WO2023227085A1 - 用于预防或治疗癌症的特异性t细胞及其制备方法 - Google Patents

用于预防或治疗癌症的特异性t细胞及其制备方法 Download PDF

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WO2023227085A1
WO2023227085A1 PCT/CN2023/096419 CN2023096419W WO2023227085A1 WO 2023227085 A1 WO2023227085 A1 WO 2023227085A1 CN 2023096419 W CN2023096419 W CN 2023096419W WO 2023227085 A1 WO2023227085 A1 WO 2023227085A1
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cells
cancer
water
components
nanoparticles
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French (fr)
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刘密
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苏州尔生生物医药有限公司
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    • CCHEMISTRY; METALLURGY
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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0634Cells from the blood or the immune system
    • C12N5/0636T lymphocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14Blood; Artificial blood
    • A61K35/17Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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    • C12N13/00Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2509/00Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present invention relates to the technical field of immunotherapy, and in particular to a specific T cell for preventing or treating cancer, its preparation method and its application.
  • Immune cells refer to cells that participate in or are related to immune responses, including innate lymphocytes, various phagocytes, and lymphocytes that can recognize antigens and produce specific immune responses, such as T cells and B cells. , NK cells, DC cells, macrophages, granulocytes, mast cells, etc.
  • Autoimmune cell therapy is used to fight tumors by isolating and culturing immune cells from one's own blood. By supplementing highly active immune cells, the number of immune cells in the body is increased, and the original immune cells in the body are activated, thereby greatly improving the ability of immune cells to kill tumor cells, bacteria, viruses, etc., to achieve the effect of preventing and fighting cancer. Purpose.
  • a detection method which can detect T cells activated by nanoparticles loaded with patient tumor components.
  • this application is a detection method rather than a treatment method. It does not sort and amplify activated T cells, nor does it involve a reinfusion step. During the detection process, T cells need to be fixed and stained, and the corresponding active T cells cannot be provided. cell. Therefore, there is no prior art report on loading nanoparticles with tumor antigens to activate and enrich specific types of T cells to exert tumor therapeutic effects.
  • cancer-specific T cells with the function of recognizing cancer cells and killing cancer cells are further stimulated by tumor antigens in vitro, and then are sorted, separated, and amplified on a large scale before being infused back to patients for use, thus achieving cancer specificity through infusion.
  • T cells are an effective method for preventing cancer occurrence, preventing cancer metastasis and treating cancer.
  • the object of the present invention is to provide a method for preparing cancer-specific T cells derived from autologous or allogeneic cells for the prevention or treatment of cancer, which specifically includes the following steps: first, isolate immune cells from peripheral blood or peripheral immune organs; Then, they are incubated with Antigen-presenting cells (APC) and nanoparticles and/or microparticles loaded with cancer whole cell components or whole cell partial antigen components for a period of time to activate cancer-specific T cells, and then separated. Cancer-specific T cells activated by cancer antigens are obtained, expanded in vitro and then infused back into the body to exert anti-cancer effects.
  • APC Antigen-presenting cells
  • the preparation method specifically includes the following steps:
  • step (3) Co-incubate the T cells expressing specific cell markers sorted in step (2) with cytokines and/or antibodies to obtain amplified specific T cells;
  • the tumor antigen component can be a whole cell lysate component of tumor tissue/cancer cells, or the antigen component can be a whole cell lysate component of tumor tissue/cancer cells. part of the components.
  • the whole cell lysate component includes a water-soluble component and a water-insoluble component.
  • the non-water-soluble component is dissolved (or solubilized) using a dissolving solution (or solubilizing solution) containing a dissolving agent (or solubilizing agent). ).
  • the tumor antigen component is obtained by lysing the whole cells of one or more cancer cells and/or tumor tissues, or is obtained by lysing the whole cells of one or more cancer cells and/or tumor tissues.
  • the component consists of one or more cancer cells and/or a part of the tumor tissue, and part of the component contains protein/polypeptide components and/or mRNA components in the lysis solution;
  • the antigen component when the whole cell antigen component is a part of the whole cell lysate component, the antigen component contains the protein and polypeptide components in the whole cell lysate component and/or mRNA components.
  • nanoparticles and/or microparticles loaded with tumor antigen components can be co-incubated with antigen-presenting cells and T cells simultaneously to activate cancer cell-specific T cells; nanoparticles loaded with tumor antigen components can also be co-incubated.
  • Particles and/or microparticles are first incubated with antigen-presenting cells to activate the antigen-presenting cells, and then the activated antigen-presenting cells are separately incubated with T cells to activate cancer cell-specific T cells; the load is Nanoparticles and/or microparticles of tumor antigen components are first co-incubated with antigen-presenting cells to activate the antigen-presenting cells.
  • the antigen-presenting cells can then be co-incubated with T cells to activate specific T cells without special treatment.
  • the antigen-presenting cells can be fixed, irradiated, irradiated, modified, inactivated, mineralized, etc., and then co-incubated with T cells to activate specific T cells.
  • step (1) the allogeneic or allogeneic immune cells of the peripheral blood or peripheral immune system are separated and extracted.
  • the above cells may be separated and extracted without any treatment, or after radiotherapy, Immunotherapy, chemotherapy, particle therapy, vaccine treatment.
  • the T cells obtained by sorting in step (1) are any one of CD3 + T cells, CD3 + CD8 + T cells, CD3 + CD4 + T cells, or a combination thereof.
  • the sorting method in step (1) and step (2) is any one of flow cytometry, magnetic bead method or a combination thereof.
  • one activation marker for activated T cells can be used, or a combination of more than one different markers can be used as an activation marker.
  • the tumor antigen component is obtained by lysing one or more cancer cells and/or whole cells of tumor tissue, or is obtained by lysing one or more cancer cells and/or whole cells of tumor tissue. Or it is obtained by processing and processing the whole cells of tumor tissue, or it is obtained by processing and processing the whole cells of one or more cancer cells and/or tumor tissues.
  • at least one of the cancer cells or tumor tissues is the same as the target disease type.
  • the antigen component is composed of one or more cancer cells and/or part of the tumor tissue, and part of the component contains protein/polypeptide components and/or mRNA components in the lysate.
  • the antigen component can be: (1) a cancer cell/tumor tissue whole cell lysate component; (2) or a protein and a protein contained in the cancer cell/tumor tissue whole cell lysate component
  • the polypeptide group is a part of the whole cell component; (3) or it is the protein polypeptide component in the whole cell component of cancer cells/tumor tissue plus the mRNA component.
  • the tumor antigen component is a cell lysis component of tumor tissue and/or cancer cells, including a water-soluble component and a water-insoluble component produced after cell lysis of tumor tissue and/or cancer cells.
  • a solubilizing solution containing a solubilizing agent to directly lyse cancer cells or tumor tissues and dissolve all the components.
  • Cell components are used to prepare nanometer or micron particles, and the water-insoluble components are dissolved in a dissolving solution containing a dissolving agent.
  • it can also be the protein and polypeptide components obtained after appropriate treatment of the above lysate components, or the protein and polypeptide components plus the mRNA component.
  • the preparation method is: (1) first lyse the cancer cells/tumor tissue, and then prepare the water-soluble component and the non-water-soluble component respectively The non-water-soluble components are then dissolved in a specific lysing agent containing the lysing agent before use; (2) The cells are lysed using a lysing agent containing the lysing agent, and then the lysed whole cell components are dissolved using a lysing agent containing the lysing agent. .
  • the preparation method of the antigen component being a part of the whole cell lysate component is: (1) first Lyse cancer cells/tumor tissue, and then separately prepare water-soluble components and non-water-soluble components. Then use the non-water-soluble component after dissolving it with a specific dissolving agent containing a dissolving solution, and then use an appropriate method from the water-soluble component.
  • Appropriate methods for separating and extracting protein and polypeptide components include but are not limited to salting out, heating, enzymatic hydrolysis, etc.
  • the nanoparticles/microparticles are Particle concentration ranges from 2.5ng/mL to 50mg/mL; total incubation time ranges from 1 to 168 hours.
  • the whole cell component can undergo inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, etc. before or (and) after lysis.
  • Nanoparticles or microparticles can be prepared after treatment; they can also be prepared directly without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, or nuclease treatment before or/and after cell lysis.
  • the tumor tissue cells have been inactivated or (and) denatured before lysis, or they can be inactivated or (and) denatured after cell lysis, or they can be before and after cell lysis. All were inactivated or/and denatured.
  • the inactivation or (and) denaturation treatment method before or (and) after cell lysis includes ultraviolet irradiation, high-temperature heating, radioactive irradiation, high pressure, solidification, biomineralization, ionization, chemical Any one of modification, nuclease treatment, collagenase treatment, freeze-drying or a combination thereof.
  • the number ratio of antigen-presenting cells to T cells is greater than 1:1; nanoparticles or microparticles are used to activate peripheral immune cells in vitro after presentation by antigen-presenting cells.
  • Cancer-specific T cells pre-existing in the 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 ⁇ m to 1000 ⁇ m.
  • 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 of the above; the co-incubated antigens Presenting cells are B cells, dendritic cells, macrophages, or any mixture of the three.
  • 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, and are preferably dendritic cells. Any one of DC, B cells, macrophages or a combination thereof; more preferably, a combination of more than one antigen-presenting cell is used.
  • the mixing and co-incubation are selected from any one of the following three methods: (a) the three are directly mixed and co-incubated for a certain period of time; (b) micron particles and/or nanoparticles and antigen extraction The presenting cells are first co-incubated for a period of time, and then T cells are added for co-incubation; (c) microparticles and/or nanoparticles and antigen-presenting cells are first co-incubated for a period of time, the incubated antigen-presenting cells are sorted, and then the antigen is extracted Both cells and T cells were co-incubated.
  • T cells Before cells are co-incubated with nanoparticles/microparticles and antigen-presenting cells, T cells can be cultured separately for a period of time or appropriately sorted; or before T cells are co-incubated with activated antigen-presenting cells , T cells can be cultured alone for a period of time, or they can be appropriately sorted.
  • the culture conditions of the mixed co-incubation are 1-168 hours of co-incubation at 30-38°C and 1-10% CO2 .
  • cytokines can be added to the mixed co-incubation; the added cytokines include but are not limited to interleukins, tumor necrosis factors, interferons, and growth factors; preferably, the added cytokines include Interleukin 7 (IL-7), interleukin 15 (IL-15).
  • IL-7 Interleukin 7
  • IL-15 interleukin 15
  • the sorting method is to use antibodies with fluorescence or magnetism or specific ligands to bind to specific cell markers on the surface of T cells and then use flow cytometry or Magnetic bead methods and other methods are used to isolate cells expressing specific cell markers from the cell population.
  • the sorted T cells expressing specific cell markers include but are not limited to 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, T
  • the concentration of the cytokine is 1-6000ng/ml, preferably 5-200ng/ml, and more preferably 10-30ng/ml.
  • the cytokines include but are not limited to interleukins, interferons, and tumor necrosis factors.
  • 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).
  • IL-2 interleukin 2
  • IL-7 interleukin 7
  • IL-12 interleukin 12
  • IL-15 interleukin 15
  • IL-17 interleukin 17
  • IL-21 interleukin 21
  • the concentration of the antibody is 1-6000ng/ml, preferably 5-100ng/ml, and more preferably 10-30ng/ml.
  • the antibody includes but is not limited to any one of ⁇ CD3 antibody, ⁇ CD28 antibody, ⁇ CD80 antibody, ⁇ CD86 antibody, ⁇ OX40 antibody or a combination thereof.
  • the incubation time of the nanoparticles/microparticles with the mixture of antigen-presenting cells and T cells is at least 4 hours, preferably 6-96 hours.
  • the incubation time of the nanoparticles/microparticles alone with the antigen-presenting cells is at least 1 hour, preferably 6-96 hours.
  • the co-incubation time of the mixture of activated antigen-presenting cells and T cells is at least 1 hour, preferably 6-96 hours.
  • the amplification culture time is at least 1 day, preferably 4-36 days.
  • the concentration of the nanoparticles/microparticles is 2.5ng/mL to 50mg/mL.
  • the concentration of the nanoparticles/microparticles is 2.5ng/mL to 50mg/mL.
  • the content of protein and polypeptide components in the antigen component loaded by the nanoparticles/microparticles is higher than 10 ng/mL.
  • the specific T cells obtained after in vitro expansion in step (3) are reinfused into the body to exert anti-cancer effects.
  • the cancer antigens loaded by the nanoparticles and/or microparticles are whole cell components of tumor tissue and/or cancer cells, including water-soluble components of tumor tissue and/or cancer cells and /or non-water soluble components.
  • the nanoparticles and/or microparticles for activating cancer-specific T cells load one and/or multiple components of tumor tissue and/or cancer cells onto the nanoparticles.
  • the loading method of the micron particles is that the water-soluble components and the water-insoluble components of the whole cells are separately or simultaneously loaded inside the particles, and/or are loaded separately or simultaneously on the surface of the particles.
  • the nanoparticles and/or microparticles used to activate cancer-specific T cells are loaded with the original non-water-soluble part of the whole cell component derived from tumor tissue or cancer cells.
  • Structural formula 1 has the following structure:
  • R1 is C, N, S or O
  • R2 to R5 are independently selected from at least one of hydrogen, alkyl, amino, carboxyl, substituted or unsubstituted guanidyl.
  • 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, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine sulfamate, guanidine succinic acid, amino acid hydrochloride Urea, carbamoylurea, acetourea, sulfonylurea compounds (glibenclamide, gliclazide, glazid
  • the surface of the nanoparticles and/or microparticles used to activate cancer-specific T cells is connected with a target that actively targets antigen-presenting cells.
  • the water-soluble component and/or the non-water-soluble component is loaded on the surface of the cancer vaccine in a manner including adsorption, covalent connection, charge interaction, hydrophobic interaction, one or more steps. At least one of step curing, mineralization and encapsulation.
  • the particle size of the nanoparticles is 1 nm-1000 nm; the particle size of the microparticles is 1 ⁇ m-1000 ⁇ m.
  • the surface of the nano-sized particles or micron-sized particles is electrically neutral, negatively charged or positively charged.
  • the nano-vaccine and/or micro-vaccine are prepared from organic synthetic polymer materials, natural polymer materials or inorganic materials.
  • the organic synthetic polymer materials are PLGA, PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acids, and synthetic polypeptides ;
  • the natural polymer materials are lecithin, cholesterol, sodium alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, and polypeptides;
  • the inorganic materials are ferric oxide, trioxide Iron, calcium carbonate, calcium phosphate.
  • the nanoparticles and/or microparticles used to activate cancer-specific T cells can combine one and/or multiple components of tumor tissue and/or cancer cells with immune Adjuvants are co-loaded on nanoparticles or microparticles.
  • both the water-soluble part and the water-insoluble part can be dissolved by a solubilizing aqueous solution containing a solubilizing agent or an organic solvent.
  • the solubilizing agent is at least one of the solubilizing agents that can increase the solubility of the protein or polypeptide in an aqueous solution;
  • the organic solvent is an organic solvent that can dissolve the protein or polypeptide.
  • the original water-insoluble part is changed from insoluble in pure water to soluble in an aqueous solution containing a solubilizing agent/dissolving agent or an organic solvent using an appropriate solubilizing method;
  • the solubilizing agent is selected from one or more compounds containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, polypeptide, amino acid, glycoside and choline. species; among them, structural formula 1 is as follows:
  • Structural formula 1 has the following structure:
  • R1 is C, N, S or O
  • R2 to R5 are independently selected from at least one of hydrogen, alkyl, amino, carboxyl, substituted or unsubstituted guanidyl.
  • 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, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine sulfamate, guanidine succinic acid, amino acid hydrochloride Urea, carbamoylurea, acetourea, sulfonylurea compounds (glibenclamide, gliclazide, glazid
  • the cellular components loaded on the nanoparticles or microparticles used to activate cancer-specific T cells are derived from one or more cancer cells and/or one or more tumor tissue whole cells.
  • the obtained components are loaded with non-water-soluble components onto the delivery particles so that the nano- or micron system contains more antigens. More preferably, both water-soluble components and non-water-soluble components are loaded onto the delivery particles at the same time.
  • the delivery particles are loaded with whole cell component antigens.
  • nanoparticles and/or microparticle-loaded cell components for activating cancer-specific T cells or a mixture thereof the mixture includes but is not limited to water-soluble components mixed with each other, or water-insoluble components components are mixed with each other, or all or part of the water-soluble components are mixed with all or part of the water-soluble components.
  • the loading method is that the water-soluble components and the non-water-soluble components of the cells are loaded inside the particles respectively or simultaneously, and/or are loaded separately or simultaneously on the particle surface, including but not limited to the water-soluble components being loaded simultaneously on the particle surface.
  • the particles are neutralized and loaded on the surface of the particles.
  • the water-insoluble components are loaded on the particles and on the surface of the particles.
  • the water-soluble components are loaded on the particles.
  • the non-water-soluble components are loaded on the surface of the particles.
  • the non-water-soluble components are loaded on the particles.
  • the water-soluble components are loaded in the particles and the water-soluble components are loaded on the surface of the particles.
  • the water-soluble components and the water-insoluble components are loaded in the particles and only the non-water-soluble components are loaded on the surface of the particles.
  • the water-soluble components and the water-insoluble components are loaded on the particles. In the particles, only the water-soluble components are loaded on the surface of the particles.
  • the water-soluble components are loaded in the particles, while the water-soluble components and the non-water-soluble components are loaded on the surface of the particles at the same time.
  • the non-water-soluble components are loaded in the particles and the water-soluble components are loaded on the particles.
  • Components and water-insoluble components are loaded on the particle surface at the same time, water-soluble components and water-insoluble components are loaded on the particles at the same time, and water-soluble components and water-insoluble components are loaded on the particle surface at the same time.
  • the interior and/or surface of the nanoparticles or microparticles used to activate cancer-specific T cells may also include immune-enhancing adjuvants.
  • the immune-enhancing adjuvants include but are not limited to immune enhancers derived from microorganisms.
  • immune-enhancing adjuvants include but are not limited to pattern recognition receptors Body agonist, Bacillus Calmette-Guerin (BCG), manganese-related adjuvant, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyantigen, mineral oil, virus-like particles, immune enhancement Reconstructed influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, miquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I: C), immune adjuvant poly ICLC, Corynebacterium parvum vaccine, hemolytic streptococcus preparation, coenzyme
  • the immune-enhancing adjuvant is preferably a Toll-like receptor agonist.
  • the immune-enhancing adjuvant is preferably a combination of two or more Toll-like receptor agonists.
  • the immune adjuvant and the cell component are co-loaded in nanoparticles or microparticles. After the nanoparticles or microparticles are phagocytosed by antigen-presenting cells, cancer-specific T cells can be better activated.
  • the microparticles or nanoparticles are loaded with substances that increase the escape of the nanoparticles and/or the microparticles or the antigens they carry from lysosomes to the cytoplasm.
  • the substances that increase lysosomal escape include amino acids, polypeptides, sugars, lipids, and inorganic salts that can produce a proton sponge effect.
  • the amino acids in the substances that increase lysosomal escape include positively charged amino acids.
  • the polypeptide species that increases lysosomal escape contains positively charged amino acids.
  • the surface of the nanoparticles or microparticles may not be connected to a target with an active targeting function, or may be connected to a target with an active targeting function,
  • Active targeting targets can be commonly used targets such as mannose, mannan, CD19 antibodies, CD20 antibodies, BCMA antibodies, CD32 antibodies, CD11c antibodies, CD103 antibodies, CD44 antibodies, etc., leading the particle system to targeted delivery to antigen presentation cell.
  • the surface of the nanoparticles and/or microparticles is connected with a target that actively targets antigen-presenting cells.
  • the nanoparticles or microparticles may not be modified during the preparation process, or appropriate modification technology may be used to increase the antigen loading capacity of the nanoparticles or microparticles.
  • Modification technologies include but are not limited to biomineralization (such as silicification, calcification, magnesization), gelation, cross-linking, chemical modification, and addition of charged substances.
  • the cell components or their mixture are loaded inside the nanoparticles or microparticles in any manner that can load the cell components or their mixture inside the nanoparticles or microparticles.
  • the cell components or mixtures thereof are loaded on the surface of nanoparticles or microparticles in a manner including but not limited to adsorption, covalent connection, charge interaction (such as adding positively charged substances, adding negatively charged substances). substances), hydrophobic interactions, one or more steps of solidification, mineralization, encapsulation, etc.
  • the water-soluble components and/or non-water-soluble components loaded on the surface of nanoparticles or microparticles are loaded into one or more layers, and the surface of the vaccine is loaded with multiple layers of water-soluble components and/or When there are non-water-soluble components, there are modifiers between the layers.
  • the particle size of the nanoparticles is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, and most preferably, the particle size is 100nm-600nm.
  • the particle size of the microparticles is 1 ⁇ m-1000 ⁇ m, more preferably, the particle size is 1 ⁇ m-100 ⁇ m, more preferably, the particle size is 1 ⁇ m-10 ⁇ m, most preferably, the particle size 1 ⁇ m-5 ⁇ m.
  • the shape of the nanoparticles or microparticles includes sphere, ellipsoid, barrel, polygon, rod, sheet, linear, worm-shaped, square, triangle, butterfly or disc. any kind.
  • the water-soluble component and/or the non-water-soluble component is loaded on the surface of the cancer vaccine in a manner including adsorption, covalent connection, charge interaction, hydrophobic interaction, one or more steps. At least one of step curing, mineralization and encapsulation.
  • the nano-vaccine and/or micro-vaccine are prepared from organic synthetic polymer materials, natural polymer materials or inorganic materials.
  • the organic synthetic polymer material is a biocompatible or degradable polymer material, including PLGA, PLA, PGA, PLGA-PEG, PLA-PEG, PGA-PEG, PEG, PCL, Any one of Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, synthetic lipid or their combination.
  • the natural polymer material is a biocompatible or degradable polymer material, including lecithin, cholesterol, sodium alginate, albumin, collagen, gelatin, cell membrane components, starch, Any one of carbohydrates and polypeptides or a combination thereof.
  • the inorganic material is a material without obvious biological toxicity, including but not limited to ferric oxide, ferric tetroxide, calcium carbonate, calcium phosphate, etc.
  • Another object of the present invention is to provide specific T cells prepared by the method of the present invention.
  • Another object of the present invention is to provide a cancer-specific T cell derived from autologous or allogeneic for preventing or treating cancer.
  • the specific T cells include but are not limited to 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 +
  • the specific T cells are obtained by co-incubating nanoparticles and/or microparticles loaded with tumor antigen components with peripheral blood mononuclear cells (PBMC) isolated from immune cells in peripheral blood or peripheral immune organs.
  • PBMC peripheral blood mononuclear cells
  • the PBMC cells are sorted and then incubated with nanoparticles and/or microparticles loaded with tumor antigen components.
  • the cells co-incubated with nanoparticles and/or microparticles loaded with tumor antigen components are sorted again to obtain specific T cells.
  • the specific T cells may also undergo an in vitro amplification step, which may be performed before or after the aforementioned sorting step, preferably after the sorting step.
  • antigen-presenting cells are also present.
  • the specific T cells are CD3 + CD8 + CD69 + cells; in some preferred embodiments, the specific T cells are CD3 + CD137 + cells; in some preferred In embodiments, the specific T cells are CD3 + CD8 + CD25 + cells and/or CD3 + CD8 + CD69 + cells; in some preferred embodiments, the specific T cells are CD3 + CD69 + cells; in some preferred embodiments, the specific T cells are CD3 + CD69 + cells; in some preferred embodiments, the specific T cells are CD3 + CD8 + CD69 + cells; in some In a preferred embodiment, the specific T cells are CD3 + CD8 + CD25 + cells and/or CD3 + CD4 + CD69 + cells; in some preferred embodiments, the specific T cells are CD8 + CD69 + cells; in some preferred embodiments, the specific T cells are CD8 + CD137 + cells; in some preferred embodiments, the specific T cells are CD8 + CD69 + cells and/or CD4 + CD69 + cells; in some preferred embodiments, the specific T cells are CD3 + CD137 + cells;
  • T cells can be used singly or in combination according to patient needs.
  • the object of the present invention is to provide a method for preparing cancer-specific T cells derived from autologous or allogeneic cells for preventing or treating cancer, which specifically includes the following steps:
  • the tumor antigen component is prepared by first isolating tumor cells or tissues, and then lysing the tumor cells or tissues to obtain any one of a water-soluble component, a water-insoluble component, a complete component, or a combination thereof;
  • the nanoparticles and/or microparticles loaded with tumor antigen components are prepared using the double emulsion method
  • T cells are selected, including but not limited to 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 + H
  • antigen-presenting cells are also added during the co-incubation process, and the antigen-presenting cells are any one of B cells, DC cells, macrophages or a combination thereof;
  • nanoparticles and/or microparticles loaded with tumor antigen components can be co-incubated with antigen-presenting cells and T cells simultaneously to activate cancer cell-specific T cells; nanoparticles loaded with tumor antigen components can also be co-incubated.
  • Particles and/or microparticles are first incubated with antigen-presenting cells to activate the antigen-presenting cells, and then the activated antigen-presenting cells are separately incubated with T cells to activate cancer cell-specific T cells; the load is Nanoparticles and/or microparticles of tumor antigen components are first co-incubated with antigen-presenting cells to activate the antigen-presenting cells.
  • the antigen-presenting cells can then be co-incubated with T cells to activate specific T cells without special treatment.
  • the antigen-presenting cells can be fixed, irradiated, irradiated, modified, inactivated, mineralized, etc., and then co-incubated with T cells to activate specific T cells;
  • nanoparticles and/or microparticles loaded with tumor antigen components are added to the culture medium, together with 1-50 million/ml PBMC cells or sorted cells, at 30-38 Incubate for 4-96 hours at °C and 1-5% CO 2 to obtain cell culture;
  • interleukin is any one of IL-2, IL-7, IL-12, IL-15, IL-17, IL-21 or any of them. combination;
  • the culture medium is any one of DMEM high-glucose complete culture medium, RPM1640 culture medium, and AIMV serum-free culture medium;
  • the co-incubation is 1-168h at 30-38°C, preferably 4-96h, more preferably 6-72h;
  • the specific T cell activity rate is greater than 60%, preferably greater than 70%, more preferably greater than 80%;
  • the amplification step is to add 1-50 million cells/mL of the specific T cells sorted in step (2) into the amplification medium, and then culture the cells at 30-38°C, 1-5% Co-incubate under CO2 conditions, change the amplification medium every 2-3 days, and obtain amplified specific T cells after a total of 5-30 days of incubation;
  • amplification is performed in an amplification medium, which is any one of DMEM high-glucose complete medium and RPM1640 medium;
  • the conditions for amplification culture are 30-38°C for 4-72 days, preferably 5-30 days;
  • the expansion medium also 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);
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the interleukin is any one of IL-2, IL-7, IL-12, IL-15, IL-17, IL-21 or a combination thereof;
  • the antibody is any one of ⁇ CD3 antibody, ⁇ CD28 antibody or a combination thereof.
  • the cells are selected from any part of an individual suffering from a tumor disease, preferably spleen cells and lymphocytes.
  • the amplified specific T cell activity rate is greater than 60%, preferably greater than 75%, and more preferably greater than 80%.
  • the sorting method is to use antibodies with fluorescence or magnetism or specific ligands to bind to specific cell markers on the surface of T cells and then use flow cytometry or Magnetic bead methods and other methods are used to isolate cells expressing specific cell markers from the cell population.
  • cytokines can be added to the mixed co-incubation; the added cytokines include but are not limited to interleukins, tumor necrosis factors, interferons, and growth factors; preferably, the added cytokines include Interleukin 7 (IL-7), interleukin 15 (IL-15).
  • IL-7 Interleukin 7
  • IL-15 interleukin 15
  • the concentration of the cytokine is 1-6000ng/ml, preferably 5-100ng/ml, and more preferably 10-30ng/ml.
  • the cytokines include but are not limited to interleukins, interferons, and tumor necrosis factors.
  • 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).
  • IL-2 interleukin 2
  • IL-7 interleukin 7
  • IL-12 interleukin 12
  • IL-15 interleukin 15
  • IL-17 interleukin 17
  • IL-21 interleukin 21
  • the concentration of the antibody is 1-6000ng/ml, preferably 5-100ng/ml, and more preferably 10-30ng/ml.
  • the antibody includes but is not limited to any one of ⁇ CD3 antibody, ⁇ CD28 antibody, ⁇ CD80 antibody, ⁇ CD86 antibody, ⁇ OX40 antibody or a combination thereof.
  • the incubation time of the nanoparticles/microparticles with the mixture of antigen-presenting cells and T cells is at least 1 hour, preferably 4-96 hours.
  • the incubation time of the nanoparticles/microparticles alone with the antigen-presenting cells is at least 1 hour, preferably 4-96 hours.
  • the co-incubation time of the mixture of activated antigen-presenting cells and T cells is at least 1 hour, preferably 6-96 hours.
  • the amplification culture time is at least 1 day, preferably 4-72 days.
  • the concentration of the nanoparticles/microparticles is 10 ng/mL to 5 mg/mL.
  • the concentration of the nanoparticles/microparticles is 2.5ng/mL to 50mg/mL.
  • the content of protein and polypeptide components in the antigen component loaded by the nanoparticles/microparticles is higher than 10 ng/mL.
  • the nanoparticles or microparticles are PLGA
  • the molecular weight is selected from 7-54KDa
  • the immune adjuvant is selected from (poly(I:C), Any one or combination of polyICLC, BCG, and CpG.
  • the nanoparticles or microparticles are PLGA
  • the molecular weight is selected from 7-51KDa
  • the immune adjuvant is selected from (poly(I:C), Any one or combination of polyICLC, BCG, and CpG.
  • the nanoparticles use PLGA with a molecular weight of 24KDa-38KDa, the immune adjuvant used is poly(I:C), and the poly(I:C) is only distributed inside the nanoparticles.
  • the nanoparticles use PLGA with a molecular weight of 7Da-17KDa, and the immune adjuvants used are poly(I:C) and CpG1018.
  • the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles use PLGA with a molecular weight of 7KDa-17KDa, and the immune adjuvants used are poly(I:C), CpG2006 and CpG2216.
  • the mass ratio of the three immune adjuvants is preferably 1: 1:1.
  • the nanoparticles use PLGA with a molecular weight of 24KDa-38KDa, and the immune adjuvant used is poly(I:C), and poly(I:C) is both distributed inside the nanoparticles and loaded on the nanoparticles.
  • Nanoparticles modified by freezing siliconization and adding cationic substances are loaded with lysate on the particle surface, inside and outside.
  • the micron particles use PLGA with a molecular weight of 24KDa-38KDa, and the immune adjuvant used is poly(I:C), and poly(I:C) is both distributed inside the micron particles and loaded on the micron particles.
  • Micron particles modified by freezing siliconization and adding cationic substances are loaded with lysate on the particle surface, both inside and outside.
  • the nanoparticles are PLA with a molecular weight of 20KDa
  • the immune adjuvants used are poly(I:C) and CpG1018.
  • the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles use PLGA with a molecular weight of 24KDa-38KDa
  • the immune adjuvants used are Poly(I:C) and CpG1018
  • the antigen components and adjuvants are simultaneously distributed inside and on the surface of the nanoparticles.
  • the micron particles use PLGA with a molecular weight of 38KDa-54KDa, CpG and Poly ICLC as immune adjuvants, and promote lysosome escape, and arginine can be added to the system.
  • the nanoparticles use PLGA with a molecular weight of 24KDa-38KDa, and the immune adjuvants are CpG and Poly(I:C), which promote lysosome escape.
  • KALA polypeptide WEAKLAKALAKALAKHLAKALAKALKACEA
  • the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles use PLGA with a molecular weight of 7KDa-17KDa
  • the immune adjuvant used is BCG
  • BCG is loaded inside the nanoparticles.
  • the nanoparticles are PLGA and mannose-modified PLGA, with a mass ratio of 4:1, a molecular weight of 7KDa-17KDa, and the immune adjuvants used are Poly(I:C) and CpG.
  • the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles use PLGA with a molecular weight of 24KDa-38KDa, and the immune adjuvants used are BCG and Poly(I:C).
  • the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles used are biologically calcified nanoparticles loaded with whole cell antigens inside and on the surface, the molecular weight of PLGA is 7KDa-17KDa, and the immune adjuvants CpG and Poly(I:C) and enhanced lysozyme are used
  • the body-escaping GALA polypeptide (WEAALAEALAEALAEHLAEALAEALEALAA) is loaded inside the nanoparticles, and the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles use PLGA with a molecular weight of 7KDa-17KDa
  • the immune adjuvants used are poly(I:C) and CpG, and they promote lysosome escape, and melittin is added to the system.
  • the adjuvant and melittin are wrapped in nanoparticles, and the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles use PLGA and mannan-modified PLGA, both of which have molecular weights of 24KDa-38KDa.
  • the mass ratio of PLGA and mannan-modified PLGA is 9:1.
  • the adjuvants are poly(I:C) and CpG, and they promote lysosome escape.
  • Polyarginine and polylysine are added to the system, and the adjuvants, polyarginine and polylysine are encapsulated in nanoparticles.
  • the mass ratio of the two immune adjuvants is preferably 1:1.
  • the micron particles use PLGA with a molecular weight of 38KDa-54KDa, and the immune adjuvants used are CpG1018 and Poly ICLC, which promote lysosome escape.
  • KALA polypeptide is added to the system, and the two immune adjuvants
  • the mass ratio of the agents is preferably 1:1.
  • the micron particle skeleton material is unmodified PLA and mannose-modified PLA, both with a molecular weight of 40KDa, and the ratio of unmodified PLA to mannose-modified PLA is 9:1.
  • the immune adjuvants used are CpG2395 and Poly ICLC, which promote lysosomal escape. Arginine and/or histidine are added to the system.
  • the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles use PLGA with a molecular weight of 7KDa-17KDa
  • the immune adjuvants used are poly(I:C) and CpG1018, which promote lysosome escape, and R8 polypeptide is added to the system.
  • the adjuvant and the R8 polypeptide are loaded in the nanoparticles, and the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles use PLGA with a molecular weight of 7KDa-17KDa, Poly(I:C) and CpG1018 as adjuvants, and promote lysosome escape.
  • NH 4 HCO 3 is added to the system, and The adjuvant and NH 4 HCO 3 are loaded into the nanoparticles, and the mass ratio of the two immune adjuvants is preferably 1:1.
  • the nanoparticles use PLGA with a molecular weight of 20KDa-40KDa, and the immune adjuvant used is poly(I:C).
  • the nanoparticles use PLA (molecular weight 30-40KDa) and mannan-PEG2000-PLA (PLA molecular weight 30-40KDa), and PLA (molecular weight 30-40KDa) and mannan-PEG2000-PLA (molecular weight 30-40KDa).
  • the mass ratio of PEG2000-PLA (PLA molecular weight is 30-40KDa) is 9:1.
  • the immune adjuvants used are CpG2006 (Class B), CpG2216 (Class A) and Poly ICLC.
  • the mass ratio of the three immune adjuvants is preferably 1:1:1.
  • the nanoparticles use PLGA with a molecular weight of 10KDa-20KDa, and the immune adjuvants used are poly(I:C), CpG 7909 and CpG2395.
  • the mass ratio of the three immune adjuvants is preferably 1 :1:1.
  • the nanoparticles adopt PLGA with a molecular weight of 10KDa-20KDa, and the loaded immune adjuvants are poly(I:C) and CpG7909.
  • the mass ratio of the two immune adjuvants is preferably 1:1.
  • the micron particles are made of PLGA with a molecular weight of 38KDa-54KDa, and the immune adjuvants used are CpG1018 and Poly ICLC, which promote lysosome escape.
  • KALA polypeptide is added to the system, and the two immune adjuvants are CpG1018 and Poly ICLC.
  • the mass ratio of adjuvants is preferably 1:1.
  • the tumor antigen component contains protein/polypeptide components in the cell lysis component of tumor tissue and/or cancer cells
  • the preparation method is: (1) first prepare the tumor tissue/cancer cells lysate, and then prepare the water-soluble components and the water-insoluble components in the lysis solution, and then prepare the protein/peptide groups in the water-soluble components and the water-insoluble components through salting out, heating, enzymatic hydrolysis, etc.
  • the protein/polypeptide components are prepared separately by methods such as solution, and loaded into nano or micro particles as antigen components.
  • the water-insoluble components and the precipitates obtained after treatments such as salting out, heating, and enzymatic hydrolysis are dissolved in a dissolving solution containing a dissolving agent.
  • the whole cell component preparation method is: (1) first lyse cancer cells/tumor tissues, then prepare water-soluble components and non-water-soluble components respectively, and then use specific Use the lysing agent containing the lysing agent after dissolving; (2) Use the lysing agent containing the lysing agent to lyse the cells, and then use the lysing agent containing the lysing agent to dissolve the lysed whole cell components.
  • the preparation method of part of the whole cell components containing whole cell proteins and polypeptide components in the whole cell components of cancer cells is: (1) first lyse the cancer cells/tumor tissue, Then prepare the water-soluble component and the non-water-soluble component separately, then dissolve the non-water-soluble component with a specific dissolving agent containing a dissolving solution, and then separate and extract the water-soluble component from the water-soluble component using an appropriate method.
  • the protein and polypeptide components are then separated and extracted from the water-soluble components together with all the non-water-soluble components to be used as antigen components; (2) first lyse the cancer cells/tumor tissues and then prepare them separately The water-soluble component and the non-water-soluble component are then used after dissolving the non-water-soluble component using a specific dissolving agent containing a dissolving solution, and then using an appropriate method to separate and extract the protein in the water-soluble component from the non-water-soluble component and peptide components, and then use the separated and extracted protein and peptide components from the non-water-soluble components together with all water-soluble components as antigen components; (3) First lyse cancer cells/tumor tissues, and then prepare water-soluble components and non-water-soluble components, and then use the non-water-soluble component to dissolve it using a specific dissolving agent containing a dissolving solution, and then separate and extract the water-soluble components from the water-soluble components and non-water-soluble components using appropriate methods.
  • Appropriate methods for separating and extracting protein and polypeptide components include but are not limited to salting out, heating, enzymatic hydrolysis, etc.
  • the tumor antigen component is prepared by first separating tumor cells or tissues, and then lysing the tumor cells or tissues to obtain water-soluble components, non-water-soluble components, Any one or combination of all components.
  • the tumor antigen component is appropriately processed by methods such as salting out, heating, and enzymatic hydrolysis.
  • both the water-soluble part and the water-insoluble part can be dissolved by a solubilizing aqueous solution containing a solubilizing agent or an organic solvent.
  • the solubilizing agent is at least one of the solubilizing agents that can increase the solubility of proteins or polypeptides in aqueous solutions;
  • the organic solvent is an organic solvent that can dissolve proteins or polypeptides.
  • the water-insoluble part is changed from insoluble in pure water to soluble in an aqueous solution containing a dissolving agent or an organic solvent using an appropriate solubilization method;
  • the dissolving agent used is selected from the group consisting of those containing the structural formula One or more compounds of structure 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, polypeptides, amino acids, glycosides and choline; wherein, structural formula 1 is as follows :
  • Structural formula 1 has the following structure:
  • R 1 is C, N, S or O
  • R 2 to R 5 are independently selected from at least one of hydrogen, alkyl, amino, carboxyl, substituted or unsubstituted guanidyl.
  • Compounds containing the structure of structural formula 1 include, but are not limited to, metformin hydrochloride, metformin sulfate, metformin sulfonate, metformin salts, metformin, urea, guanidine hydrochloride, guanidine sulfate, guanidine sulfonate, guanidine salts, other compounds containing guanidine groups, guanidine carbonate, Arginine, guanidinoacetic acid, guanidinophosphate, guanidine sulfamate, guanidinosuccinic acid, semicarbazide hydrochloride, carbamoyl urea, acetourea, sulfonylurea compounds (glibenclamide, gliclazide, Gliquidone, glimepiride, etc.), thioureas compounds (thiouracils, imidazoles, etc.), nitrosoureas, etc.
  • Urea, guanidine hydrochloride, etc. contain the structure in Structural Formula 1.
  • the immune adjuvant and the cell component are co-loaded in nanoparticles or microparticles. After the nanoparticles or microparticles are phagocytosed by antigen-presenting cells, cancer-specific T cells can be better activated.
  • the preparation method of the water-soluble component is as follows: cutting the tumor tissue or cancer cells into pieces, grinding them, filtering to obtain a single cell suspension, adding water and repeatedly freezing and thawing 1-5 times, and then ultrasonic lysis , the lysate is centrifuged at a speed of 5000-10000g for 5-10 minutes, the supernatant is taken as the water-soluble component, and the precipitated part is the non-water-soluble component.
  • the antigen component is a soluble component obtained by adding a dissolving agent to the non-water-soluble component.
  • the dissolving agent is urea, sodium deoxycholate, guanidine hydrochloride, and octyl glucose. Any one of glycosides, arginine, glycerol, semicarbazide hydrochloride, agmatine sulfate or a combination thereof.
  • the antigen component is a soluble component obtained by dissolving the non-water-soluble component by adding urea aqueous solution, and is a mixture obtained by mixing the antigen component with the water-soluble component at a ratio of 3-1:1.
  • the antigen component is a water-soluble component of tumor cells and a water-soluble component of cancer cells mixed at a ratio of 1:1, or the non-water-soluble component of tumor cells is added to a dissolving agent and then dissolved to obtain The soluble components and the water-insoluble components of the cancer cells are added to the dissolving agent and then the resulting soluble components are mixed at a ratio of 1:1.
  • the tumor tissue or cancer cells are subjected to any one of inactivation and denaturation treatment by ultraviolet high-temperature heating in advance, addition of nuclease inactivation treatment, or a combination thereof.
  • the antigen component is a component obtained by salting out and heating and precipitating a water-soluble component.
  • the antigen component is the precipitate after centrifugation of tumor tissue, that is, the precipitate obtained by adding a solubilizing agent to the non-water-soluble component and then adding a solubilizing agent for secondary dissolution.
  • Soluble component, the solubilizing agent is any one of Tween 80, guanidine sulfate or a combination thereof.
  • Another object of the present invention is to provide a method for preparing a pharmaceutical composition containing the specific T cells of the present invention, which includes the following steps. Before infusing the cancer-specific T cells back into the patient, the cancer-specific T cells can be prepared. Substances that enhance the natural immune system are added to T cells, such as albumin, NK cells, neutrophils, ⁇ T cells, and NK T cells.
  • the cell concentration of the specific T cells is (0.01-100) ⁇ 10 7 cells/ml, preferably (0.1-8) ⁇ 10 7 cells/ml.
  • the pharmaceutical composition further contains any one of hydroxyethyl starch, sugar, salt or a combination thereof.
  • Another object of the present invention is the use of the specific T cells of the present invention in the preparation of cancer treatment or preventive drugs.
  • the specific T cells are administered multiple times before the occurrence of cancer, after the occurrence of cancer, or after surgical removal of tumor tissue.
  • Another object of the present invention is the application of the specific T cells of the present invention in preparing drugs for preventing cancer recurrence or cancer metastasis.
  • Another object of the present invention is to provide the use of the specific T cells of the present invention for preparing anti-tumor immunotherapy products.
  • the tumor is selected from the group consisting of solid tumors, hematological tumors and lymphomas.
  • solid tumors e.g., adenosarcoma
  • hematological malignancies such as leukemia, brain tumors, head and neck cancer, glioma, gastric cancer, Nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal area cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, Vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma,
  • hematological malignancies such as leukemia, brain tumors, head and neck cancer, glioma, gastric cancer,
  • the tumor is selected from any one of melanoma, colon cancer, triple-negative breast cancer, pancreatic cancer, metastatic cancer, liver cancer, colon cancer, lymphoma, esophageal cancer, non-small cell lung cancer, or a combination thereof.
  • the immunotherapy is selected from any one or a combination of immunotherapy in anti-tumor treatment or immunotherapy after radical surgery.
  • the immunotherapy is selected from immunotherapy after radical resection of primary hepatocellular carcinoma.
  • the medicine is used for adult patients or pediatric patients.
  • Another object of the present invention is to provide the use of the specific T cells of the present invention for immunotherapy.
  • the administration mode of the immunotherapy is any one of intravenous injection, subcutaneous injection, intratumoral injection, intraperitoneal injection, intramuscular injection, intradermal injection or a combination thereof.
  • Another object of the present invention is to provide the use of the specific T cells of the present invention in anti-tumor or tumor immunotherapy in combination with any of radiotherapy, chemotherapy, targeted therapy, surgical therapy or immunotherapy.
  • Another object of the present invention is to provide the use of specific T cells in the preparation of drugs that enhance antiviral capabilities.
  • Another object of the present invention is to provide the use of specific T cells in the preparation of drugs to enhance the treatment of autoimmune diseases.
  • the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, juvenile diabetes, essential platelet purpura, and autoimmune hemolytic anemia. , ulcerative colitis, any of the skin diseases or their complications.
  • the percentage when the present invention relates to the percentage between liquid and liquid, the percentage is volume/volume percentage; when the present invention relates to the percentage between liquid and solid, the percentage is volume/weight percentage; the present invention When referring to percentages between solids and liquids, the percentages are weight/volume; the remainder are weight/weight.
  • the present invention has the following beneficial technical effects:
  • the nanoparticles or microparticles prepared by the present invention load tumor antigen components on the microparticles and/or nanoparticles, which can activate a broad spectrum of cancer cell-specific T cells. After the cancer cell-specific T cells are activated, After sorting and amplification, the characteristics are then infused back into the patient for cancer treatment or prevention of recurrence or metastasis.
  • the obtained cancer-specific T cells have broad spectrum and high specificity. After amplification, they can kill cancer cells to prevent or treat cancer.
  • the present invention screens the antigen components and nanoparticles obtained through specific methods, and scientifically screens, sorts and amplifies the conditions.
  • the T cells obtained have high viability, good cell stability, and strong immune cell targeting and anti-tumor activity. . After being used in the treatment of tumor patients, it can prevent tumor recurrence, prolong tumor growth, prevent cancer metastasis, and prolong survival.
  • the method of the present invention has the advantages of simple operation, controllable quality, and is suitable for industrial production.
  • Figure 1 is a schematic diagram of the representative preparation process and application fields of the T cell system of the present invention. In practical applications, it can also be other similar or improved alternative preparation processes; where a is the collection of water-soluble components and water-insoluble components respectively. and a schematic diagram of preparing nanoparticles or microparticles; b is a schematic diagram of using a solubilizing solution containing a solubilizer to dissolve whole cell components and preparing nanoparticles or microparticles; c is a schematic diagram of using the above particles prepared in a or b to assist in sorting peripheral A schematic diagram of cancer cell-specific T cells, then expanding the T cells, and using the cells to prevent or treat cancer;
  • Figures 2 to 30 are experimental results of mouse tumor growth rate and survival time when using isolated and amplified cancer-specific T cells to prevent or treat cancer, or to prevent cancer metastasis in Examples 1 to 29 of the present invention.
  • Each data point in figure a is the mean ⁇ standard error (mean ⁇ SEM); the significant difference in the tumor growth inhibition experiment in a was analyzed by ANOVA, and the significant difference in b was analyzed by Kaplan-Meier and log-rank test.
  • *** indicates that there is a significant difference at p ⁇ 0.005 compared with the PBS blank control group; ** indicates that there is a significant difference at p ⁇ 0.001 compared with the PBS blank control group; * It means p ⁇ 0.001, there is a significant difference compared with the PBS blank control group; ⁇ means p ⁇ 0.005, there is a significant difference compared with the one-step sorting cancer-specific T cell group without nanoparticle stimulation; ⁇ means Compared with the cell control group obtained by auxiliary sorting of blank nanoparticles + free lysate containing immune adjuvant, p ⁇ 0.005, there is a significant difference; ⁇ represents the cancer specificity obtained by sorting without adding IL-7 during the co-incubation process.
  • p ⁇ 0.05, there is a significant difference
  • p ⁇ 0.05, there is a significant difference compared with the cancer-specific T cell group that only uses one type of antigen-presenting cells to co-incubate, sort and amplify
  • eta represents the comparison with the sorting of nanoparticles/microparticles loaded with one adjuvant
  • $ represents p ⁇ 0.05, there is a significant difference compared with the cancer-specific T cell group obtained by auxiliary sorting of polypeptide nanoparticles or micron particles.
  • ;$$ represents p ⁇ 0.01, there is a significant difference compared with the cancer-specific T cell group obtained by assisted sorting of peptide nanoparticles or microparticles; ⁇ represents the cancer-specific T cell group obtained by assisted sorting of nanoparticles without adjuvant Compared with the T cell group, p ⁇ 0.05, there is a significant difference; ⁇ represents that compared with the CD8 + cancer-specific T cell group assisted by nanoparticle 1, p ⁇ 0.05, there is a significant difference; ⁇ represents that compared with the nanoparticle 2-assisted sorting, there is a significant difference Compared with the sorted CD8 + cancer-specific T cells + CD4 + cancer-specific T cells group, p ⁇ 0.05, there is a significant difference.
  • # represents p ⁇ 0.05, there is a significant difference; ## represents p ⁇ 0.01, there is a significant difference; ### represents p ⁇ 0.005, there is a significant difference.
  • ns represents no significant difference.
  • the antigen component is first prepared.
  • the antigen component can be (1) cancer cell whole cell component; (2) or whole cell containing whole cell protein and polypeptide components in the cancer cell whole cell component. A part of the components; (3) Or it is the protein polypeptide component in the whole cell component of cancer cells/tumor tissue plus the mRNA component.
  • the preparation method of whole cell components is: (1) First lyse cancer cells/tumor tissues, then prepare water-soluble components and non-water-soluble components respectively, and then dissolve the non-water-soluble components using a specific dissolving agent containing dissolving solution Use; (2) Use a lysing solution containing a lysing agent to lyse cells, and then use a lysing solution containing a lysing agent to dissolve the lysed whole cell components.
  • the method for preparing part of the whole cell fraction containing the whole cell protein and polypeptide components of the cancer cell whole cell fraction is: (1) first lyse the cancer cells/tumor tissue, and then separately prepare the water-soluble fraction and The non-water-soluble component is then used after being dissolved using a specific dissolving agent containing a dissolving solution, and then the protein and peptide components in the water-soluble component are separated and extracted from the water-soluble component using appropriate methods, and then The protein and peptide components separated and extracted from the water-soluble components are used as antigen components together with all non-water-soluble components; (2) first lyse cancer cells/tumor tissues, and then prepare water-soluble components and non-water-soluble components respectively components, and then dissolve the non-water-soluble components using a specific dissolving agent containing a dissolving solution, and then separate and extract the protein and peptide components in the water-soluble components from the non-water-soluble components using appropriate methods, and then use the non-water-soluble components.
  • the protein and peptide components separated and extracted from the water-soluble components are used as antigen components together with all water-soluble components; (3) Cancer cells/tumor tissues are first lysed, and then the water-soluble components and non-water-soluble components are prepared respectively. , then dissolve the non-water-soluble component with a specific dissolving agent containing a dissolving solution, and then separate and extract the protein and peptide components in the water-soluble component from the water-soluble component and the non-water-soluble component using appropriate methods.
  • the above preparation method can also add a step of isolating and extracting whole-cell mRNA, and using the whole-cell mRNA as part of the antigen component.
  • Appropriate methods for separating and extracting protein and polypeptide components include but are not limited to salting out, heating, enzymatic hydrolysis, etc.
  • the preparation method of the protein polypeptide component plus the mRNA component in the whole cell component of cancer cells/tumor tissue is: (1) first lyse the cancer cells/tumor tissue, and then prepare the water-soluble component and the water-insoluble component respectively, The non-water-soluble components are then dissolved in a specific dissolving agent containing a dissolving solution before use; then the protein and polypeptide components and the mRNA components in the water-soluble components and/or the non-water-soluble components are separated and extracted, and then the proteins and The polypeptide component and the mRNA component are mixed and used as the antigen component.
