuvant in the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 14,000 g for 30 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. The average particle size of the Nanoparticle 2 is about 300 nm, about 250 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.01 mg of poly(I:C) is carried per 1 mg of PLGA nanoparticles.

[0438] In this example, the preparation method and materials of Nanoparticle 3 are the same as those of Nanoparticle 1. First, the antigen component and the adjuvant are carried in the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 14,000 g for 30 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. The average particle size of the Nanoparticle 3 is about 300 nm, about 250 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.01 mg of poly(I:C) is carried per 1 mg of PLGA nanoparticles.

[0439] (3) Preparation of cancer-specific T cells Female C57BL / 6 mice aged 6 - 8 weeks are selected. On day 0, 1.5×10 5 B16F10 cells are subcutaneously inoculated into the mice. On days 6, 8, 10, 12, 14, 16, 18, 20, and 22, 150 μg of PD-1 antibody is intraperitoneally injected into each mouse. On day 24, the mice are sacrificed, peripheral blood is collected, and then peripheral blood mononuclear cells (PBMC) are separated from the peripheral blood. All CD69 - PBMC are sorted using flow cytometry. Next, 1 million CD69 -PBMC cells, 10 mg of nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3), were co-incubated in 2 mL of RPMI1640 complete medium for 12 hours. Next, using flow cytometry, the incubated CD3 + CD69 + T cells (cell viability 85%) were sorted. 1 million CD3 + CD69 + T cells were co-incubated with IL-2 (1000 U / mL), IL-7 (1000 U / mL), αCD3 antibody (10 ng / mL), and αCD28 antibody (10 ng / mL) in 10 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 21 days to amplify cancer cell-specific T cells (cell viability 85%).

[0440] (4) Cancer cell-specific T cells treat cancer Six- to eight-week-old female C57BL / 6 were selected as model mice. On day 0, 1.5×10 5 B16F10 cells were subcutaneously inoculated into the lower right of the back of each mouse. On days 5, 8, 11, 15, 20, and 25, 500,000 CD3 + cancer cell-specific T cells were intravenously injected respectively. The method for monitoring the tumor growth and survival period of the mice was the same as above.

[0441] (5) Experimental results As shown in FIG. 21, in the drawings, the tumor volume of the mice in the PBS group grows rapidly. The tumor growth rate of the mice treated with the cancer cell-specific T cells obtained by selection and amplification with nanoparticle, nanoparticle 2, and nanoparticle 3 is significantly slowed down, the survival period is significantly extended, and some mice are cured. Nanoparticle 3 is superior to the cancer cell-specific T cells obtained by the selection of nanoparticle 2, which indicates that the effect of the particles prepared with the antigen component separated and purified by heating is superior to the effect of those separated and purified by salting out using a specific reagent. The effect of nanoparticle 1 is superior to that of nanoparticle 2 and nanoparticle 3, which indicates that the effect of the particles prepared with the antigen component separated and purified by both salting out and heating is significantly superior to the particles prepared with the antigen component separated and purified only by salting out or only by heating.

[0442] In this example, the antigen component mainly uses the protein and polypeptide components in the whole cell component. In actual applications, after separating and extracting the mRNA in the cell lysate and mixing it with the protein / polypeptide in the whole cell component, it can be used as a mixed antigen.

[0443] Example 21, Cancer Cell-Specific T Cells for Cancer Treatment (1) Preparation of Antigen Component The cultured E.G7-OVA mouse T lymphoma cells are centrifuged at 400 g for 5 minutes, then resuspended in ultrapure water, and subsequently, the cancer cells are lysed using a 6 M guanidine sulfate aqueous solution to dissolve the lysate components. Then, a saturated ammonium sulfate aqueous solution is added. After complete precipitation, the supernatant is discarded, and the precipitate is dissolved again in a 6 M guanidine sulfate aqueous solution to obtain the protein and polypeptide components in the cancer cells dissolved in the 6 M guanidine sulfate aqueous solution, which is antigen component 1.

[0444] The cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, then resuspended in ultrapure water, and cancer cells were lysed using a 3% aqueous solution of Tween 80 to solubilize the lysate components. Next, a saturated ammonium sulfate aqueous solution was added, and after complete precipitation, the supernatant was discarded. When the precipitate was solubilized again with a 3% aqueous solution of Tween 80, protein and polypeptide components that are antigen component 2 and soluble in the 3% aqueous solution of Tween 80 were obtained.

[0445] The cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, then resuspended in ultrapure water, and subsequently, the cancer cells were lysed using a 6 M aqueous solution of guanidine sulfate to dissolve the lysate components. Then, a saturated ammonium sulfate aqueous solution was added, and after complete precipitation, the supernatant was discarded. When the precipitate was solubilized again using a 3% aqueous solution of Tween 80, protein and polypeptide components that are antigen component 3 and soluble in the 3% aqueous solution of Tween 80 were obtained.

[0446] The cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400 g for 5 minutes, then resuspended in ultrapure water, and after lysing the cancer cells using a 3% aqueous solution of Tween 80, the lysate components were lysed using a 3% aqueous solution of Tween 80. Next, a saturated ammonium sulfate aqueous solution was added, and after complete precipitation, the supernatant was discarded. When the precipitate was lysed again with a 6 M aqueous solution of guanidine sulfate, protein and polypeptide components that are antigen component 4 and soluble in the 6 M aqueous solution of guanidine sulfate were obtained.

[0447] (2) Preparation of nanoparticles In this example, nanoparticles 1 (Nanoparticle 1) are prepared by the double emulsion method. The skeletal materials of nanoparticles 1 are PLA (molecular weight 30 - 40 kDa) and mannan-PEG2000-PLA (PLA molecular weight 30 - 40 kDa), and the mass ratio of PLA (molecular weight 30 - 40 kDa) to mannan-PEG2000-PLA (PLA molecular weight 30 - 40 kDa) is 9:1. The immunoadjuvants used are CpG2006 (class B), CpG2216 (class A), and poly-ICLC. During the preparation, first, nanoparticles carrying antigen component 1 and adjuvant inside are prepared by the double emulsion method. Then, 100 mg of the nanoparticles are centrifuged at 13,000 g for 25 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then dried for 48 hours to obtain nanoparticles 1. The average particle size is about 500 nm, 5 μg of cancer cell protein and polypeptide components are carried per 1 mg of PLGA nanoparticles, and 0.02 mg of CpG2006, CpG2216, and poly-ICLC are carried respectively.

[0448] The preparation method of nanoparticles 2 (Nanoparticle 2) is the same as that of nanoparticles 1. However, what is carried inside is antigen component 2 and adjuvant. The average particle size of nanoparticles 2 is about 500 nm, 5 μg of cancer cell protein and polypeptide components are carried per 1 mg of PLGA nanoparticles 2, and 0.02 mg of CpG2006, CpG2216, and poly-ICLC are carried respectively.

[0449] The preparation method of nanoparticles 3 (Nanoparticle 3) is the same as that of nanoparticles 1. However, what is carried inside is antigen component 3, adjuvant, and R8 polypeptide. The average particle size of nanoparticles 3 is about 500 nm, 5 μg of cancer cell protein and polypeptide components are carried per 1 mg of PLGA nanoparticles 3, and 0.02 mg of CpG2006, CpG2216, and poly-ICLC are carried respectively.

[0450] The preparation method of Nanoparticle 4 is the same as that of Nanoparticle 1. However, the antigen component 4 and adjuvant are carried inside. The average particle size of Nanoparticle 4 is about 500 nm, 5 μg of cancer cell proteins and polypeptide components are carried per 1 mg of PLGA Nanoparticle 4, and 0.02 mg of CpG2006, CpG2216 and poly ICLC are carried respectively.

[0451] (3) Preparation of cancer-specific T cells Select female C57BL / 6 mice at 6-8 weeks of age. On day 0, 5×10 5 individual E.G7-OVA mouse T lymphoma cells are subcutaneously inoculated into the mice. On days 4, 6, 8, 10, 12, 14, 16, 18 and 20, 150 μg of PD-1 antibody is intraperitoneally injected into each mouse respectively. On day 22, the mice are sacrificed, peripheral blood is collected, and then peripheral blood mononuclear cells (PBMCs) are separated from the peripheral blood. Using flow cytometry, all CD25 - of PBMCs are sorted. Next, 50 million CD25 - PBMC cells and 5 μg of nanoparticles (Nanoparticle 1, or Nanoparticle 2, or Nanoparticle 3, or Nanoparticle 4) are co-incubated in 5 mL of RPMI1640 complete medium for 96 hours. Then, using flow cytometry, the incubated CD3 + CD25 + T cells (cell viability 90%), which are cancer-specific T cells that can recognize cancer antigens, are sorted. 100,000 CD8 + CD25 + T cells selected above are co-incubated with IL-2 (1000 U / mL), αCD3 antibody (10 ng / mL) and αCD28 antibody (10 ng / mL) in 20 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 28 days to amplify cancer cell-specific T cells (cell viability 90%).

[0452] (4) T cells treat cancer To prepare colon cancer mice, female C57BL / 6 at 6-8 weeks of age are selected as model mice. On day 0, 5×105 Inject a single E.G7-OVA cell subcutaneously into the lower right back of each mouse. On the 6th, 9th, 12th, 15th, 20th, and 25th days after inoculation with colon cancer cells, inject 500,000 CD3 + Cancer-specific T cells are injected intravenously, respectively. The method for monitoring the tumor growth and survival period of mice is the same as above.

[0453] (5) Experimental results As shown in Figure 22, compared with the PBS control group, the tumor growth rate of mice treated with T cells is significantly slower, and the survival period of mice is significantly extended. In addition, the effect of cancer cell-specific T cells obtained by the selection and amplification of nanoparticle 1 is superior to that of nanoparticle 2, nanoparticle 3, and nanoparticle 4, indicating that it is very important to dissolve the cancer cell antigen component for preparing the nanoparticle once or twice using an appropriate lysing agent.

[0454] Example 22, T cells treat cancer (1) Collection of antigen components 1.5×10 5 Inject 1.5×10 3 B16F10 cells subcutaneously into the back of each C57BL / 6 mouse. When the tumor reaches about 1000 mm

[0455] 1.5×10 5 B16F10 cells (1.5×10) are subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor reaches approximately 1000 mm 3 , the mouse is sacrificed and the tumor tissue is excised. After finely cutting the tumor tissue, it is ground, an appropriate amount of ultrapure water is added, and freezing and thawing are repeated 5 times. In some cases, ultrasound may also be used in combination to disrupt the lysed cells. After lysis, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as the water-soluble component soluble in pure water. An 8M urea aqueous solution is added to the obtained precipitate portion to dissolve the precipitate portion, whereby the water-insoluble antigen insoluble in pure water can be solubilized in the 8M urea aqueous solution. A saturated ammonium carbonate aqueous solution is added dropwise to the water-soluble component in the cell lysate. After precipitation is complete, the obtained sample is centrifuged at 3000 g for 5 minutes. When the precipitate is dissolved in an 8M urea aqueous solution, a part of the water-soluble component is obtained. The water-insoluble component in the cell lysate dissolved in the 8M urea aqueous solution and the component of the precipitate after salting out in the water-soluble component dissolved in the 8M urea aqueous solution are mixed at a mass ratio of 1:2 to obtain the antigen component 2 for preparing nanoparticle 2.

[0456] 1.5×10 5 B16F10 cells (1.5×10) are subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor reaches approximately 1000 mm 3 , the mouse is sacrificed and the tumor tissue is excised. After finely cutting the tumor tissue, it is ground, an appropriate amount of ultrapure water is added, and freezing and thawing are repeated 5 times. In some cases, ultrasound may also be used in combination to disrupt the lysed cells. After lysis, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as the water-soluble component soluble in pure water. An 8M urea aqueous solution is added to the obtained precipitate portion to dissolve the precipitate portion, whereby the water-insoluble antigen insoluble in pure water can be solubilized in the 8M urea aqueous solution. After mixing the water-insoluble component and the water-soluble component in the cell lysate dissolved in the 8M urea aqueous solution at a mass ratio of 1:2, the antigen component 3 for preparing nanoparticle 3 is obtained.