  • the above-mentioned dissolving agent is selected from one of the compounds containing the structure of structural formula 1, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, polypeptide, amino acid, glycoside and choline or more; wherein, Structural Formula 1 is as follows: Structural Formula 1 has the following structure:
  • R1 is C, N, S or O
  • R2 to R5 are independently selected from at least one of hydrogen, alkyl, amino, carboxyl, substituted or unsubstituted guanidyl.
  • 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, other compounds containing guanidine or urea, guanidine carbonate, arginine, guanidine acetic acid, guanidine phosphate, guanidine sulfamate, guanidine
  • Nanoparticles or microparticles loaded with cell components can be prepared by any preparation method for loading antigen components into nano/microparticles, including but not limited to solvent evaporation method, dialysis method, microfluidic method, extrusion method, thermal Any of the melting method and other methods.
  • the antigen component can be loaded inside the nanoparticles/microparticles, or on the surface of the nanoparticles/microparticles, or both inside and on the surface of the nanoparticles/microparticles.
  • the solvent evaporation method is used to illustrate the specific implementation. In practical applications, any other feasible preparation method can also be used.
  • the preparation method of the nanoparticles or microparticles includes the following steps:
  • step 2 Subject the mixed solution obtained in step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute;
  • step 4 the mixed solution that meets the predetermined stirring conditions is centrifuged at a speed greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in the fifth predetermined volume of Five predetermined concentrations of an aqueous solution containing a lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).
  • the aqueous solution can contain each component in the cancer cell lysate and the immune-enhancing adjuvant poly(I:C), BCG, manganese adjuvant, calcium adjuvant or CpG; Cancer
  • Each component in the cell lysate is a water-soluble component or an original non-water-soluble component dissolved in urea or guanidine hydrochloride during preparation.
  • the concentration of the water-soluble components from the cancer cells contained in the aqueous solution or the concentration of the original non-water-soluble components from the cancer cells dissolved in urea or guanidine hydrochloride, that is, the first predetermined concentration requires that the protein polypeptide concentration content is greater than 1ng/mL, can load enough cancer antigens to activate relevant immune responses.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the aqueous solution contains each component in the tumor tissue lysate and the immune-enhancing adjuvant poly(I:C), BCG, manganese adjuvant, calcium adjuvant or CpG; each component in the tumor tissue lysate is prepared separately It is a water-soluble component or the original non-water-soluble component dissolved in urea or guanidine hydrochloride.
  • the aqueous solution contains a concentration of water-soluble components derived from tumor tissue or a concentration of original non-water-soluble components dissolved in urea or guanidine hydrochloride from tumor tissue, that is, the first predetermined concentration requires protein polypeptide concentration content Greater than 0.01ng/mL, it can load enough cancer antigens to activate relevant immune responses.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the medical polymer material is dissolved in the organic solvent to obtain a second predetermined volume of organic phase containing the medical polymer material at a second predetermined concentration.
  • the medical polymer material is PLGA
  • the organic solvent is methylene chloride.
  • the second predetermined concentration of the medical polymer material ranges from 0.5 mg/mL to 5000 mg/mL, preferably 100 mg/mL.
  • the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase.
  • the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1:1.1-1:5000, preferably 1:10.
  • the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.
  • the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL ⁇ 100 mg/mL; when the aqueous phase solution is a lysate component/immune adjuvant solution, wherein The concentration of protein and polypeptide is greater than 1ng/mL, preferably 1mg/mL ⁇ 100mg/mL, and the concentration of immune adjuvant is greater than 0.01ng/mL, preferably 0.01mg/mL ⁇ 20mg/mL.
  • the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., with dichloromethane being preferred;
  • the concentration of the polymer material is 0.5 mg/mL ⁇ 5000mg/mL, preferably 100mg/mL.
  • the first emulsifier solution is preferably a polyvinyl alcohol aqueous solution, with a concentration of 10 mg/mL to 50 mg/mL, preferably 20 mg/mL.
  • the second emulsifier solution is preferably a polyvinyl alcohol aqueous solution with a concentration of 1 mg/mL to 20 mg/mL, preferably 5 mg/mL.
  • the dispersion liquid is PBS buffer, physiological saline or pure water.
  • Step 2 subject the mixed solution obtained in Step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute.
  • the stirring is mechanical stirring or magnetic stirring
  • the stirring speed is greater than 50 rpm
  • the stirring time is greater than 1 minute.
  • the stirring speed is 50 rpm to 1500 rpm
  • the stirring time is 0.1 hour to 24 hours
  • the ultrasonic power is greater than 5W
  • the time Greater than 0.1 seconds such as 2 to 200 seconds
  • the pressure is greater than 5 psi, such as 20 psi to 100 psi.
  • the rotation speed of the shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; the flow rate of microfluidic processing is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used for nanonization and/or micronization.
  • the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared micro-nano particles. Too large or too small will cause to changes in particle size.
  • Step 3 Add the mixture obtained after step 2 to a third predetermined volume of aqueous solution containing a third predetermined concentration of emulsifier and perform ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or perform homogenization or microfluidic treatment. deal with.
  • the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be ultrasonically or stirred to form nanometers or micrometers.
  • This step is for nanonization or micronization.
  • the length of ultrasonic time or stirring speed and time can control the size of the prepared nanoparticles or microparticles. Too long or too short will cause changes in particle size. For this reason, it is necessary to choose the appropriate ultrasound time.
  • the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds
  • the stirring speed is greater than 50 rpm, such as 50 rpm to 500 rpm
  • the stirring time is greater than 1 minute, such as 60 to 6000 seconds.
  • the stirring speed is 50 rpm ⁇ 1500 rpm and the stirring time is 0.5 hours ⁇ 5 hours; during ultrasonic treatment, the ultrasonic power is 50W ⁇ 500W.
  • the time is greater than 0.1 seconds, such as 2 to 200 seconds; when homogenizing, use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer.
  • the pressure is greater than 20 psi, such as 20 psi to 100 psi.
  • the rotation speed is greater than 1000rpm, such as 1000rpm to 5000rpm; when using microfluidic processing, the flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used to nanonize or micronize the particles. The length of ultrasonic time or stirring speed or homogenization process pressure and time can control the size of the prepared nano or micron particles. Too large or too small will cause Changes in particle size.
  • the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined volume is 5 mL
  • the third predetermined concentration is 20 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, and preferably can be 2:5.
  • the ratio of the second predetermined volume and the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles or microparticles of suitable size.
  • Step 4 Add the liquid obtained after the treatment in Step 3 to a fourth predetermined volume of the emulsifier aqueous solution with a fourth predetermined concentration, and stir until the predetermined stirring conditions are met.
  • the emulsifier aqueous solution is still PVA.
  • the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size.
  • the selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume.
  • the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10.
  • the ratio between the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.
  • the predetermined stirring condition of this step is until the volatilization of the organic solvent is completed, that is, the volatilization of methylene chloride in step 1 is completed.
  • Step 5 After centrifuging the mixed liquid that meets the predetermined stirring conditions in Step 4 at a rotation speed of greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in a fifth predetermined volume of Five predetermined concentrations of an aqueous solution containing a lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).
  • step 5 when the precipitate obtained in step 5 is resuspended in the sixth predetermined volume of PBS (or physiological saline), there is no need to freeze-dry, and the subsequent adsorption of cancer cell lysates on the surface of nanoparticles or microparticles can be directly performed.
  • PBS physiological saline
  • the precipitate obtained in step 5 needs to be freeze-dried when resuspended in an aqueous solution containing a lyoprotectant, and then freeze-dried before subsequent adsorption of cancer cell lysates on the surface of nanoparticles or microparticles. experiment.
  • Trehalose is selected as the freeze-drying protective agent.
  • the fifth predetermined concentration of the freeze-drying protective agent in this step is 4% by mass. The reason why this is set is to not affect the freeze-drying effect during subsequent freeze-drying.
  • Step 6 After freeze-drying the suspension containing the lyoprotectant obtained in Step 5, the freeze-dried material is used for later use.
  • Step 7 Resuspend a sixth predetermined volume of the nanoparticle-containing suspension obtained in Step 5 in PBS (or physiological saline) or use a sixth predetermined volume of PBS (or physiological saline) to resuspend the nanoparticle-containing suspension obtained in Step 6
  • PBS or physiological saline
  • the freeze-dried substance containing nanoparticles or microparticles and a lyoprotectant is used directly; or the above sample is mixed with a seventh predetermined volume of water-soluble components or solubilized original non-water-soluble components and used.
  • the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferred volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1 .
  • the volume of the resuspended nanoparticle suspension when the volume of the resuspended nanoparticle suspension is 10 mL, it contains cancer cell lysates or water-soluble components in tumor tissue lysates or solubilized original non-water-soluble components.
  • the volume is 1mL. In actual use, the volume and proportion of the two can be adjusted as needed.
  • the method for preparing nanoparticles or microparticles by the double emulsion method includes the following steps:
  • step 2 Subject the mixed solution obtained in step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute.
  • step 4 the mixed solution that meets the predetermined stirring conditions is centrifuged at a speed greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in the fifth predetermined volume of five predetermined concentrations of a solution containing the water-soluble and/or non-water-soluble components of the whole cell fraction, or the remaining pellet is resuspended in a fifth predetermined volume of a fifth predetermined concentration of the whole cell fraction.
  • step 5 After centrifuging the mixed liquid that meets the predetermined stirring conditions in step 5 for more than 1 minute at a rotation speed of more than 100 RPM, remove the supernatant, and resuspend the remaining sediment in the sixth predetermined volume of solidification solution.
  • the treatment reagent or mineralization treatment reagent is centrifuged and washed after acting for a certain period of time, and then the seventh predetermined substance containing positively or negatively charged substances is added and acted for a certain period of time.
  • the aqueous solution may contain each component in the cancer cell lysate and the immune-enhancing adjuvant poly(I:C), manganese adjuvant, calcium adjuvant, BCG or CpG; in the cancer cell lysate During preparation, each component is a water-soluble component or an original non-water-soluble component dissolved in urea or guanidine hydrochloride.
  • each component is a water-soluble component or an original non-water-soluble component dissolved in urea or guanidine hydrochloride.
  • the concentration of the water-soluble components from the cancer cells contained in the aqueous solution or the concentration of the original non-water-soluble components from the cancer cells dissolved in urea or guanidine hydrochloride, that is, the first predetermined concentration requires that the protein polypeptide concentration content is greater than 0.01ng/mL, it can load enough cancer antigens to activate relevant immune responses.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the aqueous solution contains each component in the tumor tissue lysate and the immune-enhancing adjuvant poly(I:C), manganese adjuvant, calcium adjuvant, BCG or CpG; each component in the tumor tissue lysate
  • the components are respectively water-soluble components or original non-water-soluble components dissolved in urea or guanidine hydrochloride during preparation.
  • the aqueous solution contains a concentration of water-soluble components derived from tumor tissue or a concentration of original non-water-soluble components dissolved in urea or guanidine hydrochloride from tumor tissue, that is, the first predetermined concentration requires protein polypeptide concentration content Greater than 0.01ng/mL, it can load enough cancer antigens to activate relevant immune responses.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the medical polymer material is dissolved in the organic solvent to obtain a second predetermined volume of organic phase containing the medical polymer material at a second predetermined concentration.
  • the medical polymer material is PLGA
  • the organic solvent is methylene chloride.
  • the second predetermined concentration of the medical polymer material ranges from 0.5 mg/mL to 5000 mg/mL, preferably 100 mg/mL.
  • PLGA or modified PLGA is selected because the material is biodegradable and has been approved by the FDA for use as a pharmaceutical dressing. Studies have shown that PLGA has certain immunomodulatory functions and is therefore suitable as an excipient in the preparation of nanoparticles or microparticles.
  • the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase.
  • the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1:1.1-1:5000, preferably 1:10.
  • the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.
  • the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL ⁇ 100 mg/mL; when the aqueous phase solution is a lysate component/immune adjuvant solution, wherein The concentration of protein and polypeptide is greater than 1ng/mL, preferably 1mg/mL ⁇ 100mg/mL, and the concentration of immune adjuvant is greater than 0.01ng/mL, preferably 0.01mg/mL ⁇ 20mg/mL.
  • the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., with dichloromethane being preferred;
  • the concentration of the polymer material is 0.5 mg/mL ⁇ 5000mg/mL, preferably 100mg/mL.
  • the first emulsifier solution is preferably a polyvinyl alcohol aqueous solution, with a concentration of 10 mg/mL to 50 mg/mL, preferably 20 mg/mL.
  • the second emulsifier solution is preferably a polyvinyl alcohol aqueous solution with a concentration of 1 mg/mL to 20 mg/mL, preferably 5 mg/mL.
  • the dispersion liquid is PBS buffer, physiological saline or pure water.
  • Step 2 subject the mixed solution obtained in Step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute.
  • the stirring is mechanical stirring or magnetic stirring
  • the stirring speed is greater than 50 rpm
  • the stirring time is greater than 1 minute.
  • the stirring speed is 50 rpm to 1500 rpm
  • the stirring time is 0.1 hour to 24 hours
  • the ultrasonic power is greater than 5W
  • the time Greater than 0.1 seconds such as 2 to 200 seconds
  • the pressure is greater than 5 psi, such as 20 psi to 100 psi.
  • the rotation speed of the shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; the flow rate of microfluidic processing is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used for nanonization and/or micronization.
  • the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared micro-nano particles. Too large or too small will cause to changes in particle size.
  • Step 3 Add the mixture obtained after step 2 to a third predetermined volume of aqueous solution containing a third predetermined concentration of emulsifier and perform ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or perform homogenization or microfluidic treatment. deal with.
  • the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be ultrasonically or stirred to form nanometers or micrometers.
  • This step is for nanonization or micronization.
  • the length of ultrasonic time or stirring speed and time can control the size of the prepared nanoparticles or microparticles. Too long or too short will cause changes in particle size. For this reason, it is necessary to choose the appropriate ultrasound time.
  • the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds
  • the stirring speed is greater than 50 rpm, such as 50 rpm to 500 rpm
  • the stirring time is greater than 1 minute, such as 60 to 6000 seconds.
  • the stirring speed is 50 rpm ⁇ 1500 rpm and the stirring time is 0.5 hours ⁇ 5 hours; during ultrasonic treatment, the ultrasonic power is 50W ⁇ 500W.
  • the time is greater than 0.1 seconds, such as 2 to 200 seconds; when homogenizing, use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer.
  • the pressure is greater than 20 psi, such as 20 psi to 100 psi.
  • the rotation speed is greater than 1000rpm, such as 1000rpm to 5000rpm; when using microfluidic processing, the flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used to nanonize or micronize the particles. The length of ultrasonic time or stirring speed or homogenization process pressure and time can control the size of the nano or micron particles prepared. Too large or too small will cause Changes in particle size.
  • the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined volume is 5 mL
  • the third predetermined concentration is 20 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, and preferably can be 2:5.
  • the ratio of the second predetermined volume and the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles or microparticles of suitable size.
  • Step 4 Add the liquid obtained after the treatment in Step 3 to a fourth predetermined volume of the emulsifier aqueous solution with a fourth predetermined concentration, and stir until the predetermined stirring conditions are met, or the subsequent treatment can be performed directly without stirring.
  • the emulsifier aqueous solution is still PVA.
  • the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size.
  • the selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume.
  • the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10.
  • the ratio of the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.
  • the predetermined stirring condition of this step is that the volatilization of the organic solvent is completed, that is, the volatilization of methylene chloride in step 1 is completed. Nor should subsequent tests be carried out without stirring.
  • Step 5 After centrifuging the mixed liquid that meets the predetermined stirring conditions in Step 4 at a rotation speed of greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in a fifth predetermined volume of five predetermined concentrations of a solution containing the water-soluble and/or non-water-soluble components of the whole cell fraction, or the remaining pellet is resuspended in a fifth predetermined volume of a fifth predetermined concentration of the whole cell fraction.
  • Step 6 After centrifuging the mixed solution that meets the predetermined stirring conditions in Step 5 at a rotation speed of greater than 100 RPM for greater than 1 minute, remove the supernatant, and resuspend the remaining sediment in a sixth predetermined volume of solidified liquid.
  • the treatment reagent or mineralization treatment reagent is centrifuged and washed after acting for a certain period of time, and then the seventh predetermined substance containing positively or negatively charged substances is added and acted for a certain period of time.
  • the precipitate obtained in step 6 does not need to be freeze-dried after being resuspended in a seventh predetermined volume of charged substance, and subsequent experiments related to loading cancer cells/tissue lysates on the surface of nanoparticles or microparticles can be directly performed.
  • the precipitate obtained in step 6 is resuspended in an aqueous solution containing a drying protective agent and then subjected to room temperature vacuum drying or freeze vacuum drying. After drying, the subsequent nanoparticles or microparticles surface adsorb cancer cell lysates. related experiments.
  • the freeze-drying protective agent is trehalose or a mixed solution of mannitol and sucrose.
  • concentration of the drying protective agent in this step is 4% by mass, which is set so as not to affect the drying effect during subsequent drying.
  • Step 7 After drying the suspension containing the drying protective agent obtained in Step 6, the dried material is used for later use.
  • Step 8 Resuspend an eighth predetermined volume of the nanoparticle-containing suspension obtained in Step 6 in PBS (or physiological saline) or use an eighth predetermined volume of PBS (or physiological saline) to resuspend the nanoparticle-containing suspension obtained in Step 7
  • PBS or physiological saline
  • the dried material containing nanoparticles or microparticles and a drying protective agent is used directly; or it is used after being mixed with a ninth predetermined volume of water-soluble components or non-water-soluble components.
  • the modification and antigen loading steps of steps 5 to 8 can be repeated multiple times to increase the loading capacity of the antigen.
  • substances with the same charge can be added multiple times or substances with different charges can be added alternately.
  • the volume of the resuspended nanoparticle suspension when the volume of the resuspended nanoparticle suspension is 10 mL, the sum of the volumes of the water-soluble components or the original non-water-soluble components in the cancer cell lysate or tumor tissue lysate is 0.1 -100mL. In actual use, the volume and proportion of the two can be adjusted as needed.
  • the water-soluble component or the original non-water-soluble component of the cancer cell lysate or tumor tissue lysate used contains poly(I:C), manganese adjuvant, Bacillus Calmette-Guérin (BCG) or CpG , and the concentration of poly(I:C), calcium adjuvant, BCG or CpG is greater than 0.01ng/mL.
  • nanoparticles and/or microparticles loaded only with water-soluble components and nanoparticles loaded only with non-water-soluble components can be used at the same time.
  • the nanoparticles or microparticles loaded with tumor antigens are prepared by any method, the nanoparticles or microparticles are simultaneously incubated with antigen-presenting cells and T cells to activate cancer cell-specific T cells.
  • the nanoparticles and/or microparticles loaded with tumor antigen components are first co-incubated with antigen-presenting cells to activate the antigen-presenting cells, and then the activated antigen-presenting cells are separately incubated with T cells. Activates cancer cell-specific T cells.
  • the T cells Before co-incubating T cells with nanoparticles/microparticles and antigen-presenting cells, the T cells can be cultured alone for a period of time or appropriately sorted; or the T cells can be incubated with activated antigen-presenting cells. Before co-incubation, T cells can be cultured alone for a period of time or appropriately sorted.
  • Nanoparticles and/or microparticles loaded with tumor antigen components are first incubated with antigen-presenting cells to activate the antigen-presenting cells.
  • the antigen-presenting cells can then be incubated with T cells to activate specificity without special treatment.
  • T cells or antigen-presenting cells can be fixed, irradiated, irradiated, modified, inactivated, mineralized, etc., and then co-incubated with T cells to activate specific T cells.
  • cancer cell separation methods such as flow cytometry or magnetic bead sorting are used to separate the activated cancer cell-specific T cells from the incubated cells, and then the cancer cell-specific T cells are separated. After the T cells are expanded in vitro for a certain period of time, the amplified cancer cell-specific T cells obtained are used to prevent or treat cancer.
  • one activation marker for activated T cells can be used, or a combination of more than one different markers can be used as an activation marker.
  • Molecules that can be used as surface markers include, but are not limited to: 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, CD11
  • Example 1 Isolation and expansion of T cells for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticles to isolate and expand peripheral cancer cell-specific T cells for cancer prevention.
  • B16F10 melanoma tumor tissue was lysed to prepare water-soluble components and non-water-soluble components of the tumor tissue.
  • the organic polymer material PLGA was used as the nanoparticle skeleton material
  • Polyinosinic-polycytidylic acid (poly(I :C)) is an immune adjuvant that uses a solvent evaporation method to prepare nanoparticles loaded with water-soluble components and non-water-soluble components of tumor tissue, and then uses the nanoparticles to assist in the isolation of cancer cell-specific T cells in the organ, and the isolation is Cancer cell-specific T cells are expanded and injected into the body to prevent melanoma.
  • This embodiment uses immune cells from mouse peripheral spleen cells. In practical applications, peripheral blood or peripheral lymph node cells can be used directly.
  • B16F10 cells 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor tissue was removed.
  • the tumor tissue is cut into pieces, ground, and passed through a cell filter to prepare a tumor tissue single cell suspension (containing cancer cells). Then add an appropriate amount of pure water to the tumor tissue single cell suspension and freeze and thaw repeatedly 5 times, and may be accompanied by ultrasound to destroy the lysed cells.
  • the nanoparticles were prepared by the double emulsion method in the solvent evaporation method. During preparation, nanoparticles loaded with water-soluble components in the whole cell component and nanoparticles loaded with non-water-soluble components in the whole cell component are prepared separately and then used together.
  • the molecular weight of PLGA, the nanoparticle preparation material used, is 24KDa-38KDa, and the immune adjuvant used is poly(I:C). The preparation method is as described above.
  • the double emulsion method is first used to load the antigen components and adjuvants inside the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose is used. Resuspend and freeze-dry for 48 hours.
  • the average particle size of the nanoparticles is about 280nm, and the surface potential of the nanoparticles is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.02 mg of poly(I:C).
  • the preparation materials and methods for blank nanoparticles are the same, and the particle size is about 260nm.
  • pure water containing an equal amount of poly(I:C) or 8M urea is used to replace the corresponding water-soluble components and non-water-soluble components. components.
  • mice Inoculate 0.5 ⁇ 10 5 B16F10 cells subcutaneously on the back of each C57BL/6 mouse.
  • the mice are killed and the mouse spleen is removed.
  • a single cell suspension of mouse spleen cells is prepared and red blood cells are analyzed. Lysis treatment to remove red blood cells from single cell suspensions.
  • the above cells were incubated with T cell magnetic bead sorting reagents (CD3 + magnetic bead sorting reagent, CD8 + magnetic bead sorting reagent) or B cell magnetic bead sorting reagents, and then used a magnetic bead sorter to CD3 + CD8 + T cells and CD19 + B cells were sorted from mouse spleen cells.
  • the CD3 + CD8 + T cells (10,000 cells, cell viability 60%) obtained by the above sorting were mixed with IL-2 (500U/mL), IL-7 (200U/mL), IL-15 (200 U/mL) and ⁇ CD3/ ⁇ CD28 (10 ng/mL) were co-incubated in 10 mL of RPMI 1640 complete medium at 37°C (5% CO 2 ) for 12 days (with the above-mentioned cytokines and antibodies every three days) Medium replacement) to expand the resulting CD8 + T cells (cell viability 60%).
  • IL-2 500U/mL
  • IL-7 200U/mL
  • IL-15 200 U/mL
  • ⁇ CD3/ ⁇ CD28 10 ng/mL
  • the sorted 1 million CD8 + T cells and 10 million B cells were combined with nanoparticles loaded with all tumor tissue antigen components (250 ⁇ g nanoparticles loaded with water-soluble components + 250 ⁇ g loaded Nanoparticles with non-water-soluble components) or blank nanoparticles (500 ⁇ g) + an equal amount of free lysate were incubated in 10 mL of DMEM high-glucose complete medium for a total of 96 hours, and then the incubated cells were separated with magnetic beads.
  • CD3 + T cell magnetic bead sorting reagent, CD8 + T cell magnetic bead sorting reagent, and CD69 magnetic bead sorting reagent are sequentially co-incubated and sorted accordingly, and the magnetic bead sorting method is used to sort the incubated T cells.
  • the CD3 + CD8 + CD69 + T cells are cancer-specific T cells activated by cancer antigens.
  • the cancer-specific T cells (10,000) obtained by the above sorting were combined with IL-2 (500U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and ⁇ CD3/ ⁇ CD28 (10ng/ mL) in RPMI 1640 complete medium and incubated for 12 days at 37°C (5% CO 2 ) (the medium containing the above cytokines and antibodies was changed every three days) to amplify the specificity of the sorted cancer cells. T cells (cell viability 60%).
  • the ratio of IFN- ⁇ + T cells to CD8 + T cells The cancer cell antigens loaded on the nanoparticles can be degraded into antigen epitopes after being engulfed by antigen-presenting cells (B cells) and presented to the surface of the antigen-presenting cells. Specific T cells that can recognize cancer cell antigens can recognize them. Cancer cells are activated after epitopes and secrete killer cytokines. IFN- ⁇ is the most important cytokine secreted by antigen-specific T cells that are activated after recognizing the antigen. However, since it is a secreted cytokine, 4% paraformaldehyde needs to be added first for cell fixation, and then a membrane-breaking agent is used.
  • the CD8 + IFN- ⁇ + T cells obtained by flow cytometry analysis are cancer cell-specific T cells that can specifically recognize cancer cell antigen epitopes.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma tumor-bearing mice.
  • the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate the recipient mice. immune cells in mice.
  • the 2 million cancer cell-specific T cells prepared by the two-step sorting method in step (3) or the 2 million CD3 + T cells expanded after the one-step sorting method were intravenously injected into the recipient mice.
  • each recipient mouse was inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 cells on the lower right side of the back. Monitor mouse tumor growth rate and mouse survival time.
  • mice in the PBS control group had the fastest tumor growth and the shortest mouse survival period.
  • the T cells obtained by the one-step sorting method and the T cells obtained by blank nanoparticle-assisted sorting and amplification were treated in the group.
  • the tumor growth rate and survival time of the mice were improved compared with the PBS control group.
  • mice treated with cancer cell-specific T cells obtained after nanoparticle-assisted sorting and amplification of whole cell components of cancer cells had the slowest tumor growth rate and the longest survival period.
  • the sorted and expanded cell system of the present invention has a good preventive effect on cancer.
  • the cancer cell-specific T cells obtained by the two-step sorting method can almost 100% recognize cancer cell antigens and be treated with them after co-incubation with antigen-presenting cells and nanoparticles loaded with cancer cell whole cell components. Cancer antigens are activated, and the CD8 + IFN- ⁇ + T cells amplified by the one-step sorting method only account for less than 5% of the CD8 + T cells after co-incubation. It can be seen that the sorting method of the present invention The method can effectively enrich cancer cell-specific T cells with killing ability.
  • a surface marker is used as a marker for T cell activation.
  • a combination of more than one different markers can also be used as an activation marker.
  • Example 2 Isolation and expansion of peripheral cancer cell-specific T cells for the prevention of melanoma
  • B16F10 melanoma tumor tissue was lysed to prepare water-soluble components and non-water-soluble components of the tumor tissue. Then, organic polymer materials were used as nanoparticle skeleton materials, and poly(I:C) and CpG1018 were used as The immune adjuvant uses a solvent evaporation method to prepare nanoparticles loaded with water-soluble components and non-water-soluble components of tumor tissue, and then uses the nanoparticles to sort and expand peripheral cancer cell-specific T cells. This embodiment uses immune cells in mouse peripheral spleen cells. In practical applications, peripheral blood or peripheral lymph node cells can be used.
  • the nanoparticles and the blank nanoparticles used as a control were prepared by the solvent evaporation method.
  • nanoparticles loaded with water-soluble components in the whole cell component and nanoparticles loaded with non-water-soluble components in the whole cell component are prepared separately and then used together.
  • the molecular weight of PLGA, the nanoparticle preparation material used, is 7KDa-17KDa, and the immune adjuvants used are poly(I:C) and CpG1018.
  • the preparation method is as described above.
  • the double emulsion method is first used to load the antigen components and adjuvants inside the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose is used. Resuspend and freeze-dry for 48 hours.
  • the average particle size of the nanoparticles is about 280nm, and the surface potential of the nanoparticles is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components, and each of poly(I:C) and CpG1018 is loaded with 0.02 mg.
  • the preparation materials and preparation methods of blank nanoparticles are the same, and the particle size is about 260 nm.
  • the blank nanoparticles are loaded with an equal amount of adjuvant but do not load any antigen components.
  • mice Inoculate 1.5 ⁇ 10 5 B16F10 cells subcutaneously on the back of each C57BL/6 mouse, and inoculate the mice on the 4th day, the 7th day, the 10th day, the 15th day, the 20th day, the 25th day and the 30th day respectively.
  • Mice were injected subcutaneously with 1 mg of PLGA nanoparticles loaded with water-soluble components and 1 mg of PLGA nanoparticles loaded with non-water-soluble components. The mice were killed on the 34th day, the spleens of the mice were collected, and a single cell suspension of mouse spleen cells was prepared.
  • the above cells were sequentially co-incubated with T magnetic bead sorting reagent and B cell magnetic bead sorting reagent and analyzed sequentially. Select and sequentially use a magnetic bead sorter to sort CD3 + T cells and CD19 + B cells from mouse spleen cells.
  • CD3 + T cells obtained by the above sorting were mixed with IL-2 (500U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and ⁇ CD3 / ⁇ CD28 (10 ng/mL each) was incubated in 10 mL of RPMI 1640 complete medium at 37°C (5% CO 2 ) for 14 days (the medium was changed every two days with medium containing the above cytokines and antibodies) to amplify The resulting T cells (cell viability 65%).
  • the sorted 5 million CD3 + T cells and 10 million B cells were combined with nanoparticles loaded with all tumor tissue antigen components (250 ⁇ g nanoparticles loaded with water-soluble components + 250 ⁇ g loaded Nanoparticles with non-water-soluble components) or blank nanoparticles (500 ⁇ g) + an equal amount of free antigen components were incubated in 10 mL of RPMI 1640 complete medium for 48 hours, and then the incubated cells were combined with the magnetic bead separation method.
  • the reagents are co-incubated, and flow cytometry is used to sort the CD3 + CD137 + T cells in the incubated T cells, which are cancer-specific T cells activated by cancer antigens (cell viability 65%).
  • the cancer cell-specific T cells (1 million) obtained by the above sorting were mixed with IL-2 (500U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and ⁇ CD3/ ⁇ CD28 (each 10 ng/mL) in 10 mL of RPMI 1640 complete medium (37°C, 5% CO 2 ) and incubated for a total of 14 days (the medium containing the above cytokines and antibodies was changed every two days) to amplify the sorted cancer cells.
  • Cell-specific T cells (cell viability 65%).
  • the T cells (500,000) expanded by the one-step sorting method in the control group or the cancer cell-specific T cells (500,000) expanded by the two-step sorting method were combined with B cells (2 million) and Nanoparticles (60 ⁇ g) loaded with whole cell components were incubated in 3 mL of RPMI 1640 complete medium for a total of 48 hours, and then the incubated cells were collected and labeled with CD3 antibodies, CD8 antibodies, and IFN- ⁇ antibodies with different fluorescent probes. After incubation, flow cytometry was used to analyze the ratio of CD3 + IFN- ⁇ + T cells to CD3 + T cells in the cells expanded after one-step sorting and two-step sorting.
  • the CD3 + IFN- ⁇ + T (cell viability rate 0%) cells obtained by flow cytometry analysis are cancer cell-specific T cells that can specifically recognize cancer cell antigens.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma tumor-bearing mice.
  • the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate the recipient mice. immune cells in mice.
  • the 2 million cancer cell-specific T cells obtained through two-step sorting and amplification in step (3) or the 2 million T cells obtained after one-step sorting and amplification were intravenously injected into the recipient mice.
  • each recipient mouse was inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 cells on the lower right side of the back.
  • the methods for monitoring tumor growth rate and survival period in mice are the same as above.
  • mice in the PBS control group had the fastest tumor growth rate and the shortest survival period.
  • the T cells obtained by the one-step sorting method and the T cells obtained by blank nanoparticle-assisted sorting were treated with The tumor growth rate and survival period of the mice were improved compared with the PBS control group.
  • mice treated with cancer cell-specific T cells obtained after sorting and amplifying nanoparticles loaded with whole-cell antigen components had the slowest tumor growth and the longest survival period.
  • the sorted and expanded cell system of the present invention has a good preventive effect on cancer.
  • the cancer cell-specific T cells obtained by the two-step sorting method can almost 100% recognize cancer cell antigens after being co-incubated with antigen-presenting cells and nanoparticles loaded with cancer cell whole cell components. Cancer cell antigens are activated, and some of the T cells amplified by the selection method only account for less than 5% of CD3 + T cells after co-incubation. It can be seen that the sorting method of the present invention The method can effectively enrich cancer cell-specific T cells with killing ability.
  • Example 3 Sorting and amplifying cancer cell-specific T cells for use in the treatment of melanoma
  • B16F10 melanoma tumor tissue and cancer cells are first lysed to prepare a mixture of water-soluble components (mass ratio 1:1) and a mixture of non-water-soluble components (mass ratio 1:1) of tumor tissue and cancer cells, Then, using PLGA as the nanoparticle skeleton material and Poly(I:C), CpG2006 and CpG2216 as adjuvants, a nanoparticle system loaded with a mixture of water-soluble components and a mixture of non-water-soluble components was prepared, and then the nanoparticles were combined with T cells Co-incubate with antigen-presenting cells in vitro to activate pre-existing cancer cell-specific T cells. After activation, cancer cell-specific T cells will highly express specific molecules. They can be sorted using flow cytometry and then expanded. Used to treat cancer.
  • B16F10 cells When collecting tumor tissue, 1.5 ⁇ 10 5 B16F10 cells were first subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to a volume of approximately 1000 mm 3 , the mice were sacrificed and the tumor tissue was removed. The tumor tissue was cut into sections. Grind, add an appropriate amount of pure water after passing through the cell filter, and freeze and thaw repeatedly 5 times, accompanied by ultrasound to destroy the lysed sample; when collecting the cultured B16F10 cancer cell line, first centrifuge to remove the medium, then wash twice with PBS and centrifuge Cancer cells were collected, resuspended in ultrapure water, frozen and thawed three times, and destroyed and lysed by ultrasound.
  • the tumor tissue or cancer cell line After the tumor tissue or cancer cell line is lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant as the water-soluble component that is soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate.
  • the non-water-soluble components that are insoluble in pure water can be converted into soluble in 8M urea aqueous solution.
  • the above are the antigen components for preparing nanoparticles.
  • the nanoparticles were prepared using the double emulsion method.
  • the molecular weight of the nanoparticle preparation material PLGA is 7KDa-17KDa, and the immune adjuvants used are poly(I:C), CpG2006 and CpG2216.
  • the preparation method is as described above, first load the antigen components and adjuvants inside the nanoparticles, then centrifuge 100 mg of the nanoparticles at 12,000 g for 25 minutes, resuspend in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dry for 48 h; Before use, resuspend it in 9 mL of PBS and then add 1 mL of lysis buffer component (protein concentration 80 mg/mL) and incubate at room temperature for 10 min to obtain nanoparticles loaded with antigen components inside and outside.
  • lysis buffer component protein concentration 80 mg/mL
  • the average particle size of the nanoparticles is about 280nm, and the surface potential of the nanoparticles is about -5mV; every 1 mg of PLGA nanoparticles is loaded with approximately 130 ⁇ g of protein or peptide components, and every 1 mg of PLGA nanoparticles is loaded with poly(I:C), CpG2006 and CpG2216 immune Each adjuvant is 0.02mg.
  • the preparation materials and methods of blank nanoparticles are the same, and the particle size is about 260 nm.
  • the blank nanoparticles are loaded with an equal amount of adjuvant but do not load any cancer cell lysate components.
  • mice Inoculate 1.5 ⁇ 10 5 B16F10 cells subcutaneously on the back of each C57BL/6 mouse, and subcutaneously inject 2 mg PLGA into the mice on day 4, day 7, day 10, day 15, day 20 and three days respectively. Nanoparticles (loaded with antigen components and adjuvants). The mice were sacrificed on the 24th day, and the peripheral blood of the mice was collected. Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of the mice using gradient centrifugation. The PBMC were incubated with CD3 antibodies and CD19 antibodies labeled with different fluorescent probes. CD3 + T cells and CD19 + B cells were then sorted using flow cytometry.
  • PBMC Peripheral blood mononuclear cells
  • the preparation method of mouse bone marrow-derived macrophages is a conventional preparation method.
  • the preparation method of BMDM is as follows: C57 mice are anesthetized and killed by dislocation. The mice are disinfected with 75% ethanol. Then use scissors to cut a small opening on the back of the mouse. Tear the skin directly to the calf joint of the mouse and remove it. Mouse foot joints and skin. Use scissors to remove the hind limbs along the greater trochanter at the root of the mouse's thigh, remove the muscle tissue and place it in a petri dish containing 75% ethanol to soak for 5 minutes. Replace the petri dish with a new one with 75% ethanol and move it to a clean bench.
  • Macrophage colony-stimulating factor stimulates bone marrow cells to differentiate into mononuclear macrophages at a concentration of 40ng/mL. After culturing for 8 days, the morphological changes of macrophages were observed under a light microscope. Digest and collect the cells after 8 days, use anti-mouse F4/80 antibody and anti-mouse CD11b antibody, incubate at 4°C for 30 minutes in the dark, and use flow cytometry to identify the proportion of successfully induced macrophages.
  • M-CSF Macrophage colony-stimulating factor
  • the sorted 5 million CD3 + T cells, 5 million B cells, 5 million BMDM, and nanoparticles (500 ⁇ g) loaded with all tumor tissue antigen components were incubated in 10 mL of RPMI 1640 complete medium for 48 hours; Or incubate the sorted 1 million CD3 + T cells, 10 million mouse BMDM and nanoparticles (500 ⁇ g) loaded with all tumor tissue antigens in 10 mL of RPMI1640 complete medium for 48 hours; or the sorted The obtained 1 million CD3 + T cells, 5 million B cells, 5 million mouse BMDM, IL-7 (10ng/mL) and nanoparticles (500 ⁇ g) loaded with all tumor tissue antigen components were completely dissolved in 10mL of RPMI1640.
  • CD3 + CD137 + T cells which are cancer-specific T cells activated by cancer cell antigens (cell viability 70%).
  • IL-2 1000U/mL
  • IL-7 1000U/mL
  • 10mL RPMI 1640 complete medium 37°C, 5% CO 2
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right side of the back of each mouse. Two million cancer-specific T cells were injected intravenously on days 4, 7, 10, 15, 20 and 25 after melanoma inoculation. The methods for monitoring mouse tumor volume and survival were as above.
  • 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 treatment group after amplification of T cells obtained by several groups of sorting were the same as those in the PBS control group.
  • the cancer-specific T cells obtained through a two-step sorting method using B cells and BMDM as mixed antigen-presenting cells have better anti-cancer effects after expansion than those obtained through a two-step sorting method using BMDM as antigen-presenting cells.
  • the anti-cancer effect of specific T cells after expansion It can be seen that when sorting cancer-specific T cells, using B cells and BMDM as mixed antigen-presenting cells is more effective than using BMDM antigen-presenting cells alone.
  • the cancer-specific T cells obtained by adding IL-7 during the co-incubation process of nanoparticles, antigen-presenting cells and T cells are better than the cancer cell-specific cells obtained by sorting without IL-7 during the incubation process. T cells.
  • Example 4 Cell system used to prevent melanoma lung metastasis
  • This example uses a mouse melanoma lung model to illustrate how to use cell systems to prevent cancer metastasis.
  • B16F10 melanoma tumor tissue is first lysed to prepare water-soluble components and non-water-soluble components of the tumor tissue; then, nanoparticles loaded with water-soluble components and non-water-soluble components of the tumor tissue are prepared.
  • a round of mineralization was carried out.
  • nanoparticles are first used to activate dendritic cells (DC) in vitro, and then the dendritic cells and cancer-specific T cells are co-incubated to activate and assist in the sorting of cancer cell-specific T cells.
  • antigen-presenting cells can be used alive, or antigen-presenting cells that have been inactivated, such as paraformaldehyde fixation or radiation inactivation.
  • B16-F10 cells 1.5 ⁇ 10 5 B16-F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor tissue was removed. Cut the tumor tissue into pieces, grind it, add collagenase (1 mg/mL), and incubate it in RPMI 1640 complete medium for 30 minutes. Then prepare a single cell suspension through a cell filter, add an appropriate amount of pure water, and freeze and thaw 5 times. There is ultrasound to destroy lysed cells.
  • the 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, the material used to prepare the nanoparticles, is 24KDa-38KDa.
  • the immune adjuvant used is poly(I:C), and poly(I:C) is both distributed inside the nanoparticles and loaded on the surface of the nanoparticles.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the cleavage components and adjuvants inside the nanoparticles, then 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and then the nanoparticles are resuspended in 7 mL of PBS and mixed with 3 mL of PBS.
  • Cell lysate 60 mg/mL was mixed with PBS solution, centrifuged at 10,000 g for 20 minutes, and then resuspended in 10 mL of silicate solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate, and 1.0 mM HCl, pH 3.0).
  • each 1 mg of PLGA nanoparticles is loaded with approximately 300 ⁇ g of protein or polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.02 mg of poly(I:C) immune adjuvant and Half and half inside and outside.
  • the steps for preparing unmodified nanoparticles are basically the same as those for preparing modified nanoparticles, except that the steps of low-temperature siliconization and addition of charged substances are not performed.
  • the double emulsion method is first used to load the antigen inside the nanoparticles. After loading the antigen (lysed component) inside, centrifuge at 10,000g for 20 minutes, then resuspend in 10 mL of ultrapure water containing 4% trehalose and then freeze-dry for 48 hours.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 150 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.02 mg of poly(I:C). Half.
  • the preparation materials and methods of blank nanoparticles are the same, and the particle size is about 300 nm.
  • the blank nanoparticles are loaded with an equal amount of adjuvant but do not load any antigen components.
  • This example takes the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare bone marrow-derived dendritic cells (BMDC).
  • BMDC bone marrow-derived dendritic cells
  • RPMI 1640 (10% FBS) medium to stop lysis, centrifuge at 400 g for 3 min, and discard the supernatant.
  • the cells were placed in a 10 mm culture dish and cultured in RPMI 1640 (10% FBS) medium with recombinant mouse GM-CSF (20 ng/mL) added at 37 degrees Celsius and 5% CO 2 for 7 days. On the third day, shake the culture bottle gently and add the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20ng/mL).
  • mice Inoculate 1.5 ⁇ 10 5 B16F10 cells subcutaneously on the back of each C57BL/6 mouse, and inject the mice subcutaneously on the 4th, 7th, 10th, 15th, 20th and 25th days respectively. 100 ⁇ L of 1 mg PLGA nanoparticles. The blank nanoparticles + free lysate control group was injected with the same dose at the corresponding time as a control. The mice were sacrificed on day 24, the mouse spleens were collected, and a single cell suspension of mouse spleen cells was prepared. The mouse spleen cells were incubated with CD3 antibodies and then flow cytometry was used to sort the CD3 + in the splenocyte single cell suspension. T cells.
  • BMDCs (1 million) prepared in step 3 with nanoparticles (40 ⁇ g) loaded with all tumor tissue antigens and incubate them in 3 mL RPMI 1640 complete medium (37°C, 5% CO 2 ) for 48 hours. Then centrifuge at 400g for 5 minutes to collect BMDC, incubate the BMDC with the sorted CD3 + T cells for 24 hours, and then use flow cytometry to sort the CD3 + CD8 + CD69 + T cells (cell viability) in the incubated cells. The rate is 75%) and CD3 + CD4 + CD25 + T cells (the cell viability rate is 75%), which are cancer-specific T cells activated by cancer antigens.
  • the 500,000 cancer cell-specific T cells obtained by the above sorting were mixed with IL-2 (200U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and ⁇ CD3/ ⁇ CD28 in 10mL of RPMI.
  • the cells were incubated in 1640 complete medium for a total of 14 days (the medium was changed every two days) to amplify the sorted cancer cell-specific T cells (cell viability 75%).
  • mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. One day before the mice were adoptively transferred cells, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. Mice were intravenously injected with 100 ⁇ L of 2 million cancer cell-specific T cells (1 million CD3 + CD8 + CD69 + T cells and 1 million CD3 + CD4 + CD25 + T cells) on day 0. At the same time, 0.5 ⁇ 10 5 B16F10 cells were intravenously injected into each mouse on the 1st day. The mice were sacrificed on the 14th day, and the number of melanoma cancer foci in the lungs of the mice was observed and recorded.
  • mice in the PBS control group had many cancer foci, while mice treated with T cells had fewer cancer foci.
  • mice treated with cancer cell-specific T cells obtained by assisted sorting and amplification of modified nanoparticles or unmodified nanoparticles had significantly fewer cancer lesions. This shows that using modified or unmodified nanoparticles to sort and expand T cells can effectively prevent cancer metastasis.
  • Example 5 Micron particle-assisted sorting and amplification of cancer cell-specific T cells for cancer prevention
  • the cultured B16F10 melanoma cancer cell line was collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, and then the cancer cells were resuspended and lysed with 6M guanidine hydrochloride and the lysed whole cell fraction was dissolved, that is To prepare the antigen component of the micron particle system.
  • the micron particles 1 are prepared by the double emulsion method, and the double emulsion method is appropriately modified and improved.
  • the molecular weight of PLGA the material used to prepare the micron particles, is 38KDa-54KDa.
  • the immune adjuvant used is CpG, and CpG is both distributed inside the micron particles and loaded on the surface of the micron particles.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the whole cell components inside the micron particles. After loading the lysis components internally, 100 mg of the micron particles are centrifuged at 10,000g for 15 minutes, and then resuspended in 7 mL of PBS.
  • micron particles were mixed with 3 mL of PBS solution containing cell lysate (50 mg/mL), followed by centrifugation at 10,000 g for 20 minutes, and then 10 mL of silicate solution (containing 120 mM NaCl, 100 mM tetramethyl orthosilicate, and 1.0 mM HCl was used). pH 3.0) and fixed at room temperature for 12 hours.
  • the lysate (50 mg/mL) was resuspended in PBS solution and incubated for 10 min, and then centrifuged at 10,000 g for 20 min. Then use 10mL silicate solution (containing 150mM NaCl, 80mM tetramethyl orthosilicate and 1.0mM HCl, pH 3.0), and fix it at room temperature for 12h, use ultrapure water to centrifuge and wash, and then use 3mL containing histone (5mg/mL ) and polyarginine (10 mg/mL) in PBS and incubated for 10 min, then centrifuged at 10,000 g for 15 min and washed, resuspended in 10 mL of PBS solution containing cell lysate (50 mg/mL) and incubated for 10 min, and then centrifuged at 10,000 g for 15 min.
  • 10mL silicate solution containing 150mM NaCl, 80mM tetramethyl orthosilicate and 1.0mM HC
  • the average particle size of the micron particles is about 1.1 ⁇ m, and the surface potential of the micron particles is about -2mV; each 1 mg of PLGA micron particles is loaded with approximately 340 ⁇ g of protein or peptide components, and each 1 mg of PLGA micron particles is loaded with 0.02 mg of CpG and half of the inside and outside.
  • the preparation materials and methods of blank microparticles 2 are the same, with a particle size of about 1.1 ⁇ m and a surface potential of about -3 mV.
  • the blank microparticles are loaded with an equal amount of adjuvant but no antigen component.
  • This example takes the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare bone marrow-derived dendritic cells (BMDC).
  • BMDC bone marrow-derived dendritic cells
  • RPMI 1640 (10% FBS) medium to stop lysis, centrifuge at 400 g for 3 min, and discard the supernatant.
  • the cells were placed in a 10 mm culture dish and cultured in RPMI 1640 (10% FBS) medium with recombinant mouse GM-CSF (20 ng/mL) added at 37 degrees Celsius and 5% CO 2 for 7 days. On the third day, shake the culture bottle gently and add the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20ng/mL).
  • mice Inoculate 1.5 ⁇ 10 5 B16F10 cells subcutaneously on the back of each C57BL/6 mouse, and inject the mice subcutaneously on the 4th day, the 7th day, the 10th day, the 15th day, the 20th day and the 25th day respectively. 100 ⁇ L of 1 mg PLGA micron particles. The mice were sacrificed on day 30, their spleens were collected, and a single cell suspension of mouse spleen cells was prepared. First, magnetic bead sorting method was used to sort CD3 + T cells in splenocyte single cell suspension.
  • cancer-specific T cells obtained by the above sorting were mixed with IL-2 (2000U/mL) and ⁇ CD3/ ⁇ CD28 antibody (20ng/mL) in 10mL of DMEM high-glucose complete medium (37°C, 5% CO 2 ) Incubate for a total of 7 days (the medium is changed once every two days) to amplify the sorted cancer cell-specific T cells (cell viability 75%).
  • Cancer cell-specific T cells are expanded and used for cancer prevention
  • mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. One day before the mice were adoptively transferred cells, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. Mice were injected intravenously with 100 ⁇ L of 2 million cancer-specific T cells on day 0. At the same time, 1.5 ⁇ 10 5 B16F10 cells were injected subcutaneously into each mouse on day 0. The methods for monitoring mouse tumor volume and survival were as above.
  • mice in the PBS control group had tumors that grew rapidly and had a short survival period, while the tumors of mice treated with T cells had a significantly slower growth rate and a significantly longer survival period. Moreover, it shows that the use of micron particles to analyze Selecting expanded T cells can effectively prevent cancer.
  • 8M urea was first used to lyse B16F10 melanoma tumor tissue and dissolve the tumor tissue lysate components. Then, PLA was used as the nanoparticle skeleton material, and Poly(I:C) and CpG1018 were used as immune adjuvants to prepare nanoparticles loaded with whole cell antigens, and the nanoparticles were used to assist in the sorting and expansion of cancer cell-specific T cells.
  • nanoparticle 1 is prepared by solvent evaporation method.
  • the molecular weight of PLA, the material for preparing nanoparticle 1, is 20KDa, and the immune adjuvants used are poly(I:C) and CpG1018.
  • the preparation method is as described above.
  • the double emulsion method is used to load the antigen components and adjuvants inside the nanoparticles.
  • 100 mg of the nanoparticles are centrifuged at 12,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then frozen. Dry for 48 hours to obtain freeze-dried powder for later use.
  • the average particle diameter of the nanoparticle 1 is about 250nm, and the surface potential is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 110 ⁇ g of protein or peptide components, and each of poly(I:C) and CpG1018 is loaded with 0.02 mg.
  • Nanoparticle 2 The preparation materials and preparation method of Nanoparticle 2 are the same as above.
  • the particle size is about 250nm and the surface potential is about -3mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 110 ⁇ g of protein or peptide components and 0.04 mg of poly(I:C).
  • mice Inoculate 1.5 ⁇ 10 5 B16F10 cells subcutaneously on the back of each C57BL/6 mouse, and subcutaneously inject 100 ⁇ L into each mouse on day 4, day 7, day 10, day 15, day 20 and day 25. 1 mg of PLA nanoparticles 1.
  • the mice were sacrificed on day 29, and the mouse spleens were collected and a single cell suspension of mouse spleen cells was prepared.
  • CD3 + T cells were sorted from mouse splenocytes using magnetic bead sorting. Then, the sorted T cells (10 million), allogeneic B cells (15 million) and 40 mg of nanoparticles (nanoparticles 1 or 2) were placed in 20 mL RPMI 1640 complete medium (37°C , 5% CO 2 ) for 48 hours.
  • CD3 antibodies and CD69 antibodies labeled with different fluorescent probes were used to mark the incubated cells, and then flow cytometry was used to sort the CD3 + CD69 + T cells (cell viability 75%) in the incubated cells, which were the cancer cells.
  • Cancer cell-specific T cells activated by cellular antigens.
  • IL-2 1000U/mL
  • ⁇ CD3/ ⁇ CD28 antibody 20ng/mL
  • 10mL high-glucose DMEM complete medium for 11 days (every two days Change the medium once a day) to amplify and sort the cancer cell-specific T cells (cell viability 75%).
  • T cells are used for cancer prevention
  • mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. One day before mouse cancer-specific T cell transplantation, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. Mice were injected subcutaneously with 100 ⁇ L of 2 million expanded cancer-specific T cells on day 0. At the same time, each mouse was subcutaneously injected with 1.5 ⁇ 10 5 B16F10 cells on day 0. The mouse tumor volume and survival period were monitored as above.
  • mice in the control group had the fastest tumor growth and longest survival.
  • Cancer-specific T cells sorted and amplified using Nanoparticle 1 and Nanoparticle 2 can slow down the growth rate and prolong the survival of mouse tumors. Mice survival period.
  • Example 7 Sorting and amplification of cancer cell-specific T cells for the treatment of colon cancer
  • colon cancer tumor tissue and lung cancer cancer cell lines were lysed to prepare a mixture of water-soluble components (mass ratio 1:1) and a mixture of non-water-soluble components (mass ratio 1:1), and the water-soluble component mixture and non-water-soluble components were mixed.
  • the mixture of sexual components is mixed in a mass ratio of 1:1.
  • the organic polymer material PLGA was used as the nanoparticle skeleton material, and CpG1018 and Poly(I:C) were used as immune adjuvants to prepare nanoparticles, and the nanoparticles were used to sort and expand cancer-specific T cells for the treatment of colon cancer.
  • the non-water-soluble components that are insoluble in pure water can be converted into soluble in 10% octyl glucoside aqueous solution.
  • the cultured LLC lung cancer cell lines were collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, and then the cells were resuspended in ultrapure water and frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells.
  • the water-soluble components from colon cancer tumor tissue and lung cancer cancer cells were mixed at a mass ratio of 1:1; the water-insoluble components dissolved in 10% octyl glucoside were also mixed at a mass ratio of 1:1. Then, the water-soluble component mixture and the water-insoluble component mixture are mixed at a mass ratio of 1:1, and this mixture is the antigen component for preparing nanoparticles.
  • the nanoparticles were prepared using the double emulsion method.
  • the molecular weight of the nanoparticle preparation material PLGA is 24KDa-38KDa
  • the immune adjuvants used are Poly(I:C) and CpG1018, and the antigen components and adjuvants are distributed both inside and on the surface of the nanoparticles.
  • the preparation method is as described above.
  • the double emulsion method is first used to load the antigen components and adjuvants inside the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose is used. Resuspend and freeze-dry for 48 hours.
  • nanoparticles Before use, resuspend 20 mg of nanoparticles in 0.9 mL of PBS, mix with 0.1 mL of sample containing antigen component (80 mg/mL) and adjuvant, and incubate at room temperature for 5 minutes before use.
  • the average particle size of the nanoparticles is about 280nm, and the surface potential is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components, and the loaded CpG1018 and Poly(I:C) immune adjuvants are 0.02 mg each.
  • the preparation materials and preparation methods of nanoparticles 2 are the same, the particle size is about 280nm, and the surface potential is -3mV; every 1 mg of PLGA nanoparticles 2 is loaded with approximately 100 ⁇ g of protein or peptide components, and the load of CpG1018 is 0.04 mg.
  • mice Inoculate 1.5 ⁇ 10 5 B16F10 cells subcutaneously on the back of each C57BL/6 mouse, and inject the mice subcutaneously on the 4th day, the 7th day, the 10th day, the 15th day, the 20th day and the 25th day respectively. 100 ⁇ L of 2 mg PLGA nanoparticles. The mice were sacrificed on the 30th day, and the spleens of mice in each group were collected to prepare a single cell suspension of mouse spleen cells, from which CD3 + CD8 + T cells and CD19 + B cells were sorted using flow cytometry.
  • CD8 + T cells and 6 million CD19 + B cells were incubated with nanoparticles (50 ⁇ g) in 2 mL RPMI complete medium for 96 hours (37°C, 5% CO 2 ), and then sorted by flow cytometry.
  • the CD3 + CD8 + CD69 + T cells (cell viability 70%) in the cells after incubation are cancer cell-specific T cells activated by cancer cell antigens.
  • the 200,000 cancer cell-specific T cells obtained by the above sorting were mixed with IL-2 (2000U/mL), IL-7 (500U/mL), IL-15 (500U/mL) and ⁇ CD3/ ⁇ CD28 antibodies in 10mL high
  • the cells were incubated in complete sugar DMEM medium (37° C., 5% CO 2 ) for 7 days (the medium was changed every two days) to amplify the sorted cancer cell-specific T cells (cell viability 70%).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer tumor-bearing mice. On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 MC38 cells, and on days 4, 7, 10, 15, and 20, mice were injected with 100 ⁇ L containing 100,000 cancer cells. specific T cells. The method for monitoring tumor growth and survival in mice is the same as above.
  • the tumors of the mice in the PBS control group grew very quickly.
  • the cancer cell-specific T cells obtained by sorting and amplifying nanoparticles 1 and 2 can effectively delay the tumor growth and improve the survival of the mice. .
  • Example 8 Sorting and amplifying T cells for the prevention of breast cancer
  • the cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water.
  • the obtained cancer cells are inactivated and denatured using ultraviolet light and high-temperature heating respectively, and then an appropriate amount of 8M urea is used to lyse the breast cancer cells and dissolve the lysate, which is the antigen component for preparing particles.
  • the double emulsion method is used to prepare micron particles.
  • the molecular weight of the micron particle 1 skeleton material PLGA is 38KDa-54KDa.
  • the immune adjuvants used are CpG and Poly ICLC.
  • the substance that increases lysosomal immune escape is arginine.
  • the double emulsion method is first used to prepare micron particles internally loaded with antigen components, adjuvants and arginine, then 100 mg of micron particles are centrifuged at 9000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose and dried. Reserve after 48 hours.
  • the average particle size of the micron particle system is about 2.1 ⁇ m, and the surface potential of the micron particles is about -5mV; each 1 mg of PLGA micron particles is loaded with approximately 110 ⁇ g of protein or peptide components, with 0.01 mg of CpG and Poly ICLC each, and 0.05 mg of arginine. .
  • the preparation materials and methods of blank microparticles 2 are the same, and the particle size is about 2.0 ⁇ m.
  • the blank microparticles are loaded with equal amounts of CpG, Poly ICLC and arginine, but do not load any antigen components.
  • mice Female BALB/c mice aged 6-8 weeks were selected and subcutaneously injected with 100 ⁇ L of micron particles 1 containing 2 mg PLGA on days 0, 4, 7, 14, 21, and 28. The mice were sacrificed on day 32, and their peripheral blood was collected, and then peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood. First, flow cytometry is used to sort out CD3 + CD8 + T cells and B220 + B cells from PBMC in the first step.
  • PBMC peripheral blood mononuclear cells
  • the sorted 200,000 CD8 + T cells, 300,000 B cells, and IL-7 (10ng/mL) and 40 ⁇ g of microparticles (microparticles 1 or microparticles 2 + equal amount of free lysate) were incubated in 2mL RPMI 1640 complete medium for 96 hours. Then flow cytometry was used to further sort the CD3 + CD8 + CD69 + T cells (cell viability 85%) in the incubated cells, which are cancer cell-specific T cells that can recognize cancer cell antigens.
  • the cancer cell-specific T cells obtained by sorting in the above two steps were incubated with IL-2 (2000U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and ⁇ CD3/ ⁇ CD28 antibodies for 7 days. Cancer cell-specific T cells obtained by amplification and sorting (cell viability 85%).
  • mice Female BALB/c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. One day before the mice were adoptively transferred cells, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. Mice were injected intravenously with 2 million expanded cancer cell-specific CD8 + T cells on day 0. At the same time, 4 ⁇ 10 5 4T1 cells were subcutaneously injected into each mouse on day 0. The methods for monitoring mouse tumor growth and survival were the same as above.
  • the tumor growth rate in the cancer cell-specific T cell treatment group was significantly slower and the survival period of mice was significantly prolonged.
  • the preventive effect of cancer cell-specific T cells obtained by assisted sorting and amplification of micron particles loaded with whole cell components is better than that of cancer cell-specific T cells obtained by assisted sorting and amplification of blank micron particles + free lysate. It can be seen that the cancer cell-specific T cells that have undergone two-step sorting and amplification according to the present invention have excellent preventive effects on breast cancer.
  • Example 9 Cancer cell-specific T cells for the prevention of cancer metastasis
  • This example uses a mouse melanoma mouse lung metastasis cancer model to illustrate the use of sorted and amplified cancer cell-specific T cells to prevent cancer metastasis.
  • the specific dosage form, adjuvant, incubation time, incubation concentration, administration time, administration frequency, and dosage regimen can be adjusted according to the situation.
  • mouse melanoma tumor tissue and cancer cell lines were lysed and dissolved with 8M urea, and then the tumor tissue lysis component and the cancer cell line lysis component were loaded on the nanoparticles at a mass ratio of 1:2, and the particles were used Activates and assists in the sorting of cancer-specific T cells, and the resulting T cells are expanded to prevent cancer metastasis in mice.
  • four polypeptide neoantigens were loaded
  • Nanoparticles of B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were used as control nanoparticles to analyze nanoparticles loaded with whole cell antigens and nanoparticles loaded with multiple peptide neoantigens to assist in sorting and amplification.
  • 8M urea was used to lyse and dissolve the tumor tissue and cancer cell whole cell components, and then the tumor tissue components and cancer cell line lysate components were mass ratio 1: 2 Miscibility is the antigen component.
  • the nanoparticles 1 are prepared by a solvent evaporation method.
  • the molecular weight of the material PLGA used to prepare the nanoparticles 1 is 24KDa-38KDa.
  • the immune adjuvants used are CpG7909 and Poly(I:C).
  • the enzyme escape substance is KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA).
  • the preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the antigen components, adjuvants and KALA polypeptides inside the nanoparticles.
  • each 1 mg of PLGA nanoparticles 1 is loaded with approximately 10 ⁇ g of protein and peptide components of the lysate, including 0.02 mg each of CpG7909 and Poly(I:C), and 0.04 mg of KALA peptide.
  • the preparation method of the control nanoparticles 2 loaded with four antigen polypeptides is the same as above.
  • the particle size of the control nanoparticles 2 is about 460 nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 10 ⁇ g of antigen polypeptides and an equal amount of adjuvant and KALA polypeptide.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected and 200 ⁇ L of 2 mg PLGA nanoparticles were subcutaneously injected on days 0, 4, 7, 14, 21, and 28 respectively. The mice were sacrificed on day 32, and their peripheral blood was collected, and then peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood.
  • PBMC peripheral blood mononuclear cells
  • First sort CD3 + T cells and CD19 + B cells from PBMC and then mix the 7 million T cells and 7 million B cells obtained in the first step with 4 mg of nanoparticle 1 or nanoparticle 2 in 10 mL of RPMI. Incubate in 1640 complete medium for a total of 96 hours.
  • CD3 + CD8 + CD69 + T cells and CD3 + CD4 + CD69 + T cells are cancer cell-specific T cells that can recognize cancer cell antigens (cell viability 80%).
  • the 1 million CD3 + CD8 + CD69 + T cells or 1 million CD3 + CD4 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-12 (1000U/mL) and ⁇ CD3/ ⁇ CD28 antibody (10 ng/mL) was incubated in 10 mL of RPMI1640 complete medium for 14 days to expand cancer cell-specific T cells (cell viability 80%).
  • mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. One day before the mice were adoptively transferred cells, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. Mice were intravenously injected with 1.5 million cancer cell-specific CD8 + T cells and 0.5 million cancer cell-specific CD4 + T cells on day 0. At the same time, each mouse was intravenously inoculated with 0.5 ⁇ 10 5 B16F10 cells on the 1st day. The mice were sacrificed on the 14th day, and the number of melanoma cancer foci in the lungs of the mice was observed and recorded.
  • the cancer cell-specific T cells obtained through nanoparticle 1 and nanoparticle 2-assisted sorting can effectively prevent cancer metastasis after expansion.
  • nanoparticles 1 loaded with whole cell components have a better effect in assisting in the sorting and expansion of cancer cell-specific T cells.
  • Example 10 Use of sorted and amplified T cells to treat pancreatic cancer
  • mouse Pan02 pancreatic cancer tumor tissue and MC38 colon cancer tumor tissue lysate components were loaded on nanoparticles at a ratio of 3:1, and the nanoparticles were used to assist in the separation of cancer cell specificities in the peripheral blood of mice.
  • the T cells are then expanded and used to treat pancreatic cancer.
  • mouse pancreatic cancer and colon cancer tumor tissues were first obtained and lysed to prepare water-soluble components and the original water-insoluble components dissolved in 6M guanidine hydrochloride.
  • the water-soluble component is a 3:1 mixture of the water-soluble component of pancreatic cancer tumor tissue and the water-soluble component of colon cancer tumor tissue; the water-insoluble component is a mixture of the water-soluble component of pancreatic cancer tumor tissue and the water-soluble component of colon cancer tumor tissue.
  • Nanoparticles were prepared using PLGA as the nanoparticle skeleton material and BCG as the adjuvant, and the nanoparticles were used to assist in the sorting and expansion of cancer cell-specific T cells.
  • Each C57BL/6 mouse was subcutaneously inoculated with 2 ⁇ 10 6 MC38 colon cancer cells or 1 ⁇ 10 6 Pan02 pancreatic cancer cells in the armpit. The mice were sacrificed when the inoculated tumors in each mouse grew to approximately 1000 mm 3 and remove tumor tissue.
  • the lysis method and the collection method of each component are the same as in Example 1, except that 6M guanidine hydrochloride is used instead of 8M urea to dissolve the non-water-soluble components.
  • the tumor tissue lysate component is the antigen component.
  • the BCG lysis method is the same as the tumor tissue lysis method.
  • the nanoparticles in this embodiment were prepared by the double emulsion method.
  • the nanoparticle preparation material PLGA used in Nanoparticle 1 has a molecular weight of 7KDa-17KDa, and the immune adjuvant used is BCG lysate component; the preparation method is as described above, first loading the antigen component and adjuvant inside the nanoparticle, and then Centrifuge 100 mg of nanoparticles at 12,000 g for 20 minutes, resuspend in 10 mL of ultrapure water containing 4% trehalose, and freeze-dry for 48 hours to obtain a freeze-dried powder for later use; before injecting nanoparticle 1, dissolve 20 mg of nanoparticles in 0.9 mL.
  • nanoparticle 1 In PBS, mix with 0.1mL sample containing antigen component (80mg/mL) and GM-CSF (50ng/mL) and incubate at room temperature for 10 minutes before use; the average particle size of nanoparticle 1 is about 160nm, and the surface potential is -4mV.
  • About 1 mg of PLGA nanoparticles can load approximately 130 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles can load 0.02 mg of BCG.
  • the preparation materials and methods used for nanoparticle 2 are the same as nanoparticle 1. Its particle size is about 160nm and its surface charge is about -4mV.
  • Each 1 mg of PLGA nanoparticle 2 loads 130 ⁇ g of protein or peptide components in tumor tissue lysate, but Does not contain any adjuvants.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected and 200 ⁇ L of 2 mg PLGA nanoparticles were subcutaneously injected on days 0, 4, 7, 14, 21, and 28 respectively. The mice were sacrificed on day 32, and their peripheral blood was collected, and then peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood.
  • PBMC peripheral blood mononuclear cells
  • CD3 + CD69 + T cells (cell viability 75%), which are cancer cell-specific T cells.
  • the 1 million CD3 + CD69 + T cells obtained by the above two steps were mixed with IL-2 (1000U/mL) and granulocyte-macrophage colony-stimulating factor (GM-CSF, 10ng/mL) in 15mL high-glucose DMEM
  • the sorted cancer cell-specific T cells were amplified by incubation in complete culture medium (37°C, 5% CO 2 ) for 14 days (the medium was changed once every two days).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare pancreatic cancer tumor-bearing mice. At the same time, each mouse was subcutaneously inoculated with 1 ⁇ 10 6 Pan02 pancreatic cancer cells on day 0. On days 6, 9, 12, 17 and 23, mice were injected intravenously with 2 million expanded cancer cell-specific T cells. The method for monitoring and recording tumor volume in mice is the same as above.
  • the cancer cell-specific T cells sorted and amplified using nanoparticles 1 and 2 according to the present invention can effectively treat pancreatic cancer.
  • This example uses mannose as an active target to illustrate how to sort and amplify cancer cell-specific T cells. It can be adjusted according to the actual application.
  • the nanoparticle system can be taken up into dendritic cells through mannose receptors on the surface of dendritic cells.
  • the antigen loaded on the particles can activate cancer cell-specific T cells after being presented by dendritic cells.
  • target-modified nanoparticles or microparticles that can target antigen-presenting cells, such as mannan, CD32 antibodies, CD19 antibodies, CD20 antibodies, B220 antibodies, and CD11c antibodies, can also be used.
  • nanoparticle 1 was prepared using the double emulsion method.
  • the nanoparticle preparation materials used are PLGA and mannose-modified PLGA. When used together to prepare nanoparticles with targets, the mass ratio of the two is 4:1, and the molecular weight is 7KDa-17KDa.
  • the immune adjuvants used were Poly(I:C) and CpG7909.
  • the preparation method is as described above. First, the antigen component and the adjuvant are loaded inside the nanoparticles, then 100 mg of the nanoparticles are centrifuged at 12,000 g for 25 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h. Backup.
  • the average particle size of nanoparticles 1 is about 120nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide components, including 0.02 mg each of Poly(I:C) and CpG7909.
  • BMDC bone marrow-derived dendritic cells
  • BMDC Spread mouse BMDC into a cell culture plate, add 5 mL RPMI 1640 (10% FBS) medium to every 100,000 BMDC cells, and then add 30 ⁇ g of nanoparticle 1 and incubate with BMDC for 48 h (37°C, 5% CO 2 ), then collect BMDC and centrifuge at 300g for 5 minutes, wash twice with phosphate buffer saline (PBS) and resuspend in PBS for later use.
  • RPMI 1640 10% FBS
  • PBS phosphate buffer saline
  • PBMC Peripheral blood mononuclear cells
  • CD3 + CD8 + T cells were sorted from the PBMC using magnetic bead sorting.
  • the sorted 2 million CD8 + T cells were incubated with 5 million BMDC prepared in step (4), 40 ⁇ g of nanoparticle 1 and 10 ng/mL of IL-7 in 4 mL of DMEM high-glucose complete medium for 18 hours.
  • CD8 + CD69 + T cells are cancer cell-specific T cells that can recognize cancer antigens.
  • the 200,000 CD8 + CD69 + T cells obtained by the above two-step separation were incubated with IL-2 (1000U/mL) and IL-7 (1000U/mL) for 10 days to expand cancer cell-specific T cells (cell viability). rate 75%).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma tumor-bearing mice.
  • the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate the recipient mice.
  • immune cells in mice.
  • the 2 million cancer-specific T cells prepared in step (5) were subcutaneously injected into the recipient mice.
  • each recipient mouse was inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 cells on the lower right side of the back.
  • the methods for monitoring mouse tumor growth rate and mouse survival are the same as above.
  • Example 12 Sorted and amplified cancer cell-specific T cells are used to prevent liver cancer
  • Hepa1-6 liver cancer cells were first lysed, PLGA was used as the nanoparticle skeleton material, Poly(I:C) and bacterial Bacillus Calmette-Guérin (BCG) were used as immune adjuvants and a solvent evaporation method was used to prepare whole cell antigen-loaded liver cancer cells. Nanoparticles are then mixed with B cells and T cells, and cancer cell-specific T cells are obtained after sorting and amplification.
  • the cultured Hepa 1-6 liver cancer cells were collected and washed twice with PBS.
  • the liver cancer cells were treated with heating and ultraviolet irradiation, and then 8M urea was used to lyse and dissolve the whole cell component of the cancer cells, which is the antigen component.
  • the BCG cleavage method is the same as above, and the BCG lysate is used as an adjuvant.
  • the nanoparticles are prepared by a solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa.
  • the immune adjuvants used are BCG and Poly(I:C).
  • the preparation method is as described above. First, load the antigen components and adjuvants inside the nanoparticles, then centrifuge 100mg of the nanoparticles at 10,000g for 25 minutes, resuspend in 10mL of ultrapure water containing 4% trehalose, and then freeze-dry for 48 hours before use. .
  • the particle size of the nanoparticles is about 270nm; each 1 mg of PLGA nanoparticles is loaded with 100 ⁇ g of protein or peptide components, and 0.02 mg each of BCG and Poly(I:C).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected and subcutaneously injected with 200 ⁇ L of 2 mg PLGA nanoparticles on days 0, 4, 7, 14, 21, and 28. The mice were sacrificed on day 32, and their peripheral blood was collected. Peripheral blood mononuclear cells (PBMC) were then isolated from the peripheral blood, and CD8 + T cells were sorted from the PBMC using magnetic bead sorting. The sorted 2 million CD8 + T cells, nanoparticles (500 ⁇ g), 8 million BAF3 mouse B cell lines, and IL-7 (10 ng/mL) were incubated in 2 mL RPMI 1640 complete medium for 48 hours.
  • PBMC Peripheral blood mononuclear cells
  • CD8 + CD69 + T cells are cancer-specific T cells that can recognize cancer antigens.
  • the CD8 + CD69 + T cells sorted above were incubated with IL-2 (1000 U/mL), IL-7 (500 U/mL), IL-15 (500 U/mL) and ⁇ CD28 antibody for 7 days to amplify the fraction.
  • the selected cancer cell-specific T cells (cell viability 80%).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare liver cancer tumor-bearing mice. One day before the mice were adoptively transferred cells, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. Mice were injected with 2 million expanded cancer cell-specific T cells on day 0. At the same time, 1.0 ⁇ 10 6 Hepa1-6 liver cancer cells were subcutaneously injected into the back of each mouse on day 0. Tumor growth and mouse survival were recorded in the same way as above.
  • Example 13 Calcified nanoparticles assist in sorting T cells for cancer prevention
  • calcified nanoparticles are used to sort cancer cell-specific T cells.
  • other biomineralization technologies, cross-linking, gelation and other modified particles can also be used.
  • the specific dosage form, adjuvant, particle size, administration time, administration frequency, administration schedule, etc. can be adjusted according to the situation.
  • mouse melanoma tumor tissue and cancer cell lines were lysed and dissolved with 8M urea, and then the tumor tissue lysis components and the cancer cell line lysis components were loaded on the nanoparticle 1 at a mass ratio of 1:1.
  • four polypeptide neoantigens were loaded
  • Nanoparticles of TRP2:180-188 were used as control nanoparticle 2.
  • 8M urea was used to lyse and dissolve the tumor tissue and the whole cell components of the cancer cell line, and then the tumor tissue components and the cancer cell line components were mass ratio 1 :1 miscible, which is the antigen component.
  • the nanoparticles are biocalcified after loading whole cell antigens inside and on the surface of the nanoparticles.
  • nanoparticle 1 is prepared by the double emulsion method.
  • the molecular weight of PLGA, the material used to prepare nanoparticle 1, is 7KDa-17KDa.
  • the immune adjuvants are CpG2006 and Poly(I:C).
  • the lysosomal escape-enhancing substance is GALA polypeptide (WEAALAEALAEALAEHLAEALAEALEALAA). .
  • the preparation method is as follows: first load the antigen component, adjuvant and GALA peptide inside the nanoparticles, then centrifuge 100mg PLGA nanoparticles at 13000g for 20 minutes and resuspend in 18mL PBS, then add 2mL of the antigen component dissolved in 8M urea (60mg/mL), incubate at room temperature for 10 minutes and then centrifuge at 12000g for 20 minutes to collect the precipitate. The 100 mg PLGA nanoparticles were then resuspended in 20 mL DMEM medium, and then 200 ⁇ L of CaCl 2 (1 mM) was added and reacted at 37°C for two hours.
  • the average particle size of the nanoparticles is about 290nm; each 1 mg of PLGA nanoparticles is loaded with approximately 230 ⁇ g of protein or peptide component in the antigen component, 0.02 mg each of CpG and Poly(I:C), and 0.001 mg of GALA peptide.
  • control nanoparticles 2 loaded with multiple antigen peptides are the same as above.
  • the particle size of the control nanoparticles is about 290nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 230 ⁇ g of antigen peptides and equal amounts of adjuvants and GALA peptides.
  • mice were intraperitoneally injected with PD-1 antibody at a dose of 10 mg/kg on each day.
  • the mice were sacrificed on day 30, and their peripheral blood was collected.
  • Peripheral blood mononuclear cells (PBMC) were then isolated from the peripheral blood.
  • CD8 + T cells and B cells were first sorted from the PBMC using flow cytometry. The sorted CD8 + T cells (5 million), nanoparticles (500 ⁇ g), B cells (20 million), and IL-7 (10 ng/mL) were incubated in 20 mL RPMI 1640 complete medium for 48 hours.
  • CD8 + CD137 + T cells are cancer-specific T cells that can recognize cancer antigens.
  • the 500,000 CD8 + CD137 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (500U/mL), IL-15 (500U/mL) and ⁇ CD3 antibody (10ng/mL).
  • CCP was incubated for 14 days to expand cancer cell-specific T cells (cell viability 75%).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected. One day before the adoptive transfer of cells, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. Mice were injected with 1 million expanded cancer cell-specific T cells on day 0. At the same time, 1.5 ⁇ 10 5 B16F10 melanoma cells were subcutaneously injected into the back of each mouse on day 0. Tumor growth and mouse survival were monitored in the same manner as above.
  • nanoparticles activated and assisted sorting of cancer cell-specific T cells could prolong the survival of mice and effectively prevent cancer.
  • the effect of nanoparticles loaded with whole cell components was better than that of nanoparticles loaded with four neoantigen peptides.
  • Example 14 Cancer cell-specific T cells for the treatment of melanoma
  • B16F10 melanoma tumor tissue was first lysed to prepare the water-soluble and non-water-soluble components of the tumor tissue, and then PLGA was used as the nanoparticle framework material, and Poly(I:C) and CpG2395 were used as immune adjuvants.
  • Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-amide) was used as a component to promote lysosome escape, and nanoparticles were prepared by solvent evaporation method.
  • the nanoparticles were prepared using the double emulsion method. During preparation, nanoparticles loaded with water-soluble components and nanoparticles loaded with non-water-soluble components are prepared separately and used together during application.
  • the molecular weight of the nanoparticle preparation material PLGA used is 7KDa-17KDa
  • the immune adjuvants used are poly(I:C) and CpG2395
  • the lysosomal escape substance is melittin.
  • the preparation method is as described above, first load the antigen components, adjuvants and melittin inside the nanoparticles, then centrifuge 100mg nanoparticles at 10000g for 20 minutes, and resuspend them in 10mL of ultrapure water containing 4% trehalose. Freeze-dry for 48 hours; resuspend it in 9 mL of PBS before use, then add 1 mL of antigen component (protein concentration 80 mg/mL) and react at room temperature for 10 min to obtain nanoparticles loaded with lysate both inside and outside.
  • the average particle size of the nanoparticles is about 290nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 140 ⁇ g of protein or peptide component in the antigen component, 0.02 mg each of poly(I:C) and CpG2395 immune adjuvant, and 0.05 mg of melittin. mg.
  • mice that are 6-8 weeks old and inoculate 1.5 ⁇ 10 5 B16F10 subcutaneously on the back of the mice on day 0, and then on days 7, 10, 15, 20 and 25
  • mice were subcutaneously injected with 1 mg of nanoparticles loaded with water-soluble components and 1 mg of nanoparticles loaded with non-water-soluble components.
  • the mice were sacrificed on day 30, and the PBMC of the mice were collected, and CD8 + T cells, CD4 + T cells, and B cells were sorted from the PBMC using flow cytometry.
  • the sorted CD8 + T cells (1 million), CD4 + T cells (1 million), nanoparticles (200 ⁇ g each of water-soluble component-loaded and water-insoluble component-loaded nanoparticles), B cells (3 million cells) and IL-15 (50ng/mL) were incubated in 2 mL RPMI 1640 complete medium for 72 hours; or the sorted CD8 + T cells (1 million cells), CD4 + T cells (1 million cells ), nanoparticles (200 ⁇ g each of water-soluble component-loaded and water-insoluble component-loaded nanoparticles), and B cells (3 million) were incubated in 2 mL RPMI 1640 complete medium for a total of 72 hours; or the sorted cells CD8 + T cells (1 million), CD4 + T cells (1 million), nanoparticles (200 ⁇ g each of nanoparticles loaded with water-soluble components and water-insoluble components), B cells (3 million) and Flt3L (50ng/mL) were incubated in 2mL RPMI 1640 complete medium for a total
  • flow cytometry was used to sort the CD8 + CD69 + T cells (cell viability rate 80%) among the incubated CD8 + T cells and the CD4 + CD69 + T cells (cell viability rate 80%) among the CD4 + T cells, that is, are cancer-specific T cells that recognize cancer antigens.
  • the 100,000 CD8 + CD69 + T cells or 100,000 CD4 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (500U/mL), IL-15 (500U/mL) mL) and ⁇ CD3 antibody (10 ng/mL) were incubated in 20 mL of RPMI 1640 complete medium (37°C, 5% CO 2 ) for a total of 11 days (the medium was changed once every two days) to expand cancer cell-specific T cells.
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right side of the back of each mouse. 800,000 cancer cell-specific CD8 + T cells and 200,000 cancer cell-specific CD4 + T cells were intravenously injected on days 4, 7, 10, 15, and 20 after melanoma inoculation. . In the experiment, the mouse tumor volume and survival period were monitored as above.
  • the cancer cell-specific T cells obtained by sorting and amplifying the present invention have good therapeutic effects on melanoma.
  • Example 15 Cancer cell-specific T cells are used in the treatment of melanoma
  • B16F10 melanoma tumor tissue was first lysed, and then PLGA was used as the nanoparticle framework material, Poly(I:C) and CpG7909 were used as immune adjuvants, and polyarginine and polylysine were used to increase lytic enzymes. Preparation of nanoparticles from bulk escape components.
  • the supernatant part is the water-soluble component; the precipitate part uses 0.1M metformin hydrochloride and 0.1M arginine aqueous solution to dissolve the non-water-soluble component.
  • the antigen component for preparing nanoparticles is obtained by mixing the water-soluble component and the non-water-soluble component dissolved in the solution at a mass ratio of 1:1.
  • nanoparticle 1 is prepared by the double emulsion method and has the ability to target dendritic cells.
  • the materials used to prepare the nanoparticle 1 are PLA and mannan-modified PLA, both of which have molecular weights of 20KDa-30KDa. When used, the mass ratio of unmodified PLA to mannan-modified PLA is 9:1.
  • the immune adjuvants used are poly(I:C) and CpG7909, and the substances that increase lysosomal immune escape are polyarginine and polylysine.
  • the preparation method is as described above. First, load the lysis solution components, adjuvants, polyarginine and polylysine inside the nanoparticles.
  • nanoparticles After loading the above components inside, 100mg nanoparticles are centrifuged at 10000g for 20 minutes, and Resuspend in 10 mL of ultrapure water containing 4% trehalose and freeze-dry for 48 h.
  • the average particle size of the nanoparticles is 360nm; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide component in the antigen component, 0.02 mg each of poly(I:C) and CpG7909 immune adjuvant, and polyarginine and Polylysine 0.01mg each.
  • CD3 + CD69 + T cells are cancer-specific T cells that can recognize cancer antigens.
  • the 300,000 CD3 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (1000U/mL), IL-15 (1000U/mL) and ⁇ CD3 antibody (20ng/mL). Incubate in 20 mL RPMI 1640 complete medium for a total of 14 days to expand cancer cell-specific T cells.
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right side of the back of each mouse. 800,000 expanded CD3 + T cells were injected intravenously on days 4, 7, 10, 15 and 20 after melanoma inoculation. The methods for monitoring mouse tumor volume and survival were as above.
  • the tumors in the PBS control group grew very quickly, and the tumor growth rate of mice treated with cancer cell-specific T cells amplified by nanoparticle sorting was significantly slower.
  • Example 16 Sorting and amplifying T cells for the prevention of breast cancer
  • breast cancer cells are first inactivated and denatured, and then the cells are lysed, and octylglucoside is used to dissolve and lyse the non-water-soluble components in the cancer cells. Then, PLGA was used as the micron particle skeleton material, and CpG1018 and Poly ICLC were used as immune adjuvants to prepare micron particles loaded with whole cell antigens.
  • the cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water.
  • the obtained cancer cells were inactivated and denatured using ultraviolet and high-temperature heating respectively, and then ultrapure water was added and repeatedly frozen and thawed 5 times, supplemented by ultrasound to lyse the cancer cells.
  • the cell lysates were centrifuged at 5000g for 10 minutes, and the supernatant was water-soluble.
  • the precipitate is dissolved with 10% octylglucoside to obtain the dissolved original non-water-soluble component.
  • the water-soluble component and the non-water-soluble component are mixed at a mass ratio of 2:1 to prepare Antigen component required for micron particles1.
  • the double emulsion method is used to prepare the microparticle 1.
  • the molecular weight of the framework material PLGA of the microparticle 1 is 38KDa-54KDa.
  • the immune adjuvants used are CpG1018 and Poly ICLC.
  • the double emulsion method is first used to prepare micron particles internally loaded with antigen component 1 and adjuvant. Then, 100 mg of micron particles are centrifuged at 9000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use.
  • the average particle size of this micron particle system is about 5.0 ⁇ m; each 1 mg of PLGA micron particles is loaded with approximately 410 ⁇ g of protein or peptide components of cancer cells, and each of CpG1018 and Poly ICLC is loaded with 0.01 mg.
  • This example uses BMDC as antigen-presenting cells.
  • the preparation method is as described above.
  • BMDC 5 million BMDC, 2 mg micron particles and IL-15 (20 ng/mL) were incubated in 5 mL RPMI 1640 (10% FBS) medium for 8 hours, and then the BMDC were collected and the activated dendritic cells were inactivated by irradiation. Dendritic cells, inactivated dendritic cells are used to activate T cells.
  • the 300,000 CD3 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (1000U/mL), IL-15 (1000U/mL) and ⁇ CD3 antibody at (10ng/mL ) were incubated in 10 mL of DMEM complete medium (37°C, 5% CO 2 ) for a total of 14 days to expand cancer cell-specific T cells (cell viability 85%).
  • mice Female BALB/c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. One day before the mice were adoptively transferred cells, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. On day 0, mice were injected subcutaneously with 100 ⁇ L containing 1.2 million expanded CD3 + T cells. At the same time, 1 ⁇ 10 6 4T1 cells were subcutaneously injected into each mouse on day 0, and the tumor volume of the mice was recorded every 3 days starting from day 3.
  • the tumor growth rate in the treatment group with cancer cell-specific T cells obtained by micron particle sorting and amplification was significantly slower and the survival period of mice was significantly prolonged. It can be seen that the cancer cell-specific T cells of the present invention have a preventive effect on breast cancer.
  • Example 17 Micron particle sorting and amplification of T cells for the prevention of breast cancer
  • 8M urea solution was first used to lyse breast cancer cells and dissolve the lysis components. Then, PLA was used as the micron particle skeleton material, and CpG2395 and Poly ICLC were used as immune adjuvants to prepare micron particles.
  • the cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water.
  • the obtained cancer cells were inactivated and denatured using ultraviolet light and high-temperature heating respectively, and then an 8M urea aqueous solution was used to lyse the cancer cells and dissolve the lysate components, which are the antigen components for preparing micron particles.
  • the double emulsion method is used to prepare micron particles 1.
  • the molecular weight of the micron particle 1 framework material PLA is 40KDa
  • CpG2395 and Poly ICLC are immune adjuvants.
  • the antigen components and adjuvants are first loaded internally, and then 100 mg of micron particles are centrifuged at 9000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use.
  • the average particle size of the micron particles is about 2.5 ⁇ m, and each 1 mg of PLGA micron particles is loaded with approximately 600 ⁇ g of protein or peptide components, and each of CpG2395 and Poly ICLC is loaded with 0.02 mg.
  • mice Select 6-8 week old female C57BL/6 mice and subcutaneously inject 100 ⁇ L of micron particles 1 containing 2 mg PLGA on days 0, 4, 7, 14, 21, and 28.
  • the mice were sacrificed on day 32, and PBMCs in the peripheral blood of the mice were collected, and flow cytometry was used to sort out CD3 + T cells and CD19 + B cells from the PBMCs.
  • the sorted CD3 + T cells (8 million), micron particles 1 (500 ⁇ g), B cells (9 million), IL-7 (10ng/mL) and IL-15 (10ng/mL) were added to 5mL RPMI1640 Incubate in complete medium for 24 hours, and then use flow cytometry to sort the CD3 + CD69 + T cells (cell viability 80%) among the incubated CD3 + T cells, which are cancer-specific T cells that can recognize cancer antigens. cell.
  • the 500,000 CD3 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (1000U/mL), IL-15 (1000U/mL) and ⁇ CD3 antibody (10ng/mL). Incubate 10 mL of DMEM complete medium (37°C, 5% CO 2 ) for 10 days to amplify cancer cell-specific T cells (cell viability 80%), and the resulting cells are T cells 1.
  • T cells 2 million
  • micron particles 1 50 ⁇ g
  • B cells 6 million
  • IL-7 10ng/mL
  • IL-15 10ng/mL
  • mice Female BALB/c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. One day before the mice were adoptively transferred cells, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. On day 0, mice were injected subcutaneously with 1 million sorted and expanded T cells 1 or T cells 2. At the same time, 1 ⁇ 10 6 4T1 cells were subcutaneously injected into each mouse on day 0. The mouse tumor volume and survival period were monitored as above.
  • the tumor growth rate in the T cell treatment group was significantly slower and the survival period was significantly prolonged.
  • the effect of T cell 1 was significantly better than that of T cell 2.
  • Example 18 Cancer cell-specific T cells for the treatment of melanoma
  • B16F10 cells When collecting tumor tissue, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. When the tumor grew to about 1000 mm 3 , the mice were killed and the tumor tissue was removed. The tumor tissue was cut into pieces and ground. Add an appropriate amount of pure water to the cell filter and freeze and thaw repeatedly 5 times (can be accompanied by ultrasound) to destroy the lysed sample. Add nuclease for 10 minutes and then heat at 95°C for 10 minutes to inactivate the nuclease; collect the cultured B16F10 cancer cell line First, centrifuge to remove the culture medium, then wash twice with PBS and centrifuge to collect the cancer cells.
  • the cancer cells Resuspend the cancer cells in ultrapure water, freeze and thaw repeatedly for 3 times, and destroy the cancer cells with ultrasound, and then add them to the sample. After 10 minutes of nuclease action, the nuclease was inactivated by heating at 95°C for 5 minutes. After the tumor tissue or cancer cell line is treated with enzyme, the lysate is centrifuged at 5000g for 5 minutes and the supernatant is taken as the water-soluble component soluble in pure water; 50% glycerol is added to the resulting precipitate. The dissolved precipitate is partially converted into soluble.
  • nanoparticle 1 is prepared by the double emulsion method.
  • nanoparticles loaded with a mixture of water-soluble components and nanoparticles loaded with a mixture of non-water-soluble components are prepared separately and used together during application.
  • the molecular weight of PLGA, the nanoparticle preparation material used, is 7KDa-17KDa
  • the immune adjuvants used are poly(I:C) and CpG1018
  • the R8 polypeptide (RRRRRRRR) is a substance that increases lysosomal escape.
  • the preparation method is as described above. First, the double emulsion method is used to load the lysis solution components, adjuvants and R8 peptides inside the nanoparticles.
  • nanoparticles are centrifuged at 12000g for 25 minutes, and 10mL of ultrapure solution containing 4% trehalose is used. Resuspend in water and freeze-dry for 48 hours; resuspend it in 9 mL PBS before use, then add 1 mL of lysate component (protein/peptide concentration 80 mg/mL) and react at room temperature for 10 min to obtain nanoparticles loaded with lysate both inside and outside.
  • lysate component protein/peptide concentration 80 mg/mL
  • the average particle size of the nanoparticles is about 290nm; each 1 mg PLGA nanoparticle is loaded with approximately 140 ⁇ g of protein or peptide component in the antigen component, 0.02 mg each of poly(I:C) and CpG1018 immune adjuvant, and 0.1 mg R8 polypeptide. .
  • Nanoparticles DC2.4 cell line (300,000 cells) and IL-7 (10ng/mL) were incubated in 2mL RPMI1640 complete medium for a total of 96 hours, and then flow cytometry was used to sort the incubated CD8 + T cells.
  • the CD8 + CD69 + T cells (cell viability rate 80%) and the CD4 + CD69 + T cells (cell viability rate 80%) in the CD4 + T cells are cancer-specific T cells that can recognize cancer antigens.
  • the 200,000 CD8+CD69+ cancer cell-specific T cells or 100,000 CD4+CD69+ cancer cell-specific T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (1000U/mL), IL-21 (1000U/mL) and ⁇ CD3 antibody (10ng/mL) were incubated in 10mL DMEM complete medium (37°C, 5% CO 2 ) for 14 days to expand cancer cell-specific T cells (cell viability 80 %).
  • CD8 + T cells or 100,000 CD4 + T cells sorted from mouse PBMCs can be directly mixed with IL-2 (1000U/mL) and IL-7 (1000U/mL) without further sorting.
  • IL-21 (1000U/mL) and ⁇ CD3 antibody (10ng/mL) were incubated in 10mL of DMEM complete medium (37°C, 5% CO 2 ) for a total of 14 days to expand cancer cell-specific T cells.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right side of the back of each mouse. Expanded 800,000 cancer-specific CD8 + T cells and 400,000 cancer-specific CD4 + were injected intravenously on days 4, 7, 10, 15, and 20 after melanoma inoculation, respectively. T cells (after two steps of sorting); or 800,000 CD8 + T cells and 400,000 CD4 + T cells (without nanoparticle sorting) expanded after only one step of sorting were injected on the above days. The method for monitoring tumor growth and survival in mice is the same as above.
  • cancer-specific T cells obtained by sorting and amplifying nanoparticles loaded with lysate components were better than T cells directly amplified without nanoparticle sorting.
  • the nanoparticles were prepared using the double emulsion method.
  • the preparation material of nanoparticle 1 is PLGA with a molecular weight of 7KDa-17KDa, Poly(I:C) and CpG1018 as adjuvants, and NH 4 HCO 3 as a substance that increases lysosomal escape.
  • the preparation method is as described above.
  • the lysis solution components, adjuvants and NH 4 HCO 3 are first loaded inside the nanoparticles. Then 100 mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and 10 mL of trehalose containing 4% trehalose is used.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein and peptide components, with 0.02 mg of poly(I:C) and 0.02 mg of CpG1018 each. NH 4 HCO 3 0.01 mg.
  • the preparation materials and preparation method of Nanoparticle 2 are the same as Nanoparticle 1, but it does not load adjuvants and only loads increased lysosomal escape substances; the particle size is about 260nm, and the surface potential is about -7mV; every 1 mg of PLGA nanoparticles is loaded with about 90 ⁇ g of protein.
  • the peptide component, each 1mg of PLGA nanoparticles is loaded with 0.01mg of NH 4 HCO 3 and does not load with adjuvants.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected and injected subcutaneously with 100 ⁇ L of nanoparticles 1 containing 2 mg PLGA on days 0, 4, 7, 14, 21, and 28. The mice were sacrificed on day 32, and the PBMCs of the mice were collected, and CD8 + T cells, CD4 + T cells, and B cells were sorted from the PBMCs using flow cytometry.
  • the sorted CD8 + T cells (2 million), CD4 + T cells (1 million), nanoparticles (50 ⁇ g), B cells (3 million) and IL-7 (10ng/mL) were added to 2mL RPMI1640 Incubate in complete medium for 48 hours, and then use flow cytometry to sort the CD8 + CD69 + T cells (cell viability 80%) among the incubated CD8 + T cells and the CD4 + CD69 + T cells among the CD4 + T cells. (cell viability 80%), which are cancer-specific T cells that can recognize cancer antigens.
  • the 200,000 CD8 + CD69 + T cells or 200,000 CD4 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (1000U/mL), IL-15 (1000U/mL) mL) and ⁇ CD3 antibody (10 ng/mL) were incubated in 10 mL of DMEM complete medium (37°C, 5% CO 2 ) for a total of 14 days to expand cancer cell-specific T cells (cell viability 80%).
  • the tumor growth rate in the treatment group with cancer-specific T cells obtained by nanoparticle sorting and amplification was significantly slower and the survival period was significantly longer.
  • CD8 + T cells and CD4 + T cells obtained by simultaneous sorting and expansion using nanoparticles are better than CD8 + T cells obtained only by using nanoparticles to assist sorting and expansion.
  • Example 20 Cancer cell-specific T cells for the treatment of melanoma
  • Dissolving the precipitated part can convert the water-insoluble components insoluble in pure water into soluble in 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution. Add saturated ammonium sulfate aqueous solution dropwise to the water-soluble components in the lysis solution. After the precipitation is complete, centrifuge the sample at 3000g for 5 minutes. Dissolve the precipitate in 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution for later use. Heat the supernatant at 100°C for 5 minutes and centrifuge the sample at 3000g for 5 minutes.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the mice were sacrificed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and then ground.
  • An appropriate amount of ultrapure water was added through a cell filter and frozen and thawed 5 times repeatedly, accompanied by ultrasound to destroy the lysed cells.
  • centrifuge the lysate at 5000g for 5 minutes and take the supernatant as the water-soluble component soluble in pure water; add 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution to the resulting precipitate.
  • Dissolving the precipitated part can convert the water-insoluble components insoluble in pure water into soluble in 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution. Add saturated ammonium sulfate aqueous solution dropwise to the water-soluble components in the lysis solution. After the precipitation is complete, centrifuge the obtained sample at 3000g for 5 minutes. Dissolve the precipitate in 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution as a water solution. used as part of the sexual component.
  • the water-insoluble components in the lysate dissolved in the above 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution and the water-soluble components in the above 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution were salted out and precipitated.
  • the obtained components are mixed at a mass ratio of 1:1, which is the antigen component 2 for preparing nanoparticles 2.
  • Dissolving the precipitated part can convert the water-insoluble components insoluble in pure water into soluble in 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution. Heat the water-soluble components in the lysate at 100°C for 5 minutes, then centrifuge the sample at 3000g for 5 minutes. Discard the supernatant and dissolve the precipitate in 2M semicarbazide hydrochloride and 0.2M agmatine sulfate aqueous solution, that is It is part of the water-soluble components.
  • Nanoparticle 1 (Nanoparticle 1) was prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of PLGA, the material used to prepare the antigen delivery nanoparticles, is 20KDa-40KDa, and the immune adjuvant used is poly(I:C).
  • the preparation method is as described above.
  • the double emulsion method is used to load the antigen component 1 and the adjuvant inside the nanoparticles.
  • 300mg of the nanoparticles are centrifuged at 14000g for 30 minutes and resuspended in 10mL of ultrapure water containing 4% trehalose. Freeze dry for 48h.
  • the average particle size of the nanoparticles 1 is about 100nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 250 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.01 mg of poly(I:C).
  • Nanoparticle 2 the preparation method and preparation materials of Nanoparticle 2 (Nanoparticle 2) are the same as Nanoparticle 1.
  • the preparation method is as described above. First, use the double emulsion method to load the antigen component 2 and the adjuvant inside the nanoparticles. Then, centrifuge 100mg of the nanoparticles at 14000g for 30 minutes, and resuspend in 10mL of ultrapure water containing 4% trehalose. Freeze dry for 48h.
  • the average particle size of the nanoparticles 2 is about 300nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 250 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.01 mg of poly(I:C).
  • Nanoparticle 3 the preparation method and preparation materials of Nanoparticle 3 (Nanoparticle 3) are the same as Nanoparticle 1.
  • the antigen components and adjuvants were loaded inside the nanoparticles, and then 100 mg of the nanoparticles was centrifuged at 14,000 g for 30 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h.
  • the average particle size of the nanoparticles 3 is about 300nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 250 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.01 mg of poly(I:C).
  • mice that are 6-8 weeks old and inoculate the mice subcutaneously with 1.5 ⁇ 10 5 B16F10 cells on day 0.
  • 150 ⁇ g PD-1 antibody was injected intraperitoneally into each mouse on days 16, 18, 20 and 22 respectively. The mice were sacrificed on day 24, and their peripheral blood was collected. Peripheral blood mononuclear cells (PBMC) were then isolated from the peripheral blood, and all CD69- PBMC were sorted using flow cytometry.
  • PBMC Peripheral blood mononuclear cells
  • CD3 + CD69 + T cells are cancer-specific T cells that can recognize cancer antigens.
  • the 1 million CD3 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (1000U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL).
  • the cells were incubated in 10 mL of DMEM complete medium (37°C, 5% CO 2 ) for 21 days to expand cancer cell-specific T cells (cell viability 85%).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right side of the back of each mouse. 500,000 CD3 + cancer-specific T cells were injected intravenously on days 5, 8, 11, 15, 20, and 25. The method for monitoring tumor growth and survival in mice is the same as above.
  • mice in the PBS group grew rapidly.
  • the tumor growth rate of mice treated with cancer cell-specific T cells obtained by sorting and amplifying nanoparticles, nanoparticles 2 and nanoparticles 3 was significantly slowed down, the survival period was significantly prolonged, and some mice were cured.
  • nanoparticle 3 is better than nanoparticle 2 in sorting cancer cell-specific T cells, indicating that the effect of particles prepared by using heating to separate and purify antigen components is better than using specific reagents to separate and purify by salting out; nanoparticles
  • the effect of particle 1 is better than that of nanoparticle 2 and nanoparticle 3, which means that the effect of particles prepared by using salting out and heating to separate and purify the antigen component is significantly better than that of particles prepared by using only salting out to separate and purify the antigen component or Particles prepared using only heat to separate and purify antigen components.
  • the antigen component mainly uses the protein and polypeptide components in the whole cell fraction.
  • the mRNA in the cell lysate can also be separated and extracted and the protein/polypeptide in the whole cell fraction can also be extracted. Mix and use as mixed antigen.
  • the cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400g for 5 minutes, then resuspended in ultrapure water, and then 6M guanidine sulfate aqueous solution was used to lyse the cancer cells and dissolve the lysate components, and then saturated ammonium sulfate was added aqueous solution until the precipitation is complete, discard the supernatant, and dissolve the precipitate in 6M guanidine sulfate aqueous solution again to obtain the protein and peptide components in cancer cells dissolved in the 6M guanidine sulfate aqueous solution, which is the antigen component 1.
  • the cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400g for 5 minutes, then resuspended in ultrapure water, and then 3% Tween 80 aqueous solution was used to lyse the cancer cells and solubilize the lysate components, and then added Saturate the ammonium sulfate aqueous solution until the precipitation is complete, discard the supernatant, and solubilize the precipitate again with 3% Tween 80 aqueous solution to obtain the protein and peptide components dissolved in the 3% Tween 80 aqueous solution, which is the antigen group. Score 2.