[0457] 1.5×10 5 B16F10 cells (1.5×10) are subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor reaches approximately 1000 mm 3When this occurs, the mouse is sacrificed and the tumor tissue is excised. After mincing the tumor tissue, it is ground, and an appropriate amount of ultrapure water is added. Freezing and thawing are repeated 5 times, and sometimes ultrasonic waves are also used in combination to disrupt the lysed cells. After lysis, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as the water-soluble components soluble in pure water. 8 M urea aqueous solution is added to the obtained precipitate portion to dissolve the precipitate portion, whereby the water-insoluble components insoluble in pure water can be solubilized in the 8 M urea aqueous solution. A saturated ammonium sulfate aqueous solution is added dropwise to the water-soluble components in the cell lysate. After precipitation is complete, the obtained sample is centrifuged at 3000 g for 5 minutes. After solubilizing the precipitate with a 5% aqueous solution of PEG5000, it is used as a part of the water-soluble components. After mixing the components of the precipitate after salting out in the water-insoluble components in the cell lysate dissolved in the above 8 M urea aqueous solution and the water-soluble components dissolved in the 5% aqueous solution of PEG5000 at a mass ratio of 1:2, the antigen component 4 for preparing nanoparticle 4 is obtained.

[0458] 1.5×10 5 1.5×10⁶ B16F10 cells are subcutaneously inoculated into the back of each C57BL / 6 mouse. When the tumor reaches approximately 1000 mm 3 When this occurs, the mouse is sacrificed and the tumor tissue is excised. After mincing the tumor tissue, it is ground, and then an appropriate amount of ultrapure water is added. Freezing and thawing are repeated 5 times, and sometimes ultrasonic waves are also used in combination to disrupt the lysed cells. After lysis, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as the water-soluble components soluble in pure water. A 5% aqueous solution of PEG5000 is added to the obtained precipitate portion to solubilize the precipitate portion, whereby components that can solubilize the water-insoluble components in the 5% aqueous solution of PEG5000 can be obtained. A saturated ammonium sulfate aqueous solution is added dropwise to the water-soluble components in the cell lysate. After precipitation is complete, the obtained sample is centrifuged at 3000 g for 5 minutes. When the precipitate is solubilized with a 5% aqueous solution of PEG5000, it is used as a part of the water-soluble components. After mixing the components of the precipitate after salting out in the water-insoluble components in the cell lysate dissolved in the above-mentioned aqueous solution of PEG5000 and the water-soluble components dissolved in the 5% aqueous solution of PEG5000 at a mass ratio of 1:2, the antigen component 5 for preparing nanoparticle 5 is obtained.

[0459] (2) Preparation of Nanoparticles In this example, Nanoparticle 1 is prepared by the double emulsion method in the solvent evaporation method. The molecular weight of PLGA, which is the material for preparing the antigen delivery nanoparticles used, is 10 KDa to 20 KDa, and the immunoadjuvant used is poly(I:C). First, using the double emulsion method, the cell antigen component 1 and the adjuvant are carried in the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 13,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. The average particle size of the Nanoparticle 1 is about 200 nm, about 15 μg of protein or polypeptide component is carried per 1 mg of the PLGA nanoparticles, and 0.2 mg of poly(I:C) is carried.

[0460] The preparation method, materials, and preparation steps of Nanoparticle 2 are the same as those of Nanoparticle 1. The preparation method is as described above. First, the cell antigen component 2 and the adjuvant are carried in the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 13,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. The average particle size of the Nanoparticle 2 is about 200 nm, about 15 μg of protein or polypeptide component is carried per 1 mg of the PLGA nanoparticles, and 0.2 mg of poly(I:C) is carried.

[0461] The preparation method, materials, and preparation steps of Nanoparticle 3 are the same as those of Nanoparticle 1. First, the cell antigen component 3 and the adjuvant are carried in the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 13,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. The average particle size of the Nanoparticle 3 is about 200 nm, about 15 μg of protein or polypeptide component is carried per 1 mg of the PLGA nanoparticles, and 0.2 mg of poly(I:C) is carried.

[0462] The preparation method, materials, and preparation steps of Nanoparticle 4 are the same as those of Nanoparticle 1. The preparation method is as described above. First, the cell antigen component 4 and an adjuvant are loaded into the nanoparticle. Next, 100 mg of the nanoparticles are centrifuged at 13,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. The average particle size of the Nanoparticle 4 is about 200 nm, about 15 μg of protein or polypeptide component is loaded per 1 mg of PLGA nanoparticles, and 0.2 mg of poly(I:C) is loaded.

[0463] The preparation method, materials, and preparation steps of Nanoparticle 5 are the same as those of Nanoparticle 1. First, the cell antigen component 5 and an adjuvant are loaded into the nanoparticle. Next, 100 mg of the nanoparticles are centrifuged at 13,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then lyophilized for 48 hours. The average particle size of the Nanoparticle 5 is about 200 nm, about 15 μg of protein or polypeptide component is loaded per 1 mg of PLGA nanoparticles, and 0.2 mg of poly(I:C) is loaded.

[0464] (3) Preparation of T cells Female C57BL / 6 mice at 6 - 8 weeks of age are selected. On day 0, 1.5×10 5 B16F10 cells are subcutaneously inoculated into the mice. On days 4, 6, 8, 10, 12, 14, 16, 18, and 20, 150 μg of PD-L1 antibody is intraperitoneally injected into each mouse respectively. On day 22, the mice are sacrificed, and the PBMC of the mice are collected. The PBMC are first cultured in vitro for 12 hours, and then all CD69 - PBMC are sorted using flow cytometry. Next, 2.5 million CD69 -PBMC cells, 500 μg of nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3, or nanoparticle 4, or nanoparticle 5) were co-incubated in 10 mL of RPMI 1640 complete medium for 36 hours, and then, using flow cytometry, incubated CD3, which is a cancer-specific T cell capable of recognizing cancer antigens + CD69 + T cells (cell viability 90%) were sorted. 100,000 CD3 + CD69 + T cells were co-incubated with IL-2 (1000 U / mL), αCD3 antibody (10 ng / mL), and αCD28 antibody (10 ng / mL) in 10 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 21 days to amplify cancer cell-specific T cells (cell viability 90%).

[0465] (4) T cells treat cancer To prepare melanoma tumor-bearing mice, 6- to 8-week-old female C57BL / 6 were selected as model mice, and on day 0, 1.5×10 5 cells of B16F10 were subcutaneously inoculated at the lower right of the mouse's back. On days 3, 6, 9, 14, and 20 before inoculating the mice with tumors, 100,000 cancer cell-specific T cells or 100 μL of PBS obtained by selection assistance with different nanoparticles were subcutaneously inoculated into the mice respectively. The tumor growth rate of the mice and the survival period of the mice were monitored.

[0466] (4) Experimental results As shown in Fig. 23, the tumor volume of the mice in the PBS group grows rapidly. The tumor growth rate of the mice treated with cancer cell-specific T cells obtained by screening and amplifying with Nanoparticle 1, Nanoparticle 2, Nanoparticle 3, Nanoparticle 4, and Nanoparticle 5 is significantly slowed down, and the survival period is significantly extended. The effect of Nanoparticle 1 is superior to those of Nanoparticle 2, Nanoparticle 3, Nanoparticle 4, and Nanoparticle 5, which indicates that the separation and purification effect of the protein and polypeptide antigen components in the water-soluble component by ammonium sulfate salting-out is superior to that of ammonium carbonate, and the effect after separating and purifying a part of the water-soluble component is superior to the effect when directly using the water-soluble component. Also, the effect of dissolving the water-soluble component with urea and generating a precipitate by salting-out treatment is superior to the effect when using PEG.

[0467] Example 23, the selected and amplified T cells kill breast cancer cells In this example, nanoparticles are prepared using tumor tissues from multiple breast cancer patients.

[0468] (1) Preparation of antigen component Collect tumor tissues obtained by surgical resection from 12 triple-negative breast cancer patients. After cutting each patient's tumor tissue into small pieces, grind it, pass it through a cell filter, add it to ultrapure water, repeat freezing and thawing 5 times, and sometimes use ultrasonic waves in combination to lyse the cancer cells in the tumor tissue. Add 1 mg / mL nuclease to the lysed cancer cells to degrade the nucleic acids in the lysate, then heat at 95 °C for 10 minutes to inactivate the nuclease, then centrifuge at 5000 g for 5 minutes, collect the supernatant as the water-soluble component, and dissolve the precipitate in an aqueous solution of 8 M urea (containing 0.01 M arginine) to obtain the water-insoluble component. After mixing the water-soluble components of the 12 breast cancer patients in a mass ratio of 1:1, a water-soluble component mixture is obtained. After mixing the water-insoluble components of the 12 breast cancer patients in a mass ratio of 1:1, a water-insoluble component mixture is obtained. When the water-soluble component mixture and the water-insoluble component mixture are mixed in a mass ratio of 5:1, antigen component 1 for preparing nanoparticle 1 (NP1) is obtained.

[0469] Collect tumor tissues obtained by surgical resection from another triple-negative breast cancer patient A. Patient A is not included in the above 12 cancer patients. After cutting the tumor tissue into small pieces, grind it, pass it through a cell filter, add it to ultrapure water, repeat freezing and thawing 5 times, and sometimes use ultrasonic waves in combination to lyse the cancer cells. Add 1 mg / mL nuclease to the lysed cancer cells to degrade the nucleic acids in the lysate, then heat at 95 °C for 10 minutes to inactivate the nuclease, then centrifuge at 5000 g for 5 minutes, collect the supernatant as the water-soluble component, and dissolve the precipitate in an aqueous solution of 8 M urea (containing 0.01 M arginine) to obtain the water-insoluble component. When the water-soluble component and the water-insoluble component are mixed in a mass ratio of 5:1, antigen component 2 for preparing nanoparticle 2 (NP2) is obtained.

[0470] (2) Preparation of Nanoparticles In this example, nanoparticles are prepared by the double emulsion method. The molecular weight of PLGA, which is the skeletal material of nanoparticle 1, is 10KDa - 20KDa, and the immune adjuvants used are CpG2395 (class C), CpG1018 (class B), and poly ICLC. During the preparation, first, antigen component 1 and the adjuvant are carried inside the particles. Next, 100 mg of the nanoparticles are centrifuged at 12000 g for 20 minutes, resuspended in ultrapure water (10 mL) containing 4% trehalose, and then dried for 48 hours before being prepared for use. The particle size of the nanoparticles is approximately 280 nm, and approximately 950 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.02 mg of CpG2395, CpG1018, and poly(I:C) are carried respectively.

[0471] The molecular weight of PLGA, which is the skeletal material of nanoparticle 2, is 10KDa - 20KDa, and the immune adjuvants used are CpG2395 (class C), CpG1018 (class B), and poly ICLC. During the preparation, nanoparticles with antigen component 2 and the adjuvant carried inside are prepared by the double emulsion method. Next, 100 mg of the nanoparticles are centrifuged at 12000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, dried for 48 hours, and prepared for use. The particle size of the nanoparticles is approximately 280 nm, and approximately 950 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.02 mg of CpG2395, CpG1018, and poly(I:C) are carried respectively.

[0472] (3) Isolation and amplification of cancer cell-specific T cells Patient A undergoes treatment after the tumor tissue is surgically removed, and the treatment effect after treatment is very good, and the tumor mass gradually becomes smaller. 10 mL of peripheral blood is collected from the patient before and after immunotherapy respectively. Next, PBMC is separated from the peripheral blood, cultured in vitro for 12 hours, and then subsequent experiments are conducted.

[0473] PBMC (6 million), IL-7 (10 ng), and IL-15 (10 ng) before and after immunotherapy are co-incubated with 20 μg of nanoparticles (nanoparticle 1 or nanoparticle 2) and 2 mL of AIMV serum-free medium (37 °C, 5% CO 2 ) for 72 hours. After incubation, the cells are collected, and CD3 + CD25 + T cells (cell viability 85%) are sorted. 300,000 T cells sorted above are co-incubated with IL-2 (1000 U / mL), αCD3 antibody (10 ng / mL), and αCD28 antibody (10 ng / mL) and 10 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 21 days to amplify cancer cell-specific T cells (cell viability 85%).

[0474] Alternatively, PBMC (6 million), IL-7 (10 ng), and IL-15 (10 ng) after immunotherapy are co-incubated with 5 ng of nanoparticles (each being nanoparticle 1 or nanoparticle 2) and 2 mL of AIMV serum-free medium (37 °C, 5% CO 2 ) for 72 hours. After incubation, the cells are collected, and CD3 + CD25 + T cells (cell viability 85%) are sorted. 300,000 T cells sorted above are co-incubated with IL-2 (1000 U / mL), αCD3 antibody (10 ng / mL), and αCD28 antibody (10 ng / mL) and 10 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 21 days to amplify cancer cell-specific T cells (cell viability 85%).

[0475] Alternatively, PBMC (6 million), IL-7 (10 ng), and IL-15 (10 ng) after immunotherapy are co-incubated with 100 mg of nanoparticles (each being nanoparticle 1 or nanoparticle 2) and 2 mL of AIMV serum-free medium (37 °C, 5% CO 2 ) for 72 hours. After incubation, the cells are collected, and CD3 + CD25 +Select T cells (cell viability 85%), and incubate 300,000 T cells selected above with IL-2 (1000 U / mL), αCD3 antibody (10 ng / mL), and αCD28 antibody (10 ng / mL) in 10 mL of complete DMEM medium (37 °C, 5% CO 2 ) for 21 days to amplify cancer cell-specific T cells (cell viability 85%).

[0476] (4) Cancer cell-specific T cells kill cancer cells Select 6- to 8-week-old nude mice, and on day 0, subcutaneously inoculate 5×10 5 cancer cells amplified from the tumor tissue obtained by surgical resection of patient A under the lower right of the back of each nude mouse. On the 3rd, 6th, 9th, 14th, and 20th days after inoculating the mice with tumors, subcutaneously inoculate 300,000 cancer cell-specific T cells obtained by sorting and amplification assisted by different nanoparticles or 100 μL of PBS into the mice respectively. Monitor the tumor growth rate of the mice.

[0477] (5) Experimental results As shown in Figure 24, compared with the control group, cancer cell-specific T cells obtained by sorting and amplification with two types of nanoparticles can both effectively slow down the tumor growth rate, and the effects of nanoparticle 1 and nanoparticle 2 are similar. Also, when using an appropriate concentration (10 μg / mL) of nanoparticles to assist sorting by co-incubation, the effect was significantly superior to that when using too low a concentration (2.5 ng / mL) or too high a concentration (50 mg / mL). Also, the effect of cancer cell-specific T cells obtained by sorting and amplification after immunotherapy was superior to that of cancer cell-specific T cells obtained by sorting and amplification before immunotherapy. Also, when sorting and amplifying with peripheral blood immune cells after immunotherapy, the effect was better.

[0478] Example 24, sorted and amplified T cells kill esophageal cancer

[0479] (1) Preparation of antigen components Collect the tumor tissues of 5 esophageal patients. After mixing the tumor tissues of the 5 patients in a mass ratio of 1:1:1:1:1, cut them finely and grind them. Then, after passing them through a cell filter, add an appropriate amount of 8M urea aqueous solution to dissolve the above cells, and use the 8M urea aqueous solution to completely dissolve the tumor tissue lysate components to obtain antigen component 1 for preparing nanoparticle 1 and nanoparticle 2.

[0480] Collect the tumor tissue of another esophageal patient A. Patient A is not included in the above 5 cancer patients. Cut the tumor tissue of patient A finely and grind it. Then, after passing it through a cell filter, add an appropriate amount of 8M urea aqueous solution to dissolve the above cells, and use the 8M urea aqueous solution to completely dissolve the tumor tissue lysate components to obtain antigen component 2 for preparing nanoparticle 3 and nanoparticle 4.

[0481] (2) Preparation of Nanoparticles In this example, nanoparticle 1 (NP1) is prepared by the double emulsion method. The molecular weight of PLGA, the material for preparing the nanoparticles used, is 10KDa - 30KDa. The adjuvants carried are poly(I:C) and CpG7909, and the substance that increases the escape from lysosomes carried is the KALA polypeptide. The preparation method is as described above. First, carry the antigen component, adjuvant and KALA polypeptide in the nanoparticles. Then, centrifuge 100 mg of the nanoparticles at 12,000 g for 25 minutes, resuspend them using ultrapure water (10 mL) containing 4% trehalose, and then lyophilize them for 48 hours. The average particle size of the nanoparticles is about 250 nm. About 100 μg of the protein or polypeptide component of the tumor tissue is carried per 1 mg of the PLGA nanoparticles, 0.01 mg of poly(I:C) and CpG7909 are carried respectively, and 0.15 mg of the KALA polypeptide is carried.

[0482] The preparation materials and methods of nanoparticles 2 (NP2) are the same, with a particle size of about 250 nm. For every 1 mg of PLGA nanoparticles, about 0.01 μg of the protein or polypeptide component of tumor tissue is carried, 0.01 mg of poly(I:C) and CpG7909 are each carried, and 0.15 mg of KALA polypeptide is carried.

[0483] The preparation materials and preparation methods of nanoparticles 3 (NP3) are the same, with a particle size of about 250 nm. For every 1 mg of PLGA nanoparticles, about 100 μg of the protein and polypeptide components of tumor tissue are carried, 0.01 mg of poly I:C and CpG7909 are each carried, and 0.15 mg of KALA polypeptide is carried.

[0484] The preparation materials and preparation methods of nanoparticles 4 (NP4) are the same, with a particle size of about 250 nm. For every 1 mg of PLGA nanoparticles, about 0.01 μg of the protein or polypeptide component of tumor tissue is carried, 0.01 mg of poly I:C and CpG7909 are each carried, and 0.15 mg of KALA polypeptide is carried.

[0485] (3) Detection of cancer cell-specific T cells Patient A undergoes cancer immunotherapy after surgical resection of the tumor tissue. The treatment effect after treatment is very good, and the tumor mass gradually becomes smaller. 12 mL of peripheral blood is collected from the patient after immunotherapy. Next, PBMC is separated from the peripheral blood. PBMC (10 million cells) and 100 μg of nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3, or nanoparticle 4) are co-incubated in 10 mL of AIMV serum-free medium (37 °C, 5% CO 2 ) for 96 hours. After incubation, the cells are collected, labeled with CD3 and HLA-DR flow cytometry antibodies, and then, using the flow cytometry method, CD3, which is a cancer cell-specific T cell + HLA-DR +Select T cells (cell viability 85%). Incubate the T cells selected above with IL-2 (1000 U / mL), αCD3 antibody (10 ng / mL), and αCD28 antibody (10 ng / mL) in 10 mL of DMEM complete medium (37 °C, 5% CO 2 ) for 21 days to amplify cancer cell-specific T cells (cell viability 85%).

[0486] (4) The selected and amplified T cells kill cancer cells Select 6- to 8-week-old nude mice. On day 0, subcutaneously inoculate 5 × 10 5 cancer cells amplified from the tumor tissue obtained by surgical resection of patient A under the lower right of the back of each nude mouse. On days 3, 6, 9, 14, and 20 after inoculating the mice with tumors, subcutaneously inject 500,000 cancer cell-specific T cells or 100 μL of PBS obtained by selection and amplification assisted by different nanoparticles into the mice respectively. Monitor the tumor growth rate of the mice.

[0487] (5) Experimental results As shown in Figure 25, the effect of cancer cell-specific T cells obtained by selection and amplification by nanoparticles carrying a mixture of tumor tissues of multiple allogeneic cancer patients is not significantly different from the effect of nanoparticles obtained by assisting selection and amplification with the autologous tumor tissue of cancer patients. In addition, nanoparticles carrying appropriate antigen components were superior to nanoparticles carrying only a small amount of antigen components.

[0488] Example 25, the selected and amplified T cells kill lung cancer In this example, the water-soluble components and water-insoluble components in several human lung cancer cell lines are carried on nanoparticles, and then the nanoparticles are used to select and amplify cancer cell-specific T cells in the peripheral immune organs of lung cancer patients.

[0489] (1) Preparation of antigen components Culture human lung cancer cell lines A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells respectively.

[0490] After collecting each of the above eight types of cells, the above A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells, and H520 cells were mixed at a cell number ratio of 20:20:2:2:2:1:1:1. Next, after centrifugation, the medium was removed, and the cell pellet was resuspended in ultrapure water. Then, freezing and thawing were repeated 5 times. During the freezing and thawing process, in order to more thoroughly lyse the cancer cells, sonication was used in combination. After lysing the cells, the lysate was centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant was taken as the water-soluble components soluble in pure water. 6 M guanidine sulfate was added to the obtained precipitate to dissolve the precipitate, whereby the water-insoluble components insoluble in pure water could be solubilized in a 6 M guanidine sulfate aqueous solution. After heating the water-soluble components in the cell lysate at 95 °C for 10 minutes, the obtained sample was centrifuged at 3000 g for 5 minutes, and the precipitated protein and polypeptide components were dissolved in a 6 M guanidine sulfate aqueous solution. The mRNA in the supernatant was extracted using an mRNA extraction kit. Next, when the mRNA component was mixed with the protein and polypeptide components dissolved in 6 M guanidine sulfate, the antigen component in the water-soluble components was obtained. Thereafter, a saturated ammonium sulfate solution was added to the water-insoluble components dissolved in 6 M guanidine sulfate to salting-out the protein and polypeptide components. The salted-out precipitate was dissolved twice in 6 M guanidine sulfate to obtain the antigen component in the water-insoluble components. When the antigen component in the above water-insoluble components and the antigen component in the water-soluble components were mixed at a mass ratio of 1:1, antigen component 1 for preparing nanoparticle 1 (NP1) was obtained.

[0491] Alternatively, a sample of surgically resected tumor tissue from non-small cell lung cancer patient A is collected. The non-small cell lung cancer patient shows a good response to immunotherapy. After cutting the tumor tissue into small pieces, it is filtered through a cell filter, and then resuspended using ultrapure water. Subsequently, freezing and thawing are repeated 5 times, and sonication may be used in combination to lyse cancer cells during the freezing and thawing process. After lysing the cells, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as the water-soluble components soluble in pure water. 6 M guanidine sulfate is added to the obtained precipitate to dissolve the precipitate, whereby the water-insoluble components insoluble in pure water can be solubilized in a 6 M guanidine sulfate aqueous solution. After heating the water-soluble components in the cell lysate at 95 °C for 10 minutes, the obtained sample is centrifuged at 3000 g for 5 minutes, and the precipitated protein and polypeptide components are dissolved in a 6 M guanidine sulfate aqueous solution. The mRNA in the supernatant is extracted using an mRNA extraction kit. Next, when the mRNA component is mixed with the protein and polypeptide components dissolved in 6 M guanidine sulfate, the antigen component in the water-soluble components is obtained. Thereafter, a saturated ammonium sulfate solution is added to the water-insoluble components dissolved in 6 M guanidine sulfate to salting out the protein and polypeptide components. The salted-out precipitate is dissolved twice with 6 M guanidine sulfate to obtain the antigen component in the water-insoluble components. When the antigen component in the water-insoluble components and the antigen component in the water-soluble components are mixed at a mass ratio of 1:1, antigen component 2 for preparing nanoparticle 2 (NP2) is obtained.