  • the cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400g for 5 minutes, then resuspended in ultrapure water, and then 6M guanidine sulfate aqueous solution was used to lyse the cancer cells and dissolve the lysate components, and then add saturated ammonium sulfate aqueous solution until the precipitation is complete, discard the supernatant, and solubilize the precipitate again with 3% Tween 80 aqueous solution to obtain the protein and peptide components dissolved in the 3% Tween 80 aqueous solution, which is the antigen component 3.
  • the cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400g for 5 minutes, then resuspended in ultrapure water, and the cancer cells were lysed using 3% Tween 80 aqueous solution and then dissolved and lysed using 3% Tween 80 aqueous solution. Then add saturated ammonium sulfate aqueous solution until the precipitation is complete, discard the supernatant, and dissolve the precipitate again with 6M guanidine sulfate aqueous solution to obtain the protein and peptide components dissolved in the 6M guanidine sulfate aqueous solution, which is the antigen.
  • Component 4 The cultured E.G7-OVA mouse T lymphoma cells were centrifuged at 400g for 5 minutes, then resuspended in ultrapure water, and the cancer cells were lysed using 3% Tween 80 aqueous solution and then dissolved and lysed using 3% Tween
  • the double emulsion method is used to prepare Nanoparticle 1 (Nanoparticle 1).
  • the skeleton materials of nanoparticle 1 are PLA (molecular weight 30-40KDa) and mannan-PEG2000-PLA (PLA molecular weight 30-40KDa), and PLA (molecular weight 30-40KDa) and mannan-PEG2000-PLA (PLA molecular weight 30-40KDa) mass ratio is 9:1.
  • the immune adjuvants used are CpG2006 (Class B), CpG2216 (Class A) and Poly ICLC.
  • the double emulsion method is first used to prepare nanoparticles internally loaded with antigen component 1 and adjuvant.
  • nanoparticles are centrifuged at 13,000g for 25 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours.
  • the obtained nanoparticles 1 have an average particle size of about 500nm.
  • Each 1 mg of PLGA nanoparticles 1 is loaded with approximately 5 ⁇ g of protein and peptide components of cancer cells, and 0.02 mg each of CpG2006, CpG2216 and Poly ICLC.
  • Nanoparticle 2 The preparation method of Nanoparticle 2 is the same as Nanoparticle 1. But the internal load is antigen component 2 and adjuvant.
  • the average particle size of nanoparticles 2 is about 500nm.
  • Each 1 mg of PLGA nanoparticles 2 is loaded with approximately 5 ⁇ g of protein and peptide components of cancer cells, and each of 0.02 mg of CpG2006, CpG2216 and Poly ICLC is loaded.
  • Nanoparticle 3 The preparation method of Nanoparticle 3 is the same as Nanoparticle 1. But the internal load is antigen component 3, adjuvant and R8 polypeptide. The average particle size of nanoparticles 3 is about 500nm. Each 1 mg of PLGA nanoparticles 3 is loaded with approximately 5 ⁇ g of protein and peptide components of cancer cells, and each of 0.02 mg of CpG2006, CpG2216 and Poly ICLC is loaded.
  • Nanoparticle 4 The preparation method of Nanoparticle 4 is the same as Nanoparticle 1. But the internal load is antigen component 4 and adjuvant.
  • the average particle size of nanoparticles 4 is about 500nm.
  • Each 1 mg of PLGA nanoparticles 4 is loaded with approximately 5 ⁇ g of protein and peptide components of cancer cells, and each of 0.02 mg of CpG2006, CpG2216 and Poly ICLC is loaded.
  • mice Female C57BL/6 mice aged 6 to 8 weeks were selected and inoculated subcutaneously with 5 ⁇ 10 5 E.G7-OVA mouse T lymphoma cells on day 0. Each mouse was intraperitoneally injected with 150 ⁇ g of PD-1 antibody on days 4, 6, 8, 10, 12, 14, 16, 18, and 20. The mice were sacrificed on day 22, and their peripheral blood was collected. Peripheral blood mononuclear cells (PBMC) were then isolated from the peripheral blood, and all CD25- PBMC were sorted using flow cytometry.
  • PBMC Peripheral blood mononuclear cells
  • CD25 - PBMC cells and 5 ⁇ g nanoparticles were incubated in 5 mL RPMI1640 complete medium for a total of 96 hours, and then flow cytometry was used CD3 + CD25 + T cells (cell viability 90%) after surgical sorting and incubation are cancer-specific T cells that can recognize cancer antigens.
  • the 100,000 CD8 + CD25 + T cells obtained above were mixed with IL-2 (1000U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 20mL of DMEM complete medium (37°C , 5% CO 2 ) for 28 days to expand cancer cell-specific T cells (cell viability 90%).
  • the tumor growth rate of mice treated with T cells was significantly slower and the survival period of mice was significantly prolonged.
  • the effect of using nanoparticle 1 to sort and amplify cancer cell-specific T cells is better than that of nanoparticle 2, nanoparticle 3, and nanoparticle 4, indicating that the use of appropriate dissolving agents for primary dissolution and secondary dissolution to prepare nanoparticles for cancer cells
  • the cellular antigen component is very critical.
  • the non-water-soluble components are converted into soluble in 8M urea aqueous solution.
  • the antigen component 1 for preparing nanoparticle 1 is obtained by mixing the non-water-soluble component in the lysate dissolved in the above 8M urea aqueous solution and the water-soluble component dissolved in 8M urea after salting out and precipitating in a mass ratio of 1:2. .
  • the non-water-soluble antigen is converted into soluble in 8M urea aqueous solution.
  • the antigen components for preparing nanoparticles 2 are obtained by mixing the non-water-soluble components in the lysate dissolved in the above 8M urea aqueous solution and the water-soluble components dissolved in 8M urea after salting out and precipitating in a mass ratio of 1:2. 2.
  • the non-water-soluble antigen is converted into soluble in 8M urea aqueous solution.
  • the antigen component 3 for preparing nanoparticles 3 is obtained by mixing the non-water-soluble components and the water-soluble components in the lysate dissolved in the above 8M urea aqueous solution at a mass ratio of 1:2.
  • the non-water-soluble components are converted into soluble in 8M urea aqueous solution.
  • the antigen for preparing nanoparticle 4 is obtained by mixing the non-water-soluble components in the lysate dissolved in the above 8M urea aqueous solution and the water-soluble components dissolved in the 5% PEG5000 aqueous solution after salting out and precipitating at a mass ratio of 1:2.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the mice were sacrificed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and ground, then an appropriate amount of ultrapure water was added and frozen and thawed 5 times repeatedly, accompanied by ultrasound to destroy the lysed cells.
  • the sexual component is soluble in 5% PEG5000 aqueous solution.
  • Nanoparticle 1 (Nanoparticle 1) was prepared by the double emulsion method in the solvent evaporation method.
  • the double emulsion method was used to load the cell antigen component 1 and the adjuvant inside the nanoparticles.
  • 100 mg of the nanoparticles was centrifuged at 13,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours.
  • the average particle size of the nanoparticles 1 is about 200nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 15 ⁇ g of protein or peptide components and 0.2 mg of poly(I:C).
  • Nanoparticle 2 The preparation method, materials and preparation steps of Nanoparticle 2 are the same as Nanoparticle 1.
  • the preparation method is as described above, first load the cell antigen component 2 and adjuvant inside the nanoparticles, then centrifuge 100mg nanoparticles at 13000g for 20 minutes, resuspend in 10mL of ultrapure water containing 4% trehalose and freeze-dry for 48h. .
  • the average particle size of the nanoparticles 2 is about 200nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 15 ⁇ g of protein or peptide components and 0.2 mg of poly(I:C).
  • Nanoparticle 3 The preparation method, materials and preparation steps of Nanoparticle 3 are the same as Nanoparticle 1. First, the cell antigen component 3 and the adjuvant were loaded inside the nanoparticles, and then 100 mg of the nanoparticles was centrifuged at 13,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours. The average particle size of the nanoparticles 3 is about 200nm. Each 1 mg of PLGA nanoparticles is loaded with approximately 15 ⁇ g of protein or peptide components and 0.2 mg of poly(I:C).
  • Nanoparticle 4 The preparation method, materials and preparation steps of Nanoparticle 4 are the same as Nanoparticle 1.
  • the preparation method is as described above. First, load the cell antigen component 4 and the adjuvant inside the nanoparticles, then centrifuge 100mg of the nanoparticles at 13000g for 20 minutes, resuspend in 10mL of ultrapure water containing 4% trehalose and freeze-dry for 48h. .
  • the average particle size of the nanoparticles 4 is about 200nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 15 ⁇ g of protein or peptide components and 0.2 mg of poly(I:C).
  • Nanoparticle 5 The preparation method, materials and preparation steps of Nanoparticle 5 are the same as Nanoparticle 1.
  • Cell antigen component 5 and adjuvant are first loaded inside the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 13,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours.
  • the average particle size of the nanoparticles 5 is about 200nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 15 ⁇ g of protein or peptide components and 0.2 mg of poly(I:C).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected, and 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the mice on day 0. Each mouse was intraperitoneally injected with 150 ⁇ g of PD-L1 antibody on days 4, 6, 8, 10, 12, 14, 16, 18, and 20. The mice were sacrificed on day 22, and their PBMCs were collected. The PBMCs were first cultured in vitro for 12 hours, and then flow cytometry was used to sort out all CD69 - PBMCs.
  • CD69 - PBMC cells 2.5 million CD69 - PBMC cells and 500 ⁇ g nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3, or nanoparticle 4, or nanoparticle 5) were incubated in 10 mL RPMI 1640 complete medium for 36 hours. , and then use flow cytometry to sort the incubated CD3 + CD69 + T cells (cell viability 90%), which are cancer-specific T cells that can recognize cancer antigens.
  • the 100,000 CD3 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 10mL DMEM complete medium (37°C , 5% CO 2 ) for 21 days to expand cancer cell-specific T cells (cell viability 90%).
  • mice were prepared by selecting 6-8-week-old female C57BL/6 model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse. On the 3rd day, the 6th day, the 9th day, the 14th day and the 20th day before the mice were inoculated with tumors, the mice were subcutaneously inoculated with 100,000 cancer cell-specific T cells or 100 ⁇ L of different nanoparticle-assisted sorting. PBS. Monitor mouse tumor growth rate and mouse survival time.
  • the tumor volume of mice in the PBS group grew rapidly.
  • Cancer cell-specific T cells obtained by sorting and amplifying Nanoparticle 1, Nanoparticle 2, Nanoparticle 3, Nanoparticle 4 and Nanoparticle 5
  • the tumor growth rate of mice treated with cells was significantly slowed down, and their survival period was significantly prolonged.
  • the effect of nanoparticle 1 is better than that of nanoparticle 2, nanoparticle 3, nanoparticle 4 and nanoparticle 5, indicating that the effect of using ammonium sulfate salting out to separate and purify protein and peptide antigen components in water-soluble components is better than using ammonium carbonate.
  • the effect of isolating and purifying a part of the water-soluble medium components is better than directly using the water-soluble components.
  • the use of urea to dissolve water-soluble components through salting out treatment produces better precipitation results than the use of PEG.
  • Example 23 Sorted and amplified T cells kill breast cancer cells
  • This example uses tumor tissue from multiple breast cancer patients to prepare nanoparticles.
  • Tumor tissue obtained through surgical resection from 12 triple-negative breast cancer patients was collected. Each patient's tumor tissue was cut into sections and ground, passed through a cell filter, added to ultrapure water, and then repeatedly frozen and thawed 5 times with ultrasound to lyse the cancer cells in the tumor tissue. Add 1 mg/mL nuclease to the lysed cancer cells to degrade the nucleic acid in the lysate, then heat at 95°C for 10 minutes to inactivate the nuclease, and then centrifuge at 5000g for 5 minutes to collect the supernatant, which is the water-soluble component, and precipitate Use 8M urea (containing 0.01M arginine) aqueous solution to dissolve the non-water-soluble component.
  • 8M urea containing 0.01M arginine
  • the water-soluble components of 12 breast cancer patients were mixed at a mass ratio of 1:1 to obtain a water-soluble component mixture; the non-water-soluble components of 12 breast cancer patients were mixed at a mass ratio of 1:1 to obtain a non-water-soluble component mixture.
  • Mixture of water-soluble components Mix the water-soluble component mixture and the water-insoluble component mixture at a mass ratio of 5:1 to prepare the antigen component 1 of nanoparticle 1 (NP1).
  • the tumor tissue obtained by surgical resection from another triple-negative breast cancer patient A was collected.
  • Patient A is not included in the above 12 cancer patients.
  • the tumor tissue was cut into pieces and ground, passed through a cell filter, added to ultrapure water, and then repeatedly frozen and thawed 5 times with ultrasound to lyse cancer cells.
  • nuclease Add 1 mg/mL nuclease to the lysed cancer cells to degrade the nucleic acid in the lysate, then heat at 95°C for 10 minutes to inactivate the nuclease, and then centrifuge at 5000g for 5 minutes to collect the supernatant, which is the water-soluble component, and precipitate Use 8M urea (containing 0.01M arginine) aqueous solution to dissolve the non-water-soluble component. Mix the water-soluble component and the non-water-soluble component at a mass ratio of 5:1 to prepare the antigen for nanoparticle 2 (NP2). Component 2.
  • the double emulsion method was used to prepare nanoparticles.
  • the molecular weight of the nanoparticle 1 skeleton material PLGA is 10KDa-20KDa, and the immune adjuvants used are CpG2395 (Category C), CpG1018 (Category B) and Poly ICLC.
  • the antigen component 1 and the adjuvant are first loaded inside the particles, and then 100 mg of the nanoparticles are centrifuged at 12,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use.
  • the particle size of the nanoparticles is about 280nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 950 ⁇ g of protein or peptide components, with 0.02 mg each of CpG2395, CpG1018, and Poly(I:C).
  • the molecular weight of the nanoparticle 2 framework material PLGA is 10KDa-20KDa, and the immune adjuvants used are CpG2395 (Class C), CpG1018 (Class B) and Poly ICLC.
  • the double emulsion method was used to prepare nanoparticles internally loaded with antigen component 2 and adjuvant. Then, 100 mg of nanoparticles were centrifuged at 12,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use.
  • the particle size of the nanoparticles is about 280nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 950 ⁇ g of protein or peptide components, with 0.02 mg each of CpG2395, CpG1018, and Poly(I:C).
  • PBMC peripheral blood mononuclear cells
  • IL-7 10 ng
  • IL-15 10 ng
  • 20 ⁇ g nanoparticles 20 ⁇ g nanoparticles (nanoparticle 1 or nanoparticle 2) before or after immunotherapy were placed in 2 mL AIM V serum-free medium. Incubate in medium medium for 72 hours (37°C, 5% CO2 ). After incubation, cells were collected and flow cytometry was used to sort CD3 + CD25 + T cells (cell viability 85%).
  • T cells with IL-2 (1000U/mL) and ⁇ CD3 antibody ( 10 ng/mL) and ⁇ CD28 antibody (10 ng/mL) were incubated in 10 mL of DMEM complete medium (37°C, 5% CO 2 ) for a total of 21 days to expand cancer cell-specific T cells (cell viability 85%).
  • PBMC peripheral blood mononuclear cells
  • IL-7 10ng
  • IL-15 10ng
  • nanoparticles 1 or nanoparticles 2 respectively 5ng nanoparticles after immunotherapy in 2mL AIM V serum-free medium.
  • the culture medium was incubated for a total of 72 hours (37°C, 5% CO2 ). After incubation, cells were collected and flow cytometry was used to sort CD3 + CD25 + T cells (cell viability 85%).
  • T cells with IL-2 (1000U/mL) and ⁇ CD3 antibody ( 10 ng/mL) and ⁇ CD28 antibody (10 ng/mL) were incubated in 10 mL of DMEM complete medium (37°C, 5% CO 2 ) for a total of 21 days to expand cancer cell-specific T cells (cell viability 85%).
  • PBMC peripheral blood mononuclear cells
  • IL-7 10ng
  • IL-15 10ng
  • 100mg nanoparticles 100mg nanoparticles (nanoparticles 1 or nanoparticles 2 respectively) after immunotherapy in 2mL AIM V serum-free medium.
  • the culture medium was incubated for a total of 72 hours (37°C, 5% CO2 ). After incubation, cells were collected and flow cytometry was used to sort CD3 + CD25 + T cells (cell viability 85%).
  • T cells with IL-2 (1000U/mL) and ⁇ CD3 antibody ( 10 ng/mL) and ⁇ CD28 antibody (10 ng/mL) were incubated in 10 mL of DMEM complete medium (37°C, 5% CO 2 ) for a total of 21 days to expand cancer cell-specific T cells (cell viability 85%).
  • Nude mice that were 6 to 8 weeks old were selected, and on day 0, 5 ⁇ 10 5 cancer cells amplified from the tumor tissue surgically resected by patient A were subcutaneously inoculated into the lower right side of each nude mouse's back.
  • the cancer cell-specific T cells obtained by sorting and amplifying the two types of nanoparticles can effectively control the growth of tumor tissue.
  • nanoparticle 1 and nanoparticle 2 have similar effects.
  • the effect of co-incubation-assisted sorting with nanoparticles at an appropriate concentration (10 ⁇ g/mL) is significantly better than using a concentration that is too low (2.5ng/mL) or a concentration that is too high (50 mg/mL).
  • the effect of cancer cell-specific T cells sorted and amplified after immunotherapy is better than that of cancer cell-specific T cells sorted and amplified before immunotherapy.
  • peripheral blood immune cells after immunotherapy for sorting and amplification.
  • Example 24 Sorted and amplified T cells kill esophageal cancer cells
  • Tumor tissues from 5 esophageal cancer patients were collected.
  • the tumor tissues of 5 patients were mixed according to the mass ratio of 1:1:1:1:1, cut into pieces, and ground. Then, after passing through the cell filter, an appropriate amount of 8M urea aqueous solution was added to lyse the above cells, and the 8M urea aqueous solution was used to completely dissolve the tumor tissue.
  • the lysate component is the antigen component 1 for preparing nanoparticles 1 and 2.
  • Tumor tissue from another esophageal cancer patient A was collected. Patient A is not included in the above five cancer patients. Cut and grind the tumor tissue of patient A, then add an appropriate amount of 8M urea aqueous solution to lyse the above cells after passing through the cell filter, and use the 8M urea aqueous solution to completely dissolve the tumor tissue lysate components, which is to prepare nanoparticles 3 and 4. Antigenic component 2.
  • nanoparticle 1 was prepared by the double emulsion method.
  • the nanoparticle preparation material used is PLGA with a molecular weight of 10KDa-30KDa.
  • the loaded adjuvants are poly I:C and CpG7909.
  • the loaded KALA polypeptide increases the lysosomal immune evasion substance.
  • the preparation method is as described above. First, load the antigen component, adjuvant and KALA peptide inside the nanoparticles, then centrifuge 100mg nanoparticles at 12000g for 25 minutes, resuspend in 10mL of ultrapure water containing 4% trehalose and freeze Dry for 48h. The average particle size of the nanoparticles is about 250nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components of tumor tissue, 0.01 mg each of poly I:C and CpG7909, and 0.15 mg of KALA peptide.
  • Nanoparticle 2 The preparation materials and methods of Nanoparticle 2 (NP2) are the same.
  • the particle size is about 250nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 0.01 ⁇ g of protein or peptide components of tumor tissue.
  • the loading of poly I:C and CpG7909 is 0.01 mg each.
  • nanoparticle 3 The preparation materials and methods of nanoparticle 3 (NP3) are the same, about 250nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 100 ⁇ g of protein and peptide components of tumor tissue, with 0.01 mg each of poly I:C and CpG7909, and 0.15 mg of KALA peptide. mg.
  • nanoparticle 4 The preparation materials and methods of nanoparticle 4 (NP4) are the same, about 250nm. Each 1mg PLGA nanoparticle is loaded with approximately 0.01 ⁇ g of protein or peptide components of tumor tissue, with 0.01mg each of poly I:C and CpG7909, and KALA peptide. 0.15 mg.
  • PBMCs are then isolated from peripheral blood. PBMC (10 million) and 100 ⁇ g nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3, or nanoparticle 4) were incubated in 10 mL AIM V serum-free medium for 96 hours (37°C, 5% CO 2 ).
  • CD3 + HLA-DR + T cells (cell viability 85%), which are cancer cell-specific T cells.
  • the T cells sorted above were co-incubated with IL-2 (1000U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 10mL DMEM complete medium (37°C, 5% CO 2 ) 21 days to expand cancer cell-specific T cells (cell viability 85%).
  • Nude mice that were 6 to 8 weeks old were selected, and on day 0, 5 ⁇ 10 5 cancer cells amplified from the tumor tissue surgically resected by patient A were subcutaneously inoculated into the lower right side of each nude mouse's back.
  • the specificity of cancer cells obtained after subcutaneous injection of 500,000 different nanoparticles into mice on days 3, 6, 9, 14 and 20 after tumor inoculation in mice. T cells or 100 ⁇ L PBS. Monitor tumor growth rate in mice.
  • the effect of sorting and amplifying cancer cell-specific T cells obtained by sorting and amplifying nanoparticles loaded with a mixture of multiple allogeneic cancer patient tumor tissues is comparable to that of nanoparticles obtained by using autologous tumor tissue from cancer patients to assist sorting and amplification. There is no significant difference in the effect. Furthermore, nanoparticles loaded with appropriate antigenic components are better than nanoparticles loaded with only low levels of antigenic components.
  • Example 25 Sorting and amplifying T cells to kill lung cancer cells
  • water-soluble components and non-water-soluble components from various human lung cancer cancer cell lines are loaded into nanoparticles, and then the nanoparticles are used to sort and amplify cancer cells in the peripheral immune organs of lung cancer patients. specific T cells.
  • Human lung cancer cell lines A549 cells, H1299 cells, PC9 cells, H1437 cells, H226 cells, HCC1588 cells, H2170 cells and H520 cells were cultured respectively.
  • tumor tissue samples from surgical resection of non-small cell lung cancer patient A This patient with non-small cell lung cancer has a good response to immunotherapy.
  • the tumor tissue was cut into pieces and filtered through a cell mesh, then resuspended in ultrapure water and repeatedly frozen and thawed 5 times. During the freezing and thawing process, ultrasonic disruption was used to more thoroughly lyse cancer cells. After the cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant, which is the water-soluble component that is soluble in pure water; add 6M guanidine sulfate to the resulting precipitate to dissolve the precipitate and remove the insoluble components.
  • the water-insoluble components of pure water are converted to soluble in 6M aqueous guanidine sulfate solution. Heat the water-soluble components in the lysate at 95°C for 10 minutes, then centrifuge the resulting sample at 3000g for 5 minutes.
  • NP2 nanoparticle 2
  • the water-insoluble components of pure water are converted to soluble in 6M aqueous guanidine sulfate solution. Heat the water-soluble components in the lysate at 95°C for 10 minutes, then centrifuge the resulting sample at 3000g for 5 minutes.
  • NP3 nanoparticle 3
  • nanoparticle 1 was prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 20KDa-40KDa, and the adjuvants are poly(I:C), CpG7909 and CpG2395.
  • the preparation method was as described above, first loading the antigen components and adjuvants inside the nanoparticles, then centrifuging 100mg of the nanoparticles at 10,000g for 20 minutes, resuspending in 10mL of ultrapure water containing 4% trehalose and freeze-drying for 48h.
  • the average particle size of the nanoparticles 1 is about 380nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 800 ⁇ g of protein or peptide components, and each of poly(I:C), CpG7909, and CpG2395 is loaded with 0.02 mg.
  • nanoparticle 2 (NP2) is the same as that of nanoparticle 1.
  • the average particle size of nanoparticle 2 is about 380nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 800 ⁇ g of protein or peptide components, including poly(I:C), CpG7909 and CpG2395. 0.02mg each.
  • nanoparticle 3 (NP3) is the same as that of nanoparticle 1.
  • the average particle size of nanoparticle 3 is about 380nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 800 ⁇ g of protein or peptide components, including poly(I:C), CpG7909 and CpG2395. 0.02mg each.
  • Non-small cell lung cancer patient A s tumor tissue shrank after immunotherapy. Two weeks after non-small cell lung cancer patient A's immunotherapy, 10 mL of peripheral blood was drawn from the patient's peripheral blood. Peripheral blood mononuclear cells (PBMC) were isolated from 10 mL of peripheral blood of patients with non-small cell lung cancer using gradient centrifugation.
  • PBMC peripheral blood mononuclear cells
  • Nanoparticle 1 (0.5 mg) or nanoparticle 2 (0.5 mg) or nanoparticle 3 (0.5 mg) and PBMC (10 million) were incubated in 1 mL of AIM V serum-free medium for 12 hours (37°C, 5% CO 2 ). The cells were then collected and centrifuged at 400g for 5 minutes. The cells were resuspended in PBS and the T cells were first treated with Fc block to avoid non-specific loading.
  • the other half of the cells were directly sorted from CD3 + FASL + T cells (cell viability 85%), and the sorted 500,000 T cells were mixed with IL-2 (1000U/mL) and ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10 ng/mL) was incubated in 10 mL of DMEM complete medium (37°C, 5% CO 2 ) for a total of 28 days to expand cancer cell-specific T cells (cell viability 85%).
  • Nude mice that were 6-8 weeks old were selected, and on day 0, 5 ⁇ 10 5 cancer cells amplified from the cancer cells in patient A were subcutaneously inoculated into the lower right side of each nude mouse's back.
  • the specificity of cancer cells obtained after subcutaneous injection of 500,000 different nanoparticles into mice on days 3, 6, 9, 14 and 20 after tumor inoculation in mice. T cells or 100 ⁇ L PBS. Monitor mouse tumor growth rate and mouse survival time.
  • nanoparticle 1 and nanoparticle 2 have the same effect on cancer cell-specific T cells obtained by sorting and amplifying them. Moreover, T cells that can secrete IFN- ⁇ after activation overlap with T cells that can express FASL. , Moreover, the cancer cell-specific T cells obtained by sorting and amplifying the present invention can effectively kill cancer cells.
  • the effect of sorting and amplifying T cells using nanoparticles prepared from the cancer patient's own tumor tissue is better than using a single tumor from other patients. Nanoparticles prepared from tissue; and the effect of using antigen components of multiple cancer cell lines to prepare nanoparticles is the same as that of nanoparticles prepared from the patient's own tumor tissue.
  • Example 26 Cancer cell-specific T cells for the treatment of melanoma
  • B16-F10, B16-F1, S91, ME, K735, B16-BL6 and MEC57 cancer cell lines Collect the cultured B16-F10, B16-F1, S91, ME, K735, B16-BL6 and MEC57 cancer cell lines, and mix the above cancer cell lines in a quantitative ratio of 1:1:1:1:1:1:1.
  • the mixed cells were then lysed using 0.2M arginine and 0.1M semicarbazide hydrochloride. After lysis, all lysis components were dissolved using 0.2M arginine and 0.1M semicarbazide hydrochloride, and then saturated aqueous ammonium sulfate solution was added dropwise. After the precipitation is complete, centrifuge the obtained sample at 3000g for 5 minutes.
  • the precipitate was dissolved in 0.2M arginine and 0.1M semicarbazide hydrochloride aqueous solution; then 0.2M arginine and 0.1M semicarbazide hydrochloride were used.
  • the dissolved precipitate after salting out and the precipitate after heating are combined to form the antigen component 2 for preparing the nanoparticle 2.
  • Nanoparticle 1 (Nanoparticle 1) is prepared by the double emulsion method.
  • the particle preparation material PLGA has a molecular weight of 10KDa-20KDa, and the immune adjuvants used are poly(I:C), CpG 7909 and CpG2395.
  • the preparation method was as described above. First, the antigen component 1 and the adjuvant were loaded inside the nanoparticles. Then, 100 mg of the nanoparticles was centrifuged at 12,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h. The average particle size of the nanoparticle 1 (nano vaccine 1) is about 280nm. Each 1 mg PLGA particle is loaded with approximately 250 ⁇ g of protein or peptide components, and each of poly(I:C), CpG7909 and CpG2395 is loaded with 0.01 mg.
  • Nanoparticle 2 (Nanoparticle 2) in this embodiment are the same as Nanoparticle 1.
  • Antigen component 2 and adjuvant are first loaded inside the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 12,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours.
  • the average particle size of the particles 2 is about 280nm.
  • Each 1 mg PLGA particle is loaded with approximately 250 ⁇ g of protein or peptide components, and each of poly(I:C), CpG7909 and CpG2395 is loaded with 0.01 mg.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected and inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 mouse melanoma cancer cells on day 0. Each mouse was intraperitoneally injected with 150 ⁇ g of PD-L1 antibody on days 6, 8, 10, 12, 14, 16, 18, and 20. The mice were sacrificed on day 22, their peripheral blood was collected, and then mouse PBMCs were prepared, which are mixed immune cells co-incubated with nanoparticles.
  • CD3 + CD69 + T cells which are those that can recognize cancer antigens.
  • Cancer-specific T cells cell viability 85%).
  • the CD3 + CD69 + T cells obtained above were mixed with IL-2 (100U/mL), IL-7 (100U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 10mL of DMEM.
  • T cells 1 were obtained after incubation in complete medium (37°C, 5% CO 2 ) for 48 days.
  • CD3 + IFN- ⁇ + T cells obtained above were mixed with IL-2 (100U/mL), IL-7 (100U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 10mL.
  • T cells 3 were obtained after incubation in DMEM complete medium (37°C, 5% CO 2 ) for 48 days.
  • CD3 + FOXP3 + T cells obtained above were mixed with IL-2 (100U/mL), IL-7 (100U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 10mL of DMEM. T cells 4 were obtained after incubation in complete medium (37°C, 5% CO 2 ) for 48 days.
  • CD3 + CD69 + T cells which are those that can recognize cancer antigens.
  • Cancer-specific T cells cell viability 85%).
  • the CD3 + CD69 + T cells obtained above were mixed with IL-2 (100U/mL), IL-7 (100U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 10mL of DMEM.
  • T cells 5 were obtained after incubation in complete medium (37°C, 5% CO 2 ) for 48 days.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse. On the 3rd day, 6th day, 9th day, 14th day and 20th day before the mice were inoculated with tumors, 1 million T cells (T cell 1, or T cell 2, or T cell 3) were inoculated subcutaneously in the mice. , or T cells 4, or T cells 5) or 100 ⁇ L PBS or 2 mg nanoparticles 1. Monitor mouse tumor growth rate and mouse survival time.
  • T cell 1 is more effective than T cell 2, T cell 3, T cell 4, T cell 5 and nanoparticle 1. This shows that it is necessary to sort out cancer cell-specific T cells before amplifying them; moreover, it is necessary to use appropriate activation markers to ensure that T cells with high viability are sorted out and then amplified.
  • T cells 1 Due to the differences in the microenvironment of cancer cells in the body and the high heterogeneity of cancer cells, the use of multiple cancer cell lines can cover a more complete antigen spectrum and more diverse antigens than a single cancer cell line, and therefore has better results.
  • the effect of injecting T cells 1 is better than the direct injection of nanoparticles (vaccine) 1, indicating that the effect of infusing cancer cell-specific T cells after sorting and amplification is better than direct injecting nanoparticles (vaccine) into the body.
  • Example 27 T cells for the treatment of colon cancer
  • methylguanidine and 0.05M polyhexamethyleneguanidine hydrochloride After cleavage, all the cleavage components were dissolved using 0.2M methylguanidine hydrochloride and 0.05M polyhexamethyleneguanidine hydrochloride, and then saturated aqueous ammonium sulfate solution was added dropwise.
  • Nanoparticle 1 or Nanovaccine 1 is prepared by the double emulsion method.
  • the particle preparation material PLGA has a molecular weight of 10KDa-20KDa, and the loaded immune adjuvants are poly(I:C) and CpG7909.
  • the preparation method was as described above. First, the antigen component 1 and the adjuvant were loaded inside the nanoparticles. Then, 100 mg of the nanoparticles was centrifuged at 13,000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours.
  • the average particle size of the nanoparticles 1 is about 280nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 25 ⁇ g of protein or peptide components. Each 1 mg of PLGA nanoparticles is loaded with 0.01 mg each of poly(I:C) and CpG7909.
  • mice Female C57BL/6 mice aged 6 to 8 weeks were selected and inoculated subcutaneously with 1 ⁇ 10 6 MC38 mouse colon cancer cells on day 0. Each mouse was intraperitoneally injected with 150 ⁇ g of PD-L1 antibody on days 6, 8, 10, 12, 14, 16, 18, 20, and 22. The mice were sacrificed on day 24, and the lymph nodes of the mice were collected, and then flow cytometry was used to sort out CD3 + CD69 - T cells, CD19 + B cells and CD11c + DCs from the lymph node cells, and then the above CD19 + B cells were Mix with CD11c + DC at a quantity ratio of 1:1 to form a mixed antigen-presenting cell co-incubated with nanoparticles.
  • CD3 + CD69 + T cells which are cancer-specific T cells that can recognize cancer antigens ( Cell viability is 90%).
  • the CD3 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (10U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 10mL of DMEM. T cells 1 were obtained after incubation in complete medium (37°C, 5% CO 2 ) for 42 days.
  • the cells were incubated in RPMI 1640 complete medium for a total of 36 hours (37°C, 5% CO 2 ), and then flow cytometry was used to sort the incubated CD3 + T cells (cell viability was 90%).
  • T cells 2 were obtained after incubation in medium (37°C, 5% CO 2 ) for 42 days.
  • the cells were incubated in RPMI 1640 complete medium for a total of 36 hours (37°C, 5% CO 2 ), and then flow cytometry was used to sort the incubated CD3 + IFN- ⁇ + T cells (cell viability 0%).
  • the CD3 + IFN- ⁇ + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (10U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 10mL.
  • T cells 3 were obtained after incubation in DMEM complete medium (37°C, 5% CO 2 ) for 42 days.
  • mice Female C57BL/6 mice aged 6 to 8 weeks were selected as model mice. On day 0, each mouse was subcutaneously inoculated with 1.0 ⁇ 10 6 MC38 colon cancer cells on the lower right side of the back. On the 3rd day, 6th day, 9th day, 14th day and 20th day before the mice were inoculated with tumors, 1 million T cells (T cell 1, or T cell 2, or T cell 3) were inoculated subcutaneously in the mice. Or 2mg nanoparticles 1) or 100 ⁇ L PBS. Monitor mouse tumor growth rate and mouse survival time.
  • T cell 1 is more effective than T cell 2, T cell 3 and nanoparticle 1. This shows that it is necessary to sort out cancer cell-specific T cells before amplifying them, and it is also necessary to use appropriate activation markers to ensure that T cells with high viability are sorted out and then amplified. Moreover, the therapeutic effect of infusing T cells after sorting and expansion is better than direct injection of nanoparticles.
  • Example 28 Sorting and amplifying T cells for the prevention of lung cancer
  • the cultured LLC cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water.
  • the obtained cancer cells were inactivated and denatured using ultraviolet and high-temperature heating respectively, and then ultrapure water was added and repeatedly frozen and thawed 5 times, supplemented by ultrasound to lyse the cancer cells.
  • the cell lysates were centrifuged at 5000g for 10 minutes, and the supernatant was water-soluble.
  • the precipitate is dissolved with 10% octylglucoside to obtain the dissolved original non-water-soluble component.
  • the water-soluble component and the non-water-soluble component are mixed at a mass ratio of 2:1 to prepare Antigen component required for micron particles1.
  • the double emulsion method is used to prepare microparticle 1.
  • the molecular weight of the microparticle skeleton material PLGA is 38KDa-54KDa.
  • the immune adjuvants used are CpG1018 and Poly ICLC.
  • the antigen component 1 and adjuvant are first loaded internally, and then 100 mg of micron particles are centrifuged at 9000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use.
  • the average particle size of this micron particle system is about 5.0 ⁇ m; each 1 mg of PLGA micron particles is loaded with approximately 410 ⁇ g of protein or peptide component of the antigen component, and each of CpG1018 and Poly ICLC is loaded with 0.01 mg.
  • BMDC 5 million BMDC, 2 mg micron particles and IL-15 (20 ng/mL) were incubated in 5 mL RPMI 1640 (10% FBS) medium for 8 hours, and then BMDC were collected and dendritic cells activated by irradiation were used to inactivate the dendritic cells. Dendritic cells, inactivated dendritic cells are used to activate T cells.
  • mice Select 6-8 week old female C57BL/6 mice and subcutaneously inject 100 ⁇ L of micron particles 1 containing 2 mg PLGA on days 0, 4, 7, 14, 21, and 28.
  • the mice were sacrificed on day 32, and their peripheral blood was collected.
  • Peripheral blood mononuclear cells (PBMC) were then isolated from the peripheral blood, and CD3 + T cells were sorted from the PBMC using flow cytometry.
  • the sorted CD3 + T cells (2 million cells), the inactivated BMDC prepared in step 4 (3 million cells), and IL-7 (10 ng/mL) were incubated in 10 mL RPMI1640 complete medium for a total of 18 hours.
  • CD3 + CD69 + T cells are cancer-specific T cells that can recognize cancer antigens.
  • the 300,000 CD3 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-7 (1000U/mL), IL-15 (1000U/mL) and ⁇ CD3 antibody at (10ng/mL ) were incubated in 10 mL of DMEM complete medium (37°C, 5% CO 2 ) for a total of 14 days to expand cancer cell-specific T cells (cell viability 85%).
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice. One day before the mice were adoptively transferred cells, the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate immune cells in the recipient mice. On day 0, mice were injected subcutaneously with 100 ⁇ L containing 1.2 million expanded CD3 + T cells. At the same time, 1 ⁇ 10 6 LLC cells were subcutaneously injected into each mouse on day 0. The tumor volume and survival period were monitored in the same way as above.
  • the lung cancer tumor growth rate in the treatment group with cancer cell-specific T cells obtained by micron particle sorting and amplification was significantly slower and the survival period of mice was significantly prolonged.
  • Example 29 Cancer cell-specific T cells for the treatment of melanoma
  • B16-F10, B16-F1, S91, ME, K735, B16-BL6 and MEC57 cancer cell lines Collect the cultured B16-F10, B16-F1, S91, ME, K735, B16-BL6 and MEC57 cancer cell lines, and mix the above cancer cell lines in a quantitative ratio of 1:1:1:1:1:1:1.
  • the mixed cells were then lysed using 8M urea aqueous solution. After lysis, all lysis components were dissolved using 8M urea aqueous solution, and then saturated ammonium sulfate aqueous solution was added dropwise. After the precipitation was complete, the resulting sample was centrifuged at 3000g for 5 minutes to dissolve the precipitate.
  • Nanoparticle 1 (Nanoparticle 1) was prepared by the double emulsion method.
  • the particle preparation material PLGA has a molecular weight of 10KDa-20KDa, and the immune adjuvants used are poly(I:C), CpG 7909 and CpG2395.
  • the preparation method was as described above. First, the antigen component 1 and the adjuvant were loaded inside the nanoparticles. Then, 100 mg of the nanoparticles was centrifuged at 12,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h.
  • the average particle size of the nanoparticle 1 is about 280 nm, and each 1 mg PLGA particle is loaded with approximately 250 ⁇ g of protein or polypeptide + components. Each 1 mg PLGA particle is loaded with 0.01 mg each of poly(I:C), CpG7909, and CpG2395.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected and inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 mouse melanoma cancer cells on day 0. Each mouse was intraperitoneally injected with 150 ⁇ g of PD-L1 antibody on days 6, 8, 10, 12, 14, 16, 18, and 20. The mice were sacrificed on day 22, their peripheral blood was collected, and then mouse PBMCs were prepared, which are mixed immune cells co-incubated with nanoparticles.
  • CD3 + CD69 + T cells which are cancer-specific T cells that can recognize cancer antigens (cell viability 80%) .
  • the CD3 + CD69 + T cells obtained above were mixed with IL-2 (100U/mL), IL-7 (100U/mL), ⁇ CD3 antibody (10ng/mL) and ⁇ CD28 antibody (10ng/mL) in 10mL of DMEM.
  • T cells (cell viability 80%) were obtained after incubation in complete medium (37°C, 5% CO 2 ) for 48 days. Among them, the T cells 1 obtained after the co-incubation time of nanoparticles and immune cells was 6 hours, sorted and amplified; the T cells 2 obtained after the co-incubation time of nanoparticles and immune cells were sorted and amplified for 1 hour; Nanoparticles and immune cells The cells were co-incubated for 168 hours and the T cells obtained by sorting and amplification were T cells 3.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse. On days 3, 6, 9, 14 and 20 after mice were inoculated with tumors, 100,000 T cells (T cell 1, or T cell 2, or T cell 3) were injected subcutaneously into the mice. , or T cells 4) or inject 100 ⁇ L PBS or inject 2 mg nanoparticle 1 (Nanoparticle 1, nanovaccine 1). Monitor mouse tumor growth rate and mouse survival time.
  • T cell 1 is more effective than T cell 2, T cell 3 and T cell 4. This shows that when nanoparticles are used to sort cancer cell-specific T cells, appropriate incubation time is critical. Moreover, proper sorting and amplification after the incubation is completed can achieve better results.
  • nanoparticles/microparticles are co-incubated with antigen-presenting cells and T cells at the same time.
  • nanoparticles/microparticles are first co-incubated with antigen-presenting cells and then the incubated antigen-presenting cells are incubated. The cells were co-incubated with T cells.
  • no other treatment is required after the co-incubation of nanoparticles/microparticles with antigen-presenting cells.
  • Directly co-incubate with T cells or the incubated antigen-presenting cells can be fixed or irradiated before being co-incubated with T cells.
  • the antigen-presenting cells that have been co-incubated with nanoparticles can be treated by fixing them with reagents such as paraformaldehyde, irradiating them with radiation, or performing other treatments to inactivate the incubated antigen-presenting cells.
  • the nanoparticles and antigen-presenting cells are co-incubated, and then when the co-incubated antigen-presenting cells are co-incubated with T cells, the antigen-presenting cells can be living cells or dead cells.
  • T cells Due to space limitations, in the examples of the present invention, only a few surface markers such as CD69, CD25, HLA-DR, CD107a, FASL, etc. are listed and used as markers of T cell activation to sort out the specificity of activated cancer cells. T cells, in practical applications, can also use any other surface markers that can be used to indicate that T cells are activated.