[0492] Alternatively, a sample of tumor tissue from another non-small cell lung cancer patient B is collected. After cutting the tumor tissue into small pieces, it is filtered through a cell filter, and then resuspended using ultrapure water. Subsequently, freezing and thawing are repeated 5 times. In some cases, sonication may be used in combination to lyse cancer cells during the freezing and thawing process. After lysing the cells, the lysate is centrifuged at a rotational speed of 5000 g for 5 minutes, and the supernatant is taken as the water-soluble components soluble in pure water. 6 M guanidine sulfate is added to the obtained precipitate to dissolve the precipitate, thereby solubilizing the water-insoluble components insoluble in pure water in a 6 M guanidine sulfate aqueous solution. After heating the water-soluble components in the cell lysate at 95 °C for 10 minutes, the obtained sample is centrifuged at 3000 g for 5 minutes. The precipitated protein and polypeptide components are dissolved in a 6 M guanidine sulfate aqueous solution, and mRNA in the supernatant is extracted using an mRNA extraction kit. Next, when the mRNA component is mixed with the protein and polypeptide components dissolved in 6 M guanidine sulfate, an antigen component in the water-soluble components is obtained. Thereafter, a saturated ammonium sulfate solution is added to the water-insoluble components dissolved in 6 M guanidine sulfate to salting out the protein and polypeptide components. The salted-out precipitate is dissolved twice with 6 M guanidine sulfate to obtain an antigen component in the water-insoluble components. When the antigen component in the water-insoluble components and the antigen component in the water-soluble components are mixed at a mass ratio of 1:1, an antigen component 3 for preparing nanoparticle 3 (NP3) is obtained.

[0493] (2) Preparation of Nanoparticles Loaded with Whole Cell Components In this example, nanoparticles 1 (NP1) are prepared by the double emulsion method in the solvent evaporation method. The molecular weight of PLGA, which is the material for preparing the nanoparticles used, is 20KDa - 40KDa, and the adjuvants are poly(I:C), CpG7909, and CpG2395. The preparation method is as described above. First, an antigen component and an adjuvant are carried in the nanoparticles. Next, 100 mg of the nanoparticles are centrifuged at 10,000 g for 20 minutes, resuspended using ultrapure water (10 mL) containing 4% trehalose, and then freeze-dried for 48 hours. The average particle size of the nanoparticles 1 is about 380 nm, about 800 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.02 mg of poly(I:C), CpG7909, and CpG2395 are carried respectively.

[0494] The preparation process of nanoparticles 2 (NP2) is the same as that of nanoparticles 1. The average particle size of the nanoparticles 2 is about 380 nm, about 800 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.02 mg of poly(I:C), CpG7909, and CpG2395 are carried respectively.

[0495] The preparation process of nanoparticles 3 (NP3) is the same as that of nanoparticles 1. The average particle size of the nanoparticles 3 is about 380 nm, about 800 μg of protein or polypeptide component is carried per 1 mg of PLGA nanoparticles, and 0.02 mg of poly(I:C), CpG7909, and CpG2395 are carried respectively.

[0496] (3) Selection and amplification of T cells The tumor tissue of non-small cell lung cancer patient A shrinks after immunotherapy. Two weeks after non-small cell lung cancer patient A receives immunotherapy, 10 mL of peripheral blood is collected from non-small cell lung cancer patient A. Peripheral blood mononuclear cells (PBMC) are separated from 10 mL of the peripheral blood of the non-small cell lung cancer patient using gradient centrifugation.

[0497] Incubate nanoparticle 1 (0.5 mg) or nanoparticle 2 (0.5 mg) or nanoparticle 3 (0.5 mg) with 1 mL of AIM V serum-free medium containing 10 million PBMCs for 12 hours (37 °C, 5% CO 2 ). Next, after collecting the cells, centrifuge them at 400 g for 5 minutes, resuspend the cells in PBS, and first treat the T cells with Fc block to avoid non-specific loading.

[0498] Subsequently, stain half of the cells extracellularly with CD3 antibody and FASL antibody, then fix the cells with 4% paraformaldehyde, disrupt the membrane with a membrane-disrupting agent, and perform intracellular st...

Claims

Claim 1 A method for preparing cancer-specific T cells derived from autologous cells or allogeneic cells for preventing or treating cancer. Specifically, first, a step of separating immune cells from peripheral blood or peripheral immune organs, and then, co-incubating them with antigen-presenting cells, and nanoparticles and / or microparticles carrying whole tumor cell components or containing a part of whole tumor cell antigen components for a certain period to activate cancer-specific T cells, and then, separating the cancer-specific T cells activated by cancer antigens, amplifying them in vitro, and then reinjecting them into the body to exert an anti-cancer effect, Preferably, the preparation method specifically includes (1) First, separating immune cells from peripheral blood or peripheral immune organs, or selecting T cells from the immune cells, (2) Mixing microparticles and / or nanoparticles carrying tumor antigen components with antigen-presenting cells and T cells, then co-incubating them for a certain period, and then selecting T cells that are activated and express specific cell markers, (3) Co-incubating the T cells expressing specific cell markers selected in step (2) with cytokines and / or antibodies to obtain amplified specific T cells, Preferably, in step (2), the tumor antigen component is the whole cell lysate component of tumor tissue / cancer cells, or a part of the whole cell lysate component of tumor tissue / cancer cells. The whole cell lysate component can be divided into a water-soluble component and a water-insoluble component dissolved in a lysate containing a lysing agent. Preferably, in step (2), the tumor antigen component is obtained by lysing one or more cancer cells and / or whole tumor tissues, or by treating them after lysing one or more cancer cells and / or whole tumor tissues, or by treating one or more cancer cells and / or whole tumor tissues and then lysing them. Preferably, at least one of the cancer cells or tumor tissues is the same as the type of the target disease, or the antigen component is composed of a part of the components of one or more cancer cells and / or tumor tissues, and the part of the components includes protein / polypeptide components and / or mRNA components in the cell lysate. Preferably, the T cells selected in step (1) are any one or a combination thereof of CD3+ T cells, CD3+CD8+ T cells, CD4 ＋ T cells, method. Claim 2 Nanoparticles and / or microparticles carrying tumor antigen components can be co-incubated with antigen-presenting cells and T cells simultaneously to activate cancer cell-specific T cells. First, nanoparticles and / or microparticles carrying tumor antigen components can be co-incubated with antigen-presenting cells to activate the antigen-presenting cells. Next, the activated antigen-presenting cells alone can be co-incubated with T cells to activate cancer cell-specific T cells. After nanoparticles and / or microparticles carrying tumor antigen components are co-incubated with antigen-presenting cells, the antigen-presenting cells are activated, and the antigen-presenting cells that do not require special treatment can be co-incubated with T cells to activate specific T cells. Or after the antigen-presenting cells are treated with fixation, radiation, irradiation, modification, inactivation, mineralization, etc., the antigen-presenting cells can be co-incubated with T cells to activate specific T cells. When selecting cancer cell-specific T cells, one marker can be selected as the activation marker, or a component of multiple markers can also be selected as the activation marker. The method according to claim 1, characterized in that.

3. The method according to any one of claims 1 to 2, characterized in that the antigen component can contain an mRNA component in addition to protein and polypeptide components.

4. In step (1) above, allogeneic or xenogeneic immune cells of peripheral blood or immune cells of the peripheral immune system can be separated, and the cells can be directly separated and extracted without any treatment, or the cells can be separated and extracted by radiotherapy, immunotherapy, chemotherapy, particle therapy, vaccine therapy, etc. The method according to any one of claims 1 to 3, characterized in that.

5. The sorting method in steps (1) and (2) above is any one or a combination thereof of flow cytometry and magnetic bead method. The method according to any one of claims 1 to 4, characterized in that.

6. The tumor antigen component is a cell lysate component of tumor tissue and / or cancer cells, and contains one or both of a water-soluble component and a water-insoluble component generated after cell lysis of tumor tissue and / or cancer cells. First, the water-soluble component and the water-insoluble component are collected respectively, and nanoparticles or microparticles are prepared respectively. The water-insoluble component is dissolved using a lysate containing a solubilizer, or the lysate containing a solubilizer is directly used to directly lyse cancer cells or tumor tissue. Also, the whole cell component can be lysed to prepare nanoparticles or microparticles. The water-insoluble component is dissolved in a lysate containing a solubilizer. Preferably, when the antigen component is the whole cell lysate component of tumor tissue / cancer cells, the preparation method is as follows: (1) First, cancer cells / tumor tissue are lysed, then the water-soluble component and the water-insoluble component are prepared respectively, and then the water-insoluble component is dissolved with a specific solubilizer containing a lysate before use; (2) Cells are lysed using a lysate containing a solubilizer, and then the whole cell component lysed using the lysate containing a solubilizer is lysed. The method according to any one of claims 1 to 5, characterized by the above.

7. When the tumor antigen component is a part of the whole cell lysate component containing tumor tissue and / or cancer cells, the preparation method is as follows: (1) First, prepare a cell lysate of tumor tissue / cancer cells. Next, prepare a water-soluble component and a water-insoluble component respectively. Then, dissolve the water-insoluble component with a specific solvent containing the lysate and use it. Subsequently, separate and extract the protein component and polypeptide component in the water-soluble component from the water-soluble component by an appropriate method. Then, use the proteins and polypeptides separated and extracted from the water-soluble component together with all the water-insoluble components as the antigen component. (2) First, prepare a cell lysate of tumor tissue / cancer cells. Next, prepare a water-soluble component and a water-insoluble component respectively. Then, dissolve the water-insoluble component with a specific solvent containing the lysate and use it. Subsequently, separate and extract the protein component and polypeptide component in the water-soluble component from the water-insoluble component by an appropriate method. Then, use the proteins and polypeptides separated and extracted from the water-insoluble component together with all the water-soluble components as the antigen component. (3) First, prepare a cell lysate of tumor tissue / cancer cells. Next, prepare a water-soluble component and a water-insoluble component respectively. Then, dissolve the water-insoluble component with a specific solvent containing the lysate and use it. Subsequently, separately separate and extract the protein component and polypeptide component in the water-soluble component from the water-soluble component and the water-insoluble component by an appropriate method. Then, use the proteins and polypeptides separated and extracted from the water-soluble component and the water-insoluble component as the antigen component. (4) Or directly use a lysate containing a solvent to directly lyse cells or tissues, and also lyse whole cell tissues. Next, prepare the protein component and polypeptide component therein by an appropriate method and use them as the antigen component. In the above preparation method, it is also possible to add a step of separating and extracting whole cell mRNA and using the whole cell mRNA as a part of the antigen component. The appropriate treatment methods include, but are not limited to, treatment methods such as salting out, heating, and enzymatic hydrolysis. The water-insoluble component, or the precipitate generated by treatments such as salting out, heating, and enzymatic hydrolysis, is dissolved using a lysate containing a solvent. The method according to any one of claims 1 to 6, characterized in that.

8. When incubating the above nanoparticles / microparticles alone with antigen-presenting cells, or when incubating the above nanoparticles / microparticles simultaneously with antigen-presenting cells and T cells, the concentration of the nanoparticles / microparticles is 2.5 ng / mL to 50 mg / mL, and the total incubation time is 1 to 168 hours. The method according to any one of claims 1 to 7, characterized in that.

9. Before and / or after lysis, after inactivating and / or denaturing, solidifying, biomineralizing, ionizing, chemically modifying, or nuclease-treating the whole cell components, nanoparticles or microparticles can be prepared. Before and / or after cell lysis, they can also be directly prepared without inactivating and / or denaturing, solidifying, biomineralizing, ionizing, chemically modifying, or nuclease-treating. The method according to any one of claims 1 to 8, characterized in that.

10. For tumor tissue cells, inactivation and / or denaturation treatment can be performed before lysis, inactivation and / or denaturation treatment can also be performed after cell lysis, or inactivation and / or denaturation treatment can be performed before and / or after cell lysis. The method according to any one of claims 1 to 9, characterized in that.

11. The inactivation and / or denaturation treatment method before and / or after cell lysis includes any one or a combination of ultraviolet irradiation, high-temperature heating, radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, and freeze-drying. The method according to any one of claims 1 to 10, characterized in that.

12. In the above step (2), the ratio of the number of antigen-presenting cells to T cells used is greater than 1:

1. The nanoparticles or microparticles are used to activate existing cancer-specific T cells in peripheral immune cells in vitro through the presentation of antigen-presenting cells. The above nanoparticles or microparticles are selected from nanoparticles with a particle size of 1 nm to 1000 nm or microparticles with a particle size of 1 μm to 1000 μm. The method according to any one of claims 1 to 11, characterized in that.

13. The antigen-presenting cells incubated with T cells and nanoparticles and / or microparticles are derived from autologous, allogeneic, cell lines, stem cells or any mixture thereof, and the antigen-presenting cells to be co-incubated are B cells, dendritic cells, macrophages or any mixture of these three, according to any one of claims 1 to 12.

14. The antigen-presenting cells are derived from autologous antigen-presenting cells, allogeneic antigen-presenting cells, antigen-presenting cell lines or antigen-presenting cells differentiated from stem cells, preferably any one of dendritic cells (DC), B cells, macrophages or a combination thereof, and more preferably, a combination of one or more antigen-presenting cells is used, according to any one of claims 1 to 13.

15. Any one of the following three methods is selected for the above co-incubation by mixing: (a) directly mixing these three and co-incubating for a certain period; (b) first co-incubating the microparticles and / or nanoparticles with the antigen-presenting cells for a certain period, and then adding T cells for co-incubation; (c) first co-incubating the microparticles and / or nanoparticles with the antigen-presenting cells for a certain period, selecting the incubated antigen-presenting cells, and then co-incubating the antigen-presenting cells with T cells, according to any one of claims 1 to 14.

16. The culture conditions for the above-mentioned co-incubation of the mixed ink are co-incubating at 30 to 38 °C and 1 to 10% CO ２ for 1 to 168 hours, and the method according to any one of claims 1 to 15, characterized in that.

17. Cytokines can be added to the above co-incubation process by mixing, and the added cytokines include, but are not limited to, interleukin, tumor necrosis factor, interferon, growth factor. Preferably, the added cytokines include interleukin 7 (IL-7) and interleukin 15 (IL-15), according to any one of claims 1 to 16.

18. In step (2) above, the selection method is to bind an antibody having fluorescence, magnetism or a specific ligand to a specific cell marker on the surface of T cells, and then use flow cytometry or magnetic beads, etc. to separate the cells expressing the specific cell marker from the cell population, according to any one of claims 1 to 17.

19. The T cells expressing the selected specific cell markers are any one or a combination thereof among CD69, CD137, CD25, CD134, CD80, CD86, OX40L, OX40, CD28, FAS-L, IL-2R, HLA-DR, CD127 (IL-7R), CD150, CD107A, CD83, CD166, CD39, CD178, CD212, CD229, CD100, CD107b, CD108, CD109, CD113, CD122, CD126, CD253, CD197, PD-1, TIM3, LAG-3, TIGIT, CD62L, CD70, CTLA-4 (CD152), CD27, CD26, CD30, TNFRSF9, CD74, PD-L1 (CD274), CD258, CD261, 4-1BB, CD154, ICAM-1, LFA-1, LFA-2, VLA-4, CD160, CD71, CXCR3, TNFRSF14, TNFRSF18, TNFSF4, TNFSF9, TNFSF14, CD11a, CD101, CD48, CD244, CD49a, CD95, CD44, CXCR1, CD103, CD45RO, ICOS (CD278), VTCN1, HLA2, LGAL59, CCR7, CD357, BCL6, TCF-1, CD38, CD27, and the method according to any one of claims 1 to 18, characterized in that it is one or a combination thereof.

20. In step (3) above, the concentration of the cytokine is 1 to 6000 ng / ml, preferably 5 to 200 ng / ml, more preferably 10 to 30 ng / ml, and the method according to any one of claims 1 to 19, characterized in that it is so.

21. In step (3) above, the cytokine includes, but is not limited to, interleukin, interferon, and tumor necrosis factor, and the method according to any one of claims 1 to 20, characterized in that it is so.

22. The interleukin includes, but is not limited to, interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 21 (IL-21), and the method according to any one of claims 1 to 21, characterized in that it is so.

23. The concentration of the above antibody is 1 to 6000 ng / ml, preferably 5 to 100 ng / ml, more preferably 10 to 30 ng / ml, and the method according to any one of claims 1 to 22 is characterized in that.

24. The above antibody contains, but is not limited to, any one or a combination thereof among αCD3 antibody, αCD28 antibody, αCD80 antibody, αCD86 antibody, αOX40 antibody, and the method according to any one of claims 1 to 23 is characterized in that.

25. The co-incubation time of the above nanoparticles / microparticles with the mixture of antigen-presenting cells and T cells is at least 4 hours, preferably 6 to 96 hours, and the method according to any one of claims 1 to 24 is characterized in that.

26. The co-incubation time of the above nanoparticles / microparticles alone with antigen-presenting cells is at least 1 hour, preferably 6 to 96 hours, and the method according to any one of claims 1 to 25 is characterized in that.

27. The co-incubation time of the above activated antigen-presenting cells with the mixture of T cells is at least 1 hour, preferably 6 to 96 hours, and the method according to any one of claims 1 to 26 is characterized in that.

28. The above amplification culture time is at least 1 day, preferably 4 to 72 days, and the method according to any one of claims 1 to 27 is characterized in that.

29. When the above nanoparticles / microparticles alone are co-incubated with antigen-presenting cells, or when the above nanoparticles / microparticles are co-cultured with antigen-presenting cells and T cells, the concentration of the nanoparticles / microparticles is 2.5 ng / mL to 50 mg / mL, and the method according to any one of claims 1 to 28 is characterized in that.

30. The content of the protein and polypeptide components in the antigen component carried by the above nanoparticles / microparticles exceeds 10 ng / mL, and the method according to any one of claims 1 to 29 is characterized in that.

31. In step (3), the specific T cells obtained after in vitro proliferation are re-injected into the body and exert an anti-cancer effect, and the method according to any one of claims 1 to 30 is characterized in that.

32. The cancer antigen carried by the above nanoparticles and / or microparticles is the whole cell component of tumor tissue and / or cancer cells, and contains the water-soluble component and / or water-insoluble component of tumor tissue and / or cancer cells, and the method according to any one of claims 1 to 31 is characterized in that.

33. The method according to any one of claims 1 to 32, wherein the method of carrying one or more tumor tissues and / or components of cancer cells on the nanoparticles and / or microparticles for activating specific T cells is to encapsulate the water-soluble components and water-insoluble components of whole cells into the particles respectively or simultaneously, and / or carry them on the particle surface respectively or simultaneously.

34. The original water-insoluble part in the whole cell components derived from tumor tissues or cancer cells carried on the nanoparticles and / or microparticles for activating cancer-specific T cells is changed from a state insoluble in pure water to a state soluble in an aqueous solution or organic solvent containing a solubilizing agent / dissolving agent by an appropriate solubilization method. The solubilizing agent / dissolving agent used is at least one selected from compounds containing the structure of Structural Formula 1, deoxycholate, lauryl sulfate, glycerol, proteolytic enzymes, albumin, lecithin, polypeptides, amino acids, glycosides, and choline. Structural Formula 1 is as follows, and the structure of Structural Formula 1 is as follows. 【Chemical 1】 R1 is C, N, S, or O, and R2 to R5 are at least one independently selected from hydrogen, alkyl, amino, carboxyl, substituted and unsubstituted guanidino. The compounds containing Structural Formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, polyhexamethylene guanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, guanidine, or other compounds containing urea, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetourea, sulfonylurea compounds (such as glibenclamide, glibencarpid, glazidone, glimepiride, etc.), thiourea compounds (such as thiouracil, imidazole, etc.), nitrosourea-based compounds containing Structural Formula 1, etc. The method according to any one of claims 1 to 33.

35. The method according to any one of claims 1 to 34, characterized in that a target head for actively targeting antigen-presenting cells is connected to the surface of the nanoparticles and / or microparticles for activating cancer-specific T cells.

36. The method in which the water-soluble component and / or water-insoluble component is carried on the surface of the cancer vaccine includes one or more of adsorption, covalent bonding, charge interaction, hydrophobic interaction, solidification, mineralization, and encapsulation in one or more steps, The method according to any one of claims 1 to 35, characterized in that.

37. The method according to any one of claims 1 to 36, characterized in that the particle size of the nanoparticles is 1 nm to 1000 nm, and the particle size of the microparticles is 1 µm to 1000 µm.

38. The method according to any one of claims 1 to 37, characterized in that the surface of the nano-sized particles or micron-sized particles may be electrically neutral, negatively charged, or positively charged.

39. The preparation material of the nano-vaccine and / or micron-vaccine is an organic synthetic polymer material, a natural polymer material, or an inorganic material, The method according to any one of claims 1 to 38, characterized in that.

40. The organic synthetic polymer material is PLGA, PLA, PGA, PEG, PCL, poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, and the natural polymer material is lecithin, cholesterol, sodium alginate, albumin, collagen, gelatin, cell membrane component, starch, sugar, polypeptide, and the inorganic material is ferric oxide, magnetite, calcium carbonate, calcium phosphate, The method according to any one of claims 1 to 39, characterized in that.

41. The nanoparticles and / or microparticles for activating cancer-specific T cells can simultaneously carry one or more tumor tissues and / or components of cancer cells together with an immune adjuvant on the nanoparticles or microparticles, The method according to any one of claims 1 to 40, characterized in that.

42. Both the water-soluble part and the water-insoluble part can be dissolved by a solubilized aqueous solution containing a solubilizing agent or an organic solvent. The solubilizing agent is at least one of the solubilizing agents that can enhance the solubility of a protein or polypeptide in an aqueous solution, and the organic solvent is an organic solvent that can dissolve the protein or polypeptide. The method according to any one of claims 1 to 41, characterized in that.

43. The original water-insoluble part is changed from an insoluble state in pure water to a soluble state in an aqueous solution or an organic solvent containing a solubilizing agent / dissolving agent by an appropriate solubilization method. The solubilizing agent / dissolving agent used is a compound containing the structure of Structural Formula 1, deoxycholate, lauryl sulfate, glycerol, protease, albumin, lecithin, polypeptide, amino acid, glycoside, and choline. At least one selected from among them, the structural formula 1 is as follows, the structure of the structural formula 1 is as follows, [Chemical 2] R1 is C, N, S or O, and R2 to R5 are at least one independently selected from hydrogen, alkyl, amino, carboxyl, substituted and unsubstituted guanidino. Compounds containing Structural Formula 1 include metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salt, metformin, polyhexamethylene guanidine hydrochloride, agmatine sulfate, methylguanidine hydrochloride, tetramethylguanidine hydrochloride, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salt, guanidine or other compounds containing urea, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetourea, sulfonylurea compounds (such as glibenclamide, glibencarpid, glazidone, glimepiride, etc.), thiourea compounds (such as thiouracil, imidazole, etc.), nitrosourea-based compounds containing Structural Formula 1, etc., but are not limited thereto. The method according to any one of claims 1 to 42, characterized in that.

44. The cell components carried by nanoparticles or microparticles for activating cancer-specific T cells are derived from components obtained from all cells of one or more cancer cells and / or one or more tumor tissues. To include more antigens in the nano or micron system, water-insoluble components are carried by delivery particles, and more preferably, water-soluble components and water-insoluble components are simultaneously carried by delivery particles. Therefore, the delivery particles carry whole cell component antigens. The method according to any one of claims 1 to 43, characterized in that.

45. In cell components or mixtures thereof carried by nanoparticles and / or microparticles for activating cancer-specific T cells, the mixture includes, but is not limited to, those in which water-soluble components are mixed with each other, those in which water-insoluble components are mixed with each other, or those in which all or part of the water-soluble components are mixed with all or part of the water-soluble components. The method according to any one of claims 1 to 44, characterized in that.