  • Molecules that can be used as surface markers include but are not limited to: 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

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Abstract

本发明涉及一种用于预防或治疗癌症的基于外周血或外周免疫器官的癌症特异性T细胞的制备方法,具体包括如下步骤:先分离得到外周血或者外周免疫器官的免疫细胞,然后与负载癌症全细胞抗原的纳米粒子和/或微米粒子共孵育一段时间激活癌症特异性T细胞,尔后分离得到被癌症抗原激活的癌症特异性T细胞,经过体外扩增后回输机体内发挥抗癌作用。本发明制备得到的纳米粒子或微米粒子,将肿瘤抗原组分负载在微米粒子和/或纳米粒子,用于激活癌症特异性T细胞,然后经过扩增后回输患者用于癌症的治疗或者预防复发或转移。分选法分离得到的癌症特异性T细胞特异性高,经扩增后杀伤癌细胞效果即可预防或治疗癌症。

Description

用于预防或治疗癌症的特异性T细胞及其制备方法 技术领域
本发明涉及免疫治疗技术领域,尤其涉及一种用于预防或治疗癌症的特异性T细胞及其制备方法和其应用。
背景技术
免疫细胞(immune cell)是指参与免疫应答或与免疫应答相关的细胞,包括先天性淋巴细胞、各种吞噬细胞以及能识别抗原、产生特异性免疫应答的淋巴细胞等,如T细胞、B细胞、NK细胞、DC细胞、巨噬细胞、粒细胞、肥大细胞等。自身免疫细胞治疗剂是通过从自身血液中分离和培养免疫细胞而用于抗肿瘤。通过补充高活性的免疫细胞,增加了体内免疫细胞的数量,同时激活了体内原有的免疫细胞,从而大幅提升了免疫细胞杀灭肿瘤细胞、细菌、病毒等的能力,达到防癌抗癌的目的。
在本申请发明团队的在先申请CN202011027741.0中,公开了一种检测方法,该检测方法可检测用患者肿瘤组分负载的纳米粒子激活的T细胞。但该申请为检测方法而非治疗方法,未对激活的T细胞进行分选、扩增,也未涉及回输步骤,在检测过程中需要对T细胞进行固定染色,未能提供相应的活性T细胞。因此,通过肿瘤抗原负载纳米粒子从而激活、富集特定类型的T细胞从而发挥肿瘤治疗效果未见现有技术报道。
由于癌症患者尤其是身体机能状态较差的癌症患者体内的癌症特异性T细胞数量较少且活性较低,其癌症特异性T细胞存在活化困难、扩增慢、培养周期长、细胞活率低等影响抗肿瘤免疫治疗的问题。本发明将识别癌细胞和杀伤癌细胞功能的癌症特异性T细胞经过体外肿瘤抗原的进一步刺激,再经分选分离、大规模扩增后回输给患者使用,实现了通过回输癌症特异性T细胞用于预防癌症发生、预防癌症转移和治疗癌症的有效方法。
中国专利申请(202011027741.0)所公开的纳米粒子激活肿瘤特异性T细胞等技术内容作为本发明必不可少的组成部分。
发明内容
本发明的目的在于提供一种来源于自体或同种异体的用于预防或治疗癌症的癌症特异性T细胞的制备方法,具体包括如下步骤:先分离得到外周血或者外周免疫器官的免疫细胞,然后与抗原提呈细胞(Antigen-presenting cells,APC)以及负载癌症全细胞组分或含全细胞部分抗原组分的纳米粒子和/或微米粒子共孵育一段时间激活癌症特异性T细胞,尔后分离得到被癌症抗原激活的癌症特异性T细胞,经过体外扩增后回输机体内发挥抗癌作用。
本发明优选的技术方案中,所述制备方法具体包括如下步骤:
(1)先分离得到外周血或者外周免疫器官的免疫细胞,或从所述免疫细胞中分选得到T细胞;
(2)将负载有肿瘤抗原组分的微米粒子和/或纳米粒子与抗原提呈细胞以及T细胞混合后共孵育,分选出被激活并表达特定细胞标志物的T细胞;
(3)将步骤(2)分选得到的表达特定细胞标志物的T细胞与细胞因子和/或抗体共孵育,获得扩增后的特异性T细胞;
优选地,步骤(2)中,所述肿瘤抗原组分可以为肿瘤组织/癌细胞的全细胞裂解物组分,或者所述抗原组分可以为肿瘤组织/癌细胞的全细胞裂解物组分中的一部分组分。
所述全细胞裂解物组分包含水溶性组分和非水溶性组分,非水溶性组分使用含有溶解剂(或称增溶剂)的溶解液(或称增溶液)溶解(或称增溶)。
优选地,步骤(2)中,所述肿瘤抗原组分由一种或多种癌细胞和/或肿瘤组织的全细胞裂解得到,或者由一种或多种癌细胞和/或肿瘤组织的全细胞裂解后加工得到,或者由一种或多种癌细胞和/或肿瘤组织的全细胞加工后裂解得到,优选所述癌细胞或肿瘤组织中至少有一种与目标疾病类型相同;或者所述抗原组分由一种或多种癌细胞和/或肿瘤组织中的一部分组分组成,一部分组分中含有裂解液中的蛋白质/多肽组分和/或mRNA组分;
本发明优选的技术方案中,所述全细胞抗原组分为全细胞裂解物组分中的一部分组分时,抗原组分中含有全细胞裂解物组分中的蛋白质和多肽组分和/或mRNA组分。
优选地,负载有肿瘤抗原组分的纳米粒子和/或微米粒子可以与抗原提呈细胞和T细胞三者同时共孵育以激活癌细胞特异性T细胞;也可以负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞,然后将被激活的抗原提呈细胞再单独与T细胞二者一起共孵育以激活癌细胞特异性T细胞;负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞后,抗原提呈细胞可以不经过特殊处理再去与T细胞二者共孵育激活特异性T细胞,或者抗原提呈细胞可以经过固定、辐射、照射、修饰、灭活、矿化等处理后再与T细胞二者共孵育激活特异性T细胞。
本发明优选的技术方案中,所述步骤(1),分离得到外周血或者外周免疫系统的免疫细胞的同种同体或同种异体在分离提取上述细胞时可以不经任何处理,或者经过放疗、免疫治疗、化疗、粒子治疗、疫苗治疗处理。
本发明优选的技术方案中,所述步骤(1)分选得到的T细胞为CD3+T细胞、CD3+CD8+T细胞、CD3+CD4+T细胞中的任一种或其组合。
本发明优选的技术方案中,所述步骤(1)和步骤(2)中的分选方法为流式细胞术、磁珠法的任一种或其组合。在分选被激活的癌细胞特异性T细胞时,可以使用一种T细胞被激活的激活标志物,也可以使用一种以上的不同标志物的组合作为激活标志物。
本发明优选的技术方案中,步骤(2)中,所述肿瘤抗原组分由一种或多种癌细胞和/或肿瘤组织的全细胞裂解得到,或者由一种或多种癌细胞和/或肿瘤组织的全细胞裂解后加工得到,或者由一种或多种癌细胞和/或肿瘤组织的全细胞加工后裂解得到,优选所述癌细胞或肿瘤组织中至少有一种与目标疾病类型相同;或者所述抗原组分由一种或多种癌细胞和/或肿瘤组织中的一部分组分组成,一部分组分中含有裂解液中的蛋白质/多肽组分和/或mRNA组分。
本发明优选的技术方案中,抗原组分可以为:(1)癌细胞/肿瘤组织全细胞裂解物组分;(2)或者为含有癌细胞/肿瘤组织全细胞裂解物组分中的蛋白质和多肽组构成的的全细胞组分中的一部分组分;(3)或者为癌细胞/肿瘤组织全细胞组分中的蛋白质多肽组分加上mRNA组分。
本发明优选的技术方案中,所述肿瘤抗原组分为肿瘤组织和/或癌细胞的细胞裂解组分,包含有肿瘤组织和/或癌细胞细胞裂解后产生的水溶性组分和非水溶性组分的一种或两种,先分别收集水溶性组分和水不溶性组分并分别制备纳米或微米粒子;或者也可以直接采用含有增溶剂的增溶液直接裂解癌细胞或肿瘤组织并溶解全细胞组分并制备纳米或微米粒子,所述非水溶性组分由含有溶解剂的溶解液溶解。或者也可以为上述裂解液组分经过适当处理后所得蛋白质和多肽组分,或者蛋白质和多肽组分加上mRNA组分。
本发明优选的技术方案中,所述抗原组分为全细胞裂解物组分时,其制备方法为:(1)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用;(2)使用含有溶解剂的溶解液裂解细胞,然后使用含有溶解剂的溶解液溶解裂解后的全细胞组分。
本发明优选的技术方案中,所述抗原组分为全细胞裂解物组分中的一部分组分(含有癌细胞全细胞组分中的蛋白质和多肽组分)的制备方法为:(1)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中分离提取的蛋白质和多肽组分与所有非水溶性组分一起作为抗原组分使用;(2)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从非水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将非水溶性组分中分离提取的蛋白质和多肽组分与所有水溶性组分一起作为抗原组分使用;(3)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分和非水溶性组分中使用适当方法分别分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中和非水溶性组分中分离提取的蛋白质和多肽组分一起作为抗原组分使用;(4)使用含有溶解剂的溶解液裂解细胞,然后使用含有溶解剂的溶解液溶解裂解后的全细胞组分,然后使用适当方法分离提取其中的蛋白质和多肽组分。上述制备方法中还可以增加分离提取全细胞mRNA的步骤,并将全细胞mRNA作为抗原组分的一部分使用。
上述分离提取蛋白质和多肽组分的适当方法包括但不限于盐析、加热和酶解等。
上述蛋白质和多肽组分分离提取后重新溶解于含有溶解剂的溶解液中。
本发明优选的技术方案中,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时,或者所述纳米粒子/微米粒子与抗原提呈细胞和T细胞同时共孵育时,纳米粒子/微米粒子浓度为2.5ng/mL到50mg/mL;共孵育时间为1-168小时。
本发明优选的技术方案中,所述全细胞组分在裂解前或(和)裂解后既可经过灭活或(和)变性、固化、生物矿化、离子化、化学修饰、核酸酶处理等处理后再制备纳米粒子或微米粒子;也可细胞裂解前或(和)裂解后不经过任何灭活或(和)变性、固化、生物矿化、离子化、化学修饰、核酸酶处理直接制备。
本发明优选的技术方案中,肿瘤组织细胞在裂解前经过了灭活或(和)变性处理,也可以在细胞裂解后做灭活或(和)变性处理,或者也可以细胞裂解前和裂解后均做灭活或(和)变性处理。
本发明优选的技术方案中,细胞裂解前或(和)裂解后的灭活或(和)变性处理方法包括紫外照射、高温加热、放射线辐照、高压、固化、生物矿化、离子化、化学修饰、核酸酶处理、胶原酶处理、冷冻干燥中的任一种或其组合。
本发明优选的技术方案中,所述步骤(2),所用抗原提呈细胞与T细胞数量比大于1:1;利用纳米粒子或微米粒子通过抗原提呈细胞的提呈后体外激活外周免疫细胞中预存的癌症特异性T细胞,所述纳米粒子或微米粒子选自粒径1nm-1000nm的纳米粒子或粒径1μm-1000μm的微米粒子。
本发明优选的技术方案中,所述与T细胞和纳米粒子和/或微米粒子共孵育的抗原提呈细胞来源于自体、同种异体、细胞系、干细胞或以上的任意混合物;共孵育的抗原提呈细胞为B细胞、树突状细胞、巨噬细胞或者三者的任意混合物。
本发明优选的技术方案中,所述抗原提呈细胞源于自体抗原提呈细胞、同种异体抗原提呈细胞、抗原提呈细胞系或者干细胞分化而来的抗原提呈细胞,优选为树突状细胞(DC)、B细胞、巨噬细胞中的任一种或其组合;更优选的使用多于一种抗原提呈细胞的组合。
本发明优选的技术方案中,所述混合共孵育选自以下三种方式中的任一种:(a)三者直接混合共孵育一定时间;(b)微米粒子和/或纳米粒子与抗原提呈细胞先共孵育一段时间,再加入T细胞共孵育;(c)微米粒子和/或纳米粒子与抗原提呈细胞先共孵育一段时间,分选出孵育后的抗原提呈细胞后将抗原提呈细胞与T细胞二者共孵育。
细胞与纳米粒子/微米粒子和抗原提呈细胞一起共孵育前,T细胞可以先单独静息培养一段时间,或者进行适当分选;或者在将T细胞与被激活的抗原提呈细胞共孵育前,T细胞可以先单独静息培养一段时间,或者进行适当分选。
本发明优选的技术方案中,所述混合共孵育的培养条件为在30-38℃、1-10%CO2条件下共孵育1-168h。
本发明优选的技术方案中,所述混合共孵育中可加入细胞因子;所加入的细胞因子包括但不限于白介素、肿瘤坏死因子、干扰素、生长因子;优选的,所加入的细胞因子中包含白介素7(IL-7)、白介素15(IL-15)。
本发明优选的技术方案中,所述步骤(2),所述分选方法为使用带有荧光或者磁性或者特定配体的抗体与T细胞表面的特定细胞标志物结合后利用流式细胞术或者磁珠法等从细胞群中分离表达特定细胞标志物的细胞。
本发明优选的技术方案中,所述步骤(2),所述分选得到的表达特定细胞标志物的T细胞包括但不限于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、CXCR 1、CD103、CD45RO、ICOS(CD278)、VTCN1、HHLA2、LGAL59、CCR7、CD357、BCL6、TCF-1、CD38、CD27等中的任一种或其组合。
本发明优选的技术方案中,步骤(3)中,所述细胞因子的浓度为1-6000ng/ml,优选为5-200ng/ml,更优选10-30ng/ml。
本发明优选的技术方案中,所述步骤(3),所述细胞因子包括但不限于白介素、干扰素、肿瘤坏死因子。
本发明优选的技术方案中,所述白介素包括但不限于白介素2(IL-2)、白介素7(IL-7)、白介素12(IL-12)、白介素15(IL-15)、白介素17(IL-17),白介素21(IL-21)。
本发明优选的技术方案中,所述抗体的浓度为1-6000ng/ml,优选为5-100ng/ml,更优选10-30ng/ml。
本发明优选的技术方案中,所述抗体包括但不限于αCD3抗体、αCD28抗体、αCD80抗体、αCD86抗体、αOX40抗体中的任一种或其组合。
本发明优选的技术方案中,所述纳米粒子/微米粒子与抗原提呈细胞和T细胞混合物共孵育时间为至少4小时,优选为6-96小时。
本发明优选的技术方案中,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时间为至少1小时,优选为6-96小时。
本发明优选的技术方案中,所述激活的抗原提呈细胞和T细胞混合物共孵育时间为至少1小时,优选为6-96小时。
本发明优选的技术方案中,所述扩增培养时间为至少1天,优选为4–36天。
本发明优选的技术方案中,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时,纳米粒子/微米粒子浓度为2.5ng/mL到50mg/mL。
本发明优选的技术方案中,所述纳米粒子/微米粒子与抗原提呈细胞和T细胞共孵育时,纳米粒子/微米粒子浓度为2.5ng/mL到50mg/mL。
本发明优选的技术方案中,所述纳米粒子/微米粒子负载的抗原组分中的蛋白质和多肽组分含量高于10ng/mL。本发明优选的技术方案中,步骤(3)得到的体外扩增后得到的特异性T细胞,回输机体内发挥抗癌作用。
本发明优选的技术方案中,所述纳米粒子和/或微米粒子所负载的癌症抗原为肿瘤组织和/或癌细胞的全细胞组分,含有肿瘤组织和/或癌细胞的水溶性组分和/或非水溶性组分。
本发明优选的技术方案中,所述用于激活癌症特异性T细胞的纳米粒子和/或微米粒子,将一种和/或多种的肿瘤组织和/或癌细胞的组分负载到纳米粒子或微米粒子的负载方式为全细胞的水溶性成分和非水溶性成分分别或同时被包载于粒子内部,和/或分别或同时负载于粒子表面。
本发明优选的技术方案中,所述用于激活癌症特异性T细胞的纳米粒子和/或微米粒子,其所负载的来源于肿瘤组织或癌细胞的全细胞组分中的原非水溶性部分采用适当增溶方法由在纯水中不溶变为在含增溶剂/溶解剂的水溶液中或有机溶剂中可溶;采用的增溶剂/溶解剂选自含有结构式1结构的化合物、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、多肽、氨基酸、糖苷和胆碱中的一种或多种;
其中,结构式1如下:结构式1结构如下:
,R1为C、N、S或O,R2~R5独立地选自氢、烷基、氨基、羧基、取代或未取代胍基的至少一种。
含有结构式1的化合物包括但不限于盐酸二甲双胍、硫酸二甲双胍、磺酸二甲双胍、二甲双胍盐、二甲双胍、盐酸聚六亚甲基胍、硫酸胍基丁胺、盐酸甲基胍、盐酸四甲基胍、尿素、盐酸胍、硫酸胍、磺酸胍、胍盐、其他含有胍基或脲的化合物、碳酸胍、精氨酸、胍基乙酸、胍基磷酸、氨基磺酸胍、胍基琥珀酸、盐酸氨基脲、氨基甲酰脲、乙酰脲、磺酰脲类化合物(格列本脲、格列齐特、格列喹酮、格列美脲等)、硫脲类化合物(硫氧嘧啶类、咪唑类等)、亚硝基脲类等含有结构式1结构的化合物。
本发明优选的技术方案中,所述用于激活癌症特异性T细胞的纳米粒子和/或微米粒子的表面连接有主动靶向抗原提呈细胞的靶头。
本发明优选的技术方案中,所述水溶性组分和/或非水溶性组分负载于所述癌症疫苗的表面的方式包括吸附、共价连接、电荷相互作用、疏水相互作用、一步或多步的固化、矿化和包裹中的至少一种。
本发明优选的技术方案中,所述纳米粒子的粒径为1nm-1000nm;所述微米粒子的粒径为1μm-1000μm。
本发明优选的技术方案中,所述的纳米尺寸粒子或微米尺寸粒子表面为电中性,带负电或者带正电。
本发明优选的技术方案中,所述纳米疫苗和/或微米疫苗的制备材料为有机合成高分子材料、天然高分子材料或者无机材料。
本发明优选的技术方案中,所述有机合成高分子材料为PLGA、PLA、PGA、PEG、PCL、Poloxamer、PVA、PVP、PEI、PTMC、聚酸酐、PDON、PPDO、PMMA、聚氨基酸、合成多肽;所述的天然高分子材料为卵磷脂、胆固醇、海藻酸钠、白蛋白、胶原蛋白、明胶、细胞膜成分、淀粉、糖类、多肽;所述的无机材料为三氧化二铁、四氧化三铁、碳酸钙、磷酸钙。
本发明优选的技术方案中,所述的用于激活癌症特异性T细胞的纳米粒子和/或微米粒子,可将一种和/或多种的肿瘤组织和/或癌细胞的组分与免疫佐剂一起共负载于纳米粒子或微米粒子。
本发明优选的技术方案中,所述水溶性部分和非水溶性部分都可以被含增溶剂的增溶水溶液或有机溶剂溶解。增溶剂为可以增加蛋白质或多肽在水溶液中溶解性的增溶剂中的至少一种;有机溶剂为可以溶解蛋白质或多肽的有机溶剂。
本发明优选的技术方案中,所述的原非水溶性部分采用适当增溶方法由在纯水中不溶变为在含增溶剂/溶解剂的水溶液中或有机溶剂中可溶;采用的增溶剂/溶解剂选自含有结构式1结构的化合物、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、多肽、氨基酸、糖苷和胆碱中的一种或多种;其中,结构式1如下:结构式1结构如下:
,R1为C、N、S或O,R2~R5独立地选自氢、烷基、氨基、羧基、取代或未取代胍基的至少一种。
含有结构式1的化合物包括但不限于盐酸二甲双胍、硫酸二甲双胍、磺酸二甲双胍、二甲双胍盐、二甲双胍、盐酸聚六亚甲基胍、硫酸胍基丁胺、盐酸甲基胍、盐酸四甲基胍、尿素、盐酸胍、硫酸胍、磺酸胍、胍盐、其他含有胍基或脲的化合物、碳酸胍、精氨酸、胍基乙酸、胍基磷酸、氨基磺酸胍、胍基琥珀酸、盐酸氨基脲、氨基甲酰脲、乙酰脲、磺酰脲类化合物(格列本脲、格列齐特、格列喹酮、格列美脲等)、硫脲类化合物(硫氧嘧啶类、咪唑类等)、亚硝基脲类等含有结构式1结构的化合物。
本发明优选的技术方案中,用于激活癌症特异性T细胞的纳米粒子或微米粒子所负载的细胞组分来源于一种或多种癌细胞和/或一种或多种肿瘤组织全细胞中得到的组分,将非水溶性组分负载到递送粒子上,使该纳米或微米系统中含有更多的抗原,更优选地,将水溶性组分和非水溶性组分同时负载到递送粒子上,使递送粒子上负载了全细胞组分抗原。
本发明优选的技术方案中,用于激活癌症特异性T细胞的纳米粒子和/或微米粒子负载的细胞组分或其混合物,混合物包括但不限于水溶性组分互相混合,或者非水溶性组分互相混合,或者全部或部分水溶性组分与全部或部分水溶性组分互相混合。
本发明优选的技术方案中,在纳米粒子(Nanoparticle,NP)和/或微米粒子(Microparticle,MP)中,细胞组分或其混合物被负载于纳米粒子或微米粒子内部和/或表面,具体的,所述负载方式为细胞的水溶性组分和非水溶性组分分别或同时被包载于粒子内部,和/或分别或同时负载于粒子表面,包括但不限于水溶性组分同时装载于粒子中和负载于粒子表面,非水溶性组分同时装载于粒子中和负载于粒子表面,水溶性组分装载于粒子中而非水溶性组分负载于粒子表面,非水溶性组分装载于粒子中而水溶性组分负载于粒子表面,水溶性组分和非水溶性组分装载于粒子中而只有非水溶性组分负载于粒子表面,水溶性组分和非水溶性组分装载于粒子中而只有水溶性组分负载于粒子表面,水溶性组分装载于粒子中而水溶性组分和非水溶性组分同时负载于粒子表面,非水溶性组分装载于粒子中而水溶性组分和非水溶性组分同时负载于粒子表面,水溶性组分和非水溶性组分同时装载于粒子中而且水溶性组分和非水溶性组分同时负载于粒子表面。
本发明优选的技术方案中,用于激活癌症特异性T细胞的纳米粒子或微米粒子的内部和/或表面还可包括免疫增强佐剂,免疫增强佐剂包括但不限于微生物来源的免疫增强剂、人或动物免疫系统的产物、固有免疫激动剂、适应性免疫激动剂、化学合成药物、真菌多糖类、中药及其他类中的至少一类;免疫增强佐剂包括但不限于模式识别受体激动剂、卡介苗(BCG)、锰相关佐剂、卡介苗细胞壁骨架、卡介苗甲醇提取残余物、卡介苗胞壁酰二肽、草分枝杆菌、多抗甲素、矿物油、病毒样颗粒、免疫增强的再造流感病毒小体、霍乱肠毒素、皂苷及其衍生物、Resiquimod、胸腺素、新生牛肝活性肽、米喹莫特、多糖、姜黄素、免疫佐剂CpG、免疫佐剂poly(I:C)、免疫佐剂poly ICLC、短小棒状杆菌苗、溶血性链球菌制剂、辅酶Q10、左旋咪唑、聚胞苷酸、锰佐剂、铝佐剂、钙佐剂、各种细胞因子、白细胞介素、干扰素、聚肌苷酸、聚腺苷酸、明矾、磷酸铝、羊毛脂、角鲨烯、细胞因子、植物油、内毒素、脂质体佐剂、MF59、双链RNA、双链DNA、铝相关佐剂、CAF01、人参、黄芪的有效成分中的至少一种。本领域技术人员可以理解,此处为列举并非穷举,免疫增强佐剂也可采用其他可使免疫反应增强的物质。
本发明优选的技术方案中,免疫增强佐剂优选Toll样受体激动剂。
本发明优选的技术方案中,免疫增强佐剂优选两种以上Toll样受体激动剂联用。
本发明优选的技术方案中,将免疫佐剂与细胞组分共负载于纳米粒子或微米粒子中,在纳米粒子或微米粒子被抗原提呈细胞吞噬后可以更好的癌症特异性T细胞。
本发明优选的技术方案中,微米粒子或纳米粒子中负载增加纳米粒子和/或微米粒子或其负载的抗原从溶酶体中逃逸至细胞质的物质。所述增加溶酶体逃逸的物质包括氨基酸、多肽、糖类、脂类、可以产生质子海绵效应的无机盐,优选地,所述增加溶酶体逃逸的物质中的氨基酸包含带正电的氨基酸,优选地,所述增加溶酶体逃逸的多肽种包含带正电的氨基酸。
本发明优选的技术方案中,纳米粒子或微米粒子的表面可以不连接具有主动靶向功能的靶头,或者连接有主动靶向功能的靶头,
主动靶向靶头可为甘露糖、甘露聚糖、CD19抗体、CD20抗体、BCMA抗体、CD32抗体、CD11c抗体、CD103抗体、CD44抗体等常用的靶头,带领粒子系统靶向输送到抗原提呈细胞。
本发明优选的技术方案中,所述纳米粒子和/或微米粒子的表面连接有主动靶向抗原提呈细胞的靶头。
本发明优选的技术方案中,纳米粒子或微米粒子在制备过程中可以不做修饰处理,也可以采用适当的修饰技术以提高纳米粒子或微米粒子的抗原负载量。修饰技术包括但不限于生物矿化(如硅化、钙化、镁化)、凝胶化、交联、化学修饰、添加带电物质。
本发明优选的技术方案中,细胞组分或其混合物被负载于纳米粒子或微米粒子内部的形式为任何可以将细胞组分或其混合物负载于纳米粒子或微米粒子内部的方式。
本发明优选的技术方案中,细胞组分或其混合物被负载于纳米粒子或微米粒子表面的方式包括但不限于吸附、共价连接、电荷相互作用(如添加带正电的物质、添加带负电的物质)、疏水相互作用、一步或多步的固化、矿化、包裹等。
本发明优选的技术方案中,负载于纳米粒子或微米粒子表面的水溶性组分和/或非水溶性组分负载后为一层或多层,疫苗表面负载多层水溶性组分和/或非水溶性组分时,层与层之间为修饰物。
本发明优选的技术方案中,所诉纳米粒子的粒径大小为1nm-1000nm,更优选地,粒径大小为30nm-1000nm,最优选地,粒径大小为100nm-600nm。
本发明优选的技术方案中,微米粒子的粒径大小为1μm-1000μm,更优选地,粒径大小为1μm-100μm,更优选地,粒径大小为1μm-10μm,最优选地,粒径大小为1μm-5μm。
本发明优选的技术方案中,所述纳米粒子或微米粒子的形状包括球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形或圆盘形中的任一种。
本发明优选的技术方案中,所述水溶性组分和/或非水溶性组分负载于所述癌症疫苗的表面的方式包括吸附、共价连接、电荷相互作用、疏水相互作用、一步或多步的固化、矿化和包裹中的至少一种。
本发明优选的技术方案中,所述纳米疫苗和/或微米疫苗的制备材料为有机合成高分子材料、天然高分子材料或者无机材料。
本发明优选的技术方案中,所述有机合成高分子材料为生物相容或可降解的高分子材料,包括PLGA、PLA、PGA、PLGA-PEG、PLA-PEG、PGA-PEG、PEG、PCL、Poloxamer、PVA、PVP、PEI、PTMC、聚酸酐、PDON、PPDO、PMMA、聚氨基酸、合成多肽、合成脂质中的任一种或其组合。
本发明优选的技术方案中,所述天然高分子材料为生物相容或可降解的高分子材料,包括为卵磷脂、胆固醇、海藻酸钠、白蛋白、胶原蛋白、明胶、细胞膜成分、淀粉、糖类、多肽中的任一种或其组合。
本发明优选的技术方案中,所述无机材料为无明显生物毒性的材料,包括但不限于三氧化二铁、四氧化三铁、碳酸钙、磷酸钙等。
本发明的另一目的在于提供由本发明所述方法制备得到的特异性T细胞。
本发明的另一目的在于提供一种来源于自体或同种异体的用于预防或治疗癌症的癌症特异性T细胞,所述特异性T细胞包括但不限于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+等T细胞的任一种或其组合;所述特异性T细胞活率大于60%,优选大于70%,更优选大于80%;
所述特异性T细胞由负载有肿瘤抗原组分的纳米粒子和/或微米粒子,与从外周血或外周免疫器官的免疫细胞中分离的外周血单个核细胞(PBMC)共孵育获得。
在一些实施方式中,所述PBMC细胞经过分选后,再与负载有肿瘤抗原组分的纳米粒子和/或微米粒子共孵育。
在一些实施方式中,与负载有肿瘤抗原组分的纳米粒子和/或微米粒子共孵育后的细胞再次经过分选,获得特异性T细胞。
在一些实施方式中,所述特异性T细胞还可经过体外扩增的步骤,该步骤可以在前述分选步骤之前,也可在前述分选步骤之后,优选在分选步骤之后进行。
在一些实施方式中,所述的PBMC细胞与所述的纳米/微米粒子共孵育时,还存在抗原递呈细胞(APC)。
在一些优选的实施方式中,所述的特异性T细胞为CD3+CD8+CD69+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD3+CD137+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD3+CD8+CD25+细胞和/或CD3+CD8+CD69+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD3+CD69+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD3+CD69+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD3+CD8+CD69+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD3+CD8+CD25+细胞和/或CD3+CD4+CD69+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD8+CD69+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD8+CD137+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD8+CD69+细胞和/或CD4+CD69+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD3+CD25+细胞;在一些优选的实施方式中,所述的特异性T细胞为CD3+HLA-DR+;在一些优选的实施方式中,所述的特异性T细胞为CD3+FASL+
所述各种类型的T细胞可以单一使用,也可以根据患者需要组合使用。
本发明的目的在于提供一种来源于自体或同种异体的用于预防或治疗癌症的癌症特异性T细胞的制备方法,具体包括如下步骤:
(1)从外周血或外周免疫器官的免疫细胞中分离单个核细胞PBMC;优选地,所述PBMC经过第一步分选,获得以下至少一种效应性T细胞:CD3+CD8+T细胞、CD19+B细胞、CD3+T细胞、CD8+T细胞、CD4+T细胞、B220+B细胞、CD69-的PBMC细胞、CD25-的PBMC细胞、CD3+CD69+T细胞、CD11c+DC细胞;在将T细胞与纳米粒子/微米粒子和抗原提呈细胞一起共孵育前,T细胞可以先单独静息培养一段时间,或者进行适当分选;或者在将T细胞与被激活的抗原提呈细胞共孵育前,T细胞可以先单独静息培养一段时间,或者进行适当分选。
(2)制备负载有肿瘤抗原组分的纳米粒子和/或微米粒子;
优选地,所述肿瘤抗原组分的制备为先分离得到肿瘤细胞或组织,裂解所述肿瘤细胞或组织获得水溶性组分、非水溶性组分、全组分的任一种或其组合;
优选地,负载有肿瘤抗原组分的纳米粒子和/或微米粒子利用复乳法制备;
(3)在培养基中加入负载有肿瘤抗原组分的纳米粒子和/或微米粒子,与(1)所述的PBMC细胞或T细胞共孵育,得细胞培养物,从细胞培养物中进一步分选出特异性T细胞,所述特异性T细胞包括但不限于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+等T细胞的任一种或其组合;表面激活标志物可以使用一种,或者多种表面激活标志物的组分。
优选地,共孵育过程还加入10-5000万个/ml的抗原提呈细胞,所述抗原提呈细胞为B细胞、DC细胞、巨噬细胞中的任一种或其组合;
优选地,负载有肿瘤抗原组分的纳米粒子和/或微米粒子可以与抗原提呈细胞和T细胞三者同时共孵育以激活癌细胞特异性T细胞;也可以负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞,然后将被激活的抗原提呈细胞再单独与T细胞二者一起共孵育以激活癌细胞特异性T细胞;负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞后,抗原提呈细胞可以不经过特殊处理再去与T细胞二者共孵育激活特异性T细胞,或者抗原提呈细胞可以经过固定、辐射、照射、修饰、灭活、矿化等处理后再与T细胞二者共孵育激活特异性T细胞;
优选地,在培养基中加入2.5ng-50mg/ml的负载有肿瘤抗原组分的纳米粒子和/或微米粒子,与1-5000万个/ml的PBMC细胞或分选细胞,在30-38℃、1-5%CO2条件下共孵育4-96h,得细胞培养物;
优选地,共孵育过程还加入10-500ng/ml的白介素,所述白介素为IL-2、IL-7、IL-12、IL-15、IL-17、IL-21中的任一种或其组合;
所述培养基为DMEM高糖完全培养基、RPM1640培养基、AIMV无血清培养基中的任一种;
所述的共孵育是在30-38℃条件下孵育1-168h,优选为4-96h,更优选为6-72h;
所述特异性T细胞活率大于60%,优选大于70%,更优选大于80%;
(4)扩增(3)获得的特异性T细胞;
优选地,所述扩增步骤为,在扩增培养基中,加入1-5000万个细胞/mL的步骤(2)分选得到的特异性T细胞,在30-38℃、1-5%CO2条件下共孵育,每2-3天使用扩增培养基换液,共孵育5-30天后,获得扩增后的特异性T细胞;
优选地,在扩增培养基中扩增,所述扩增培养基为DMEM高糖完全培养基、RPM1640培养基中的任一种;
优选地,扩增培养的条件为30-38℃培养4-72天,优选为5-30天;
优选地,所述扩增培养基还包含200-1000U/ml的白介素、10-200ng/ml的抗体和/或1-10ng/mL粒细胞-巨噬细胞集落刺激因子(GM-CSF);
优选地,所述白介素为IL-2、IL-7、IL-12、IL-15、IL-17、IL-21中的任一种或其组合;
优选地,所述抗体为αCD3抗体、αCD28抗体中的任一种或其组合。
本发明优选的技术方案中,所述步骤(1),所述细胞选自患肿瘤疾病个体的任何部位,优选为脾脏细胞、淋巴细胞。
所述扩增后的特异性T细胞活率大于60%,优选大于75%,更优选大于80%。
本发明优选的技术方案中,所述步骤(2),所述分选方法为使用带有荧光或者磁性或者特定配体的抗体与T细胞表面的特定细胞标志物结合后利用流式细胞术或者磁珠法等从细胞群中分离表达特定细胞标志物的细胞。
本发明优选的技术方案中,所述混合共孵育中可加入细胞因子;所加入的细胞因子包括但不限于白介素、肿瘤坏死因子、干扰素、生长因子;优选的,所加入的细胞因子中包含白介素7(IL-7)、白介素15(IL-15)。
本发明优选的技术方案中,步骤(3)中,所述细胞因子的浓度为1-6000ng/ml,优选为5-100ng/ml,更优选10-30ng/ml。
本发明优选的技术方案中,所述步骤(3),所述细胞因子包括但不限于白介素、干扰素、肿瘤坏死因子。
本发明优选的技术方案中,所述白介素包括但不限于白介素2(IL-2)、白介素7(IL-7)、白介素12(IL-12)、白介素15(IL-15)、白介素17(IL-17),白介素21(IL-21)。
本发明优选的技术方案中,所述抗体的浓度为1-6000ng/ml,优选为5-100ng/ml,更优选10-30ng/ml。
本发明优选的技术方案中,所述抗体包括但不限于αCD3抗体、αCD28抗体、αCD80抗体、αCD86抗体、αOX40抗体中的任一种或其组合。
本发明优选的技术方案中,所述纳米粒子/微米粒子与抗原提呈细胞和T细胞混合物共孵育时间为至少1小时,优选为4-96小时。
本发明优选的技术方案中,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时间为至少1小时,优选为4-96小时。
本发明优选的技术方案中,所述激活的抗原提呈细胞和T细胞混合物共孵育时间为至少1小时,优选为6-96小时。
本发明优选的技术方案中,所述扩增培养时间为至少1天,优选为4–72天。
本发明优选的技术方案中,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时,纳米粒子/微米粒子浓度为10ng/mL到5mg/mL。
本发明优选的技术方案中,所述纳米粒子/微米粒子与抗原提呈细胞和T细胞共孵育时,纳米粒子/微米粒子浓度为2.5ng/mL到50mg/mL。
本发明优选的技术方案中,所述纳米粒子/微米粒子负载的抗原组分中的蛋白质和多肽组分含量高于10ng/mL。
本发明优选的技术方案中,步骤(2)和(3)中,所述纳米粒子或微米粒子为PLGA,分子量选自7-54KDa,所述免疫佐剂选自(poly(I:C)、polyICLC、BCG、以及CpG的任一种或其组合。
本发明优选的技术方案中,步骤(2)和(3)中,所述纳米粒子或微米粒子为PLGA,分子量选自7-51KDa,所述免疫佐剂选自(poly(I:C)、polyICLC、BCG、以及CpG的任一种或其组合。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为poly(I:C)且poly(I:C)只分布于纳米粒子内部。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为7Da-17KDa,所采用的免疫佐剂为poly(I:C)和CpG1018,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为poly(I:C)、CpG2006和CpG2216,三种免疫佐剂的质量比优选为1:1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为poly(I:C)且poly(I:C)既分布于纳米粒子内部也负载于纳米粒子表面,内外都负载裂解物的经冷冻硅化和添加阳离子物质的修饰的纳米粒子。
本发明优选的技术方案中,所述微米粒子采用PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为poly(I:C)且poly(I:C)既分布于微米粒子内部也负载于微米粒子表面,内外都负载裂解物的经冷冻硅化和添加阳离子物质的修饰的微米粒子。
本发明优选的技术方案中,所述纳米粒子采用PLA分子量为20KDa,所采用的免疫佐剂为poly(I:C)和CpG1018,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为Poly(I:C)和CpG1018,且抗原组分和佐剂同时分布于纳米粒子内部和表面。
本发明优选的技术方案中,所述微米粒子采用PLGA分子量为38KDa-54KDa,以CpG和Poly ICLC为免疫佐剂,且为促溶酶体逃逸,体系中可添加精氨酸。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为24KDa-38KDa,免疫佐剂为CpG和Poly(I:C),且为促溶酶体逃逸,体系中可添加KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA),两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为BCG,且BCG负载于纳米粒子内部。
本发明优选的技术方案中,所述纳米粒子采用PLGA和甘露糖修饰的PLGA,质量比为4:1,分子量为7KDa-17KDa,所采用的免疫佐剂为Poly(I:C)和CpG,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为BCG和Poly(I:C),两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所采用的纳米粒子内部和表面负载全细胞抗原后生物钙化纳米粒子,PLGA分子量为7KDa-17KDa,所采用免疫佐剂CpG和Poly(I:C)以及增强溶酶体逃逸的GALA多肽(WEAALAEALAEALAEHLAEALAEALEALAA)负载于纳米粒子内部,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为poly(I:C)和CpG,且为促溶酶体逃逸,体系中添加蜂毒肽,且佐剂和蜂毒肽包裹于纳米粒子内,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA和甘露聚糖修饰的PLGA,二者分子量都为24KDa-38KDa,PLGA和甘露聚糖修饰PLGA的质量比为9:1,所采用的免疫佐剂为poly(I:C)和CpG,且为促溶酶体逃逸,体系中添加聚精氨酸和聚赖氨酸,且佐剂、聚精氨酸和聚赖氨酸包载于纳米粒子内,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述微米粒子采用PLGA分子量为38KDa-54KDa,所采用的免疫佐剂为CpG1018和Poly ICLC,且为促溶酶体逃逸,体系中添加KALA多肽,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述微米粒子骨架材料为未修饰的PLA和甘露糖修饰的PLA,分子量都为40KDa,未修饰的PLA和甘露糖修饰的PLA的比例为9:1。所采用的免疫佐剂为CpG2395和Poly ICLC,且为促溶酶体逃逸,体系中添加精氨酸和/或组氨酸,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为poly(I:C)和CpG1018,且为促溶酶体逃逸,体系中添加R8多肽,且佐剂和R8多肽负载于纳米粒子内,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为7KDa-17KDa,以Poly(I:C)和CpG1018为佐剂,且为促溶酶体逃逸,体系中添加NH4HCO3,且佐剂和NH4HCO3负载于纳米粒子内,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为20KDa-40KDa,所采用的免疫佐剂为poly(I:C)。
本发明优选的技术方案中,所述纳米粒子采用PLA(分子量30-40KDa)和甘露聚糖-PEG2000-PLA(PLA分子量为30-40KDa),且PLA(分子量30-40KDa)和甘露聚糖-PEG2000-PLA(PLA分子量为30-40KDa)质量比为9:1,所采用的免疫佐剂为CpG2006(B类)、CpG2216(A类)和Poly ICLC,三种免疫佐剂的质量比优选为1:1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为10KDa-20KDa,所采用的免疫佐剂为poly(I:C)、CpG 7909和CpG2395,三种免疫佐剂的质量比优选为1:1:1。
本发明优选的技术方案中,所述纳米粒子采用PLGA分子量为10KDa-20KDa,负载的免疫佐剂为poly(I:C)和CpG7909,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述微米粒子采用PLGA分子量为38KDa-54KDa,所采用的免疫佐剂为CpG1018和Poly ICLC,且为促溶酶体逃逸,体系中添加为KALA多肽,两种免疫佐剂的质量比优选为1:1。
本发明优选的技术方案中,所述肿瘤抗原组分含有肿瘤组织和/或癌细胞的细胞裂解组分中的蛋白质/多肽组分,其制备方法为:(1)先制备肿瘤组织/癌细胞的裂解液,然后制备裂解液中的水溶性组分和非水溶性组分,再通过盐析、加热、酶解等方法分别制备水溶性组分和非水溶性组分中的蛋白质/多肽组分,然后将其作为抗原组分负载于纳米或微米粒子;(2)或者也可以直接采用含有增溶剂的增溶液直接裂解细胞或组织并溶解全细胞组分,然后通过盐析、加热、酶解等方法分别制备其中的蛋白质/多肽组分,并将其作为抗原组分负载于并纳米或微米粒子。所述非水溶性组分以及盐析、加热和酶解等处理后得到的沉淀由含有溶解剂的溶解液溶解。
本发明优选的技术方案中,全细胞组分制备方法为:(1)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用;(2)使用含有溶解剂的溶解液裂解细胞,然后使用含有溶解剂的溶解液溶解裂解后的全细胞组分。
本发明优选的技术方案中,含有癌细胞全细胞组分中的全细胞蛋白质和多肽组分的全细胞组分中的一部分组分的制备方法为:(1)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中分离提取的蛋白质和多肽组分与所有非水溶性组分一起作为抗原组分使用;(2)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从非水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将非水溶性组分中分离提取的蛋白质和多肽组分与所有水溶性组分一起作为抗原组分使用;(3)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分和非水溶性组分中使用适当方法分别分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中和非水溶性组分中分离提取的蛋白质和多肽组分一起作为抗原组分使用;(4)使用含有溶解剂的溶解液裂解细胞,然后使用含有溶解剂的溶解液溶解裂解后的全细胞组分,然后使用适当方法分离提取其中的蛋白质和多肽组分。
上述分离提取蛋白质和多肽组分的适当方法包括但不限于盐析、加热和酶解等。
上述蛋白质和多肽组分分离提取后重新溶解于含有溶解剂的溶解液中。
本发明优选的技术方案中,步骤(2)中,所述肿瘤抗原组分的制备为先分离得到肿瘤细胞或组织,裂解所述肿瘤细胞或组织获得水溶性组分、非水溶性组分、全组分的任一种或其组合。
本发明优选的技术方案中,步骤(2)中,所述肿瘤抗原组分经过盐析、加热和酶解等方法进行适当处理。
本发明优选的技术方案中,所述水溶性部分和非水溶性部分都可以被含增溶剂的增溶水溶液或有机溶剂溶解。
本发明优选的技术方案中,所述增溶剂为可以增加蛋白质或多肽在水溶液中溶解性的增溶剂中的至少一种;有机溶剂为可以溶解蛋白质或多肽的有机溶剂。
本发明优选的技术方案中,所述的非水溶性部分采用适当增溶方法由在纯水中不溶变为在含溶解剂的水溶液中或有机溶剂中可溶;采用的溶解剂选自含有结构式1结构的化合物、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、多肽、氨基酸、糖苷和胆碱中的一种或多种;其中,结构式1如下:结构式1结构如下:
,R1为C、N、S或O,R2~R5独立地选自氢、烷基、氨基、羧基、取代或未取代胍基中的至少一种。
含有结构式1结构的化合物包括但不限于盐酸二甲双胍、硫酸二甲双胍、磺酸二甲双胍、二甲双胍盐、二甲双胍、尿素、盐酸胍、硫酸胍、磺酸胍、胍盐、其他含有胍基的化合物、碳酸胍、精氨酸、胍基乙酸、胍基磷酸、氨基磺酸胍、胍基琥珀酸、盐酸氨基脲、氨基甲酰脲、乙酰脲、磺酰脲类化合物(格列本脲、格列齐特、格列喹酮、格列美脲等)、硫脲类化合物(硫氧嘧啶类、咪唑类等)、亚硝基脲类等。
尿素和盐酸胍等含有结构式1中的结构,发明人发现具有结构式1结构的物质可以作为溶解液中的溶解剂溶解细胞或者肿瘤组织中的非水溶性组分,因而除了尿素和盐酸胍、二甲双胍等常见的含有胍基结构的化合物外,其他含有改结构的化合物也具有作为溶解剂溶解非水溶性组分的能力。
本发明优选的技术方案中,将免疫佐剂与细胞组分共负载于纳米粒子或微米粒子中,在纳米粒子或微米粒子被抗原提呈细胞吞噬后可以更好的癌症特异性T细胞。
本发明优选的技术方案中,所述水溶性组分的制备方法为:将肿瘤组织或癌细胞切块后研磨,过滤制得单细胞悬液,加水反复冻融1-5次后,超声裂解,裂解物以5000-10000g的转速离心5-10分钟后,取上清液即为水溶性组分,沉淀部分为非水溶性组分。
本发明优选的技术方案中,所述抗原组分为非水溶性组分中加入溶解剂溶解后得到的可溶组分,所述溶解剂为尿素、脱氧胆酸钠、盐酸胍、辛基葡萄糖苷、精氨酸、甘油、盐酸氨基脲、硫酸胍基丁胺中的任一种或其组合。
本发明优选的技术方案中,所述抗原组分为非水溶性组分中加入尿素水溶液溶解后得到的可溶组分,与水溶性组分按3-1:1混合得到的混合物。
本发明优选的技术方案中,所述抗原组分为肿瘤细胞的水溶性组分和癌细胞的水溶性组分按1:1混合,或肿瘤细胞的非水溶性组分加入溶解剂后溶解得到的可溶组分和癌细胞的非水溶性组分加入溶解剂后溶解得到的可溶组分按1:1混合。
本发明优选的技术方案中,所述肿瘤组织或癌细胞提前进行紫外高温加热进行灭活和变性处理、加入核酸酶灭活处理中的任一种或其组合。
本发明优选的技术方案中,所述抗原组分为水溶性组分经盐析和加热沉淀后得到的组分。
本发明优选的技术方案中,所述抗原组分为肿瘤组织裂解离心后的沉淀部分即非水溶性组分中加入溶解剂溶解后得到的沉淀物中,再加入增溶剂进行二次溶解得到的可溶组分,所述增溶剂为吐温80、硫酸胍中的任一种或其组合。
本发明的另一目的在于提供一种含有本发明所述的特异性T细胞的药物组合物的制备方法,包括下述步骤,在将癌症特异性T细胞回输给患者前可以在癌症特异性T细胞中添加具有增强天然免疫系统的物质,如白蛋白、NK细胞、中性粒细胞、γδT细胞、NK T细胞。
本发明的优选技术方案中,所述特异性T细胞的细胞浓度为(0.01-100)×107个/ml,优选为(0.1-8)×107个/ml。
本发明的优选技术方案中,所述药物组合物中还包含羟乙基淀粉、糖、盐中的任一种或其组合。
本发明的另一目的在于本发明的特异性T细胞在制备癌症治疗或预防药物中的应用。
本发明优选的技术方案中,所述特异性T细胞在癌症发生前、癌症发生后或手术切除肿瘤组织后多次给药。
本发明的另一目的在于本发明的特异性T细胞在制备预防癌症复发或预防癌症转移的药物中的应用。
本发明的另一目的在于提供本发明的特异性T细胞用于制备抗肿瘤免疫治疗的制品中的应用。
本发明优选的技术方案中,所述肿瘤选自实体瘤、血液瘤和淋巴瘤。包括但不限于肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液瘤如白血病、脑肿瘤、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤、骨肉瘤、骨癌、胰腺癌、皮肤癌、前列腺癌、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、霍奇金病、非霍奇金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症、转移癌、循环肿瘤细胞的任一种或其组合。
优选地,所述肿瘤选自黑色素瘤、结肠癌、三阴性乳腺癌、胰腺癌、转移癌、肝癌、结肠癌、淋巴瘤、食管癌、非小细胞肺癌中的任一种或其组合。
本发明优选的技术方案中,所述免疫治疗选自抗肿瘤治疗中的免疫治疗或根治术后免疫治疗的任一种或其组合。
本发明优选的技术方案中,所述免疫治疗选自原发性肝细胞癌根治术后免疫治疗。
本发明优选的技术方案中,所述药物用于成年患者或儿童患者。
本发明的另一目的在于提供本发明的特异性T细胞用于免疫治疗的应用。
本发明的优选技术方案中,所述免疫治疗的给药方式为静脉注射、皮下注射、瘤内注射、腹腔注射、肌肉注射、皮内注射中的任一种或其组合。
本发明的另一目的在于提供本发明的特异性T细胞与放疗、化疗、靶向治疗、手术治疗或免疫治疗的任一种联用于抗肿瘤或肿瘤免疫治疗中的应用。
本发明的另一目的在于提供特异性T细胞用于制备增强抗病毒能力的药物中的应用。
本发明的另一目的在于提供特异性T细胞用于制备增强治疗自身免疫性疾病的药物中的应用。
本发明的优选技术方案中,所述自身免疫性疾病选自系统性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺机能亢进、青少年糖尿病、原发性血小板紫癜、自身免疫性溶血性贫血、溃疡性结肠炎、皮肤病的任一种或其并发症。
除非另有说明,本发明涉及液体与液体之间的百分比时,所述的百分比为体积/体积百分比;本发明涉及液体与固体之间的百分比时,所述百分比为体积/重量百分比;本发明涉及固体与液体之间的百分比时,所述百分比为重量/体积百分比;其余为重量/重量百分比。
与现有技术相比,本发明具有下述有益技术效果:
1.本发明制备得到的纳米粒子或微米粒子,将肿瘤抗原组分负载在微米粒子和/或纳米粒子,可以激活广谱的癌细胞特异性T细胞,利用癌细胞特异性T细胞被激活后的特性分选后经过扩增后回输患者用于癌症的治疗或者预防复发或转移。所得到的癌症特异性T细胞广谱而且特异性高,经扩增后杀伤癌细胞即可预防或治疗癌症。
2.本发明通过特定方法筛选得到的抗原组分、纳米粒子,并科学筛选分选扩增条件,得到的T细胞活率高、细胞稳定性好、免疫细胞的靶向性和抗肿瘤活性强。用于肿瘤患者的治疗后,可以预防肿瘤复发,延长肿瘤生长速度,预防癌症转移、延长生存期。
3、本发明的方法具有操作简便、质量可控,适宜工业化生产等优点。
附图说明
图1为本发明T细胞系统的代表性制备过程及应用领域示意图,在实际应用中还可以为其他类似或改良的替代制备过程;其中,a为水溶性组分和非水溶性组分分别收集和制备纳米粒子或微米粒子的示意图;b为采用含有增溶剂的增溶液溶解全细胞组分和制备纳米粒子或微米粒子的示意图;c为使用a或b中制备的上述粒子辅助分选外周的癌细胞特异性T细胞,尔后扩增该类T细胞,并用该类细胞预防或治疗癌症的示意图;
图2-图30为根据本发明的实施例1-29中用分离扩增的癌症特异性T细胞预防或治疗癌症时小鼠肿瘤生长速度和生存期实验结果,或者预防癌症转移时癌症转移灶的数量监测结果,或者癌症特异性T细胞占T细胞比例的结果;其中,a或b为预防或治疗癌症时的肿瘤生长速和小鼠生存期实验结果图(n≥8);c,使用流式细胞术分析比较特定T细胞亚群实验结果(n≥8)。a图中每个数据点为平均值±标准误差(mean±SEM);a中肿瘤生长抑制实验的显著性差异采用ANOVA法分析,b中显著性差异采用Kaplan-Meier和log-rank test分析。
如无特殊说明,以上附图中,***表示与PBS空白对照组相比p<0.005,有显著性差异;**表示与PBS空白对照组相比p<0.001,有显著性差异;*表示与PBS空白对照组相比p<0.001,有显著性差异;τττ代表不经纳米粒子刺激而一步分选的癌症特异性T细胞组相比p<0.005,有显著性差异;■■■表示与含免疫佐剂的空白纳米粒+游离裂解液辅助分选得到的细胞对照组相比p<0.005,有显著性差异;λ代表与共孵育过程中未加入IL-7分选得到的癌症特异性T细胞组相比p<0.05,有显著性差异;ε代表与只使用一种抗原提呈细胞共孵育分选扩增的癌症特异性T细胞组相比p<0.05,有显著性差异;εε代表与只使用一种抗原提呈细胞共孵育分选扩增的癌症特异性T细胞组相比p<0.01,有显著性差异;η代表与负载一种佐剂的纳米粒子/微米粒子分选扩增的癌症特异性T细胞组相比p<0.05,有显著性差异;$代表与多肽纳米粒或微米粒辅助分选得到的癌症特异性T细胞组相比p<0.05,有显著性差异;$$代表与多肽纳米粒或微米粒辅助分选得到的癌症特异性T细胞组相比p<0.01,有显著性差异;δ代表与无佐剂的纳米粒辅助分选得到的癌症特异性T细胞组相比p<0.05,有显著性差异;σ代表与纳米粒子1辅助分选的CD8+癌症特异性T细胞组相比p<0.05,有显著性差异;Δ代表与纳米粒子2辅助分选的CD8+癌症特异性T细胞+CD4+癌症特异性T细胞组相比p<0.05,有显著性差异。#代表p<0.05,有显著性差异;##代表p<0.01,有显著性差异;###代表p<0.005,有显著性差异。ns代表无显著性差异。
具体实施方式
以下实施例中,首先制备抗原组分,抗原组分可以为(1)癌细胞全细胞组分;(2)或者为含有癌细胞全细胞组分中的全细胞蛋白质和多肽组分的全细胞组分中的一部分组分;(3)或者为癌细胞/肿瘤组织全细胞组分中的蛋白质多肽组分加上mRNA组分。
全细胞组分制备方法为:(1)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用;(2)使用含有溶解剂的溶解液裂解细胞,然后使用含有溶解剂的溶解液溶解裂解后的全细胞组分。
含有癌细胞全细胞组分中的全细胞蛋白质和多肽组分的全细胞组分中的一部分组分的制备方法为:(1)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中分离提取的蛋白质和多肽组分与所有非水溶性组分一起作为抗原组分使用;(2)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从非水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将非水溶性组分中分离提取的蛋白质和多肽组分与所有水溶性组分一起作为抗原组分使用;(3)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分和非水溶性组分中使用适当方法分别分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中和非水溶性组分中分离提取的蛋白质和多肽组分一起作为抗原组分使用;(4)使用含有溶解剂的溶解液裂解细胞,然后使用含有溶解剂的溶解液溶解裂解后的全细胞组分,然后使用适当方法分离提取其中的蛋白质和多肽组分。上述制备方法中还可以增加分离提取全细胞mRNA的步骤,并将全细胞mRNA作为抗原组分的一部分使用。
上述分离提取蛋白质和多肽组分的适当方法包括但不限于盐析、加热和酶解等。
上述蛋白质和多肽组分分离提取后重新溶解于含有溶解剂的溶解液中。
癌细胞/肿瘤组织全细胞组分中的蛋白质多肽组分加上mRNA组分的制备方法为:(1)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用;然后分别分离提取水溶性组分和/或非水溶性组分中蛋白质和多肽组分以及mRNA组分,然后将蛋白质和多肽组分与mRNA组分混合后作为抗原组分使用。
上述所述溶解剂选自含有结构式1结构的化合物、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、多肽、氨基酸、糖苷和胆碱中的一种或多种;其中,结构式1如下:结构式1结构如下:
,R1为C、N、S或O,R2~R5独立地选自氢、烷基、氨基、羧基、取代或未取代胍基的至少一种。含有结构式1的化合物包括但不限于盐酸二甲双胍、硫酸二甲双胍、磺酸二甲双胍、二甲双胍盐、二甲双胍、盐酸聚六亚甲基胍、硫酸胍基丁胺、盐酸甲基胍、盐酸四甲基胍、尿素、盐酸胍、硫酸胍、磺酸胍、胍盐、其他含有胍基或脲的化合物、碳酸胍、精氨酸、胍基乙酸、胍基磷酸、氨基磺酸胍、胍基琥珀酸、盐酸氨基脲、氨基甲酰脲、乙酰脲、磺酰脲类化合物(格列本脲、格列齐特、格列喹酮、格列美脲等)、硫脲类化合物(硫氧嘧啶类、咪唑类等)、亚硝基脲类等含有结构式1结构的化合物。