46. In nanoparticles and / or microparticles, cell components or mixtures thereof are carried inside and / or on the surface of the nanoparticles or microparticles. Specifically, the above-mentioned loading method is to encapsulate the water-soluble and water-insoluble components of the cells inside the particles respectively or simultaneously, and / or carry them on the particle surface respectively or simultaneously. It includes encapsulating the water-soluble components inside the particles and carrying them on the particle surface simultaneously, encapsulating the water-insoluble components inside the particles and carrying them on the particle surface simultaneously, encapsulating the water-soluble components inside the particles and carrying the water-insoluble components on the particle surface, encapsulating the water-insoluble components inside the particles and carrying the water-soluble components on the particle surface, encapsulating the water-soluble and water-insoluble components inside the particles but only carrying the water-insoluble components on the particle surface, encapsulating the water-soluble and water-insoluble components inside the particles but only carrying the water-soluble components on the particle surface, encapsulating the water-soluble components inside the particles but carrying the water-soluble and water-insoluble components on the particle surface simultaneously, encapsulating the water-insoluble components inside the particles but carrying the water-soluble and water-insoluble components on the particle surface simultaneously, and encapsulating the water-soluble and water-insoluble components inside the particles simultaneously and carrying the water-soluble and water-insoluble components on the particle surface simultaneously. However, it is characterized in that it is not limited to these. The method according to any one of claims 1 to 45, characterized in that.

47. The interior and / or surface of the nanoparticles or microparticles for activating cancer-specific T cells may further contain an immune-enhancing adjuvant, and the immune-enhancing adjuvant includes, but is not limited to, at least one of immune enhancers derived from microorganisms, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicines, and other types. The immune-enhancing adjuvant includes, but is not limited to, pattern recognition receptor agonists, live tuberculosis vaccine (BCG) for tuberculosis prevention, manganese-related adjuvants, cell wall skeletons of live tuberculosis vaccine for tuberculosis prevention, methanol extraction residues of live tuberculosis vaccine for tuberculosis prevention, muramyl dipeptide of live tuberculosis vaccine for tuberculosis prevention, Corynebacterium parvum, polyactin A, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virions, cholera enterotoxin, saponin and its derivatives, resiquimod, thymosin, newborn calf liver active peptide, imiquimod, polysaccharides, curcumin, immune adjuvant CpG, poly(I:C) of immune adjuvant, polyICLC of immune adjuvant, Corynebacterium parvum vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvant, aluminum adjuvant, calcium adjuvant, various cytokines, interleukins, interferons, 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, ginseng, and at least one of the active ingredients of lotus. Those skilled in the art can understand that the enumeration herein is not exhaustive, and other substances that can enhance the immune response can also be used as immune-enhancing adjuvants. A method according to any one of claims 1 to 46, characterized in that.

48. The method according to any one of claims 1 to 47, characterized in that the immune-enhancing adjuvant is preferably a Toll-like receptor agonist.

49. The method according to any one of claims 1 to 48, characterized in that the immune-enhancing adjuvant is preferably a combination of two or more Toll-like receptor agonists.

50. The method according to any one of claims 1 to 49, characterized in that when an immune adjuvant and cell components are co-carried on nanoparticles or microparticles, after the nanoparticles or microparticles are engulfed by antigen-presenting cells, cancer-specific T cells can be more effectively activated.

51. The microparticles or nanoparticles carry a substance that increases the escape of the nanoparticles and / or microparticles or the antigens carried thereon from lysosomes to the cytoplasm. The substance that increases the escape from lysosomes includes amino acids, polypeptides, sugars, lipids, and inorganic salts capable of producing a proton sponge effect. Preferably, the amino acids in the substance that increases the escape from lysosomes include positively charged amino acids. Preferably, the polypeptide that increases the escape from lysosomes includes positively charged amino acids. The method according to any one of claims 1 to 50.

52. The surface of the nanoparticles or microparticles may or may not be connected to a target head having an active targeting function. The target head of active targeting can be a normal target head such as mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, etc. The method according to any one of claims 1 to 51.

53. The surface of the above-mentioned nanoparticles and / or microparticles is characterized in that it is connected to a target that actively targets antigen-presenting cells. The method according to any one of claims 1 to 52.

54. The nanoparticles or microparticles may not be modified during the preparation process, or appropriate modification techniques may be used to increase the antigen-carrying capacity of the nanoparticles or microparticles. The modification techniques include, but are not limited to, biomineralization (e.g., silicification, calcification, magnetization), gelation, crosslinking, chemical modification, addition of charged substances, etc. The method according to any one of claims 1 to 53.

55. The form of carrying cell components or their mixtures inside nanoparticles or microparticles is any method capable of carrying cell components or their mixtures inside nanoparticles or microparticles. The method according to any one of claims 1 to 54.

56. The method of supporting a cell component or a mixture thereof on the surface of nanoparticles or microparticles includes, but is not limited to, adsorption, covalent bonding, charge interaction (e.g., addition of a positively charged substance, addition of a negatively charged substance), hydrophobic interaction, solidification in one or more steps, mineralization, encapsulation, etc., and is characterized by the method according to any one of claims 1 to 55.

57. The water-soluble component and / or water-insoluble component supported on the surface of the nanoparticles or microparticles forms one or more layers after being supported. When a plurality of layers of water-soluble components and / or water-insoluble components are supported on the surface of the vaccine, there are modifiers between the layers, and is characterized by the method according to any one of claims 1 to 56.

58. The size of the particle diameter of the above-mentioned nanoparticles is 1 nm to 1000 nm, more preferably, the size of the particle diameter is 30 nm to 1000 nm, and most preferably, the size of the particle diameter is 100 nm to 600 nm, and is characterized by the method according to any one of claims 1 to 57.

59. The size of the particle diameter of the microparticles is 1 μm to 1000 μm, more preferably, the size of the particle diameter is 1 μm to 10 μm, and most preferably, the size of the particle diameter is 1 μm to 5 μm, and is characterized by the method according to any one of claims 1 to 58.

60. The shape of the above-mentioned nanoparticles or microparticles includes any one of spherical, elliptical, barrel-shaped, polygonal, rod-shaped, sheet-shaped, linear, worm-shaped, square, triangular, butterfly-shaped or disc-shaped, and is characterized by the method according to any one of claims 1 to 59.

61. The method by which the above-mentioned water-soluble component and / or water-insoluble component is supported on the surface of the cancer vaccine includes one or more of adsorption, covalent bonding, charge interaction, hydrophobic interaction, solidification in one or more steps, mineralization and encapsulation, and is characterized by the method according to any one of claims 1 to 60.

62. The preparation materials of the nano-vaccine and / or micron-vaccine are organic synthetic polymer materials, natural polymer materials or inorganic materials, and is characterized by the method according to any one of claims 1 to 61.

63. The above-mentioned organic synthetic polymer material is a biocompatible or degradable polymer material, which includes any one or a combination thereof among PLGA, PLA, PGA, PLGA-PEG, PLA-PEG, PGA-PEG, PEG, PCL, poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, and synthetic lipid. The method according to any one of claims 1 to 62 is characterized by this.

64. The above-mentioned natural polymer material is a biocompatible or degradable polymer material, which includes any one or a combination thereof among lecithin, cholesterol, sodium alginate, albumin, collagen, gelatin, cell membrane components, starch, sugar, and polypeptide. The method according to any one of claims 1 to 63 is characterized by this.

65. The above-mentioned inorganic material is a non-explicitly biotoxic material, which includes, but is not limited to, ferric oxide, magnetite, calcium carbonate, calcium phosphate, etc. The method according to any one of claims 1 to 64 is characterized by this.

66. Specifically, Step of separating mononuclear cells PBMC from immune cells in peripheral blood or peripheral immune organs, preferably, the PBMC is, by first selection, CD3 ＋ CD8 ＋ T cells, CD19 ＋ B cells, CD3 ＋ T cells, CD8+ T cells, CD4 ＋ T cells, B220 ＋ B cells, CD69 － PBMC cells of, CD25 － PBMC cells of, CD3 ＋ CD69 ＋ T cells, CD11c ＋ Step of obtaining at least one of effector cells such as DC cells (2) A step of preparing nanoparticles and / or microparticles carrying a tumor antigen component, Preferably, in the preparation of the tumor antigen component, first, tumor cells or tissues are separated, and the tumor cells or tissues are lysed to obtain any one or a combination thereof among water-soluble components, water-insoluble components, and total components. Preferably, a step of preparing nanoparticles and / or microparticles carrying a tumor antigen component by the double emulsion method, (3) Adding nanoparticles and / or microparticles carrying tumor antigen components to a medium, co-incubating with the PBMC cells or T cells described in (1) to obtain a cell culture, and further selecting specific T cells from the cell culture, wherein the specific T cells are CD3 ＋ CD69 ＋ 、CD3 ＋ CD8 ＋ CD69 ＋ 、CD3 ＋ CD4 ＋ CD69 +, CD3 ＋ CD137 ＋ 、CD3 ＋ CD4 ＋ CD137 ＋ 、CD3 ＋ CD8 ＋ CD137 ＋ 、CD3 ＋ CD25 ＋ 、CD3 ＋ CD8 ＋ CD25 ＋ 、CD3 ＋ CD4 ＋ CD25 ＋ 、CD3 ＋ CD134 ＋ 、CD3 ＋ CD8 ＋ CD134 ＋ 、CD3 ＋ CD4 ＋ CD134 ＋ 、CD3 ＋ IL-2R ＋ 、CD3 ＋ CD8 ＋ IL-2R ＋ 、CD3 ＋ CD4 ＋ IL-2R ＋ 、CD3 ＋ HLA-DR ＋ 、CD3 ＋ CD8 ＋ HLA-DR ＋ 、CD3 ＋ CD4 ＋ HLA-DR ＋ 、CD3 ＋ FASL ＋ CD3 ＋ CD8 ＋ FASL ＋ , CD3 ＋ CD4 ＋ FASL ＋ , CD3 ＋ OX40 ＋ , CD3 ＋ CD8 ＋ OX40 ＋ , CD3 ＋ CD4 ＋ OX40 ＋ , CD3 ＋ TCF-1 ＋ , CD3 ＋ CD8 ＋ TCF-1 ＋ , CD3 ＋ CD4 ＋ TCF-1 ＋ , CD3 ＋ PD-1 ＋ , CD3 ＋ CD8 ＋ PD-1 ＋ , CD3 ＋ CD4 ＋ PD-1 ＋ , CD3 ＋ CD39 ＋ , CD3 ＋ CD8 ＋ CD39 ＋ , CD3 ＋ CD4 ＋ CD39 ＋ , CD3 ＋ CD38 ＋ , CD3 ＋ CD8 ＋ CD38 ＋ , CD3 ＋ CD4 ＋ CD38 ＋ , CD3 ＋ CD28 ＋ , CD3 ＋ CD8 ＋ CD28 ＋ , CD3 ＋ CD4 ＋ CD28 ＋ , CD3 ＋ CD71 ＋ , CD3 ＋ CD8 ＋ CD71 ＋ , CD3 ＋ CD4 ＋ CD71 ＋ , CD3 ＋ CD44 ＋ , CD3 ＋ CD8 ＋ CD44 ＋ , CD3 ＋ CD4 ＋ CD44 ＋ , CD3 ＋ CXCR3 ＋ , CD3 ＋ CD8 ＋ CXCR3 ＋ , CD3 ＋ CD4 ＋ CXCR3 ＋ , CD3 ＋ CXCR1 ＋ , CD3 ＋ CD8 ＋ CXCR1 ＋ , CD3 ＋ CD4 ＋ CXCR1 ＋ , CD3 ＋ ICAM-1 ＋ , CD3 ＋ CD8 ＋ ICAM-1 ＋ , CD3 ＋ CD4 ＋ ICAM-1 ＋ , CD3 ＋ CD70 ＋ , CD3 ＋ CD8 ＋ CD70 ＋ , CD3 ＋ CD4 ＋ CD70 ＋ , CD3 ＋ CD154 ＋ , CD3 ＋ CD8 ＋ CD154 ＋ , CD3 ＋ CD4 ＋ CD154 ＋ , CD3 ＋ CD62L ＋ , CD3 ＋ CD8 ＋ CD62L ＋ , CD3 ＋ CD4 ＋ CD62L ＋ , CD3 ＋ CD154 ＋ , CD3 ＋ CD8 ＋ CD154 ＋ , CD3 ＋ CD4 ＋ CD154 ＋ , CD3 ＋ CD160 ＋ , CD3 ＋ CD8 ＋ CD160 ＋ , CD3 ＋ CD4 ＋ CD160 ＋ , CD3 ＋ CD160 ＋ , CD3 ＋ CD8 ＋ CD160 ＋ , CD3 ＋ CD4 ＋ CD160 ＋ , CD3 ＋ ICOS ＋ , CD3 ＋ CD8 ＋ ICOS ＋ , CD3 ＋ CD4 ＋ ICOS ＋ , CD3 ＋ CD27 ＋ , CD3 ＋ CD8 ＋ CD27 ＋ , CD3 ＋ CD4 ＋ CD27 ＋ , CD3 ＋ CD107A ＋ , CD3 ＋ CD8 ＋ CD107A ＋ , CD3 ＋ CD4 ＋ CD107A ＋ including, but not limited to, any one or a combination of these specific T cells such as Preferably, in the co-incubation process, antigen-presenting cells at a concentration of 5 to 50 million / ml are also added, and the antigen-presenting cells are any one or a combination thereof among B cells, DC cells, and macrophages. Preferably, the nanoparticles and / or microparticles carrying the tumor antigen component can be co-incubated with antigen-presenting cells and T cells simultaneously to activate cancer cell-specific T cells. First, the nanoparticles and / or microparticles carrying the tumor antigen component can be co-incubated with antigen-presenting cells to activate the antigen-presenting cells. Next, the activated antigen-presenting cells alone can be co-incubated with T cells to activate cancer cell-specific T cells. After the nanoparticles and / or microparticles carrying the tumor antigen component are co-incubated with antigen-presenting cells, the antigen-presenting cells are activated, and the antigen-presenting cells that do not require special treatment can be co-incubated with T cells to activate specific T cells. Alternatively, after the antigen-presenting cells are treated with fixation, radiation, irradiation, modification, inactivation, mineralization, etc., the antigen-presenting cells can be co-incubated with T cells to activate specific T cells. Preferably, nanoparticles and / or microparticles carrying a tumor antigen component at 2.5 ng to 50 mg / ml are added to a medium, and 1 to 50 million / ml of PBMC cells or sorted cells are co-incubated at 30 to 38 °C and 1 to 5% CO ２ under the conditions of 2 for 4 to 96 hours to obtain a cell culture, Preferably, during the co-incubation process, 10 - 500 ng / ml of interleukin is also added, and the interleukin is any one or a combination of IL-2, IL-7, IL-12, IL-15, IL-17, IL-21. The medium is any one of DMEM high-glucose complete medium, RPM1640 medium, and AIMV serum-free medium. The co-incubation is carried out by incubating for 1 - 168 hours under the condition of 30 - 38°C, preferably 4 - 96 hours, more preferably 6 - 72 hours. The survival rate of the specific T cells exceeds 60%, preferably exceeds 70%, and more preferably exceeds 80%. (4) A step of amplifying the specific T cells obtained in (3), characterized by comprising a method for preparing cancer-specific T cells derived from autologous cells or allogeneic cells for preventing or treating cancer according to claim 1.

67. The amplification step is as follows: Add the specific T cells selected in step (2) at 1 to 50 million cells / mL to the amplification medium, and co-incubate under the conditions of 30 to 38 °C and 1 to 5% CO ２ 2, and replace the amplification medium every 2 to 3 days. After co-incubating for 5 to 30 days, the amplified specific T cells are obtained. The preparation method according to claim 66, characterized in that.

68. Preferably, amplification is carried out in an amplification medium, and the amplification medium is any one of DMEM high-glucose complete medium and RPM1640 medium. Preferably, the conditions for amplification culture are culturing at 30 - 38°C for 4 - 72 days. Preferably, the amplification medium further contains 200 - 1000 U / ml of interleukin, 10 - 200 ng / ml of antibody, and / or 1 - 10 ng / mL of granulocyte-macrophage colony-stimulating factor (GM-CSF). Preferably, the interleukin is any one or a combination thereof selected from IL-2, IL-7, IL-12, IL-15, IL-17, IL-21, Preferably, the antibody is any one or a combination thereof selected from an αCD3 antibody and an αCD28 antibody, and the method according to any one of claims 66 to 67 is characterized in that.

69. In the above step (1), the cell is selected from any part of an individual suffering from a tumor disease, preferably spleen cells and lymphocytes, and the method according to any one of claims 66 to 68 is characterized in that.

70. The survival rate of the amplified specific T cells exceeds 60%, preferably exceeds 75%, and more preferably exceeds 80%, and the method according to any one of claims 66 to 69 is characterized in that.

71. In the above step (2), the selection method binds an antibody having fluorescence, magnetism or a specific ligand to a specific cell marker on the surface of T cells, and then uses flow cytometry or magnetic beads, etc. to separate cells expressing the specific cell marker from the cell population, and the method according to any one of claims 66 to 70 is characterized in that.

72. Cytokines can be added to the above mixed co-incubation process, and the added cytokines include, but are not limited to, interleukin, tumor necrosis factor, interferon, growth factor. Preferably, the added cytokines include interleukin 7 (IL-7) and interleukin 15 (IL-15), and the method according to any one of claims 66 to 71 is characterized in that.

73. In step (3), the concentration of the cytokine is 1 to 6000 ng / ml, preferably 5 to 100 ng / ml, and more preferably 10 to 30 ng / ml, and the method according to any one of claims 66 to 72 is characterized in that.

74. In the above step (3), the cytokine includes, but is not limited to, interleukin, interferon, tumor necrosis factor, and the method according to any one of claims 66 to 73 is characterized in that.

75. The interleukin includes, but is not limited to, interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 17 (IL-17), and interleukin 21 (IL-21). The method according to any one of claims 66 to 74, characterized in that

76. The concentration of the antibody is 1 to 6000 ng / ml, preferably 5 to 100 ng / ml, more preferably 10 to 30 ng / ml. The method according to any one of claims 66 to 75, characterized in that

77. The antibody includes, but is not limited to, any one or a combination of αCD3 antibody, αCD28 antibody, αCD80 antibody, αCD86 antibody, and αOX40 antibody. The method according to any one of claims 66 to 76, characterized in that

78. The co-incubation time of the above-mentioned nanoparticles / microparticles with the mixture of antigen-presenting cells and T cells is at least 1 hour, preferably 6 to 96 hours. The method according to any one of claims 66 to 77, characterized in that

79. The co-incubation time of the above-mentioned nanoparticles / microparticles alone with antigen-presenting cells is at least 1 hour, preferably 6 to 96 hours. The method according to any one of claims 66 to 78, characterized in that

80. The co-incubation time of the above-mentioned activated antigen-presenting cells with the mixture of T cells is at least 1 hour, preferably 6 to 96 hours. The method according to any one of claims 66 to 79, characterized in that

81. The amplification culture time is at least 1 day, preferably 4 to 36 days. The method according to any one of claims 66 to 80, characterized in that

82. When the above-mentioned nanoparticles / microparticles are co-incubated with antigen-presenting cells alone, or when the above-mentioned nanoparticles / microparticles are co-incubated with antigen-presenting cells and T cells, the concentration of the nanoparticles / microparticles is 2.5 ng / mL to 50 mg / mL. The method according to any one of claims 66 to 81, characterized in that

83. The content of the protein and polypeptide components in the antigen component carried by the above-mentioned nanoparticles / microparticles exceeds 10 ng / mL. The method according to any one of claims 66 to 82, characterized in that

84. When the antigen component is the whole cell lysate component, the preparation method is as follows: (1) First, cancer cells / tumor tissues are lysed. Next, a water-soluble component and a water-insoluble component are prepared respectively. Then, the water-insoluble component is dissolved with a specific solvent containing a lysing solution and then used. (2) A lysing solution containing a solvent is used to lyse cells. Next, the whole cell components lysed with the lysing solution containing a solvent are lysed. The method according to any one of claims 66 to 83, characterized in that.

85. When the tumor antigen component is a part of the whole cell component, the preparation method is as follows: (1) First, a cell lysate of tumor tissue / cancer cells is prepared. Next, a water-soluble component and a water-insoluble component are prepared respectively. Then, the water-insoluble component is dissolved with a specific solvent containing a lysing solution and then used. Subsequently, the protein component and polypeptide component in the water-soluble component are separated and extracted from the water-soluble component by an appropriate method. Then, the proteins and polypeptide components separated and extracted from the water-soluble component are used as antigen components together with all the water-insoluble components. (2) First, a cell lysate of tumor tissue / cancer cells is prepared. Next, a water-soluble component and a water-insoluble component are prepared respectively. Then, the water-insoluble component is dissolved with a specific solvent containing a lysing solution and then used. Subsequently, the protein component and polypeptide component in the water-soluble component are separated and extracted from the water-insoluble component by an appropriate method. Then, the proteins and polypeptide components separated and extracted from the water-insoluble component are used as antigen components together with all the water-soluble components. (3) First, a cell lysate of tumor tissue / cancer cells is prepared. Next, a water-soluble component and a water-insoluble component are prepared respectively. Then, the water-insoluble component is dissolved with a specific solvent containing a lysing solution and then used. Subsequently, the protein component and polypeptide component in the water-soluble component are separated and extracted from the water-soluble component and the water-insoluble component respectively by an appropriate method. Then, the proteins and polypeptide components separated and extracted from the water-soluble component and the water-insoluble component are used as antigen components. (4) Or a lysing solution containing a solvent is directly used to directly lyse cells or tissues, and the whole cell tissue is lysed. Next, the protein component and polypeptide component therein are prepared by an appropriate method and can be used as antigen components. Preferably, the appropriate treatment method includes, but is not limited to, treatment methods such as salting out, heating, and enzymatic hydrolysis. The water-insoluble component or the precipitate generated by treatment such as salting out, heating, and enzymatic hydrolysis is dissolved using a lysate containing a solubilizing agent. Preferably, in the above preparation method, it is also possible to add a step of separating and extracting total cell mRNA and using the total cell mRNA as part of the antigen component. The method according to any one of claims 66 to 84.

86. In step (2), in the preparation of the tumor antigen component, first, tumor cells or tissues are separated, and the tumor cells or tissues are lysed to obtain any one or a combination of a water-soluble component, a water-insoluble component, and a total component. The method according to any one of claims 66 to 85.

87. In step (2), the tumor antigen component is appropriately treated by methods such as salting out, heating, and enzymatic hydrolysis. The method according to any one of claims 66 to 86.

88. Both the water-soluble part and the water-insoluble part can be dissolved by a solubilized aqueous solution or an organic solvent containing a solubilizing agent. The method according to any one of claims 66 to 87.

89. The solubilizing agent is at least one of the solubilizing agents that can enhance the solubility of proteins or polypeptides in an aqueous solution, and the organic solvent is an organic solvent that dissolves proteins or polypeptides. The method according to any one of claims 66 to 88.

90. The water-insoluble part changes from an insoluble state in pure water to a soluble state in an aqueous solution or an organic solvent containing a solubilizing agent by an appropriate solubilization method. The solubilizing agent used is at least one selected from compounds containing the structure of Structural Formula 1, deoxycholate, lauryl sulfate, glycerol, proteolytic enzymes, albumin, lecithin, polypeptides, amino acids, glycosides, and choline. Structural Formula 1 is as follows. The structure of Structural Formula 1 is as follows. 【Chemical Formula 3】 R1 is C, N, S, or O, and R2 to R5 are each independently selected from at least one of hydrogen, alkyl, amino, carboxyl, substituted and unsubstituted guanidino. The compounds containing the structure of Structural Formula 1 include metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, other compounds containing guanidine, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoylurea, acetourea, sulfonylurea compounds (such as glyburide, glibencramide, glipizide, glimepiride), thiourea compounds (such as thiouracil, imidazole), nitrosoureas, etc., but are not limited thereto. The method according to any one of claims 66 to 89.