然后将抗原组分负载于纳米粒子或微米粒子。负载细胞组分的纳米粒子或微米粒子可以采用任何将抗原组分负载到纳米/微米粒子的制备方法制备得到,包括但不限于溶剂挥发法、透析法、微流控法、挤出法、热熔法、等方法中的任一种。
抗原组分可以负载于纳米粒子/微米粒子内部,或者负载于纳米/微米粒子表面,或者同时负载于纳米/微米粒子内部和表面。
在本发明的实施例中,以溶剂挥发法来说明具体如何实施,在实际应用中也可以采用任何其他可行的制备方法。
所述纳米粒子或微米粒子的制备方法,包括如下步骤:
(1)将第一预定体积的含有第一预定浓度的水相溶液加入第二预定体积的含有第二预定浓度医用高分子材料的有机相中;
(2)将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌或均质处理或微流控处理;
(3)将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于2秒的超声处理或大于1分钟的搅拌或进行均质处理或微流控处理;
(4)将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中,并进行搅拌直至满足预定搅拌条件。
(5)将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后,去除上清液,并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有冻干保护剂的水溶液中或者第六预定体积的PBS(或生理盐水)中。
(6)将步骤5得到的含有冻干保护剂的混悬液进行冷冻干燥处理后,将冻干物质备用。
(7)将第六预定体积的步骤5中得到的重悬于PBS(或生理盐水)中的含纳米粒的混悬液或者采用第六预定体积的PBS(或生理盐水)重悬步骤6得到的冷冻干燥后的含有纳米粒或微米粒和冻干保护剂的冻干物质直接使用;或者上述样品与第七预定体积的水溶性组分或者增溶的原非水溶性组分混合后使用。
所述纳米粒子或微米粒子的制备方法,水相溶液可含有癌细胞裂解物中的各组分以及免疫增强佐剂poly(I:C)、BCG、锰佐剂、钙佐剂或CpG;癌细胞裂解物中的各组分在制备时分别为水溶性组分或者是溶于尿素或盐酸胍中的原非水溶性组分。水相溶液所含有来自癌细胞的水溶性组分的浓度或者是来自癌细胞的溶于尿素或盐酸胍中的原非水溶性组分的浓度,也即第一预定浓度要求蛋白质多肽浓度含量大于1ng/mL,能负载足够癌症抗原以激活相关免疫反应。免疫增强佐剂在初始水相中的浓度为大于0.01ng/mL。
水相溶液含有肿瘤组织裂解物中的各组分以及免疫增强佐剂poly(I:C),BCG、锰佐剂、钙佐剂或CpG;肿瘤组织裂解物中的各组分在制备时分别为水溶性组分或者是溶于尿素或盐酸胍中的原非水溶性组分。水相溶液所含有得来自肿瘤组织的水溶性组分的浓度或者是来自肿瘤组织的溶于尿素或盐酸胍中的原非水溶性组分的浓度,也即第一预定浓度要求蛋白质多肽浓度含量大于0.01ng/mL,能负载足够癌症抗原以激活相关免疫反应。免疫增强佐剂在初始水相中的浓度为大于0.01ng/mL。
将医用高分子材料溶解于有机溶剂中,得到第二预定体积的含有第二预定浓度医用高分子材料的有机相。在一些实施例中,医用高分子材料为PLGA,有机溶剂选用二氯甲烷。另外,在一些实施例中,医用高分子材料的第二预定浓度的范围为0.5mg/mL-5000mg/mL,优选为100mg/mL。
实际中,有机相的第二预定体积根据其和水相的第一预定体积的比例进行设定,在本发明中,水相的第一预定体积和有机相的第二预定体积之比的范围为1:1.1-1:5000,优先地为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整以调整制备的纳米粒或微米粒的尺寸大小。
优选的,水相溶液为裂解物组分溶液时,其中蛋白质和多肽的浓度大于1ng/mL,优选1mg/mL~100mg/mL;水相溶液为裂解物组分/免疫佐剂溶液时,其中蛋白质和多肽的浓度大于1ng/mL,优选1mg/mL~100mg/mL,免疫佐剂的浓度大于0.01ng/mL,优选0.01mg/mL~20mg/mL。高分子材料有机相溶液中,溶剂为DMSO、乙腈、乙醇、氯仿、甲醇、DMF、异丙醇、二氯甲烷、丙醇、乙酸乙酯等,优选二氯甲烷;高分子材料的浓度为0.5mg/mL~5000mg/mL,优选为100mg/mL。第一乳化剂溶液优选为聚乙烯醇水溶液,浓度为10mg/mL~50mg/mL,优选20mg/mL。第二乳化剂溶液优选为聚乙烯醇水溶液,浓度为1mg/mL~20mg/mL,优选5mg/mL。分散液为PBS缓冲液或生理盐水或纯水。
步骤2,将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌或均质处理或微流控处理。优选的,搅拌为机械搅拌或者磁力搅拌时,搅拌速度大于50rpm,搅拌时间大于1分钟,比如搅拌速度为50rpm~1500rpm,搅拌时间为0.1小时~24小时;超声处理时,超声功率大于5W,时间大于0.1秒,比如2~200秒;均质处理时使用高压/超高压均质机或高剪切均质机,使用高压/超高压均质机时压力大于5psi,比如20psi~100psi,使用高剪切均质机时转速大于100rpm,比如1000rpm~5000rpm;使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化和/或微米化,超声时间长短或搅拌速度或均质处理压力及时间能控制制备的微纳粒子大小,过大或过小都会带来粒径大小的变化。
步骤3,将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于2秒的超声处理或大于1分钟的搅拌或进行均质处理或微流控处理。该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌纳米化或微米化。该步骤是为了进行纳米化或微米化,超声时间长短或搅拌速度及时间能控制制备的纳米粒子或微米粒子大小,过长或过短都会带来粒径大小的变化,为此,需要选择合适的超声时间。在本发明中,超声时间大于0.1秒,比如2~200秒,搅拌速度大于50rpm,比如50rpm~500rpm,搅拌时间大于1分钟,比如60~6000秒。优选的,搅拌为机械搅拌或者磁力搅拌时,搅拌速度大于50rpm,搅拌时间大于1分钟,比如搅拌速度为50rpm~1500rpm,搅拌时间为0.5小时~5小时;超声处理时,超声功率为50W~500W,时间大于0.1秒,比如2~200秒;均质处理时使用高压/超高压均质机或高剪切均质机,使用高压/超高压均质机时压力大于20psi,比如20psi~100psi,使用高剪切均质机时转速大于1000rpm,比如1000rpm~5000rpm;使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化或微米化,超声时间长短或搅拌速度或均质处理压力及时间能控制制备的纳米或微米粒子大小,过大或过小都会带来粒径大小的变化。
在本发明中,乳化剂水溶液为聚乙烯醇(PVA)水溶液,第三预定体积为5mL,第三预定浓度为20mg/mL。第三预定体积根据其与第二预定体积的比例进行调整。在本发明中,第二预定体积与第三预定体积之的范围为1:1.1-1:1000进行设定,优先地可以为2:5。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸,可以对第二预定体积和第三预定体积之比进行调整。同样地,本步骤的超声时间或搅拌时间、乳化剂水溶液的体积以及浓度的取值根据,均为了得到尺寸大小合适的纳米粒或微米粒。
步骤4,将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中,并进行搅拌直至满足预定搅拌条件。
本步骤中,乳化剂水溶液依然为PVA。
第四预定浓度为5mg/mL,第四预定浓度的选择,以得到尺寸大小合适的纳米粒或微米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本发明中,第三预定体积与第三预定体积之比为范围为1:1.5-1:2000,优先地为1:10。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。
在本发明中,本步骤的预定搅拌条件为直至有机溶剂挥发完成,也即步骤1中的二氯甲烷挥发完成。
步骤5,将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后,去除上清液,并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有冻干保护剂的水溶液中或者第六预定体积的PBS(或生理盐水)中。
在本发明一些实施方案中,步骤5所得沉淀重新混悬于第六预定体积的PBS(或生理盐水)中时不需要冻干,可直接进行后续纳米粒子或微米粒子表面吸附癌细胞裂解物的相关实验。
在本发明一些实施方案中,步骤5所得沉淀重新混悬于含有冻干保护剂的水溶液中时需进行冷冻干燥,再冷冻干燥以后再进行后续纳米粒子或微米粒子表面吸附癌细胞裂解物的相关实验。
在本发明中,所述冻干保护剂选用海藻糖(Trehalose)。
在本发明中,该步骤的冻干保护剂的第五预定浓度为质量百分比4%,之所以如此设定,是为了在后续进行冷冻干燥中不影响冻干效果。
步骤6,将步骤5得到的含有冻干保护剂的混悬液进行冷冻干燥处理后,将冻干物质备用。
步骤7,将第六预定体积的步骤5中得到的重悬于PBS(或生理盐水)中的含纳米粒的混悬液或者采用第六预定体积的PBS(或生理盐水)重悬步骤6得到的冷冻干燥后的含有纳米粒或微米粒和冻干保护剂的冻干物质直接使用;或者上述样品与第七预定体积的水溶性组分或者增溶的原非水溶性组分混合后使用。
在本发明中,第六预定体积与第七预定体积的体积比为1:10000到10000:1,优先体积比为1:100到100:1,最优体积比为1:30到30:1。
在一些实施例中,所述重悬的纳米粒子混悬液体积为10mL时,含有癌细胞裂解物或含有肿瘤组织裂解物中的水溶性组分或者增溶的的原非水溶性组分的体积与为1mL。在实际使用时可将二者体积和比例根据需要进行调整。
本发明优选的技术方案中,所述复乳法制备纳米粒子或微米粒子的方法,包括如下步骤:
(1)将第一预定体积的含有第一预定浓度的水相溶液加入第二预定体积的含有第二预定浓度医用高分子材料的有机相中。
(2)将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌或均质处理或微流控处理。
(3)将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于2秒的超声处理或大于1分钟的搅拌或进行均质处理或微流控处理。
(4)将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中,并进行搅拌直至满足预定搅拌条件或者也可不进行搅拌直接进行后续处理。
(5)将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后,去除上清液,并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有全细胞组分中水溶性和/或非水溶性组分的溶液中,或者将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有全细胞组分中水溶性和/或非水溶性组分与佐剂混合的溶液中。
(6)将步骤5处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后,去除上清液,并将剩下的沉淀物重新混悬于第六预定体积的固化处理试剂或矿化处理试剂,作用一定时间后离心洗涤,然后加入第七预定提交的含有带正电或者带负电的物质并作用一定时间。
(7)将步骤6得到的含有干燥保护剂的混悬液进行干燥处理后,将干燥后的物质备用。
(8)将第八预定体积的步骤6中得到的重悬于PBS(或生理盐水)中的含纳米粒的混悬液或者采用第八预定体积的PBS(或生理盐水)重悬步骤7得到的干燥后的含有纳米粒或微米粒和干燥保护剂的干燥后物质直接使用;或者与第九预定体积的水溶性组分或者非水溶性组分混合后使用。
在一些实施例中,水相溶液可含有癌细胞裂解物中的各组分以及免疫增强佐剂poly(I:C)、锰佐剂、钙佐剂、BCG或CpG;癌细胞裂解物中的各组分在制备时分别为水溶性组分或者是溶于尿素或盐酸胍中的原非水溶性组分。水相溶液所含有来自癌细胞的水溶性组分的浓度或者是来自癌细胞的溶于尿素或盐酸胍中的原非水溶性组分的浓度,也即第一预定浓度要求蛋白质多肽浓度含量大于0.01ng/mL,能负载足够癌症抗原以激活相关免疫反应。免疫增强佐剂在初始水相中的浓度为大于0.01ng/mL。
在一些实施例中,水相溶液含有肿瘤组织裂解物中的各组分以及免疫增强佐剂poly(I:C),锰佐剂、钙佐剂、BCG或CpG;肿瘤组织裂解物中的各组分在制备时分别为水溶性组分或者是溶于尿素或盐酸胍中的原非水溶性组分。水相溶液所含有得来自肿瘤组织的水溶性组分的浓度或者是来自肿瘤组织的溶于尿素或盐酸胍中的原非水溶性组分的浓度,也即第一预定浓度要求蛋白质多肽浓度含量大于0.01ng/mL,能负载足够癌症抗原以激活相关免疫反应。免疫增强佐剂在初始水相中的浓度为大于0.01ng/mL。
在本发明中,将医用高分子材料溶解于有机溶剂中,得到第二预定体积的含有第二预定浓度医用高分子材料的有机相。在一些实施例中,医用高分子材料为PLGA,有机溶剂选用二氯甲烷。另外,在一些实施例中,医用高分子材料的第二预定浓度的范围为0.5mg/mL-5000mg/mL,优选为100mg/mL。
在本发明中,之所以选择PLGA或修饰的额PLGA,是由于该材料为生物可降解材料且已被FDA批准用作药物敷料。研究表明PLGA具有一定的免疫调节功能,因而适合作为纳米粒子或微米粒子制备时的辅料。
实际中,有机相的第二预定体积根据其和水相的第一预定体积的比例进行设定,在本发明中,水相的第一预定体积和有机相的第二预定体积之比的范围为1:1.1-1:5000,优先地为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整以调整制备的纳米粒或微米粒的尺寸大小。
优选的,水相溶液为裂解物组分溶液时,其中蛋白质和多肽的浓度大于1ng/mL,优选1mg/mL~100mg/mL;水相溶液为裂解物组分/免疫佐剂溶液时,其中蛋白质和多肽的浓度大于1ng/mL,优选1mg/mL~100mg/mL,免疫佐剂的浓度大于0.01ng/mL,优选0.01mg/mL~20mg/mL。高分子材料有机相溶液中,溶剂为DMSO、乙腈、乙醇、氯仿、甲醇、DMF、异丙醇、二氯甲烷、丙醇、乙酸乙酯等,优选二氯甲烷;高分子材料的浓度为0.5mg/mL~5000mg/mL,优选为100mg/mL。第一乳化剂溶液优选为聚乙烯醇水溶液,浓度为10mg/mL~50mg/mL,优选20mg/mL。第二乳化剂溶液优选为聚乙烯醇水溶液,浓度为1mg/mL~20mg/mL,优选5mg/mL。分散液为PBS缓冲液或生理盐水或纯水。
步骤2,将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌或均质处理或微流控处理。优选的,搅拌为机械搅拌或者磁力搅拌时,搅拌速度大于50rpm,搅拌时间大于1分钟,比如搅拌速度为50rpm~1500rpm,搅拌时间为0.1小时~24小时;超声处理时,超声功率大于5W,时间大于0.1秒,比如2~200秒;均质处理时使用高压/超高压均质机或高剪切均质机,使用高压/超高压均质机时压力大于5psi,比如20psi~100psi,使用高剪切均质机时转速大于100rpm,比如1000rpm~5000rpm;使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化和/或微米化,超声时间长短或搅拌速度或均质处理压力及时间能控制制备的微纳粒子大小,过大或过小都会带来粒径大小的变化。
步骤3,将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于2秒的超声处理或大于1分钟的搅拌或进行均质处理或微流控处理。该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌纳米化或微米化。该步骤是为了进行纳米化或微米化,超声时间长短或搅拌速度及时间能控制制备的纳米粒子或微米粒子大小,过长或过短都会带来粒径大小的变化,为此,需要选择合适的超声时间。在本发明中,超声时间大于0.1秒,比如2~200秒,搅拌速度大于50rpm,比如50rpm~500rpm,搅拌时间大于1分钟,比如60~6000秒。优选的,搅拌为机械搅拌或者磁力搅拌时,搅拌速度大于50rpm,搅拌时间大于1分钟,比如搅拌速度为50rpm~1500rpm,搅拌时间为0.5小时~5小时;超声处理时,超声功率为50W~500W,时间大于0.1秒,比如2~200秒;均质处理时使用高压/超高压均质机或高剪切均质机,使用高压/超高压均质机时压力大于20psi,比如20psi~100psi,使用高剪切均质机时转速大于1000rpm,比如1000rpm~5000rpm;使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化或微米化,超声时间长短或搅拌速度或均质处理压力及时间能控制制备的纳米或微米粒子大小,过大或过小都会带来粒径大小的变化。
在本发明中,乳化剂水溶液为聚乙烯醇(PVA)水溶液,第三预定体积为5mL,第三预定浓度为20mg/mL。第三预定体积根据其与第二预定体积的比例进行调整。在本发明中,第二预定体积与第三预定体积之的范围为1:1.1-1:1000进行设定,优先地可以为2:5。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸,可以对第二预定体积和第三预定体积之比进行调整。同样地,本步骤的超声时间或搅拌时间、乳化剂水溶液的体积以及浓度的取值根据,均为了得到尺寸大小合适的纳米粒或微米粒。
步骤4,将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中,并进行搅拌直至满足预定搅拌条件或者也可不进行搅拌直接进行后续处理。
本步骤中,乳化剂水溶液依然为PVA。
第四预定浓度为5mg/mL,第四预定浓度的选择,以得到尺寸大小合适的纳米粒或微米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本发明中,第三预定体积与第三预定体积之比为范围为1:1.5-1:2000,优先地为1:10。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。
在本发明中,本步骤的预定搅拌条件为有机溶剂挥发完成,也即步骤1中的二氯甲烷挥发完成。也不不经搅拌进行后续试验。
步骤5,将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后,去除上清液,并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有全细胞组分中水溶性和/或非水溶性组分的溶液中,或者将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有全细胞组分中水溶性和/或非水溶性组分与佐剂混合的溶液中。
步骤6,将步骤5处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后,去除上清液,并将剩下的沉淀物重新混悬于第六预定体积的固化处理试剂或矿化处理试剂,作用一定时间后离心洗涤,然后加入第七预定提交的含有带正电或者带负电的物质并作用一定时间。
在本发明一些实施方案中,步骤6所得沉淀重新混悬于第七预定体积的带电物质后可不需要冻干,可直接进行后续纳米粒子或微米粒子表面负载癌细胞/组织裂解物的相关实验。
在本发明一些实施方案中,步骤6所得沉淀重新混悬于含有干燥保护剂的水溶液中后进行室温真空干燥或者冷冻真空干燥,在干燥以后再进行后续纳米粒子或微米粒子表面吸附癌细胞裂解物的相关实验。
在本发明中,所述冻干保护剂选用海藻糖(Trehalose),或者甘露醇与蔗糖的混合溶液。在本发明中,该步骤的干燥保护剂的浓度为质量百分比4%,之所以如此设定,是为了在后续进行干燥中不影响干燥效果。
步骤7,将步骤6得到的含有干燥保护剂的混悬液进行干燥处理后,将干燥后的物质备用。
步骤8,将第八预定体积的步骤6中得到的重悬于PBS(或生理盐水)中的含纳米粒的混悬液或者采用第八预定体积的PBS(或生理盐水)重悬步骤7得到的干燥后的含有纳米粒或微米粒和干燥保护剂的干燥后物质直接使用;或者与第九预定体积的水溶性组分或者非水溶性组分混合后使用。
在本发明中,步骤5-步骤8的修饰和抗原负载步骤可重复多次以提高抗原的负载量。而且在添加带正电或带负电的物质时可以多次添加带同种电荷的或者也可以交替添加带不同电荷的物质。
在一些实施例中,所述重悬的纳米粒子混悬液体积为10mL时,含有癌细胞裂解物或含有肿瘤组织裂解物中的水溶性组分或者原非水溶性组分的体积与为0.1-100mL。在实际使用时可将二者体积和比例根据需要进行调整。
在本发明中,所采用的含有癌细胞裂解物或含有肿瘤组织裂解物中水溶性组分或者原非水溶性组分中含有poly(I:C)、锰佐剂、卡介苗(BCG)或CpG,且poly(I:C)、钙佐剂、BCG或CpG的浓度为大于0.01ng/mL。
进一步地,本发明所述纳米粒子或微米粒子在制备时,在体外激活癌症特异性T细胞时可以同时使用只负载水溶性组分的纳米粒子和/或微米粒子和只负载非水溶性组分的纳米粒子和/或微米粒子、使用只负载水溶性组分的纳米粒子和/或微米粒子、使用只负载非水溶性组分的纳米粒子和/或微米粒子或者使用同时负载水溶性组分和非水溶性组分的纳米粒子和/或微米粒子。
通过任何一种方法制备得到负载肿瘤抗原的纳米粒子或微米粒子后,将纳米粒子或微米粒子与抗原提呈细胞和T细胞三者同时共孵育以激活癌细胞特异性T细胞。或者将负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞,然后将被激活的抗原提呈细胞再单独与T细胞二者一起共孵育以激活癌细胞特异性T细胞。
在将T细胞与纳米粒子/微米粒子和抗原提呈细胞一起共孵育前,T细胞可以先单独静息培养一段时间,或者进行适当分选;或者在将T细胞与被激活的抗原提呈细胞共孵育前,T细胞可以先单独静息培养一段时间,或者进行适当分选。
负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞后,抗原提呈细胞可以不经过特殊处理再去与T细胞二者共孵育激活特异性T细胞,或者抗原提呈细胞可以经过固定、辐射、照射、修饰、灭活、矿化等处理后再与T细胞二者共孵育激活特异性T细胞。
当癌细胞特异性T细胞被激活后,使用流式细胞术或者磁珠分选法等细胞分离方法从孵育后的细胞中分选出被激活的癌细胞特异性T细胞,然后将癌细胞特异性T细胞在体外扩增一定时间后,所得扩增后的癌细胞特异性T细胞用于预防或者治疗癌症。
在分选被激活的癌细胞特异性T细胞时,可以使用一种T细胞被激活的激活标志物,也可以使用一种以上的不同标志物的组合作为激活标志物。
可以用来作为表面标志物的分子包括但不限于: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、CXCR 1、CD103、CD45RO、ICOS(CD278)、VTCN1、HLA2、LGAL59、CCR7、CD357、BCL6、TCF-1、CD38、CD27等当中的任一种或其任意组合。
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1分离扩增T细胞用于黑色素瘤的预防
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用纳米粒子分离扩增外周的癌细胞特异性T细胞后用于预防癌症。本实施例中,裂解B16F10黑色素瘤肿瘤组织以制备肿瘤组织的水溶性组分和非水溶性组分,然后,以有机高分子材料PLGA为纳米粒骨架材料,以Polyinosinic-polycytidylic acid(poly(I:C))为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织的水溶性组分和非水溶性组分的纳米粒子,然后使用纳米粒子辅助分离器官中的癌细胞特异性T细胞,分离得到的癌细胞特异性T细胞经扩增后注射到体内预防黑色素瘤。本实施例使用小鼠外周脾细胞中的免疫细胞,在实际应用时可以直接使用外周血或者外周淋巴结细胞。
(1)抗原组分的制备
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到体积分别为约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网后制备得到肿瘤组织单细胞悬液(含有癌细胞)。然后在肿瘤组织单细胞悬液中加入适量纯水并反复冻融5次,并可伴有超声以破坏裂解细胞。待细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为水溶性组分;在所得沉淀部分中加入8M尿素水溶液(含500mM氯化钠)溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。以上即为制备纳米粒子的抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒采用溶剂挥发法中的复乳法制备。在制备时负载全细胞组分中水溶性组分的纳米粒子和负载全细胞组分中非水溶性组分的纳米粒子分别制备,然后使用时一起使用。所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为poly(I:C)。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载抗原组分和佐剂,然后将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子平均粒径为280nm左右,纳米粒子表面电位为-3mV左右;每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分,每1mg PLGA纳米粒负载poly(I:C)0.02mg。空白纳米粒制备材料和制备方法相同,粒径为260nm左右,空白纳米粒制备时分别采用含有等量poly(I:C)的纯水或8M尿素代替相对应的水溶性组分和非水溶性组分。
(3)癌症特异性T细胞的分选和扩增
在每只C57BL/6小鼠背部皮下接种0.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取小鼠脾脏,制备小鼠脾脏细胞单细胞悬液,并进行红细胞裂解处理以去除单细胞悬液中的红细胞。先将上述细胞分别依次与T细胞磁珠分选试剂(CD3+磁珠分选试剂、CD8+磁珠分选试剂)或者B细胞磁珠分选试剂共孵育,然后使用磁珠分选仪从小鼠脾脏细胞中分选得到CD3+CD8+T细胞和CD19+B细胞。
一步分选法对照组中,将上述分选得到的CD3+CD8+T细胞(1万个,细胞活率60%)与IL-2(500U/mL)、IL-7(200U/mL)、IL-15(200U/mL)和αCD3/αCD28(10ng/mL)在10mL的RPMI 1640完全培养基中在37℃(5%CO2)共孵育12天(每三天使用含有上述细胞因子和抗体的培养基换液)以扩增所得CD8+T细胞(细胞活率60%)。
在两步分选法中,将分选得到的100万个CD8+T细胞、1000万个B细胞与负载肿瘤组织全组分抗原的纳米粒子(250μg负载水溶性组分的纳米粒子+250μg负载非水溶性组分的纳米粒子)或者空白纳米粒(500μg)+等量游离裂解液在10mL的DMEM高糖完全培养基中共孵育96小时,然后将孵育后的细胞与磁珠分选法中的CD3+T细胞磁珠分选试剂、CD8+T细胞磁珠分选试剂、以及CD69磁珠分选试剂依次共孵育并进行相应分选,使用磁珠分选法分选孵育后的T细胞中的CD3+CD8+CD69+T细胞(细胞活率60%),即为被癌症抗原激活的癌症特异性T细胞。将上述分选得到的癌症特异性T细胞(1万个)与IL-2(500U/mL)、IL-7(200U/mL)、IL-15(200U/mL)和αCD3/αCD28(10ng/mL)在RPMI 1640完全培养基中在37℃(5%CO2)共孵育12天(每三天使用含有上述细胞因子和抗体的培养基换液)以扩增分选得到的癌细胞特异性T细胞(细胞活率60%)。
将对照组一步分选法扩增后的CD8+T细胞(50万个)或者两步分选法得到的扩增后的癌症特异性T细胞(50万个)与B细胞(200万个)和负载全细胞组分的纳米粒子(30μg负载水溶性组分的纳米粒子+30μg负载非水溶性组分的纳米粒子)在3mL DMEM高糖完全培养基中共孵育48小时,然后收集孵育后的细胞,依次使用带有不同荧光探针的CD3抗体、CD8抗体、IFN-γ抗体标记孵育后的细胞,尔后使用流式细胞术分析一步分选后和两步分选后扩增所得细胞中CD8+IFN-γ+T细胞占CD8+T细胞的比例。纳米粒子所负载的癌细胞抗原在被抗原提呈细胞(B细胞)吞噬后可被降解成抗原表位被提呈到抗原提呈细胞表面,可以识别癌细胞抗原的特异性T细胞即可以识别癌细胞抗原表位后被激活并分泌杀伤性细胞因子。IFN-γ是抗原特异性T细胞识别抗原后被激活所分泌的最主要的细胞因子,但是由于其为分泌性细胞因子,所以需要先加入4%多聚甲醛进行细胞固定,然后使用破膜剂破膜后再使用抗体进行细胞内染色(分析后细胞为死细胞),这样经过多聚甲醛固定后的细胞即为四细胞。使用流式细胞术分析所得到的CD8+IFN-γ+T细胞(细胞活率0%)即为可以特异性识别癌细胞抗原表位的癌细胞特异性T细胞。
(4)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠,在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。然后,将步骤(3)中两步分选法制备得到的200万个癌细胞特异性T细胞或者一步分选法后扩增得到的200万个CD3+T细胞静脉注射给受体小鼠。隔天,给每只受体小鼠背部右下方皮下接种1.5×105个B16F10细胞。监测小鼠肿瘤生长速度和小鼠生存期。在实验中,从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v=0.52×a×b2计算,其中v为肿瘤体积,a为肿瘤长度,b为肿瘤宽度。出于动物实验伦理,在小鼠生存期实验中当小鼠肿瘤体积超过2000mm3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
图2中a和b所示,PBS对照组小鼠的肿瘤生长速度最快,小鼠生存期最短,一步分选法得到的T细胞和空白纳米粒辅助分选扩增得到的T细胞处理组的小鼠的肿瘤生长速度和生存期与PBS对照组相比都有改善。与上述三组相比,经过负载癌细胞全细胞组分的纳米粒子辅助分选和扩增后得到的癌细胞特异性T细胞处理的小鼠体内的肿瘤生长速度最慢,小鼠生存期最长,这本发明所述的分选和扩增后的细胞系统对癌症具有良好的预防效果。
如图2中c所示,两步分选法得到癌细胞特异性T细胞在和抗原提呈细胞以及负载癌细胞全细胞组分的纳米粒共孵育后几乎100%可以识别癌细胞抗原并被癌症抗原激活,而一步分选法扩增得到的T细胞在共孵育后CD8+IFN-γ+T细胞仅占CD8+T细胞的不到5%,由此可见,本发明所述的分选方法可以有效的富集具有杀伤能力的癌细胞特异性T细胞。
本实施例中使用了一种表面标志物作为T细胞被激活的标志物,在实际应用中,也可以使用一种以上的多个不同标志物的组合作为激活标志物。
实施例2外周的癌细胞特异性T细胞分离扩增后用于黑色素瘤的预防
本实施例中,裂解B16F10黑色素瘤肿瘤组织以制备肿瘤组织的水溶性组分和非水溶性组分,然后,以有机高分子材料为纳米粒骨架材料,以poly(I:C)和CpG1018为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织的水溶性组分和非水溶性组分的纳米粒子,然后使用纳米粒子分选扩增外周的癌细胞特异性T细胞。本实施例使用小鼠外周脾细胞中的免疫细胞,在实际应用时可以使用外周血或者外周淋巴结细胞。
(1)抗原组分的制备
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网后制备得到单细胞悬液,然后加入适量纯水并反复冻融5次,并伴有超声以破坏裂解细胞。待细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入8M尿素溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。以上即为制备纳米粒子的抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒子及作为对照的空白纳米粒采用溶剂挥发法制备。在制备时负载全细胞组分中水溶性组分的纳米粒子和负载全细胞组分中非水溶性组分的纳米粒子分别制备,然后使用时一起使用。所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为poly(I:C)和CpG1018。制备方法如前所述,在制备过程中先采用复乳法在纳米粒子内部负载抗原组分和佐剂,尔后将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子平均粒径为280nm左右,纳米粒子表面电位为-3mV左右;每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分,负载poly(I:C)和CpG1018各0.02mg。空白纳米粒制备材料和制备方法相同,粒径为260nm左右,空白纳米粒负载等量佐剂但是不负载任何抗原组分。
(3)癌症特异性T细胞的分离和扩增
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在第4天,第7天,第10天,第15天,第20天、第25天和第30天分别给小鼠别皮下注射负载水溶性组分的1mg PLGA纳米粒子和负载非水溶性组分的1mg PLGA纳米粒子。在第34天处死小鼠,收集小鼠的脾脏,制备小鼠脾脏细胞单细胞悬液,将上述细胞依次与T磁珠分选试剂和B细胞磁珠分选试剂分别共孵育并依次进行分选,依次使用磁珠分选仪从小鼠脾脏细胞中分选得到CD3+T细胞和CD19+B细胞。
一步分选法对照组中,将上述分选得到的100万个CD3+T细胞与IL-2(500U/mL)、IL-7(200U/mL)、IL-15(200U/mL)和αCD3/αCD28(各10ng/mL)在10mL的RPMI 1640完全培养基中在37℃(5%CO2)共孵育14天(每两天使用含有上述细胞因子和抗体的培养基换液)以扩增所得T细胞(细胞活率65%)。
在两步分选法中,将分选得到的500万个CD3+T细胞、1000万个B细胞与负载肿瘤组织全组分抗原的纳米粒子(250μg负载水溶性组分的纳米粒子+250μg负载非水溶性组分的纳米粒子)或者空白纳米粒(500μg)+等量游离抗原组分在10mL的RPMI 1640完全培养基中共孵育48小时,然后将孵育后的细胞与磁珠分选法中的试剂共孵育,使用流式细胞术分选孵育后的T细胞中的CD3+CD137+T细胞,即为被癌症抗原激活的癌症特异性T细胞(细胞活率65%)。将上述分选得到的癌细胞特异性T细胞(100万个)与IL-2(500U/mL)、IL-7(200U/mL)、IL-15(200U/mL)和αCD3/αCD28(各10ng/mL)在10mL的RPMI 1640完全培养基(37℃,5%CO2)中共孵育14天(每两天使用含有上述细胞因子和抗体的培养基换液)以扩增分选得到的癌细胞特异性T细胞(细胞活率65%)。
将对照组一步分选法扩增后的T细胞(50万个)或者两步分选法得到的扩增后的癌细胞特异性T细胞(50万个)与B细胞(200万个)和负载全细胞组分的纳米粒子(60μg)在3mL RPMI 1640完全培养基中共孵育48小时,然后收集孵育后的细胞,一次使用带有不同荧光探针的CD3抗体、CD8抗体、IFN-γ抗体标记孵育后的细胞,尔后使用流式细胞术分析一步分选后和两步分选后扩增所得细胞中CD3+IFN-γ+T细胞占CD3+T细胞的比例。使用流式细胞术分析所得到的CD3+IFN-γ+T(细胞活率0%)细胞即为可以特异性识别癌细胞抗原的癌细胞特异性T细胞。
(4)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠,在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。然后,将步骤(3)经过两步分选和扩增得到的200万个癌细胞特异性T细胞或者一步分选后扩增得到的200万个T细胞静脉注射给受体小鼠。隔天,给每只受体小鼠背部右下方皮下接种1.5×105个B16F10细胞。小鼠肿瘤生长速度和生存期监测方法同上。
(4)实验结果
如图3中a和b所示,PBS对照组小鼠的肿瘤生长速度最快,小鼠生存期最短,一步分选法得到的T细胞和空白纳米粒辅助分选得到的T细胞处理组的小鼠的肿瘤生长速度和生存期与PBS对照组相比都有改善。与上述三组相比,经过负载全细胞抗原组分的纳米粒子分选和扩增后得到的癌细胞特异性T细胞处理的小鼠体内的肿瘤生长速度最慢,小鼠生存期最长,这本发明所述的分选和扩增后的细胞系统对癌症具有良好的预防效果。
如图3中c所示,两步分选法得到癌细胞特异性T细胞在和抗原提呈细胞以及负载癌细胞全细胞组分的纳米粒共孵育后几乎100%可以识别癌细胞抗原并被癌细胞抗原激活,而一部分选法扩增得到的T细胞在共孵育后CD3+IFN-γ+T细胞仅占CD3+T细胞的不到5%,由此可见,本发明所述的分选方法可以有效的富集具有杀伤能力的癌细胞特异性T细胞。
实施例3分选扩增的癌细胞特异性T细胞后用于黑色素瘤的治疗
本实施例中,首先裂解B16F10黑色素瘤肿瘤组织和癌细胞以制备肿瘤组织和癌细胞的水溶性组分混合物(质量比1:1)和非水溶性组分混合物(质量比1:1),然后,以PLGA为纳米粒骨架材料,以Poly(I:C)、CpG2006和CpG2216为佐剂制备负载水溶性组分混合物和非水溶性组分混合物的纳米粒子系统,然后将纳米粒子与T细胞和抗原提呈细胞在体外共孵育,激活预存的癌细胞特异性T细胞,癌细胞特异性T细胞被激活后会高表达特定分子,使用流式细胞术即可进行分选,然后扩增后用于治疗癌症。
(1)抗原组分的制备
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到体积分别为约1000mm3时处死小鼠并摘取肿瘤组织,将肿瘤组织切块后研磨,通过细胞过滤网后加入适量纯水并反复冻融5次,并伴有超声以破坏裂解所得样品;收集培养的B16F10癌细胞系时,先离心去除培养基后使用PBS洗涤两次并离心收集癌细胞,将癌细胞在超纯水中重悬,反复冻融3次,并伴有超声破坏裂解癌细胞。待肿瘤组织或癌细胞系裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入8M尿素溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。将肿瘤组织的水溶性组分和癌细胞系的水溶性组分按质量比1:1混合;肿瘤组织的非水溶性组分和癌细胞系的非水溶性组分按质量比1:1混合。以上即为制备纳米粒子的抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒子采用复乳法制备。纳米粒子制备材料PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为poly(I:C)、CpG2006和CpG2216。制备方法如前所述,先在纳米粒子内部负载抗原组分和佐剂,然后将100mg纳米粒子在12000g离心25分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h;在使用前将其用9mL PBS重悬然后加入1mL的裂解液组分(蛋白质浓度80mg/mL)并室温作用10min,得到内外都负载抗原组分的纳米粒子。该纳米粒子平均粒径为280nm左右,纳米粒子表面电位为-5mV左右;每1mg PLGA纳米粒子约负载130μg蛋白质或多肽组分,每1mg PLGA纳米粒子负载poly(I:C)、CpG2006和CpG2216免疫佐剂各0.02mg。空白纳米粒制备材料和制备方法相同,粒径为260nm左右,空白纳米粒负载等量佐剂但是不负载任何癌细胞裂解液组分。
(3)癌症特异性T细胞的分离和扩增
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在第4天,第7天,第10天,第15天,第20天和分分别给小鼠别皮下注射2mg PLGA纳米粒子(负载抗原组分和佐剂)。在第24天处死小鼠,收集小鼠外周血,从小鼠外周血中使用梯度离心法分离外周血单个核细胞(PBMC),将PBMC与不同荧光探针标记的CD3抗体以及CD19抗体共孵育,然后使用流式细胞术分选得到CD3+T细胞和CD19+B细胞。
小鼠骨髓来源巨噬细胞(BMDM)制备方法为常规制备方法。BMDM制备方法如下:将C57小鼠麻醉后脱臼处死,将小鼠使用75%乙醇的消毒,然后用剪刀在小鼠背部剪开一小口,用手直接撕开皮肤至小鼠小腿关节处,去除小鼠足关节以及皮肤。用剪刀沿着小鼠大腿根部大转子将后肢拆下来,去掉肌肉组织后放置在含有75%乙醇的培养皿内浸泡5min,更换新的75%乙醇的培养皿移入超净台中。将乙醇浸泡的腿骨移入冷的PBS浸泡,洗去胫骨、股骨表面的乙醇,此过程可重复3次。将清洗好的股骨、胫骨分开,并用剪刀分别将股骨、胫骨两端剪断,使用1mL注射器吸取冷的诱导培养基将骨髓从股骨、胫骨中吹出,反复吹洗3次,直至腿骨内看不到明显的红色为止。用5mL移液枪将含有骨髓细胞的培养基反复吹打,使细胞团块分散,然后使用70μm细胞滤器将细胞过筛,转移至15mL离心管内,1500rpm/min离心5min,弃上清,加入红细胞裂解液重悬静置5min后1500rpm/min离心5min,弃上清用冷的配置好的骨髓巨噬细胞诱导培养基(含有15%L929培养基的DMEM高糖培养基)重悬,铺板。将细胞培养过夜,以去除贴壁较快的其他杂细胞如纤维细胞等等。收集未贴壁细胞按实验设计安排种入皿或细胞培养板内。巨噬细胞集落刺激因子(M-CSF)以40ng/mL浓度刺激使骨髓细胞向单核巨噬细胞分化。培养8天,光镜下观察巨噬细胞形态变化。8天后消化收集细胞,用抗小鼠F4/80抗体和抗小鼠CD11b抗体,4℃避光孵育30min后,使用流式细胞术鉴定所诱导成功的巨噬细胞的比例即可。
将分选得到的500万个CD3+T细胞、500万个B细胞、500万个BMDM以及负载肿瘤组织全组分抗原的纳米粒子(500μg)在10mL的RPMI 1640完全培养基中共孵育48小时;或者将分选得到的100万个CD3+T细胞、1000万个小鼠BMDM与负载肿瘤组织全组分抗原的纳米粒子(500μg)在10mL的RPMI1640完全培养基中共孵育48小时;或者将分选得到的100万个CD3+T细胞、500万个B细胞、500万个小鼠BMDM、IL-7(10ng/mL)与负载肿瘤组织全组分抗原的纳米粒子(500μg)在10mL的RPMI1640完全培养基中共孵育48小时。然后使用流式细胞术分选出CD3+CD137+T细胞,即为被癌细胞抗原激活的癌症特异性T细胞(细胞活率70%)。将上述分选得到的100万个癌症特异性T细胞与IL-2(1000U/mL)、IL-7(1000U/mL)在10mLRPMI 1640完全培养基(37℃,5%CO2)中共孵育14天(每两天更换培养基)以扩增分选得到的癌细胞特异性T细胞。
(4)癌细胞特异性T细胞用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。在接种黑色素瘤后第4天、第7天、第10天、第15天、第20天和第25天分别静脉注射200万个癌症特异性T细胞。小鼠肿瘤体积和生存期监测方法同上。
(5)实验结果
如图4所示,PBS对照组小鼠的肿瘤生长速度最快,小鼠生存期最短,几组分选得到的T细胞扩增后处理组的小鼠的肿瘤生长速度和生存期与PBS对照组相比都明显改善。以B细胞和BMDM作为混合抗原提呈细胞通过两步分选法得到的癌症特异性T细胞经过扩增后的抗癌效果好于以BMDM作为抗原提呈细胞通过两步分选法得到的癌症特异性T细胞经过扩增后的抗癌效果。由此可见,在分选癌症特异性T细胞时,以B细胞和BMDM作为混合抗原提呈细胞效果好于单用BMDM抗原提呈细胞。而且,在纳米粒子与抗原提呈细胞以及T细胞共孵育过程中加入IL-7所分选得到的癌症特异性T细胞效果好于孵育过程中不加IL-7分选得到的癌细胞特异性T细胞。
实施例4细胞系统用于黑色素瘤肺转移的预防
本实施例以小鼠黑色素瘤肺模型来说明如何使用细胞系统预防癌症转移。本实施例中,首先裂解B16F10黑色素瘤肿瘤组织以制备肿瘤组织的水溶性组分和非水溶性组分;然后,制备负载有肿瘤组织的水溶性组分和非水溶性组分的纳米粒子。在本实施例中进行了一轮矿化处理。本实施例中,先使用纳米粒子在体外激活树突状细胞(DC),再将树突状细胞与癌症特异性T细胞共孵育激活和辅助分选癌细胞特异性T细胞。在实际使用中,抗原提呈细胞可以使用活的,或者使用经过灭活处理的抗原提呈细胞,比如可以使用多聚甲醛固定处理或者使用射线辐射灭活处理等。
(1)抗原组分的制备
在每只C57BL/6小鼠背部皮下接种1.5×105个B16-F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨并加入胶原酶(1mg/mL)在RPMI 1640完全培养基中孵育30min,然后通过细胞过滤网制备单细胞悬液并加入适量纯水并反复冻融5次,并伴有超声以破坏裂解细胞。待细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入10%脱氧胆酸钠(含0.8M精氨酸)溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为10%脱氧胆酸钠水溶液中可溶,将水溶性组分和非水溶性组分按质量比3:1混合,即为抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒子采用溶剂挥发法中的复乳法制备,对复乳法进行了适当的修饰改进。所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为poly(I:C)且poly(I:C)既分布于纳米粒子内部也负载于纳米粒子表面。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载裂解组分和佐剂,然后将100mg纳米粒子在10000g离心20分钟,然后使用7mL PBS重悬纳米粒子并与3mL含有细胞裂解物(60mg/mL)的PBS溶液混合,尔后在10000g离心20分钟,然后采用10mL硅酸盐溶液(含150mM NaCl、80mM原硅酸四甲酯和1.0mM HCl,pH 3.0)重悬,并在室温固定10min,尔后在-80℃固定24h,使用超纯水离心洗涤后使用3mL含鱼精蛋白(5mg/mL)和聚赖氨酸(10mg/mL)的PBS重悬并作用10min,然后10000g离心20min洗涤,采用10mL含有细胞裂解物(50mg/mL)的PBS溶液重悬并作用10min,然后在10000g离心20分钟并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h;在粒子使用前将其用7mL PBS重悬然后加入3mL含佐剂的癌组织裂解液组分(蛋白质浓度50mg/mL)并室温作用10min,得到内外都负载裂解物的经冷冻硅化和添加阳离子物质的修饰的纳米粒子。该纳米粒子平均粒径为350nm左右,纳米粒子表面电位为-3mV左右;每1mg PLGA纳米粒子约负载300μg蛋白质或多肽组分,每1mgPLGA纳米粒负载poly(I:C)免疫佐剂0.02mg且内外各半。
未经修饰处理的纳米粒子制备方法步骤基本与修饰处理的纳米粒子的制备相同,只是未经过低温硅化和添加带电物质处理这些步骤。在制备过程中首先采用复乳法在纳米粒子内部负载抗原,在内部负载抗原(裂解组分)后在10000g离心20分钟,然后使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h,在粒子使用前将其用7mL PBS重悬然后加入含佐剂的3mL癌组织裂解液组分(蛋白质浓度50mg/mL)并室温作用10min,得到内外都负载裂解物的纳米粒子。该纳米粒子平均粒径为320nm左右,纳米粒子表面电位为-5mV左右;每1mg PLGA纳米粒子约负载150μg蛋白质或多肽组分,每1mgPLGA纳米粒负载poly(I:C)为0.02mg且内外各半。
空白纳米粒制备材料和制备方法相同,粒径为300nm左右,空白纳米粒负载等量佐剂但是不负载任何抗原组分。
(3)树突状细胞的制备
本实施例以从小鼠骨髓细胞制备树突状细胞为例来说明如何制备骨髓来源的树突状细胞(BMDC)。首先,取1只6-8周龄C57小鼠颈椎脱臼处死,手术取出后腿的胫骨和股骨放入PBS中,用剪刀和镊子将骨周围的肌肉组织剔除干净。用剪刀剪去骨头两端,再用注射器抽取PBS溶液,针头分别从骨头两端插入骨髓腔,反复冲洗骨髓到培养皿中。收集骨髓溶液,400g离心3min后加入1mL红细胞裂解液裂红。加入3mL RPMI 1640(10%FBS)培养基终止裂解,400g离心3min,弃上清。将细胞放置10mm培养皿中培养,使用RPMI 1640(10%FBS)培养基,同时加入重组小鼠GM-CSF(20ng/mL),37度,5%CO2培养7天。第3天轻轻摇晃培养瓶,补充同样体积含有GM-CSF(20ng/mL)RPMI 1640(10%FBS)培养基。第6天,对培养基进行半量换液处理。第7天,收集少量悬浮及半贴壁细胞,通过流式检测,当CD86+CD80+细胞在CD11c+细胞中的比例为15-20%之间,诱导培养的BMDC即可被用来做下一步实验。
(4)癌细胞特异性T细胞的分离和扩增
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在第4天,第7天,第10天,第15天、第20天和第25天分别给小鼠别皮下注射100μL的1mgPLGA纳米粒子。空白纳米粒+游离裂解液对照组在相应时间注射相同剂量作为对照。在第24天处死小鼠,收集小鼠脾脏,制备小鼠脾脏细胞单细胞悬液,将小鼠脾脏细胞与CD3抗体孵育后使用流式细胞术分选脾细胞单细胞悬液中的CD3+T细胞。将步骤3所制备的BMDC(100万个)与负载肿瘤组织全组分抗原的纳米粒子(40μg)混合后在3mL RPMI 1640完全培养基中(37℃,5%CO2)共孵育48小时,然后在400g离心5分钟收集BMDC,将BMDC与分选所得CD3+T细胞并共孵育24小时,尔后采用流式细胞术分选孵育后的细胞中的CD3+CD8+CD69+T细胞(细胞活率75%)和CD3+CD4+CD25+T细胞(细胞活率75%),即为被癌症抗原激活的癌症特异性T细胞。将上述分选得到的50万个癌细胞特异性T细胞与IL-2(200U/mL)、IL-7(200U/mL)、IL-15(200U/mL)和αCD3/αCD28在10mL的RPMI 1640完全培养基中共孵育14天(每两天换液)以扩增分选得到的癌细胞特异性T细胞(细胞活率75%)。
(5)同种异体细胞系统用于癌症转移的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠静脉注射100μL的200万个癌细胞特异性T细胞(100万个CD3+CD8+CD69+T细胞和100万个CD3+CD4+CD25+T细胞)。同时在第1天给每只小鼠静脉注射0.5×105个B16F10细胞,第14天处死小鼠,观察记录小鼠肺部黑色素瘤癌灶数量。
(6)实验结果
如图5所示,PBS对照组小鼠的癌灶很多,而经T细胞处理的小鼠癌灶都变少。与对照组相比,使用修饰过的纳米粒子或者未经修饰的纳米粒子辅助分选扩增得到的癌细胞特异性T细胞处理过的小鼠都癌灶都明显减少。说明,使用修饰或者未经修的纳米粒子分选扩增的T细胞都能有效预防癌症转移。
实施例5微米粒子辅助分选和扩增的癌细胞特异性T细胞用于癌症的预防
在本实施例中微米粒子制备时进行了两轮硅化处理。使用负载抗原的微米粒子分选扩增得到癌细胞特异性T细胞后,注射给小鼠预防癌症。
(1)抗原组分的制备
将培养的B16F10黑色素瘤癌细胞系收集后在350g离心5分钟,然后弃去上清并用PBS洗涤两遍,然后采用6M盐酸胍重悬和裂解癌细胞并溶解裂解后的全细胞组分,即为制备微米粒子系统的抗原组分。
(2)微米粒子的制备
本实施例中微米粒子1采用复乳法制备,对复乳法进行了适当的修饰改进。所采用的微米粒子制备材料PLGA分子量为38KDa-54KDa,所采用的免疫佐剂为CpG且CpG既分布于微米粒子内部也负载于微米粒子表面。制备方法如前所述,在制备过程中首先采用复乳法在微米粒子内部负载全细胞组分,在内部负载裂解组分后,将100mg微米粒子在10000g离心15分钟,然后使用7mL PBS重悬微米粒子并与3mL含有细胞裂解物(50mg/mL)的PBS溶液混合,尔后在10000g离心20分钟,然后采用10mL硅酸盐溶液(含120mM NaCl、100mM原硅酸四甲酯和1.0mM HCl,pH 3.0)重悬,并在室温固定12h,使用超纯水离心洗涤后使用3mL含聚天冬氨酸(10mg/mL)的PBS重悬并作用10min,然后10000g离心15min洗涤,采用10mL含有细胞裂解物(50mg/mL)的PBS溶液重悬并作用10min,然后在10000g离心20分钟。然后采用10mL硅酸盐溶液(含150mM NaCl、80mM原硅酸四甲酯和1.0mM HCl,pH 3.0),并在室温固定12h,使用超纯水离心洗涤后使用3mL含组蛋白(5mg/mL)和聚精氨酸(10mg/mL)的PBS重悬并作用10min,然后10000g离心15min洗涤,采用10mL含有细胞裂解物(50mg/mL)的PBS溶液重悬并作用10min,然后在10000g离心15分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h;在粒子使用前将其用7mL PBS重悬然后加入3mL含佐剂的癌细胞裂解液组分(蛋白质浓度50mg/mL)并室温作用10min,得到内外都负载裂解物的经两轮冷冻硅化、添加阳离子物质和阴离子物质的修饰的微米粒子。该微米粒子平均粒径为1.1μm左右,微米粒子表面电位为-2mV左右;每1mg PLGA微米粒子约负载340μg蛋白质或多肽组分,每1mgPLGA微米粒负载CpG为0.02mg且内外各半。
空白微米粒2制备材料和制备方法相同,粒径为1.1μm左右,表面电位为-3mV左右,空白微米粒负载等量佐剂但是不负载抗原组分。
(3)树突状细胞的制备
本实施例以从小鼠骨髓细胞制备树突状细胞为例来说明如何制备骨髓来源的树突状细胞(BMDC)。首先,取1只6-8周龄C57小鼠颈椎脱臼处死,手术取出后腿的胫骨和股骨放入PBS中,用剪刀和镊子将骨周围的肌肉组织剔除干净。用剪刀剪去骨头两端,再用注射器抽取PBS溶液,针头分别从骨头两端插入骨髓腔,反复冲洗骨髓到培养皿中。收集骨髓溶液,400g离心3min后加入1mL红细胞裂解液裂红。加入3mL RPMI 1640(10%FBS)培养基终止裂解,400g离心3min,弃上清。将细胞放置10mm培养皿中培养,使用RPMI 1640(10%FBS)培养基,同时加入重组小鼠GM-CSF(20ng/mL),37度,5%CO2培养7天。第3天轻轻摇晃培养瓶,补充同样体积含有GM-CSF(20ng/mL)RPMI 1640(10%FBS)培养基。第6天,对培养基进行半量换液处理。第7天,收集少量悬浮及半贴壁细胞,通过流式检测,当CD86+CD80+细胞在CD11c+细胞中的比例为15-20%之间,诱导培养的BMDC即可被用来做下一步实验。
(4)癌细胞特异性T细胞的分离和扩增
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在第4天,第7天,第10天,第15天,第20天和第25天分别给小鼠别皮下注射100μL的1mgPLGA微米粒子。在第30天处死小鼠,收集小鼠的脾脏,制备小鼠脾脏细胞单细胞悬液。首先使用磁珠分选法分选脾细胞单细胞悬液中的CD3+T细胞。将分选所得T细胞(2000万个)、步骤3所制备的BMDC(2000万个)与20μg微米粒子1(或者空白微米粒子2+等量游离裂解液)混合后在10mLRPMI完全培养基(含5%FSBS)中共孵育24小时,尔后采用磁珠分选法分选CD3+CD69+T细胞,即为被癌症抗原激活的癌症特异性T细胞(细胞活率75%)。将上述分选得到的100万个癌症特异性T细胞与IL-2(2000U/mL)和αCD3/αCD28抗体(20ng/mL)在10mL的DMEM高糖完全培养基中(37℃,5%CO2)共孵育7天(两天换液一次)以扩增分选得到的癌细胞特异性T细胞(细胞活率75%)。