91. When an immunoadjuvant and cell components are co-loaded on nanoparticles or microparticles, after the nanoparticles or microparticles are engulfed by antigen-presenting cells, cancer-specific T cells can be more effectively activated. The method according to any one of claims 66 to 90.

92. The preparation method of the above water-soluble component is as follows. The tumor tissue or cancer cells are finely cut, ground, filtered to obtain a single-cell suspension, water is added, and freezing and thawing are repeated 1 to 5 times, dissolved by ultrasonic waves, and the dissolved product is centrifuged at a rotation speed of 5000 to 10000 g for 5 to 10 minutes. Then, the supernatant is used as the water-soluble component, and the precipitate part is used as the water-insoluble component. The method according to any one of claims 66 to 91.

93. The above antigen component is a soluble component obtained by adding a solubilizer to the water-insoluble component. The above solubilizer is at least one selected from compounds containing the structure of Structural Formula 1, deoxycholate, lauryl sulfate, glycerol, proteolytic enzymes, albumin, lecithin, polypeptides, amino acids, glycosides, and choline. Structural Formula 1 is as follows, the structure of Structural Formula 1 is as follows, [Chemical Formula 4] R1 is C, N, S or O, and R2 to R5 are each independently selected from at least one of hydrogen, alkyl, amino, carboxyl, substituted and unsubstituted guanidino. The compounds containing the structure of Structural Formula 1 include metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, other compounds containing guanidine, guanidine carbonate, arginine, guanidinoacetic acid, guanidinophosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoylurea, acetourea, sulfonylurea compounds (such as glibencramide, glicladide, glazidone, glimepiride, etc.), thiourea compounds (such as thiouracil, imidazole, etc.), nitrosourea systems, etc., but are not limited thereto. The method according to any one of claims 66 to 92.

94. The method according to any one of claims 66 to 93, characterized in that inactivation and denaturation treatment by ultraviolet high-temperature heating and nuclease inactivation are performed in advance on tumor tissue or cancer cells, either arbitrarily or in combination.

95. The method according to any one of claims 66 to 94, characterized in that the antigen component is a component obtained by salting out and heat-precipitating a water-soluble component.

96. The antigen component is a soluble component obtained by adding a solubilizing agent to a precipitate obtained by adding a lysing agent to the precipitate part after lysing tumor tissue and centrifuging, that is, a water-insoluble component, and dissolving it twice. The solubilizing agent is any one or a combination of Tween 80 and guanidine sulfate. The method according to any one of claims 66 to 95.

97. Specific T cells obtained by the method for preparing cancer-specific T cells derived from autologous cells or allogeneic cells for preventing or treating cancer according to any one of claims 1 to 96.

98. Cancer-specific T cells derived from autologous cells or allogeneic cells for preventing or treating cancer, wherein said specific T cells are CD3 ＋ CD69 ＋ 、CD3 ＋ CD8 ＋ CD69 ＋ 、CD3 ＋ CD4 ＋ CD69 +, CD3 ＋ CD137 ＋ 、CD3 ＋ CD4 ＋ CD137 ＋ 、CD3 ＋ CD8 ＋ CD137 ＋ 、CD3 ＋ CD25 ＋ 、CD3 ＋ CD8 ＋ CD25 ＋ 、CD3 ＋ CD4 ＋ CD25 ＋ 、CD3 ＋ CD134 ＋ 、CD3 ＋ CD8 ＋ CD134 ＋ 、CD3 ＋ CD4 ＋ CD134 ＋ 、CD3 ＋ IL-2R ＋ 、CD3 ＋ CD8 ＋ IL-2R ＋ 、CD3 ＋ CD4 ＋ IL-2R ＋ 、CD3 ＋ HLA-DR ＋ 、CD3 ＋ CD8 ＋ HLA-DR ＋ 、CD3 ＋ CD4 ＋ HLA-DR ＋ 、CD3 ＋ FASL ＋ CD3 ＋ CD8 ＋ FASL ＋ 、CD3 ＋ CD4 ＋ FASL ＋ 、CD3 ＋ OX40 ＋ , CD3 ＋ CD8 ＋ OX40 ＋ , CD3 ＋ CD4 ＋ OX40 ＋ , CD3 ＋ TCF-1 ＋ , CD3 ＋ CD8 ＋ TCF-1 ＋ , CD3 ＋ CD4 ＋ TCF-1 ＋ , CD3 ＋ PD-1 ＋ , CD3 ＋ CD8 ＋ PD-1 ＋ , CD3 ＋ CD4 ＋ PD-1 ＋ , CD3 ＋ CD39 ＋ , CD3 ＋ CD8 ＋ CD39 ＋ , CD3 ＋ CD4 ＋ CD39 ＋ , CD3 ＋ CD38 ＋ , CD3 ＋ CD8 ＋ CD38 ＋ , CD3 ＋ CD4 ＋ CD38 ＋ , CD3 ＋ CD28 ＋ , CD3 ＋ CD8 ＋ CD28 ＋ , CD3 ＋ CD4 ＋ CD28 ＋ , CD3 ＋ CD71 ＋ , CD3 ＋ CD8 ＋ CD71 ＋ , CD3 ＋ CD4 ＋ CD71 ＋ , CD3 ＋ CD44 ＋ , CD3 ＋ CD8 ＋ CD44 ＋ 、CD3 ＋ CD4 ＋ CD44 ＋ 、CD3 ＋ CXCR3 ＋ 、CD3 ＋ CD8 ＋ CXCR3 ＋ 、CD3 ＋ CD4 ＋ CXCR3 ＋ 、CD3 ＋ CXCR1 ＋ 、CD3 ＋ CD8 ＋ CXCR1 ＋ 、CD3 ＋ CD4 ＋ CXCR1 ＋ 、CD3 ＋ ICAM-1 ＋ 、CD3 ＋ CD8 ＋ ICAM-1 ＋ 、CD3 ＋ CD4 ＋ ICAM-1 ＋ 、CD3 ＋ CD70 ＋ 、CD3 ＋ CD8 ＋ CD70 ＋ 、CD3 ＋ CD4 ＋ CD70 ＋ 、CD3 ＋ CD154 ＋ 、CD3 ＋ CD8 ＋ CD154 ＋ 、CD3 ＋ CD4 ＋ CD154 ＋ 、CD3 ＋ CD62L ＋ 、CD3 ＋ CD8 ＋ CD62L ＋ 、CD3 ＋ CD4 ＋ CD62L ＋ 、CD3 ＋ CD154 ＋ 、CD3 ＋ CD8 ＋ CD154 ＋ 、CD3 ＋ CD4 ＋ CD154 ＋ , CD3 ＋ CD160 ＋ , CD3 ＋ CD8 ＋ CD160 ＋ , CD3 ＋ CD4 ＋ CD160 ＋ , CD3 ＋ CD160 ＋ , CD3 ＋ CD8 ＋ CD160 ＋ , CD3 ＋ CD4 ＋ CD160 ＋ , CD3 ＋ ICOS ＋ , CD3 ＋ CD8 ＋ ICOS ＋ , CD3 ＋ CD4 ＋ ICOS ＋ , CD3 ＋ CD27 ＋ , CD3 ＋ CD8 ＋ CD27 ＋ , CD3 ＋ CD4 ＋ CD27 ＋ , CD3 ＋ CD107A ＋ , CD3 ＋ CD8 ＋ CD107A ＋ , CD3 ＋ CD4 ＋ CD107A ＋ including, but not limited to, any one or a combination thereof of the T cells, wherein the specific T cell survival rate exceeds 60%, preferably exceeds 70%, more preferably exceeds 80% Preferably, the specific T cells are obtained by co-incubating nanoparticles and / or microparticles carrying a tumor antigen component with mononuclear cells (PBMC) separated from immune cells in peripheral blood or peripheral immune organs. Cancer-specific T cells derived from autologous cells or allogeneic cells for preventing or treating cancer.

99. After sorting, the above PBMC cells are co-incubated with nanoparticles and / or microparticles carrying a tumor antigen component. Preferably, the cells co-incubated with the nanoparticles and / or microparticles carrying the tumor antigen component are sorted again to obtain specific T cells. Preferably, the above specific T cells can also undergo an in vitro amplification step, which can be performed before or after the aforementioned sorting step, preferably before the sorting step. Preferably, when the above PBMC cells are co-incubated with the nano / microparticles, antigen-presenting cells (APCs) also exist. Preferably, the specific T cells according to claim 98, characterized in that various types of T cells can be used alone or in combination according to the needs of the patient.

100. A pharmaceutical composition containing the specific T cells according to any one of claims 97 to 99, and its preparation method includes a step of adding natural immune system enhancing substances such as albumin, NK cells, neutrophils, γδ T cells, and NK T cells to the cancer-specific T cells before re-injecting the cancer-specific T cells into the patient. Preferably, the cell concentration of the specific T cells is (0.01 to 100) × 10 ７ cells / ml, preferably (0.1 to 8) × 10 ７ cells / ml, and Preferably, the pharmaceutical composition further includes any one or a combination of hydroxyethyl starch, sugar, and salt.

101. A pharmaceutical composition containing the specific T cells according to claim 100, and its preparation method includes a step of adding natural immune system enhancing substances such as albumin, NK cells, neutrophils, γδ T cells, and NK T cells to the cancer-specific T cells before re-injecting the cancer-specific T cells into the patient. Preferably, the cell concentration of the specific T cells is (0.01 to 100) × 10 ７ cells / ml, preferably (0.1 to 8) × 10 ７ cells / ml, and Preferably, the pharmaceutical composition further includes any one or a combination of hydroxyethyl starch, sugar, and salt.

102. Application of the specific T cells according to any one of claims 97 to 99 in the preparation of a drug for treating or preventing cancer.

103. The application according to claim 102, characterized in that the above specific T cells are administered multiple times before the occurrence of cancer, after the occurrence of cancer, or after surgical removal of the tumor tissue.

104. Application of the specific T cells according to any one of claims 97 to 99 in the preparation of a drug for preventing cancer recurrence or metastasis.

105. Application of the specific T cells according to any one of claims 97 to 99 in the preparation of an anti-tumor immunotherapy product.

106. The above-mentioned tumors are selected from solid tumors, hematological tumors and lymphomas, including any one or a combination of lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, hematological malignancies such as lymphoma, leukemia, brain tumors, head and neck cancers, gliomas, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumors, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancer, metastatic cancer, circulating tumor cells, but not limited to these. Preferably, the above-mentioned tumor is selected from any one or a combination of melanoma, colon cancer, triple-negative breast cancer, pancreatic cancer, metastatic cancer, liver cancer, colon cancer, lymphoma, esophageal cancer, non-small cell lung cancer, and the application according to claim 105 is characterized in that.

107. The above-mentioned immunotherapy is selected from any one or a combination of immunotherapy in anti-tumor treatment and immunotherapy after radical surgery. Preferably, the above-mentioned immunotherapy is selected from immunotherapy after radical surgery for primary hepatocellular carcinoma, and the application according to claim 105 is characterized in that.

108. The above-mentioned drug is used for adult or pediatric patients, and the application according to any one of claims 102 to 107 is characterized in that.

109. It is the application of the specific T cells described in any one of claims 97 to 99 in immunotherapy.

110. The administration method of the above-mentioned immunotherapy is characterized in that it is one or a combination of intravenous injection, subcutaneous injection, intratumoral injection, intraperitoneal injection, intramuscular injection, intradermal injection, and the application according to claim 109.

111. It is the application of the specific T cells described in any one of claims 97 to 99 in a combination with any one of radiotherapy, chemotherapy, targeted therapy, surgical treatment or immunotherapy for anti-tumor or tumor immunotherapy.

112. It is the application of the specific T cells according to any one of claims 97 to 99 in the preparation of a drug for enhancing antiviral ability.

113. It is the application of the specific T cells according to any one of claims 97 to 99 in the preparation of a drug for enhancing the treatment of autoimmune diseases.

114. The autoimmune disease is characterized by being selected from any one of systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, skin diseases, or a complication thereof, and the application according to claim 113.

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