(5)癌细胞特异性T细胞扩增后用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠静脉注射100μL的200万个癌症特异性T细胞。同时在第0天给每只小鼠皮下注射接种1.5×105个B16F10细胞。小鼠肿瘤体积和生存期监测方法同上。
(6)实验结果
如图6所示,PBS对照组小鼠的肿瘤生长速度很快生存期很短,而经T细胞处理的小鼠的肿瘤生长速度明显变慢且生存期明显延长,而且,说明使用微米粒子分选扩增的T细胞可以有效预防癌症。
实施例6癌细胞特异性T细胞用于癌症的预防
本实施例中,首先使用8M尿素裂解B16F10黑色素瘤肿瘤组织,并溶解肿瘤组织裂解物组分。然后,以PLA为纳米粒骨架材料,以Poly(I:C)和CpG1018为免疫佐剂制备负载有全细胞抗原的纳米粒子,并使用纳米粒子辅助分选和扩增癌细胞特异性T细胞。
(1)抗原组分的制备
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网后在单细胞悬液中加入适量8M尿素水溶液裂解细胞并溶解细胞裂解物。以上即为制备纳米粒子的抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒子1采用溶剂挥发法制备。纳米粒子1制备材料PLA分子量为20KDa,所采用的免疫佐剂为poly(I:C)和CpG1018。制备方法如前所述,先采用复乳法在纳米粒子内部负载抗原组分和佐剂,然后将100mg纳米粒子在12000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h,得冻干粉后备用。该纳米粒子1平均粒径为250nm左右,表面电位为-3mV左右;每1mg PLGA纳米粒子约负载110μg蛋白质或多肽组分,负载poly(I:C)和CpG1018各0.02mg。
纳米粒2的制备材料和制备方法同上,粒径为250nm左右,表面电位为-3mV左右;每1mg PLGA纳米粒子约负载110μg蛋白质或多肽组分,负载poly(I:C)0.04mg。
(3)癌细胞特异性T细胞的分离和扩增
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在第4天,第7天,第10天,第15天,第20天和第25分别给小鼠别皮下注射100μL的1mgPLA纳米粒子1。在第29天处死小鼠,收集小鼠脾脏并制备小鼠脾脏细胞单细胞悬液。使用磁珠分选法分选得到小鼠脾细胞中的CD3+T细胞。然后,将分选得到的T细胞(1000万个)与来源于同种异体的B细胞(1500万个)以及40mg的纳米粒子(纳米粒子1或者2)在20mL RPMI 1640完全培养基(37℃,5%CO2)中共孵育48小时。然后使用不同荧光探针标记的CD3抗体和CD69抗体标记孵育后的细胞,尔后采用流式细胞术分选孵育后细胞中的CD3+CD69+T细胞(细胞活率75%),即为被癌细胞抗原激活的癌细胞特异性T细胞。将上述分选得到的100万个癌细胞特异性T细胞与IL-2(1000U/mL)、和αCD3/αCD28抗体(20ng/mL)在10mL高糖DMEM完全培养基中共孵育11天(每两天换液一次)以扩增分选得到的癌细胞特异性T细胞(细胞活率75%)。
(4)T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在小鼠癌症特异性T细胞移植前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠皮下注射100μL的200万个扩增的癌症特异性T细胞。同时在第0天给每只小鼠皮下注射接种1.5×105个B16F10细胞,小鼠肿瘤体积和生存期监测方法同上。
(5)实验结果
如图6所示,对照组小鼠的肿瘤生长速度最快生存期最快,使用纳米粒子1和纳米粒子2分选和扩增的癌症特异性T细胞都能减缓小鼠肿瘤生长速度和延长小鼠生存期。
实施例7分选扩增癌细胞特异性T细胞用于治疗结肠癌
首先裂解结肠癌肿瘤组织和肺癌癌细胞系以制备水溶性组分混合物(质量比1:1)和非水溶性组分(质量比1:1)混合物,并将水溶性组分混合物和非水溶性组分混合物按质量比1:1混合。然后,以有机高分子材料PLGA为纳米粒骨架材料,以CpG1018和Poly(I:C)为免疫佐剂制备纳米粒子,并用该纳米粒子分选扩增癌症特异性T细胞用于治疗结肠癌。
(1)抗原组分的制备
在每只C57BL/6小鼠背部皮下接种2×106个MC38细胞在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网制备成单细胞悬液后加入适量纯水并反复冻融5次,并伴有超声以破坏裂解细胞。待细胞裂解后,将裂解物以大于5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入10%辛基葡萄糖苷水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在10%辛基葡萄糖苷水溶液中可溶。将培养的LLC肺癌细胞系收集后在350g离心5分钟,然后弃去上清并用PBS洗涤两遍,然后采用超纯水重悬细胞并反复冻融5次,并伴有超声以破坏裂解细胞。待细胞裂解后,将裂解物以3000g的转速离心6分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入10%辛基葡萄糖苷水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在10%辛基葡萄糖苷水溶液中可溶。
将来自结肠癌肿瘤组织的和肺癌癌细胞的的水溶性组分按质量比1:1混合;溶解于10%辛基葡萄糖苷中的非水溶性组分也按质量比1:1混合。然后将水溶性组分混合物和非水溶性组分混合物按质量比1:1混合,该混合物为制备纳米粒子的抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒子采用复乳法制备。纳米粒子制备材料PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为Poly(I:C)和CpG1018,且抗原组分和佐剂同时分布于纳米粒子内部和表面。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载抗原组分和佐剂,然后将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。使用前将20mg纳米粒重悬于0.9mL PBS中,并于0.1mL含有抗原组分(80mg/mL)和佐剂的样品室温混合孵育5分钟后即可使用。该纳米粒子平均粒径为280nm左右,表面电位为-3mV左右;每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分,负载的CpG1018和Poly(I:C)免疫佐剂各0.02mg。
纳米粒子2制备材料和制备方法相同,粒径为280nm左右,表面电位为-3mV;每1mg PLGA纳米粒子2约负载100μg蛋白质或多肽组分,负载CpG1018为0.04mg。
(3)癌细胞特异性T细胞的分选和扩增
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在第4天,第7天,第10天,第15天,第20天和第25天分别给小鼠别皮下注射100μL的2mg PLGA纳米粒子。在第30天处死小鼠,分别收集各组小鼠的脾脏,制备小鼠脾脏细胞单细胞悬液,并使用流式细胞术从中分选出CD3+CD8+T细胞和CD19+B细胞。将300万个CD8+T细胞、600万个CD19+B细胞与纳米粒子(50μg)在2mL RPMI完全培养基中共孵育96小时(37℃,5%CO2),然后采用流式细胞术分选孵育后的细胞中的CD3+CD8+CD69+T细胞(细胞活率70%),即为被癌细胞抗原激活的癌细胞特异性T细胞。将上述分选得到的20万个癌细胞特异性T细胞与IL-2(2000U/mL)、IL-7(500U/mL)、IL-15(500U/mL)和αCD3/αCD28抗体在10mL高糖DMEM完全培养基(37℃,5%CO2)中共孵育7天(每两天换液一次)以扩增分选得到的癌细胞特异性T细胞(细胞活率70%)。
(4)癌细胞特异性T细胞用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备结肠癌荷瘤小鼠。在第0天给每只小鼠皮下接种2×106个MC38细胞,在第4、第7天、第10天、第15天和第20天分别给小鼠注射100μL含10万个癌细胞特异性T细胞。小鼠肿瘤生长和生存期监测方法同上。
(5)实验结果
如图7所示,PBS对照组小鼠肿瘤生长速度很快,使用纳米粒子1和纳米粒子2分选扩增得到的癌细胞特异性T细胞都可以有效延缓肿瘤生长速度和提高小鼠生存期。
实施例8分选扩增的T细胞用于乳腺癌的预防
(1)抗原组分的制备
将培养的4T1细胞在400g离心5分钟,然后用PBS洗涤两遍后重悬于超纯水中。所得癌细胞分别采用紫外线和高温加热进行灭活和变性处理,然后采用适量8M尿素裂解乳腺癌细胞并溶解裂解物即为制备粒子的抗原组分。
(2)微米粒子的制备
本实施例中制备微米粒子采用复乳法。微米粒子1骨架材料PLGA分子量为38KDa-54KDa,所采用的免疫佐剂为CpG和Poly ICLC,增加溶酶体免疫逃逸的物质为精氨酸。制备时先采用复乳法制备内部负载抗原组分、佐剂和精氨酸的微米粒子,然后将100mg微米粒子在9000g离心20分钟,使用10mL含4%海藻糖的超纯水重悬后干燥48h后备用。该微米粒子系统平均粒径为2.1μm左右,微米粒子表面电位为-5mV左右;每1mg PLGA微米粒子约负载110μg蛋白质或多肽组分,负载CpG和Poly ICLC各0.01mg,负载精氨酸0.05mg。空白微米粒子2制备材料和制备方法相同,粒径为2.0μm左右,空白微米粒负载等量CpG、Poly ICLC和精氨酸,但不负载任何抗原组分。
(3)癌细胞特异性T细胞的分离和扩增
选取6-8周的雌性BALB/c小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射100μL含2mg PLGA的微米粒子1。在第32天处死小鼠,收集小鼠的外周血,然后从外周血中分离外周血单个核细胞(PBMC)。首先从PBMC中利用流式细胞术先第一步分选出CD3+CD8+T细胞和B220+B细胞,将分选得到的20万个CD8+T细胞,30万个B细胞、IL-7(10ng/mL)与40μg的微米粒子(微米粒子1或者微米粒子2+等量游离裂解物)在2mL RPMI 1640完全培养基中共孵育96小时。然后采用流式细胞术进一步分选孵育后的细胞中CD3+CD8+CD69+T细胞(细胞活率85%),即为可识别癌细胞抗原的癌细胞特异性T细胞。将上述两步分选得到的癌细胞特异性T细胞与IL-2(2000U/mL)、IL-7(200U/mL)、IL-15(200U/mL)和αCD3/αCD28抗体共孵育7天以扩增分选得到的癌细胞特异性T细胞(细胞活率85%)。
(4)分选扩增的T细胞用于癌症的预防
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠静脉注射注射200万个扩增得到的癌细胞特异性CD8+T细胞。同时在第0天给每只小鼠皮下注射接种4×105个4T1细胞,小鼠肿瘤生长和生存期监测方法同上。
(5)实验结果
如图9所示,与对照组相比,癌细胞特异性T细胞处理组肿瘤生长速度明显变慢且小鼠生存期明显延长。而且采用负载全细胞组分的微米粒子辅助分选扩增得到的癌细胞特异性T细胞的预防效果好于经过空白微米粒子+游离裂解液辅助分选扩增得到的癌细胞特异性T细胞。由此可见,本发明所述的经过两步分选和扩增的癌细胞特异性T细胞对乳腺癌具有优异的预防效果。
实施例9癌细胞特异性T细胞用于癌症转移的预防
本实施例以小鼠黑色素瘤小鼠肺转移癌症模型来说明使用分选扩增后的癌细胞特异性T细胞预防癌症转移。在实际应用时具体剂型,佐剂,孵育时间、孵育浓度、给药时间、给药次数、给药方案可根据情况调整。本实施例中,将小鼠黑色素瘤肿瘤组织和癌细胞系以8M尿素裂解后溶解,然后肿瘤组织裂解组分和癌细胞系裂解组分按质量比1:2负载于纳米粒子,并用该粒子激活和辅助分选癌症特异性T细胞,所得T细胞经扩增后预防小鼠体内的癌症转移。在本实施例中,采用负载四种多肽新生抗原
B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM),
B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD),
B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和TRP2:180-188(SVYDFFVWL)的纳米粒子作为对照纳米粒子使用,以分析负载全细胞抗原的纳米粒子和负载多种多肽新生抗原的纳米粒子辅助分选和扩增的癌症特异性T细胞预防癌症肺转移的功效。
(1)抗原组分的制备
收集小鼠B16F10黑色素瘤肿瘤组织和培养的B16F10癌细胞系后采用8M尿素裂解和溶解肿瘤组织和癌细胞全细胞组分,然后肿瘤组织组分和癌细胞系裂解物组分按质量比1:2混溶即为抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒子1采用溶剂挥发法制备,所采用的纳米粒子1制备材料PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为CpG7909和Poly(I:C),所采用的的增加溶酶体逃逸物质为KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)。制备方法如前所述,在制备过程中首先采用复乳法在纳米粒子内部负载抗原组分组分、佐剂和KALA多肽,在内部负载抗原组分和佐剂后,将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h后备用。该纳米粒子1平均粒径为470nm左右;每1mg PLGA纳米粒子1约负载10μg裂解物的蛋白质和多肽组分,含CpG7909和Poly(I:C)各0.02mg,含有KALA多肽0.04mg。负载四种抗原多肽的对照纳米粒子2制备方法同上,对照纳米粒子2粒径为460nm左右,每1mg PLGA纳米粒子约负载10μg抗原多肽和等量佐剂和KALA多肽。
(3)癌细胞特异性T细胞的分选和扩增
选取6-8周的雌性C57BL/6小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射200μL的2mg PLGA纳米粒子。在第32天处死小鼠,收集小鼠的外周血,然后从外周血中分离外周血单个核细胞(PBMC)。先从PBMC中分选得到CD3+T细胞和CD19+B细胞,然后将第一步分选得到的700万个T细胞和700万个B细胞与4mg纳米粒子1或纳米粒子2在10mL的RPMI 1640完全培养基中共孵育96小时。然后采用流式细胞术分选孵育后的细胞中的CD3+CD8+CD69+T细胞和CD3+CD4+CD69+T细胞,即为可识别癌细胞抗原的癌细胞特异性T细胞(细胞活率80%)。将上述分选得到的100万个CD3+CD8+CD69+T细胞或者100万个CD3+CD4+CD69+T细胞与IL-2(1000U/mL)、IL-12(1000U/mL)和αCD3/αCD28抗体(10ng/mL)在10mL的RPMI1640完全培养基中共孵育14天扩增癌细胞特异性T细胞(细胞活率80%)。
(4)癌细胞特异性T细胞用于癌症转移的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠静脉注射150万个癌细胞特异性CD8+T细胞和50万个癌细胞特异性CD4+T细胞。同时在第1天给每只小鼠静脉注射接种0.5×105个B16F10细胞,第14天处死小鼠,观察记录小鼠肺部黑色素瘤癌灶数量。
(5)实验结果
如图10所示,纳米粒子1和纳米粒子2辅助分选得到的癌细胞特异性T细胞在扩增后可有效预防癌症转移。而且,与负载四种新生抗原多肽的纳米粒子2相比,负载全细胞组分的纳米粒子1辅助分选扩增的癌细胞特异性T细胞效果更好。
实施例10分选扩增的T细胞用于治疗胰腺癌
本实施例中,将小鼠Pan02胰腺癌肿瘤组织和MC38结肠癌肿瘤组织裂解组分按3:1的比例负载于纳米粒子,并使用该纳米粒子辅助分离小鼠外周血中的癌细胞特异性T细胞,然后扩增后用于治疗胰腺癌。实验中,先取得小鼠胰腺癌和结肠癌肿瘤组织并将其裂解以制备水溶性组分和溶于6M盐酸胍中的原非水溶性组分。在制备粒子时,水溶性组分为胰腺癌肿瘤组织水溶性组分和结肠癌肿瘤组织水溶性组分3:1的混合物;非水溶性组分为胰腺癌肿瘤组织非水溶性组分和结肠癌肿瘤组织非水溶性组分3:1的混合物。以PLGA为纳米粒子骨架材料,以BCG为佐剂制备纳米粒子,使用纳米粒子辅助分选扩增癌细胞特异性T细胞。
(1)抗原组分的制备
在每只C57BL/6小鼠腋下皮下接种2×106个MC38结肠癌细胞或接种1×106个Pan02胰腺癌细胞,在各只小鼠所接种肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。裂解方法及各组分的收集方法同实施例1,只是使用6M盐酸胍而非8M尿素溶解非水溶性组分,肿瘤组织裂解物组分即为抗原组分。BCG的裂解方法同肿瘤组织裂解方法。
(2)纳米粒子的制备
本实施例中纳米粒,采用复乳法制备。纳米粒1所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为BCG裂解物组分;制备方法如前所述,先在纳米粒子内部负载抗原组分和佐剂,然后将100mg纳米粒子在12000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h,得冻干粉后备用;在纳米粒子1注射前将20mg纳米粒子溶于0.9mL PBS中,与0.1mL含抗原组分(80mg/mL)和GM-CSF(50ng/mL)的样品混合并在室温作用10min后使用;纳米粒1平均粒径为160nm左右,表面电位为-4mV左右;每1mg PLGA纳米粒1约负载130μg蛋白质或多肽组分,每1mg PLGA纳米粒1负载BCG0.02mg。纳米粒2使用的制备材料和制备方法同纳米粒1,其粒径为160nm左右,表面电荷为-4mV左右;每1mg PLGA纳米粒2负载130μg肿瘤组织裂解物中的蛋白质或多肽组分,但是不负载任何佐剂。
(3)癌细胞特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射200μL的2mg PLGA纳米粒子。在第32天处死小鼠,收集小鼠的外周血,然后从外周血中分离外周血单个核细胞(PBMC)。先使用磁珠分选法从PBMC中分选得到CD3+T细胞和CD19+B细胞,将第一步分选得到的900万个CD3+T细胞和1200万个CD19+B细胞与50μg纳米粒子(纳米粒子1或者纳米粒子2)共孵育6小时。然后采用流式细胞术分选出CD3+CD69+T细胞(细胞活率75%),即为癌细胞特异性T细胞。将上述两步分选得到的100万个CD3+CD69+T细胞与IL-2(1000U/mL)和粒细胞-巨噬细胞集落刺激因子(GM-CSF,10ng/mL)在15mL高糖DMEM完全培养基(37℃,5%CO2)中共孵育14天(两天换液一次)扩增分选得到的癌细胞特异性T细胞。
(4)癌细胞特异性T细胞用于治疗胰腺癌
选取6-8周的雌性C57BL/6为模型小鼠制备胰腺癌荷瘤小鼠。同时在第0天给每只小鼠皮下注射接种1×106个Pan02胰腺癌细胞。在第6天、第9天、第12天、第17天和第23天分别给小鼠静脉注射注射200万个扩增得到的癌细胞特异性T细胞。小鼠肿瘤体积监测和记录方法同上。
(5)实验结果
如图11所示,本发明所述使用纳米粒子1和纳米粒子2分选和扩增的癌细胞特异性T细胞都能有效治疗胰腺癌。
实施例11分选扩增T细胞用于治疗癌症
本实施例以甘露糖为主动靶向的靶头为例说明如何分选扩增癌细胞特异性T细胞。在实际应用时可根据情况调整。该纳米粒子系统可通过树突状细胞表面的甘露糖受体摄取进入树突状细胞,粒子所负载的抗原被树突状细胞提呈后可激活癌细胞特异性T细胞。在实际应用中也可以使用甘露聚糖、CD32抗体、CD19抗体、CD20抗体、B220抗体、CD11c抗体等可以靶向抗原提呈细胞的靶头修饰纳米粒子或者微米粒子。
(1)抗原组分的制备
收集培养的B16F10癌细胞后,采用8M尿素水溶液裂解癌细胞,然后使用8M尿素水溶液溶解裂解的癌细胞全细胞组分,即为抗原组分
(2)纳米粒子的制备
本实施例中纳米粒子1使用复乳法制备。所采用的纳米粒子制备材料为PLGA和甘露糖修饰的PLGA,制备带有靶头的纳米粒子时二者一起使用时质量比为4:1,分子量都为7KDa-17KDa。所采用的免疫佐剂为Poly(I:C)和CpG7909。制备方法如前所述,先将抗原组分和佐剂共负载于纳米粒子内部,然后将100mg纳米粒子在12000g离心25分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h后备用。纳米粒子1平均粒径均为120nm左右,每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分,含Poly(I:C)和CpG7909各0.02mg。
(3)树突状细胞(DC)的制备
本实施例使用骨髓来源的树突状细胞(BMDC)作为抗原提呈细胞。制备方法同上。
(4)树突状细胞的激活
将小鼠BMDC铺到细胞培养板中,在每10万个BMDC细胞中加入5mL RPMI 1640(10%FBS)培养基,尔后加入30μg的纳米粒子1与BMDC共孵育48h(37℃,5%CO2),尔后收集BMDC后在300g离心5分钟,用磷酸盐缓冲液(PBS)洗涤两次后重悬于PBS中备用。
(5)癌细胞特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射200μL的2mg纳米粒子1。在第32天处死小鼠,收集小鼠的外周血,然后从外周血中分离外周血单个核细胞(PBMC),从PBMC中利用磁珠分选法分选得到CD3+CD8+T细胞。将分选得到的200万个CD8+T细胞与500万个步骤(4)所制备的BMDC、40μg纳米粒子1以及10ng/mL的IL-7在4mL DMEM高糖完全培养基中共孵育18小时。然后采用流式细胞术分选孵育后的CD8+T细胞中CD8+CD69+T细胞(细胞活率75%),即为可识别癌症抗原的癌细胞特异性T细胞。将上述两步分选得到的20万个CD8+CD69+T细胞与IL-2(1000U/mL)和IL-7(1000U/mL)共孵育10天扩增癌细胞特异性T细胞(细胞活率75%)。
(6)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠,在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。然后,将步骤(5)制备得到的200万个癌症特异性T细胞皮下注射给受体小鼠。隔天,给每只受体小鼠背部右下方皮下接种1.5×105个B16F10细胞。小鼠肿瘤生长速度和小鼠生存期监测方法同上。
(7)实验结果
如12图所示,与PBS对照组相比,T细胞处理的小鼠肿瘤生长速度明显变慢。
实施例12分选扩增的癌细胞特异性T细胞用于预防肝癌
本实施例中,首先裂解Hepa1-6肝癌细胞,以PLGA为纳米粒子骨架材料,以Poly(I:C)和细菌卡介苗(BCG)为免疫佐剂采用溶剂挥发法制备负载肝癌细胞全细胞抗原的纳米粒子,然后将该粒子与B细胞和T细胞混合,分选扩增后得到癌细胞特异性T细胞。
(1)抗原组分的制备
收集培养的Hepa 1-6肝癌细胞后使用PBS洗涤两遍,使用加热和紫外照射处理肝癌细胞,尔后采用8M尿素裂解和溶解癌细胞全细胞组分,即为抗原组分。BCG的裂解方法同上,BCG的裂解物作为佐剂使用。
(2)纳米粒子的制备
本实施例中纳米粒子采用溶剂挥发法制备,所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa,所采用的免疫佐剂为BCG和Poly(I:C)。制备方法如前所述,先在纳米粒子内部负载抗原组分和佐剂,然后将100mg纳米粒子在10000g离心25分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h后备用。该纳米粒子粒径为270nm左右;每1mg PLGA纳米粒子负载100μg蛋白质或多肽组分,负载BCG和Poly(I:C)各0.02mg。
(3)癌细胞特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射200μL的2mg的PLGA纳米粒子。在第32天处死小鼠,收集小鼠的外周血,然后从外周血中分离外周血单个核细胞(PBMC),从PBMC中利用磁珠分选法分选得到CD8+T细胞。将分选得到的200万个CD8+T细胞、纳米粒子(500μg)、800万个BAF3小鼠B细胞系以及IL-7(10ng/mL)在2mL RPMI 1640完全培养基中共孵育48小时。然后采用流式细胞术分选孵育后的CD8+T细胞中CD8+CD69+T细胞(细胞活率80%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的CD8+CD69+T细胞与IL-2(1000U/mL)、IL-7(500U/mL)、IL-15(500U/mL)以及αCD28抗体共孵育7天以扩增分选得到的癌细胞特异性T细胞(细胞活率80%)。
(4)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备肝癌荷瘤小鼠。在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠注射200万个扩增得到的癌细胞特异性T细胞。同时在第0天给每只小鼠后背皮下注射接种1.0×106个Hepa1-6肝癌细胞,肿瘤生长和小鼠生存期记录方式同上。
(5)实验结果
如图13所示,与对照组相比,癌细胞特异性T细胞处理的小鼠肿瘤生长速度明显变慢。
实施例13钙化的纳米粒子辅助分选T细胞用于癌症的预防
本实施例使用钙化的纳米粒子分选癌细胞特异性T细胞,在实际使用时也可以使用其他生物矿化技术、交联、凝胶化等修饰粒子。在实际应用时具体剂型,佐剂,粒径大小、给药时间、给药次数、给药方案等都可根据情况调整。本实施例中,将小鼠黑色素瘤肿瘤组织和癌细胞系以8M尿素裂解后溶解,然后肿瘤组织裂解组分和癌细胞系裂解组分按质量比1:1负载于纳米粒子1。在本实施例中,采用负载四种多肽新生抗原
B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM),
B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD),
B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ)和
TRP2:180-188(SVYDFFVWL)的纳米粒子作为对照纳米粒子2使用。
(1)抗原组分的制备
收集小鼠B16F10黑色素瘤肿瘤组织和培养的B16F10癌细胞系后采用8M尿素裂解和溶解肿瘤组织和癌细胞系的全细胞组分,然后肿瘤组织组分和癌细胞系的组分按质量比1:1混溶,即为抗原组分。
(2)纳米粒子的制备
本实施例在纳米粒子内部和表面负载全细胞抗原后生物钙化纳米粒子。本实施例中纳米粒子1采用复乳法制备,纳米粒子1制备材料PLGA分子量为7KDa-17KDa,免疫佐剂为CpG2006和Poly(I:C),增强溶酶体逃逸物质为GALA多肽(WEAALAEALAEALAEHLAEALAEALEALAA)。制备方法如下所述,先在纳米粒子内部负载抗原组分、佐剂和GALA多肽,然后将100mg PLGA纳米粒子在13000g离心20min后使用18mL PBS重悬,然后加入2mL溶解于8M尿素的抗原组分(60mg/mL),在室温作用10分钟后在12000g离心20分钟后收集沉淀。然后将该100mg PLGA纳米粒子重悬于20mL DMEM培养基中,然后加入200μL of CaCl2(1mM)并在37℃反应两小时。然后在10000g离心20分钟后收集沉淀,并采用超纯水重悬后离心洗涤两遍。该纳米粒子平均粒径为290nm左右;每1mg PLGA纳米粒子约负载230μg抗原组分中的蛋白质或多肽组分,负载CpG和Poly(I:C)各0.02mg,负载GALA多肽0.001mg。
负载多种抗原多肽的对照纳米粒子2制备材料和制备方法同上,对照纳米粒子粒径为290nm左右,每1mg PLGA纳米粒子约负载230μg抗原多肽,负载等量佐剂和GALA多肽。
(3)癌症特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天给小鼠背部皮下接种1.5×105个B16F10,然后在第10天、第13天、第17天、第21天和第28天分别给小鼠腹腔注射10mg/kg剂量的PD-1抗体。在第30天处死小鼠,收集小鼠的外周血,然后从外周血中分离外周血单个核细胞(PBMC),使用流式细胞术先从PBMC中分选得到CD8+T细胞和B细胞。将分选得到的CD8+T细胞(500万个)、纳米粒子(500μg)、B细胞(2000万个)以及IL-7(10ng/mL)在20mL RPMI 1640完全培养基中共孵育48小时。然后采用流式细胞术分选孵育后的CD8+T细胞中CD8+CD137+T细胞(细胞活率75%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的50万个CD8+CD137+T细胞与IL-2(1000U/mL)、IL-7(500U/mL)、IL-15(500U/mL)以及αCD3抗体(10ng/mL)中共孵育14天以扩增癌细胞特异性T细胞(细胞活率75%)。
(4)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6小鼠,在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠注射100万个扩增得到的癌细胞特异性T细胞。同时在第0天给每只小鼠后背皮下注射接种1.5×105个B16F10黑色素瘤细胞,肿瘤生长和小鼠生存期监测方式同上。
(5)实验结果
如图14所示,与对照组相比,钙化纳米粒子激活和辅助分选的癌细胞特异性T细胞可以延长小鼠生存期有效预防癌症。而且,负载全细胞组分纳米粒子效果好于负载4种新生抗原多肽的纳米粒子。
实施例14癌细胞特异性T细胞用于黑色素瘤的治疗
本实施例中,首先裂解B16F10黑色素瘤肿瘤组织制备肿瘤组织的水溶性组分和非水溶性组分,然后以PLGA为纳米粒骨架材料,以Poly(I:C)和CpG2395为免疫佐剂,以蜂毒肽(GIGAVLKVLTTGLPALISWIKRKRQQ-amide)为促进溶酶体逃逸组分,采用溶剂挥发法制备纳米粒子。
(1)抗原组分的制备
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织,将肿瘤组织切块后研磨,通过细胞过滤网制备成单细胞悬液后加入适量纯水并反复冻融5次,并伴有超声以破坏裂解所得样品。待肿瘤组织裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入2.0M的精氨酸和10%脱氧胆酸钠的氯化钠水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在水溶液中可溶。以上即为制备纳米粒子系统的抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒采用复乳法制备。在制备时负载水溶性组分的纳米粒子和负载非水溶性组分的纳米粒子分别制备,应用时一起使用。所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为poly(I:C)和CpG2395,溶酶体逃逸物质为蜂毒肽。制备方法如前所述,先在纳米粒子内部负载抗原组分、佐剂和蜂毒肽,然后将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h;在使用前将其用9mL PBS重悬然后加入1mL的抗原组分(蛋白质浓度80mg/mL)并室温作用10min,得到内外都负载裂解物的纳米粒子。该纳米粒子平均粒径为290nm左右,每1mg PLGA纳米粒子约负载140μg抗原组分中的蛋白质或多肽组分,负载poly(I:C)和CpG2395免疫佐剂各0.02mg,负载蜂毒肽0.05mg。
(3)癌细胞特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天给小鼠背部皮下接种1.5×105个B16F10,然后在第7天、第10天、第15天、第20天和第25天分别给小鼠皮下注射1mg负载水溶性组分的纳米粒子和1mg负载非水溶性组分的纳米粒子。在第30天处死小鼠,收集小鼠的PBMC,使用流式细胞术从PBMC中分选得到CD8+T细胞、CD4+T细胞和B细胞。将分选得到的CD8+T细胞(100万个)、CD4+T细胞(100万个)、纳米粒子(负载水溶性组分的和负载非水溶性组分的纳米粒子各200μg)、B细胞(300万个)以及IL-15(50ng/mL)在2mL RPMI 1640完全培养基中共孵育72小时;或者将分选得到的CD8+T细胞(100万个)、CD4+T细胞(100万个)、纳米粒子(负载水溶性组分的和负载非水溶性组分的纳米粒子各200μg)、B细胞(300万个)在2mL RPMI 1640完全培养基中共孵育72小时;或者将分选得到的CD8+T细胞(100万个)、CD4+T细胞(100万个)、纳米粒子(负载水溶性组分的和负载非水溶性组分的纳米粒子各200μg)、B细胞(300万个)以及Flt3L(50ng/mL)在2mL RPMI 1640完全培养基中共孵育72小时。然后采用流式细胞术分选孵育后的CD8+T细胞中CD8+CD69+T细胞(细胞活率80%)以及CD4+T细胞中CD4+CD69+T细胞(细胞活率80%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的10万个CD8+CD69+T细胞或者10万个CD4+CD69+T细胞与IL-2(1000U/mL)、IL-7(500U/mL)、IL-15(500U/mL)以及αCD3抗体(10ng/mL)在20mL的RPMI 1640完全培养基(37℃,5%CO2)中共孵育11天(两天换液一次)以扩增癌细胞特异性T细胞。
(4)使用扩增的癌细胞特异性T细胞治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别静脉注射80万个癌细胞特异性CD8+T细胞和20万个癌细胞特异性CD4+T细胞。在实验中,小鼠肿瘤体积和生存期监测方法同上。
(5)实验结果
如图15所示,本发明分选和扩增得到的癌细胞特异性T细胞对黑色素瘤具有良好的治疗效果。
实施例15癌细胞特异性T细胞后用于黑色素瘤的治疗
本实施例中,先裂解B16F10黑色素瘤肿瘤组织,然后以PLGA为纳米粒骨架材料,以Poly(I:C)和CpG7909为免疫佐剂,以聚精氨酸和聚赖氨酸为增加溶酶体逃逸组分制备纳米粒子。
(1)抗原组分的制备
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织,将肿瘤组织使用组织匀浆机搅碎组织,然后加入适量超纯水后反复冻融裂解细胞,然后加入核酸酶作用5分钟,再在95℃作用10分钟灭活核酸酶以得到去除全细胞核酸成分的全细胞抗原组分(主要为蛋白质和多肽)。尔后在8000g离心3分钟,上清液部分即为水溶性组分;沉淀部分使用0.1M盐酸二甲双胍和0.1M精氨酸水溶液溶解非水溶性组分。将水溶性组分和溶解液溶解的非水溶性组分按质量比1:1混溶即为制备纳米粒子的抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒1采用复乳法制备,具有靶向树突状细胞的能力。所采用的纳米粒子1制备材料为PLA和甘露聚糖修饰的PLA,二者分子量都为20KDa-30KDa,使用时未修饰PLA和甘露聚糖修饰PLA的质量比为9:1。所采用的免疫佐剂为poly(I:C)和CpG7909,增加溶酶体免疫逃逸的物质为聚精氨酸和聚赖氨酸。制备方法如前所述,先在纳米粒子内部负载裂解液组分、佐剂、聚精氨酸和聚赖氨酸,在内部负载上述组分后,将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子平均粒径为360nm;每1mg PLGA纳米粒子约负载100μg抗原组分中的蛋白质或多肽组分,负载poly(I:C)和CpG7909免疫佐剂各0.02mg,负载聚精氨酸和聚赖氨酸各0.01mg。
(3)癌细胞特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天给小鼠背部皮下接种1.5×105个B16F10,然后在第7天、第10天、第15天、第20天和第25天分别给小鼠皮下注射2mg的纳米粒子1。在第30天处死小鼠,收集小鼠的PBMC,使用流式细胞术从PBMC中分选得到CD3+T细胞、和B细胞。将分选得到的CD3+T细胞(200万个)、纳米粒子1(40μg)、B细胞(300万个)以及IL-15(10ng/mL)在2mL RPMI1640完全培养基中共孵育6小时。然后采用流式细胞术分选孵育后的T细胞中的CD3+CD69+T细胞(细胞活率85%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的30万个CD3+CD69+T细胞与IL-2(1000U/mL)、IL-7(1000U/mL)、IL-15(1000U/mL)以及αCD3抗体(20ng/mL)在20mL RPMI 1640完全培养基中共孵育14天以扩增癌细胞特异性T细胞。
(4)T细胞用于治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别静脉注射80万个扩增后的CD3+T细胞。小鼠肿瘤体积和生存期监测方法同上。
(5)实验结果
如图16所示,PBS对照组的肿瘤都很快长大,纳米粒子分选扩增的癌细胞特异性T细胞处理的小鼠肿瘤生长速度都明显变慢。
实施例16分选扩增T细胞用于乳腺癌的预防
本实施例中,首先对乳腺癌细胞进行灭活和变性处理,尔后裂解细胞,并以辛基葡萄糖苷溶解裂解癌细胞中的非水溶性组分。然后,以PLGA为微米粒子骨架材料,以CpG1018和Poly ICLC为免疫佐剂,制备负载有全细胞抗原的微米粒子。
(1)抗原组分的制备
将培养的4T1细胞在400g离心5分钟,然后用PBS洗涤两遍后重悬于超纯水中。所得癌细胞分别采用紫外线和高温加热进行灭活和变性处理,然后加入超纯水并反复冻融5次辅以超声裂解癌细胞,将细胞裂解物在5000g离心10分钟,上清液即为水溶性组分,将沉淀物使用10%辛基葡萄糖苷溶解后即为溶解后的原非水溶性组分,将水溶性组分和非水溶性组分按质量比2:1混合,即为制备微米粒子所需的抗原组分1。
(2)微米粒子的制备
本实施例中制备微米粒子1采用复乳法,微米粒子1骨架材料PLGA分子量为38KDa-54KDa,所采用的免疫佐剂为CpG1018和Poly ICLC。制备时先采用复乳法制备内部负载抗原组分1和佐剂的微米粒子,然后将100mg微米粒子在9000g离心20分钟,使用10mL含4%海藻糖的超纯水重悬后干燥48h后备用。该微米粒子系统平均粒径为5.0μm左右;每1mg PLGA微米粒子约负载410μg癌细胞的蛋白质或多肽组分,负载CpG1018和Poly ICLC各0.01mg。
(3)树突状细胞的制备
本实施例使用BMDC作为抗原提呈细胞。制备方法如前所述。
(4)树突状细胞的激活
将500万个BMDC、2mg微米粒子和IL-15(20ng/mL)在5mL RPMI 1640(10%FBS)培养基中共孵育8h,尔后收集BMDC后使用射线辐照激活的树突状细胞以灭活树突状细胞,将灭活后的树突状细胞用于激活T细胞。
(5)癌细胞特异性T细胞的制备
选取6-8周的雌性BALB/c小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射100μL的2mg微米粒子1。在第32天处死小鼠,收集小鼠的PBMC,使用流式细胞术从PBMC中分选得到CD3+T细胞。将分选得到的CD3+T细胞(200万个)、步骤4制备的灭活的BMDC(300万个)以及IL-7(10ng/mL)在10mLRPMI1640完全培养基中共孵育18小时。然后采用流式细胞术分选孵育后的T细胞中的CD3+CD69+T细胞(细胞活率85%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的30万个CD3+CD69+T细胞与IL-2(1000U/mL)、IL-7(1000U/mL)、IL-15(1000U/mL)以及αCD3抗体在(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育14天以扩增癌细胞特异性T细胞(细胞活率85%)。
(6)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠皮下注射100μL含120万个扩增得到的CD3+T细胞。同时在第0天给每只小鼠皮下注射接种1×106个4T1细胞,从第3天开始每3天记录一次小鼠肿瘤体积的大小。
(7)实验结果
如图17所示,与对照组相比,微米粒分选扩增得到的癌细胞特异性T细胞处理组肿瘤生长速度明显变慢且小鼠生存期明显延长。由此可见,本发明所述的癌细胞特异性T细胞对乳腺癌具有预防效果。
实施例17微米粒子分选扩增T细胞用于乳腺癌的预防
本实施例中,先使用8M尿素溶液对乳腺癌细胞进行裂解并溶解裂解组分。然后,以PLA为微米粒子骨架材料,以CpG2395和Poly ICLC为免疫佐剂,制备微米粒子。
(1)抗原组分的制备
将培养的4T1细胞在400g离心5分钟,然后用PBS洗涤两遍后重悬于超纯水中。所得癌细胞分别采用紫外线和高温加热进行灭活和变性处理,然后使用8M尿素水溶液裂解癌细胞并溶解裂解物组分,即为制备微米粒子的抗原组分。
(2)微米粒子的制备
本实施例中制备微米粒子1采用复乳法。微米粒子1骨架材料PLA分子量为40KDa,CpG2395和Poly ICLC为免疫佐剂。制备时先在内部负载抗原组分和佐剂,尔后,将100mg微米粒子在9000g离心20分钟,使用10mL含4%海藻糖的超纯水重悬后干燥48h后备用。该微米粒子平均粒径为2.5μm左右,每1mg PLGA微米粒子约负载600μg蛋白质或多肽组分,负载CpG2395和Poly ICLC各0.02mg。
(3)癌细胞特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射100μL含2mg PLGA的微米粒子1。在第32天处死小鼠,收集小鼠的外周血中的PBMC,使用流式细胞术从PBMC中分选得到CD3+T细胞、CD19+B细胞。将分选得到的CD3+T细胞(800万个)、微米粒子1(500μg)、B细胞(900万个)、IL-7(10ng/mL)和IL-15(10ng/mL)在5mL RPMI1640完全培养基中共孵育24小时,然后采用流式细胞术分选孵育后的CD3+T细胞中的CD3+CD69+T细胞(细胞活率80%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的50万个CD3+CD69+T细胞与IL-2(1000U/mL)、IL-7(1000U/mL)、IL-15(1000U/mL)以及αCD3抗体(10ng/mL)10mL的DMEM完全培养基(37℃,5%CO2)中共孵育10天以扩增癌细胞特异性T细胞(细胞活率80%),所得细胞为T细胞1。
或者将分选得到的CD3+T细胞(200万个)、微米粒子1(50μg)、B细胞(600万个)、IL-7(10ng/mL)和IL-15(10ng/mL)在5mL RPMI1640完全培养基中共孵育24小时,然后分离出其中的T细胞,不进行任何分选和扩增,所得细胞为T细胞2(细胞活率80%)。
(4)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠皮下注射100万个分选扩增后的T细胞1或者T细胞2。同时在第0天给每只小鼠皮下注射接种1×106个4T1细胞,小鼠肿瘤体积以及生存期监测方法同上。
(5)实验结果
如图18所示,与对照组相比,T细胞处理组肿瘤生长速度明显变慢且生存期明显延长,而且,T细胞1的效果明显好于T细胞2。
实施例18癌细胞特异性T细胞后用于黑色素瘤的治疗
(1)抗原组分的制备
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织,将肿瘤组织切块后研磨,通过细胞过滤网加入适量纯水并反复冻融5次(可伴有超声)以破坏裂解所得样品,加入核酸酶作用10分钟后在95℃加热10分钟灭活核酸酶;收集培养的B16F10癌细胞系时,先离心去除培养基后使用PBS洗涤两次并离心收集癌细胞,将癌细胞在超纯水中重悬,反复冻融3次,并伴有超声破坏裂解癌细胞,尔后在样品中加入核酸酶作用10分钟后在95℃加热5分钟灭活核酸酶。将肿瘤组织或癌细胞系经酶作用处理后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入50%甘油溶解沉淀部分转化为可溶。将肿瘤组织的水溶性组分和癌细胞系的水溶性组分按质量比1:1混合;肿瘤组织的非水溶性组分和癌细胞系的非水溶性组分按质量比1:1混合。以上即为制备纳米粒子的抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒子1采用复乳法制备。在制备时负载水溶性组分混合物的纳米粒子和负载非水溶性组分混合物的纳米粒子分别制备,应用时一起使用。所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa,所采用的免疫佐剂为poly(I:C)和CpG1018,R8多肽(RRRRRRRR)为增加溶酶体逃逸的物质。制备方法如前所述,先采用复乳法在纳米粒子内部负载裂解液组分、佐剂和R8多肽,然后将100mg纳米粒子在12000g离心25分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h;在使用前将其用9mL PBS重悬然后加入1mL的裂解液组分(蛋白质/多肽浓度80mg/mL)并室温作用10min,得到内外都负载裂解物的纳米粒子。该纳米粒子平均粒径为290nm左右;每1mg PLGA纳米粒子约负载140μg抗原组分中的蛋白质或多肽组分,负载poly(I:C)和CpG1018免疫佐剂各0.02mg,负载0.1mg R8多肽。
(3)癌症特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射100μL的含负载水溶性组分的1mg PLGA纳米粒子和负载非水溶性组分的1mg PLGA纳米粒子。在第32天处死小鼠,收集小鼠的PBMC,使用流式细胞术从PBMC中分选得到CD8+T细胞、CD4+T细胞。将分选得到的CD8+T细胞(200万个)、CD4+T细胞(100万个)、纳米粒子(50μg;其中25μg为负载水溶性组分的纳米粒子,25μg为负载非水溶性组分的纳米粒子)、DC2.4细胞系(30万个)以及IL-7(10ng/mL)在2mL RPMI1640完全培养基中共孵育96小时,然后采用流式细胞术分选孵育后的CD8+T细胞中的CD8+CD69+T细胞(细胞活率80%)以及CD4+T细胞中CD4+CD69+T细胞(细胞活率80%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的20万个CD8+CD69+癌细胞特异性T细胞或者10万个CD4+CD69+癌细胞特异性T细胞与IL-2(1000U/mL)、IL-7(1000U/mL)、IL-21(1000U/mL)以及αCD3抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育14天以扩增癌细胞特异性T细胞(细胞活率80%)。
或者将从小鼠PBMC中分选得到的20万个CD8+T细胞或者10万个CD4+T细胞未经进一步分选直接分别与IL-2(1000U/mL)、IL-7(1000U/mL)、IL-21(1000U/mL)以及αCD3抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育14天以扩增癌细胞特异性T细胞。
(6)T细胞治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠。在第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别静脉注射扩增后的80万个癌症特异性CD8+T细胞和40万个癌症特异性CD4+T细胞(经过了两步分选);或者在上述天数注射只经过一步分选后扩增的80万个CD8+T细胞和40万个CD4+T细胞(未经过纳米粒子分选)。小鼠肿瘤生长和生存期监测方法同上。
(7)实验结果
如图19所示,与对照组相比,T细胞处理组肿瘤生长速度明显变慢且生存期明显延长。而且,使用负载裂解物组分的纳米粒粒子分选扩增得到的癌症特异性T细胞(经过了两步分选)好于未经纳米粒子分选直接扩增的T细胞。
实施例19癌细胞特异性T细胞用于结肠癌的治疗
(1)抗原组分的制备
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种2×106个MC38结肠癌细胞,在肿瘤长到体积分别为约1000mm3时处死小鼠并摘取肿瘤组织,将肿瘤组织切块后研磨,通过细胞过滤网加入8M尿素水溶液理解肿瘤组织并溶解裂解后组分。以上即为制备纳米粒子的抗原组分。
(2)纳米粒子的制备
本实施例中纳米粒子采用复乳法制备。纳米粒子1的制备材料PLGA分子量为7KDa-17KDa,以Poly(I:C)和CpG1018为佐剂,以NH4HCO3为增加溶酶体逃逸物质。制备方法如前所述,在制备过程中首先在纳米粒子内部负载裂解液组分、佐剂和NH4HCO3,然后将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h后备用;该纳米粒子平均粒径为260nm左右;每1mg PLGA纳米粒子约负载90μg蛋白质和多肽组分,负载poly(I:C)和CpG1018各0.02mg,负载NH4HCO3 0.01mg。纳米粒子2的制备材料和制备方法同纳米粒子1,但是不负载佐剂只负载增加溶酶体逃逸物质;粒径为260nm左右,表面电位为-7mV左右;每1mg PLGA纳米粒子约负载90μg蛋白质和多肽组分,每1mg PLGA纳米粒负载NH4HCO3 0.01mg,不负载佐剂。
(3)癌症特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射100μL含2mg PLGA的纳米粒子1。在第32天处死小鼠,收集小鼠的PBMC,使用流式细胞术从PBMC中分选得到CD8+T细胞、CD4+T细胞以及B细胞。将分选得到的CD8+T细胞(200万个)、CD4+T细胞(100万个)、纳米粒子(50μg)、B细胞(300万个)以及IL-7(10ng/mL)在2mL RPMI1640完全培养基中共孵育48小时,然后采用流式细胞术分选孵育后的CD8+T细胞中的CD8+CD69+T细胞(细胞活率80%)以及CD4+T细胞中CD4+CD69+T细胞(细胞活率80%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的20万个CD8+CD69+T细胞或者20万个CD4+CD69+T细胞与IL-2(1000U/mL)、IL-7(1000U/mL)、IL-15(1000U/mL)以及αCD3抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育14天以扩增癌细胞特异性T细胞(细胞活率80%)。
(4)T细胞用于治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备结肠癌小鼠。在第0天给每只小鼠背部右下方皮下接种2×106个MC38细胞。在接种结肠癌细胞后第6天、第9天、第12天、第15天、第20天和第25天分别静脉注射100万个CD8+癌症特异性T细胞和50万个CD4+癌症特异性T细胞;或者在上述天数注射150万个CD8+癌症特异性T细胞。小鼠肿瘤生长和生存期监测方法同上。
(5)实验结果
如图20所示,与对照组相比,纳米粒子分选扩增得到的癌症特异性T细胞处理组肿瘤生长速度明显变慢且生存期明显延长。而且,同时使用纳米粒子分选扩增得到的CD8+T细胞和CD4+T细胞好于只使用纳米粒子辅助分选和扩增得到的CD8+T细胞。
实施例20癌细胞特异性T细胞用于治疗黑色素瘤
(1)抗原组分的制备
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网加入适量超纯水并反复冻融5次,并伴有超声以破坏裂解细胞。裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液中可溶。在裂解液中的水溶性组分中逐滴加入饱和硫酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀溶解于2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液备用,将上清液在100℃加热5分钟后将所得样品在3000g离心5分钟,弃去上清液后将沉淀溶解于2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液;然后将使用2M盐酸氨基脲和0.2M硫酸胍基丁胺溶解的盐析后的沉淀和溶解的加热后的沉淀合并后作为水溶性组分中的一部分组分使用。将以上2M盐酸氨基脲水溶液溶解的裂解液中非水溶性组分与以上2M盐酸氨基脲和0.2M硫酸胍基丁胺溶解的水溶性组分中盐析和加热后沉淀所得的组分按质量比1:1混合,即为制备纳米粒子1的抗原组分1。
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到体积约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网加入适量超纯水并反复冻融5次,并伴有超声以破坏裂解细胞。裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液中可溶。在裂解液中的水溶性组分中逐滴加入饱和硫酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀溶解于2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液作为水溶性组分中的一部分组分使用。将以上2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液溶解的裂解液中非水溶性组分和2M盐酸氨基脲与以上0.2M硫酸胍基丁胺溶解的水溶性组分中盐析后沉淀所得的组分按质量比1:1混合,即为制备纳米粒子2的抗原组分2。
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,通过细胞过滤网加入适量超纯水并反复冻融5次,并伴有超声以破坏裂解细胞。裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液中可溶。将裂解液中的水溶性组分在100℃加热5分钟后将所得样品在3000g离心5分钟,弃去上清液后将沉淀溶解于2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液,即为水溶性组分中的一部分组分。将以上2M盐酸氨基脲和0.2M硫酸胍基丁胺水溶液溶解的裂解液中非水溶性组分与以上2M盐酸氨基脲和0.2M硫酸胍基丁胺溶解的水溶性组分中加热后沉淀所得的组分按质量比1:1混合,即为制备纳米粒子3的抗原组分3。
(2)纳米粒子的制备
本实施例中纳米粒子1(Nanoparticle 1)采用溶剂挥发法中的复乳法制备。所采用的抗原递送纳米粒子制备材料PLGA分子量为20KDa-40KDa,所采用的免疫佐剂为poly(I:C)。制备方法如前所述,先采用复乳法在纳米粒子内部负载抗原组分1和佐剂,然后将300mg纳米粒子在14000g离心30分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1平均粒径为100nm左右,每1mg PLGA纳米粒子约负载250μg蛋白质或多肽组分,每1mgPLGA纳米粒负载poly(I:C)0.01mg。
本实施例中纳米粒子2(Nanoparticle 2)制备方法和制备材料同纳米粒子1。制备方法如前所述,先采用复乳法在纳米粒子内部负载抗原组分2和佐剂,然后将100mg纳米粒子在14000g离心30分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子2平均粒径为300nm左右,每1mg PLGA纳米粒子约负载250μg蛋白质或多肽组分,每1mgPLGA纳米粒负载poly(I:C)0.01mg。
本实施例中纳米粒子3(Nanoparticle 3)制备方法和制备材料同纳米粒子1。首先在纳米粒子内部负载抗原组分和佐剂,然后将100mg纳米粒子在14000g离心30分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子3平均粒径为300nm左右,每1mg PLGA纳米粒子约负载250μg蛋白质或多肽组分,每1mgPLGA纳米粒负载poly(I:C)0.01mg。
(3)癌症特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天给小鼠皮下接种1.5×105个B16F10细胞,在第6天、第8天、第10天、第12天、第14天、第16天、第18天、第20天和第22天分别给每只小鼠腹腔注射150μg PD-1抗体。在第24天处死小鼠,收集小鼠的外周血,然后从外周血中分离外周血单个核细胞(PBMC),使用流式细胞术分选出所有CD69-的PBMC。然后将100万个CD69-的PBMC细胞、10mg纳米粒子(纳米粒子1,或者纳米粒子2,或者纳米粒子3)在2mL RPMI1640完全培养基中共孵育12小时,然后采用流式细胞术分选孵育后的CD3+CD69+T细胞(细胞活率85%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的100万个CD3+CD69+T细胞与IL-2(1000U/mL)、IL-7(1000U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育21天以扩增癌细胞特异性T细胞(细胞活率85%)。
(4)癌细胞特异性T细胞治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠。在第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。在第5天、第8天、第11天、第15天、第20天和第25天分别静脉注射50万个CD3+癌症特异性T细胞。小鼠肿瘤生长和生存期监测方法同上。
(5)实验结果
如图21所示,图中PBS组小鼠肿瘤体积快速长大。使用纳米粒子、纳米粒子2和纳米粒子3分选扩增得到的癌细胞特异性T细胞治疗的小鼠其肿瘤生长速度都明显变慢,生存期明显延长,且部分小鼠痊愈。其中,纳米粒子3好于纳米粒子2分选得到的癌细胞特异性T细胞,说明使用加热进行分离纯化的抗原组分制备的粒子效果好于使用特定试剂进行盐析进行分离纯化的效果;纳米粒子1效果好于纳米粒子2和纳米粒子3,说明同时使用盐析和加热进行分离纯化的抗原组分制备的粒子其效果明显好于只使用盐析进行分离纯化的抗原组分制备的粒子或者只使用加热进行分离纯化的抗原组分制备的粒子。
本实施例中,抗原组分主要使用了全细胞组分中的蛋白质和多肽组分,在实际应用中也可以将细胞裂解物中的mRNA分离提取出来之后和全细胞组分中的蛋白质/多肽混合后作为混合抗原使用。
实施例21癌细胞特异性T细胞用于癌症的治疗
(1)抗原组分的制备
将培养的E.G7-OVA小鼠T淋巴瘤细胞在400g离心5分钟,然后重悬于超纯水中,然后使用6M硫酸胍水溶液裂解癌细胞并溶解裂解物组分,然后加入饱和硫酸铵水溶液,直到沉淀完全后弃去上清液,将沉淀再次溶解于6M硫酸胍水溶液,即得到溶解于6M硫酸胍水溶液中的癌细胞中的蛋白质和多肽组分,即为抗原组分1。
将培养的E.G7-OVA小鼠T淋巴瘤细胞在400g离心5分钟,然后重悬于超纯水中,再使用3%吐温80水溶液裂解癌细胞并增溶裂解物组分,然后加入饱和硫酸铵水溶液,直到沉淀完全后弃去上清液,将沉淀再次使用3%吐温80水溶液增溶,即得到溶解于3%吐温80水溶液中的蛋白质和多肽组分,即为抗原组分2。
将培养的E.G7-OVA小鼠T淋巴瘤细胞在400g离心5分钟,然后重悬于超纯水中,再使用6M硫酸胍水溶液裂解癌细胞并溶解裂解物组分,然后加入饱和硫酸铵水溶液,直到沉淀完全后弃去上清液,将沉淀再次使用3%吐温80水溶液增溶,即得到溶解于3%吐温80水溶液中的蛋白质和多肽组分,即为抗原组分3。
将培养的E.G7-OVA小鼠T淋巴瘤细胞在400g离心5分钟,然后重悬于超纯水中,再使用3%吐温80水溶液裂解癌细胞后使用3%吐温80水溶液溶解裂解物组分,然后加入饱和硫酸铵水溶液,直到沉淀完全后弃去上清液,将沉淀再次使用6M硫酸胍水溶液溶解,即得到溶解于6M硫酸胍水溶液中的蛋白质和多肽组分,即为抗原组分4。
(2)纳米粒子的制备
本实施例中制备纳米粒子1(Nanoparticle 1)采用复乳法。纳米粒子1骨架材料为PLA(分子量30-40KDa)和甘露聚糖-PEG2000-PLA(PLA分子量为30-40KDa),且PLA(分子量30-40KDa)和甘露聚糖-PEG2000-PLA(PLA分子量为30-40KDa)质量比为9:1。所采用的免疫佐剂为CpG2006(B类)、CpG2216(A类)和Poly ICLC。制备时先采用复乳法制备内部负载抗原组分1和佐剂的纳米粒子,尔后,将100mg纳米粒子在13000g离心25分钟,使用10mL含4%海藻糖的超纯水重悬后干燥48h后即得纳米粒子1,平均粒径为500nm左右,每1mg PLGA纳米粒子1约负载5μg癌细胞的蛋白质和多肽组分,负载CpG2006,CpG2216和Poly ICLC各0.02mg。
纳米粒子2(Nanoparticle 2)制备方法同纳米粒子1。但是内部负载的是抗原组分2和佐剂。纳米粒子2平均粒径为500nm左右,每1mg PLGA纳米粒子2约负载5μg癌细胞的蛋白质和多肽组分,负载CpG2006,CpG2216和Poly ICLC各0.02mg。
纳米粒子3(Nanoparticle 3)制备方法同纳米粒子1。但是内部负载的是抗原组分3、佐剂和R8多肽。纳米粒子3平均粒径为500nm左右,每1mg PLGA纳米粒子3约负载5μg癌细胞的蛋白质和多肽组分,负载CpG2006,CpG2216和Poly ICLC各0.02mg。
纳米粒子4(Nanoparticle 4)制备方法同纳米粒子1。但是内部负载的是抗原组分4和佐剂。纳米粒子4平均粒径为500nm左右,每1mg PLGA纳米粒子4约负载5μg癌细胞的蛋白质和多肽组分,负载CpG2006,CpG2216和Poly ICLC各0.02mg。
(3)癌症特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天给小鼠皮下接种5×105个E.G7-OVA小鼠T淋巴瘤细胞。在第4天、第6天、第8天、第10天、第12天、第14天、第16天、第18天和第20天分别给每只小鼠腹腔注射150μg PD-1抗体。在第22天处死小鼠,收集小鼠的外周血,然后从外周血中分离外周血单个核细胞(PBMC),使用流式细胞术分选出所有CD25-的PBMC。然后将5000万个CD25-的PBMC细胞、5μg纳米粒子(纳米粒子1,或者纳米粒子2,或者纳米粒子3,或者纳米粒子4)在5mL RPMI1640完全培养基中共孵育96小时,然后采用流式细胞术分选孵育后的CD3+CD25+T细胞(细胞活率90%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的10万个CD8+CD25+T细胞与IL-2(1000U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在20mL的DMEM完全培养基(37℃,5%CO2)中共孵育28天以扩增癌细胞特异性T细胞(细胞活率90%)。
(4)T细胞治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备结肠癌小鼠。在第0天给每只小鼠背部右下方皮下接种5×105个E.G7-OVA细胞。在接种结肠癌细胞后第6天、第9天、第12天、第15天、第20天和第25天分别静脉注射50万个CD3+癌症特异性T细胞。小鼠肿瘤生长和生存期监测方法同上。
(5)实验结果
如图22所示,与PBS对照组相比,T细胞处理的小鼠,其肿瘤生长速度明显变慢且小鼠生存期明显延长。而且,使用纳米粒子1分选扩增得到的癌细胞特异性T细胞效果好于纳米粒子2、纳米粒子3和纳米粒子4,说明使用适当的溶解剂初次溶解和二次溶解制备纳米粒子的癌细胞抗原组分是非常关键的。
实施例22 T细胞治疗癌症
(1)抗原组分的收集
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,加入适量超纯水并反复冻融5次,并伴有超声以破坏裂解细胞。裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入8M尿素水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。在裂解液中的水溶性组分中逐滴加入饱和硫酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀溶解于8M尿素水溶液即为水溶性组分中的一部分组分。以上8M尿素水溶液溶解的裂解液中非水溶性组分与8M尿素溶解的水溶性组分中盐析后沉淀所得的组分按质量比1:2混合即为制备纳米粒子1的抗原组分1。
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,加入适量超纯水并反复冻融5次,并伴有超声以破坏裂解细胞。裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入8M尿素水溶液溶解沉淀部分即可将不溶于纯水的非水溶性抗原转化为在8M尿素水溶液中可溶。在裂解液中的水溶性组分中逐滴加入饱和碳酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀溶解于8M尿素水溶液即为水溶性组分中的一部分组分。以上8M尿素水溶液溶解的裂解液中非水溶性组分与8M尿素溶解的水溶性组分中盐析后沉淀所得的组分按照质量比1:2混合后即为制备纳米粒子2的抗原组分2。
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,加入适量超纯水并反复冻融5次,并伴有超声以破坏裂解细胞。裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入8M尿素水溶液溶解沉淀部分即可将不溶于纯水的非水溶性抗原转化为在8M尿素水溶液中可溶。以上8M尿素水溶液溶解的裂解液中非水溶性组分与水溶性组分按质量比1:2混合后即为制备纳米粒子3的抗原组分3。
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,在肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,加入适量超纯水并反复冻融5次,并伴有超声以破坏裂解细胞。裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入8M尿素水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。在裂解液中的水溶性组分中逐滴加入饱和硫酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀使用5%PEG5000水溶液增溶后即为水溶性组分中的一部分组分使用。以上8M尿素水溶液溶解的裂解液中非水溶性组分与5%PEG5000水溶液溶解的水溶性组分中盐析后沉淀所得的组分按质量比1:2混合后即为制备纳米粒子4的抗原组分4。
在每只C57BL/6小鼠背部皮下接种1.5×105个B16F10细胞,肿瘤长到约1000mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨,然后加入适量超纯水并反复冻融5次,并伴有超声以破坏裂解细胞。裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入5%PEG5000水溶液增溶沉淀部分即可得非水溶性组分在5%PEG5000水溶液中可溶的组分。在裂解液中的水溶性组分中逐滴加入饱和硫酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀使用5%PEG5000水溶液增溶即为水溶性组分中的一部分组分使用。以上5%PEG5000水溶液溶解的裂解液中非水溶性组分与5%PEG5000水溶液溶解的水溶性组分中盐析后沉淀所得的组分按质量比1:2混合后即为制备纳米粒子5的抗原组分5。
(2)纳米粒子的制备
本实施例中纳米粒子1(Nanoparticle 1)采用溶剂挥发法中的复乳法制备。所采用的抗原递送纳米粒子制备材料PLGA分子量为10KDa-20KDa,所采用的免疫佐剂为poly(I:C)。先采用复乳法在纳米粒子内部负载细胞抗原组分1和佐剂,然后将100mg纳米粒子在13000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1平均粒径为200nm左右,每1mg PLGA纳米粒子约负载15μg蛋白质或多肽组分,负载poly(I:C)0.2mg。
纳米粒子2(Nanoparticle 2)制备方法、材料和制备步骤同纳米粒子1。制备方法如前所述,先在纳米粒子内部负载细胞抗原组分2和佐剂,然后将100mg纳米粒子在13000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子2平均粒径为200nm左右,每1mg PLGA纳米粒子约负载15μg蛋白质或多肽组分,负载poly(I:C)0.2mg。
纳米粒子3(Nanoparticle 3)制备方法、材料和制备步骤同纳米粒子1。首先在纳米粒子内部负载细胞抗原组分3和佐剂,然后将100mg纳米粒子在13000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子3平均粒径为200nm左右,每1mg PLGA纳米粒子约负载15μg蛋白质或多肽组分,负载poly(I:C)0.2mg。
纳米粒子4(Nanoparticle 4)制备方法、材料和制备步骤同纳米粒子1。制备方法如前所述,先在纳米粒子内部负载细胞抗原组分4和佐剂,然后将100mg纳米粒子在13000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子4平均粒径为200nm左右,每1mg PLGA纳米粒子约负载15μg蛋白质或多肽组分,负载poly(I:C)0.2mg。
纳米粒子5(Nanoparticle 5)制备方法、材料和制备步骤同纳米粒子1。先在纳米粒子内部负载细胞抗原组分5和佐剂,然后将100mg纳米粒子在13000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子5平均粒径为200nm左右,每1mg PLGA纳米粒子约负载15μg蛋白质或多肽组分,负载poly(I:C)0.2mg。
(3)T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天给小鼠皮下接种1.5×105个B16F10细胞。在第4天、第6天、第8天、第10天、第12天、第14天、第16天、第18天和第20天分别给每只小鼠腹腔注射150μg PD-L1抗体。在第22天处死小鼠,收集小鼠的PBMC,将PBMC先在体外培养12小时,然后使用流式细胞术分选出所有CD69-的PBMC。然后将250万个CD69-的PBMC细胞、500μg纳米粒子(纳米粒子1,或者纳米粒子2,或者纳米粒子3,或者纳米粒子4,或者纳米粒子5)在10mL RPMI 1640完全培养基中共孵育36小时,然后采用流式细胞术分选孵育后的CD3+CD69+T细胞(细胞活率90%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的10万个CD3+CD69+T细胞与IL-2(1000U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育21天以扩增癌细胞特异性T细胞(细胞活率90%)。
(4)T细胞治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠,第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。在小鼠接种肿瘤前第3天,第6天,第9天,第14天和第20天分别在小鼠皮下接种10万个不同纳米粒子辅助分选得到的癌细胞特异性T细胞或者100μL PBS。监测小鼠肿瘤生长速度和小鼠生存期。
(4)实验结果
如图23所示,PBS组小鼠肿瘤体积快速长大。纳米粒子1(Nanoparticle 1)、纳米粒子2(Nanoparticle 2)、纳米粒子3(Nanoparticle 3)、纳米粒子4(Nanoparticle 4)和纳米粒子5(Nanoparticle 5)分选扩增得到的癌细胞特异性T细胞处理的小鼠,其肿瘤生长速度都明显变慢,生存期明显延长。其中,纳米粒子1效果好于纳米粒子2、纳米粒子3、纳米粒子4和纳米粒子5,说明使用硫酸铵盐析分离纯化水溶性组分中蛋白质和多肽抗原组分的效果好于使用碳酸铵,而且,分离纯化水溶性中组分中一部分组分后的效果,好于直接使用水溶性组分。而且,使用尿素溶解水溶性组分经过盐析处理产生的沉淀好效果好于使用PEG。
实施例23分选扩增的T细胞杀伤乳腺癌细胞
本实施例使用来自多个乳腺癌癌症患者的肿瘤组织制备纳米粒子。
(1)抗原组分的制备
收取12位三阴性乳腺癌患者手术切除得到的肿瘤组织。将每位患者的肿瘤组织切块后研磨,通过细胞过滤网后加入超纯水中,然后反复冻融5遍并伴有超声以裂解肿瘤组织中的癌细胞。在所裂解癌细胞中加入1mg/mL的核酸酶降解裂解物中的核酸,然后在95℃加热10分钟灭活核酸酶,然后在5000g离心5分钟收集上清液即为水溶性组分,沉淀使用8M尿素(含0.01M精氨酸)水溶液溶解即为非水溶性组分。将12位乳腺癌患者的水溶性组分都按照质量比1:1混合后得到水溶性组分混合物;将12位乳腺癌患者的非水溶性组分都按照质量比1:1混合后得到非水溶性组分混合物。将水溶性组分混合物与非水溶性组分混合物按质量比5:1混合即为制备纳米粒子1(NP1)的抗原组分1。
收取另一位三阴性乳腺癌患者A手术切除得到的肿瘤组织。A患者不包括在上述12位癌症患者内。肿瘤组织切块后研磨,通过细胞过滤网后加入超纯水中,然后反复冻融5遍并伴有超声以裂解癌细胞。在所裂解癌细胞中加入1mg/mL的核酸酶降解裂解物中的核酸,然后在95℃加热10分钟灭活核酸酶,然后在5000g离心5分钟收集上清液即为水溶性组分,沉淀使用8M尿素(含0.01M精氨酸)水溶液溶解即为非水溶性组分,将水溶性组分与非水溶性组分按质量比5:1混合即为制备纳米粒子2(NP2)的抗原组分2。
(2)纳米粒子的制备
本实施例中制备纳米粒子采用复乳法。纳米粒子1骨架材料PLGA分子量为10KDa-20KDa,所采用的免疫佐剂为CpG2395(C类)、CpG1018(B类)和Poly ICLC。制备时先在粒子内部负载抗原组分1和佐剂,然后将100mg纳米粒子在12000g离心20分钟,使用10mL含4%海藻糖的超纯水重悬后干燥48h后备用。该纳米粒子粒径为280nm左右,每1mg PLGA纳米粒子约负载950μg蛋白质或多肽组分,负载CpG2395、CpG1018和Poly(I:C)各0.02mg。
纳米粒子2骨架材料PLGA分子量为10KDa-20KDa,所采用的免疫佐剂为CpG2395(C类)、CpG1018(B类)和Poly ICLC。制备时采用复乳法制备内部负载抗原组分2和佐剂的纳米粒子,然后将100mg纳米粒子在12000g离心20分钟,使用10mL含4%海藻糖的超纯水重悬后干燥48h后备用。该纳米粒子粒径为280nm左右,每1mg PLGA纳米粒子约负载950μg蛋白质或多肽组分,负载CpG2395、CpG1018和Poly(I:C)各0.02mg。
(3)癌细胞特异性T细胞的分离和扩增
患者A在手术切除肿瘤组织后经过了治疗,且经过治疗后疗效很好,肿瘤块逐渐变小。分别在免疫治疗前或者免疫治疗后抽取患者10mL外周血。然后从外周血中分离出PBMC,将PBMC先在体外培养12小时,然后进行后续实验。
将免疫治疗前或者免疫治疗后的PBMC(600万个)、IL-7(10ng)、IL-15(10ng)与20μg纳米粒子(纳米粒子1或者纳米粒子2)在2mL AIM V无血清培养基中培养基中共孵育72小时(37℃,5%CO2)。孵育后收集细胞并使用流式细胞术分选出CD3+CD25+T细胞(细胞活率85%),将上述分选得到30万个T细胞与IL-2(1000U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育21天以扩增癌细胞特异性T细胞(细胞活率85%)。
或者将免疫治疗后的PBMC(600万个)、IL-7(10ng)、IL-15(10ng)与5ng纳米粒子(分别为纳米粒子1或者纳米粒子2)在2mL AIM V无血清培养基中培养基中共孵育72小时(37℃,5%CO2)。孵育后收集细胞并使用流式细胞术分选出CD3+CD25+T细胞(细胞活率85%),将上述分选得到30万个T细胞与IL-2(1000U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育21天以扩增癌细胞特异性T细胞(细胞活率85%)。
或者将免疫治疗后的PBMC(600万个)、IL-7(10ng)、IL-15(10ng)与100mg纳米粒子(分别为纳米粒子1或者纳米粒子2)在2mL AIM V无血清培养基中培养基中共孵育72小时(37℃,5%CO2)。孵育后收集细胞并使用流式细胞术分选出CD3+CD25+T细胞(细胞活率85%),将上述分选得到30万个T细胞与IL-2(1000U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育21天以扩增癌细胞特异性T细胞(细胞活率85%)。
(4)癌细胞特异性T细胞杀伤癌细胞
选取6-8周的裸鼠,第0天给每只裸鼠背部右下方皮下接种5×105个患者A手术切除得到的肿瘤组织扩增得到的癌细胞。在小鼠接种肿瘤后第3天,第6天,第9天,第14天和第20天分别在小鼠皮下注射30万个不同纳米粒子辅助分选和扩增后得到的癌细胞特异性T细胞或者100μL PBS。监测小鼠肿瘤生长速度。
(5)实验结果
如图24所示,与对照组相比,2种纳米粒子分选和扩增得到的癌细胞特异性T细胞均可以有效控制肿瘤组织生长,其中,纳米粒子1纳米粒子2效果相似。而且,使用适当浓度(10μg/mL)纳米粒子共孵育辅助分选的效果明显好于使用太低浓度(2.5ng/mL)或者太高浓度(50mg/mL)。而且,免疫治疗后分选扩增得到的癌细胞特异性T细胞效果好于免疫治疗前分选扩增得到的癌细胞特异性T细胞。而且,使用经过免疫治疗后的外周血免疫细胞进行分选扩增效果更好。
实施例24分选扩增的T细胞杀伤食管癌细胞
(1)抗原组分的制备
收集5位食管癌患者的肿瘤组织。将5位患者的肿瘤组织按照质量比1:1:1:1:1混合后切块并研磨,然后通过细胞过滤网后加入适量8M尿素水溶液裂解上述细胞,并使用8M尿素水溶液完全溶解肿瘤组织裂解物组分,即为制备纳米粒子1和纳米粒子2的抗原组分1。
收集另外一位食管癌患者A的肿瘤组织。A患者不包括在上述5位癌症患者内。将患者A的肿瘤组织切块并研磨,然后通过细胞过滤网后加入适量8M尿素水溶液裂解上述细胞,并使用8M尿素水溶液完全溶解肿瘤组织裂解物组分,即为制备纳米粒子3和纳米粒子4的抗原组分2。
(2)纳米粒子的制备
本实施例中纳米粒1(NP1)采用复乳法制备。所采用的纳米粒子制备材料为PLGA分子量都为10KDa-30KDa,负载的佐剂为poly I:C和CpG7909,负载的增加溶酶体免疫逃逸物质KALA多肽。制备方法如前所述,先在纳米粒子内部负载抗原组分、佐剂和KALA多肽,然后将100mg纳米粒子在12000g离心25分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子平均粒径为250nm左右,每1mg PLGA纳米粒子约负载100μg肿瘤组织的蛋白质或多肽组分,负载poly I:C和CpG7909各0.01mg,负载KALA多肽0.15mg。
纳米粒2(NP2)的制备材料和方法相同,其粒径为250nm左右,每1mg PLGA纳米粒子约负载0.01μg肿瘤组织的蛋白质或多肽组分,负载poly I:C和CpG7909各0.01mg,负载KALA多肽0.15mg。
纳米粒子3(NP3)的制备材料和制备方法相同,为250nm左右,每1mg PLGA纳米粒子约负载100μg肿瘤组织的蛋白质和多肽组分,负载poly I:C和CpG7909各0.01mg,负载KALA多肽0.15mg。
纳米粒子4(NP4)的制备材料和制备方法相同,为250nm左右,每1mg PLGA纳米粒子约负载0.01μg肿瘤组织的蛋白质或多肽组分,负载poly I:C和CpG7909各0.01mg,负载KALA多肽0.15mg。
(3)癌细胞特异性T细胞的检测
患者A在手术切除肿瘤组织后经过了癌症免疫治疗,且经过治疗后疗效很好,肿瘤块逐渐变小。在免疫治疗后抽取患者12mL外周血。然后从外周血中分离出PBMC。将PBMC(1000万个)和100μg纳米粒子(纳米粒子1,或者纳米粒子2,或者纳米粒子3,或者纳米粒子4)在10mL AIM V无血清培养基中培养基中共孵育96小时(37℃,5%CO2)。孵育后收集细胞并使用CD3和HLA-DR流式抗体标记细胞,然后使用流式细胞术分选出CD3+HLA-DR+T细胞(细胞活率85%),即为癌细胞特异性T细胞。将上述分选得到T细胞与IL-2(1000U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育21天以扩增癌细胞特异性T细胞(细胞活率85%)。
(4)分选扩增的T细胞杀伤癌细胞
选取6-8周的裸鼠,第0天给每只裸鼠背部右下方皮下接种5×105个患者A手术切除得到的肿瘤组织扩增得到的癌细胞。在小鼠接种肿瘤后第3天,第6天,第9天,第14天和第20天分别在小鼠皮下注射50万个不同纳米粒子辅助分选和扩增后得到的癌细胞特异性T细胞或者100μL PBS。监测小鼠肿瘤生长速度。
(4)实验结果
如图25所示,负载多个异体癌症患者肿瘤组织混合物制备的纳米粒子分选扩增得到的癌细胞特异性T细胞的效果与使用癌症患者自体肿瘤组织辅助分选扩增后得到的纳米粒子的效果无显著性差异。而且,负载适当抗原组分的纳米粒子好于只负载低含量抗原组分的纳米粒子。
实施例25分选扩增T细胞杀伤肺癌细胞
本实施例中,将多种人肺癌的癌细胞系中的水溶性组分和非水溶性组分负载到纳米粒子,然后,以该纳米粒子分选扩增肺癌患者外周免疫器官中的癌细胞特异性T细胞。
(1)抗原组分的制备
分别培养人肺癌细胞系A549细胞、H1299细胞、PC9细胞、H1437细胞、H226细胞、HCC1588细胞、H2170细胞和H520细胞。
将上述8种细胞分别收集后,将上述A549细胞、H1299细胞、PC9细胞、H1437细胞、H226细胞、HCC1588细胞、H2170细胞和H520细胞按照细胞数量比20:20:2:2:2:1:1:1:1混合,然后离心后去除培养基,将细胞沉淀使用超纯水重悬后反复冻融5次,在冻融过程中辅以超声破碎以更彻底的裂解癌细胞。待细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入6M硫酸胍溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在6M硫酸胍水溶液中可溶。将裂解液中的水溶性组分在95℃加热10分钟后将所得样品在3000g离心5分钟,将沉淀的蛋白质和多肽组分溶解于6M硫酸胍水溶液,将上清液中的mRNA使用mRNA提取试剂盒提取出来,然后将mRNA组分与6M硫酸胍溶解的蛋白质多肽组分混合即为水溶性中组分中的抗原组分;然后在6M硫酸胍溶解的非水溶性组分中加入饱和硫酸铵溶液盐析蛋白质和多肽组分,将盐析出的沉淀使用6M硫酸胍二次溶解后即为非水溶性组分中抗原组分。将以上非水溶性组分中的抗原组分和水溶性组分中的抗原组分按质量比1:1混合,即为制备纳米粒子1(NP1)的抗原组分1。
或者收集该非小细胞肺癌患者A本人手术切除的肿瘤组织样本。该非小细胞肺癌患者免疫治疗疗效很好。将该肿瘤组织剪碎后通过细胞筛网过滤,然后使用超纯水重悬后反复冻融5次,在冻融过程中辅以超声破碎以更彻底的裂解癌细胞。待细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入6M硫酸胍溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在6M硫酸胍水溶液中可溶。将裂解液中的水溶性组分在95℃加热10分钟后将所得样品在3000g离心5分钟,将沉淀的蛋白质和多肽组分溶解于6M硫酸胍水溶液,将上清液中的mRNA使用mRNA提取试剂盒提取出来,然后将mRNA组分与6M硫酸胍溶解的蛋白质多肽组分混合即为水溶性中组分中的抗原组分;然后在6M硫酸胍溶解的非水溶性组分中加入饱和硫酸铵溶液盐析蛋白质和多肽组分,将盐析出的沉淀使用6M硫酸胍二次溶解后即为非水溶性组分中抗原组分。将以上非水溶性组分中的抗原组分和水溶性组分中的抗原组分按质量比1:1混合,即为制备纳米粒子2(NP2)的抗原组分2。
或者收集另一个非小细胞肺癌患者B的肿瘤组织样本。将该肿瘤组织剪碎后通过细胞筛网过滤,然后使用超纯水重悬后反复冻融5次,在冻融过程中辅以超声破碎以更彻底的裂解癌细胞。待细胞裂解后,将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分;在所得沉淀部分中加入6M硫酸胍溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在6M硫酸胍水溶液中可溶。将裂解液中的水溶性组分在95℃加热10分钟后将所得样品在3000g离心5分钟,将沉淀的蛋白质和多肽组分溶解于6M硫酸胍水溶液,将上清液中的mRNA使用mRNA提取试剂盒提取出来,然后将mRNA组分与6M硫酸胍溶解的蛋白质多肽组分混合即为水溶性中组分中的抗原组分;然后在6M硫酸胍溶解的非水溶性组分中加入饱和硫酸铵溶液盐析蛋白质和多肽组分,将盐析出的沉淀使用6M硫酸胍二次溶解后即为非水溶性组分中的抗原组分。将以上非水溶性组分中的抗原组分和水溶性组分中的抗原组分按质量比1:1混合,即为制备纳米粒子3(NP3)的抗原组分3。
(2)负载全细胞组分的纳米粒子的制备
本实施例中纳米粒子1(NP1)采用溶剂挥发法中的复乳法制备。所采用的纳米粒子制备材料PLGA分子量为20KDa-40KDa,佐剂为poly(I:C)、CpG7909和CpG2395。制备方法如前所述,先在纳米粒子内部负载抗原组分和佐剂,然后将100mg纳米粒子在10000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1平均粒径为380nm左右,每1mg PLGA纳米粒子约负载800μg蛋白质或多肽组分,负载poly(I:C)、CpG7909和CpG2395各0.02mg。
纳米粒子2(NP2)的制备过程同纳米粒子1,该纳米粒子2平均粒径为380nm左右,每1mg PLGA纳米粒子约负载800μg蛋白质或多肽组分,负载poly(I:C)、CpG7909和CpG2395各0.02mg。
纳米粒子3(NP3)的制备过程同纳米粒子1,该纳米粒子3平均粒径为380nm左右,每1mg PLGA纳米粒子约负载800μg蛋白质或多肽组分,负载poly(I:C)、CpG7909和CpG2395各0.02mg。
(3)T细胞的分选和扩增
非小细胞肺癌患者A在在经过免疫治疗后肿瘤组织缩小。在非小细胞肺癌患者A免疫治疗后两周抽取非小细胞肺癌癌症患者A的外周血10mL。从10mL非小细胞肺癌患者外周血中使用梯度离心法分离得到外周血单个核细胞(PBMC)。
将纳米粒子1(0.5mg)或者纳米粒子2(0.5mg)或者纳米粒子3(0.5mg)与PBMC(1000万个)在1mL的AIM V无血清培养基中共孵育12小时(37℃,5%CO2)。然后收集细胞后在400g离心5分钟,将细胞在PBS中重悬后先采用Fc block处理T细胞以避免非特异性负载。
然后一半细胞采用CD3抗体以及FASL抗体对细胞进行细胞外染色,尔后采用4%多聚甲醛固定细胞和使用破膜剂对细胞进行破膜,并采用FN-γ抗体对T细胞进行细胞内染色。尔后采用流式细胞仪检测样本T细胞。分析CD3+T细胞中被激活后可以分泌IFN-γ的T细胞以及可以表达FASL的T细胞在所有CD3+T细胞中所占的比例。
另外一半细胞从中直接分选出CD3+FASL+T细胞(细胞活率85%),并将分选的50万个T细胞与IL-2(1000U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育28天以扩增癌细胞特异性T细胞(细胞活率85%)。
(4)分选扩增的T细胞杀伤癌细胞
选取6-8周的裸鼠,第0天给每只裸鼠背部右下方皮下接种5×105个患者A体内的癌细胞经过扩增得到的癌细胞。在小鼠接种肿瘤后第3天,第6天,第9天,第14天和第20天分别在小鼠皮下注射50万个不同纳米粒子辅助分选和扩增后得到的癌细胞特异性T细胞或者100μL PBS。监测小鼠肿瘤生长速度和小鼠生存期。
(5)实验结果
如图26所示,纳米粒子1和纳米粒子2分选扩增得到的到癌细胞特异性T细胞效果相同,而且,被激活后可以分泌IFN-γ的T细胞和可以表达FASL的T细胞重合,而且,本发明分选扩增得到的癌细胞特异性T细胞可以有效杀伤癌细胞,使用癌症患者自身的肿瘤组织制备的纳米粒子分选扩增的T细胞效果好于使用单个其他患者的肿瘤组织制备的纳米粒子;而且使用多个癌细胞系的抗原组分制备纳米粒子效果与患者自身肿瘤组织制备的纳米粒子效果一样。
实施例26癌细胞特异性T细胞用于黑色素瘤的治疗
(1)抗原组分的收集
收集培养的B16-F10、B16-F1、S91、ME、K735、B16-BL6和MEC57癌细胞系,并将上述癌细胞系按数量比1:1:1:1:1:1:1混合,然后将混合细胞使用0.2M精氨酸和0.1M盐酸氨基脲裂解,裂解后将所有裂解组分使用0.2M精氨酸和0.1M盐酸氨基脲溶解,然后在其中逐滴加入饱和硫酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀溶解于0.2M精氨酸和0.1M盐酸氨基脲水溶液备用,将上清液在95℃加热10分钟后将所得样品在3000g离心5分钟,弃去上清液后将沉淀溶解于0.2M精氨酸和0.1M盐酸氨基脲水溶液;然后将使用0.2M精氨酸和0.1M盐酸氨基脲溶解的盐析后的沉淀和加热后的沉淀合并后即为制备纳米粒子1的抗原组分1。
或者收集培养的B16-F10癌细胞系,并将上述癌细胞使用0.2M精氨酸和0.1M盐酸氨基脲裂解,裂解后将所有裂解组分使用0.2M精氨酸和0.1M盐酸氨基脲溶解,然后在其中逐滴加入饱和硫酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀溶解于0.2M精氨酸和0.1M盐酸氨基脲水溶液备用,将上清液在95℃加热10分钟后将所得样品在3000g离心5分钟,弃去上清液后将沉淀溶解于0.2M精氨酸和0.1M盐酸氨基脲水溶液;然后将使用0.2M精氨酸和0.1M盐酸氨基脲溶解的盐析后的沉淀和加热后的沉淀合并后即为制备纳米粒子2的抗原组分2。
(2)纳米粒子的制备
本实施例中纳米粒子1(Nanoparticle 1)采用复乳法制备。粒子制备材料PLGA分子量为10KDa-20KDa,所采用的免疫佐剂为poly(I:C)、CpG 7909和CpG2395。制备方法如前所述,先在纳米粒子内部负载抗原组分1和佐剂,然后将100mg纳米粒子在12000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1(纳米疫苗1)平均粒径为280nm左右,每1mg PLGA粒子约负载250μg蛋白质或多肽组分,负载poly(I:C)、CpG7909和CpG2395各0.01mg。
本实施例中纳米粒子2(Nanoparticle 2)的制备材料和制备方法同纳米粒子1。先在纳米粒子内部负载抗原组分2和佐剂,然后将100mg纳米粒子在12000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该粒子2平均粒径为280nm左右,每1mg PLGA粒子约负载250μg蛋白质或多肽组分,负载poly(I:C)、CpG7909和CpG2395各0.01mg。
(3)癌症特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天给小鼠皮下接种1.5×105个B16F10小鼠黑色素瘤癌细胞。在第6天、第8天、第10天、第12天、第14天、第16天、第18天、第20天分别给每只小鼠腹腔注射150μg PD-L1抗体。在第22天处死小鼠,收集小鼠的外周血,然后制备小鼠PBMC,即为与纳米粒子共孵育的混合免疫细胞。
将5000万个上述混合免疫细胞、4mg纳米粒子1在10mL RPMI 1640完全培养基中共孵育6小时,然后采用流式细胞术分选孵育后的CD3+CD69+T细胞,即为可识别癌症抗原的癌症特异性T细胞(细胞活率为85%)。将上述分选得到的CD3+CD69+T细胞与IL-2(100U/mL)、IL-7(100U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育48天后得到T细胞1。
或者将5000万个上述混合免疫细胞、4mg纳米粒子1在10mL RPMI 1640完全培养基中共孵育6小时,然后采用流式细胞术分选孵育后的CD3+T细胞(细胞活率为85%)。将上述分选得到的CD3+T细胞与IL-2(100U/mL)、IL-7(100U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育48天后得到T细胞2。
或者将5000万个上述混合免疫细胞、4mg纳米粒子1在10mL RPMI 1640完全培养基中共孵育6小时,然后采用流式细胞术分选孵育后的CD3+IFN-γ+T细胞(细胞活率为0%)。将上述分选得到的CD3+IFN-γ+T细胞与IL-2(100U/mL)、IL-7(100U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育48天后得到T细胞3。
或者将5000万个上述混合免疫细胞、4mg纳米粒子1在10mL RPMI 1640完全培养基中共孵育6小时,然后采用流式细胞术分选孵育后的CD3+FOXP3+细胞(细胞活率为85%)。将上述分选得到的CD3+FOXP3+T细胞与IL-2(100U/mL)、IL-7(100U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育48天后得到T细胞4。
将5000万个上述混合免疫细胞、4mg纳米粒子2在10mL RPMI 1640完全培养基中共孵育6小时,然后采用流式细胞术分选孵育后的CD3+CD69+T细胞,即为可识别癌症抗原的癌症特异性T细胞(细胞活率为85%)。将上述分选得到的CD3+CD69+T细胞与IL-2(100U/mL)、IL-7(100U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育48天后得到T细胞5。
(4)纳米疫苗治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠,第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。在小鼠接种肿瘤前第3天,第6天,第9天,第14天和第20天分别在小鼠皮下接种100万个T细胞(T细胞1,或者T细胞2,或者T细胞3,或者T细胞4,或者T细胞5)或者100μL PBS或者2mg纳米粒子1。监测小鼠肿瘤生长速度和小鼠生存期。
(4)实验结果
如图27所示,PBS组小鼠肿瘤体积快速长大。T细胞和纳米粒子1处理的小鼠,其肿瘤生长速度都明显变慢,生存期明显延长,其中一部分小鼠无瘤痊愈。而且,T细胞1效果好于T细胞2、T细胞3、T细胞4和T细胞5和纳米粒子1。这说明,先将癌细胞特异性T细胞分选出来再扩增是必须的;而且,使用适当的激活标志物确保分选出高活率的T细胞后再进行扩增是必须的。由于体内癌细胞微环境的差异以及癌细胞的高度异质性,使用多种癌细胞系相比单一癌细胞系可以覆盖更全的抗原谱和更多样的抗原,因而效果更好。注射T细胞1效果好于直接注射纳米粒子(疫苗)1,说明分选扩增后回输癌细胞特异性T细胞效果好于直接体内注射纳米粒子(疫苗)。
实施例27T细胞用于结肠癌的治疗
(1)抗原组分的收集
收集培养的CMT93、Colon26(C26)、CT26、MC38、MC26癌细胞系,并将上述5种癌细胞系按数量比1:1:1:4:1混合,然后将混合细胞使用0.2M盐酸甲基胍和0.05M盐酸聚六亚甲基胍裂解,裂解后将所有裂解组分使用0.2M盐酸甲基胍和0.05M盐酸聚六亚甲基胍溶解,然后在其中逐滴加入饱和硫酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀溶解于0.2M盐酸甲基胍和0.05M盐酸聚六亚甲基胍水溶液备用,将上清液在95℃加热10分钟后将所得样品在3000g离心5分钟,弃去上清液后将沉淀溶解于0.2M盐酸甲基胍和0.05M盐酸聚六亚甲基胍水溶液;然后将使用0.2M盐酸甲基胍和0.05M盐酸聚六亚甲基胍溶液的盐析后的沉淀和加热后的沉淀合并后即为制备纳米粒子1的抗原组分1。
(2)纳米粒子的制备
本实施例中纳米粒子1(Nanoparticle 1)或称纳米疫苗1(Nanovacccine 1)采用复乳法制备。粒子制备材料PLGA分子量为10KDa-20KDa,负载的免疫佐剂为poly(I:C)和CpG7909。制备方法如前所述,先在纳米粒子内部负载抗原组分1和佐剂,然后将100mg纳米粒子在13000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1平均粒径为280nm左右,每1mg PLGA纳米粒子约负载25μg蛋白质或多肽组分,每1mgPLGA纳米粒负载poly(I:C)和CpG7909各0.01mg。
(3)癌症特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天给小鼠皮下接种1×106个MC38小鼠结肠癌细胞。在第6天、第8天、第10天、第12天、第14天、第16天、第18天、第20天和第22天分别给每只小鼠腹腔注射150μg PD-L1抗体。在第24天处死小鼠,收集小鼠的淋巴结,然后使用流式细胞术从淋巴结细胞中分选出CD3+CD69-T细胞、CD19+B细胞和CD11c+DC,然后将上述CD19+B细胞和CD11c+DC按数量比1:1混合,即为与纳米粒子共孵育的混合抗原提呈细胞。
将500万个上述混合抗原提呈细胞、100μg纳米粒子1在10mL RPMI 1640完全培养基中共孵育4小时(37℃,5%CO2),然后再400g离心4分钟去除上清液后(含纳米粒子)收集孵育后的混合抗原提呈细胞。将孵育后的混合抗原提呈细胞在4%的多聚甲醛溶液中固定30分钟后洗涤,然后将固定后的抗原提呈细胞与CD3+CD69-T细胞按数量比2:1混合后在10mL RPMI 1640完全培养基中共孵育36小时(37℃,5%CO2),然后采用流式细胞术分选孵育后的CD3+CD69+T细胞,即为可识别癌症抗原的癌症特异性T细胞(细胞活率为90%)。将上述分选得到的CD3+CD69+T细胞与IL-2(1000U/mL)、IL-7(10U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育42天后得到T细胞1。
将500万个上述混合抗原提呈细胞、100μg纳米粒子1在10mL RPMI 1640完全培养基中共孵育4小时(37℃,5%CO2),然后再400g离心4分钟去除上清液后(含纳米粒子)收集孵育后的混合抗原提呈细胞。将孵育后的混合抗原提呈细胞在4%的多聚甲醛溶液中固定30分钟后洗涤,然后将固定后的抗原提呈细胞与CD3+CD69-T细胞按数量比2:1混合后在10mL RPMI 1640完全培养基中共孵育36小时(37℃,5%CO2),然后采用流式细胞术分选孵育后的CD3+T细胞(细胞活率为90%)。将上述分选得到的CD3+T细胞与IL-2(1000U/mL)、IL-7(10U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育42天后得到T细胞2。
将500万个上述混合抗原提呈细胞、100μg纳米粒子1在10mL RPMI 1640完全培养基中共孵育4小时(37℃,5%CO2),然后再400g离心4分钟去除上清液后(汉纳米粒子)收集孵育后的混合抗原提呈细胞。将孵育后的混合抗原提呈细胞在4%的多聚甲醛溶液中固定30分钟后洗涤,然后将固定后的抗原提呈细胞与CD3+CD69-T细胞按数量比2:1混合后在10mL RPMI 1640完全培养基中共孵育36小时(37℃,5%CO2),然后采用流式细胞术分选孵育后的CD3+IFN-γ+T细胞(细胞活率为0%)。将上述分选得到的CD3+IFN-γ+T细胞与IL-2(1000U/mL)、IL-7(10U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育42天后得到T细胞3。
(4)纳米疫苗治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠,第0天给每只小鼠背部右下方皮下接种1.0×106个MC38结肠癌细胞。在小鼠接种肿瘤前第3天,第6天,第9天,第14天和第20天分别在小鼠皮下接种100万个T细胞(T细胞1,或者T细胞2,或者T细胞3或者2mg纳米粒子1)或者100μL PBS。监测小鼠肿瘤生长速度和小鼠生存期。
(4)实验结果
如图28所示,PBS组小鼠肿瘤体积快速长大,小鼠很快死亡。T细胞处理的小鼠,其肿瘤生长速度都明显变慢,生存期明显延长,其中一部分小鼠无瘤痊愈。而且,T细胞1效果好于T细胞2、T细胞3和纳米粒子1。这说明,先将癌细胞特异性T细胞分选出来再扩增是必须的,而且,使用适当的激活标志物确保分选出高活率的T细胞后再进行扩增是必须的。而且,分选扩增后回输T细胞治疗效果好于直接注射使用纳米粒子。
实施例28分选扩增T细胞用于肺癌的预防
(1)癌细胞的裂解
将培养的LLC细胞在400g离心5分钟,然后用PBS洗涤两遍后重悬于超纯水中。所得癌细胞分别采用紫外线和高温加热进行灭活和变性处理,然后加入超纯水并反复冻融5次辅以超声裂解癌细胞,将细胞裂解物在5000g离心10分钟,上清液即为水溶性组分,将沉淀物使用10%辛基葡萄糖苷溶解后即为溶解后的原非水溶性组分,将水溶性组分和非水溶性组分按质量比2:1混合,即为制备微米粒子所需的抗原组分1。
(2)微米粒子的制备
本实施例中制备微米粒子1采用复乳法,微米粒子骨架材料PLGA分子量为38KDa-54KDa,所采用的免疫佐剂为CpG1018和Poly ICLC。制备时先在内部负载抗原组分1和佐剂,然后将100mg微米粒子在9000g离心20分钟,使用10mL含4%海藻糖的超纯水重悬后干燥48h后备用。该微米粒子系统平均粒径为5.0μm左右;每1mg PLGA微米粒子约负载410μg抗原组分的蛋白质或多肽组分,负载CpG1018和Poly ICLC各0.01mg。
(3)树突状细胞的制备
同实施例16。
(4)树突状细胞的激活
将500万个BMDC、2mg微米粒子和IL-15(20ng/mL)在5mL RPMI 1640(10%FBS)培养基中共孵育8h,尔后收集BMDC使用射线辐照激活的树突状细胞以没活树突状细胞,将灭活的树突状细胞用于激活T细胞。
(5)癌细胞特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天、第4天、第7天、第14天、第21天和第28天分别皮下注射100μL含2mg PLGA的微米粒子1。在第32天处死小鼠,收集小鼠的外周血,然后从外周血中分离外周血单个核细胞(PBMC),使用流式细胞术从PBMC中分选得到CD3+T细胞。将分选得到的CD3+T细胞(200万个)、步骤4制备的灭活的BMDC(300万个)以及IL-7(10ng/mL)在10mL RPMI1640完全培养基中共孵育18小时。然后采用流式细胞术分选孵育后的T细胞中的CD3+CD69+T细胞(细胞活率85%),即为可识别癌症抗原的癌症特异性T细胞。将上述分选得到的30万个CD3+CD69+T细胞与IL-2(1000U/mL)、IL-7(1000U/mL)、IL-15(1000U/mL)以及αCD3抗体在(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育14天以扩增癌细胞特异性T细胞(细胞活率85%)。
(6)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠。在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠皮下注射100μL含120万个扩增得到的CD3+T细胞。同时在第0天给每只小鼠皮下注射接种1×106个LLC细胞,肿瘤体积和生存期监测方法同上。
(7)实验结果
如图29所示,与对照组相比,微米粒分选扩增得到的癌细胞特异性T细胞处理组肺癌肿瘤生长速度明显变慢且小鼠生存期明显延长。
实施例29癌细胞特异性T细胞用于黑色素瘤的治疗
(1)抗原组分的收集
收集培养的B16-F10、B16-F1、S91、ME、K735、B16-BL6和MEC57癌细胞系,并将上述癌细胞系按数量比1:1:1:1:1:1:1混合,然后将混合细胞使用8M尿素水溶液裂解,裂解后将所有裂解组分使用8M尿素水溶液溶解,然后在其中逐滴加入饱和硫酸铵水溶液,待沉淀完全后将所得样品在3000g离心5分钟,将沉淀溶解于8M尿素水溶液备用,将上清液在95℃加热10分钟后将所得样品在3000g离心5分钟,弃去上清液后将沉淀溶解于8M尿素水溶液;然后将使用8M尿素水溶液溶解的盐析后的沉淀和溶解的加热后的沉淀合并后即为制备纳米粒子1的抗原组分1。
(2)纳米粒子的制备
本实施例中纳米粒子1(Nanoparticle 1)采用复乳法制备。粒子制备材料PLGA分子量为10KDa-20KDa,所采用的免疫佐剂为poly(I:C)、CpG 7909和CpG2395。制备方法如前所述,先在纳米粒子内部负载抗原组分1和佐剂,然后将100mg纳米粒子在12000g离心20分钟,并使用10mL含4%海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1(纳米疫苗1)平均粒径为280nm左右,每1mg PLGA粒子约负载250μg蛋白质或多肽+组分,每1mgPLGA粒子负载poly(I:C)、CpG7909和CpG2395各0.01mg。
(3)癌症特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠,在第0天给小鼠皮下接种1.5×105个B16F10小鼠黑色素瘤癌细胞。在第6天、第8天、第10天、第12天、第14天、第16天、第18天、第20天分别给每只小鼠腹腔注射150μg PD-L1抗体。在第22天处死小鼠,收集小鼠的外周血,然后制备小鼠PBMC,即为与纳米粒子共孵育的混合免疫细胞。
将5000万个上述混合免疫细胞、4mg纳米粒子1、IL-2(10ng/mL)、IL-7(10ng/mL)和IL-15(10ng/mL)在10mL RPMI 1640完全培养基中共孵育一定时间(1小时,6小时或者168小时),然后采用流式细胞术分选孵育后的CD3+CD69+T细胞,即为可识别癌症抗原的癌症特异性T细胞(细胞活率为80%)。将上述分选得到的CD3+CD69+T细胞与IL-2(100U/mL)、IL-7(100U/mL)以及αCD3抗体(10ng/mL)和αCD28抗体(10ng/mL)在10mL的DMEM完全培养基(37℃,5%CO2)中共孵育48天后得到T细胞(细胞活率为80%)。其中,纳米粒子与免疫细胞共孵育时间为6小时分选扩增得到的为T细胞1;纳米粒子与免疫细胞共孵育时间为1小时分选扩增得到的为T细胞2;纳米粒子与免疫细胞共孵育时间为168小时分选扩增得到的为T细胞3。
或者直接将分选得到的5000万个上述混合免疫细胞、4mg纳米粒子1、IL-2(10ng/mL)、IL-7(10ng/mL)和IL-15(10ng/mL)在10mL RPMI 1640完全培养基中共孵育6小时,然后不进行任何分选和扩增,所得细胞为混合细胞T细胞4(细胞活率80%)。
(4)纳米疫苗治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠,第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。在小鼠接种肿瘤后第3天,第6天,第9天,第14天和第20天分别在小鼠皮下注射10万个T细胞(T细胞1,或者T细胞2,或者T细胞3,或者T细胞4)或者注射100μL PBS或者注射2mg纳米粒子1(Nanoparticle 1,nanovaccine 1)。监测小鼠肿瘤生长速度和小鼠生存期。
(4)实验结果
如图27所示,PBS组小鼠肿瘤体积快速长大。T细胞处理的小鼠,其肿瘤生长速度都明显变慢。而且,T细胞1效果好于T细胞2、T细胞3和T细胞4。这说明,纳米粒子在分选癌细胞特异性T细胞时,孵育适当时间非常关键。而且,在孵育完成后进行适当的分选和扩增才能达到更佳效果。
在部分实施例中,纳米粒子/微米粒子与抗原提呈细胞和T细胞同时共孵育,在部分实施例中,纳米粒子/微米粒子先与抗原提呈细胞共孵育然后再将孵育后的抗原提呈细胞与T细胞共孵育。当使用纳米粒子/微米粒子先与抗原提呈细胞共孵育然后再将孵育后的抗原提呈细胞与T细胞共孵育时,纳米粒子/微米粒子与抗原提呈细胞共孵育后可以不做其他处理直接与T细胞共孵育,也可以将孵育后的抗原提呈细胞先进行固定处理或者射线辐照处理后再与T细胞共孵育。与纳米粒子共孵育后的抗原提呈细胞的处理方式既可以是使用多聚甲醛等试剂固定,也可以进行射线照射处理,或者进行其他灭活孵育后的抗原提呈细胞的处理。先将纳米粒子与抗原提呈细胞共孵育,再将共孵育后的抗原提呈细胞再与T细胞共孵育时,抗原提呈细胞可以是活细胞,也可以是死细胞。
研究者发现,具有结构式1所示结构的含有胍基或者脲结构的化合物(比如本发明所述尿素、盐酸胍、二甲双胍和盐酸甲基胍等化合物)都能够作为溶解液中的溶解剂使用来溶解癌细胞/肿瘤组织中的非水溶性组分或者经过盐析等处理产生的沉淀组分。在实际应用中,其他含有结构式1中所列结构的化合物理论上也应该可以作为溶解剂使用,也在本发明覆盖范围内。
由于篇幅所限,本发明实施例中仅列举和使用了CD69、CD25、HLA-DR、CD107a、FASL等几种表面标志物作为T细胞被激活的标志物来分选被激活的癌细胞特异性T细胞,在实际应用中也可以使用任何其他可以用来表示T细胞被激活的表面标志物,可以用来作为表面标志物的分子包括但不限于: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、CXCR 1、CD103、CD45RO、ICOS(CD278)、VTCN1、HLA2、LGAL59、CCR7、CD357、BCL6、TCF-1、CD38、CD27等当中的任一种或其任意组合。上述任一种可以表示T细胞被激活的表面标志物或者一种以上表面标志物的组分也在本发明覆盖范围内。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (114)

  1. 一种来源于自体或同种异体的用于预防或治疗癌症的癌症特异性T细胞的制备方法,具体包括如下步骤:先分离得到外周血或者外周免疫器官的免疫细胞,然后与抗原提呈细胞以及负载癌症全细胞组分或含全细胞部分抗原组分的纳米粒子和/或微米粒子共孵育一段时间激活癌症特异性T细胞,尔后分离得到被癌症抗原激活的癌症特异性T细胞,经过体外扩增后回输机体内发挥抗癌作用;
    优选地,所述制备方法具体包括如下步骤:
    (1)先分离得到外周血或者外周免疫器官的免疫细胞,或者从所述免疫细胞中分选得到T细胞;
    (2)将负载有肿瘤抗原组分的微米粒子和/或纳米粒子与抗原提呈细胞以及T细胞混合后共孵育一定时间,然后分选出被激活并表达特定细胞标志物的T细胞;
    (3)将步骤(2)分选得到的表达特定细胞标志物的T细胞与细胞因子和/或抗体共孵育,获得扩增后的特异性T细胞;
    优选地,步骤(2)中,所述肿瘤抗原组分为肿瘤组织/癌细胞全细胞裂解物组分或者肿瘤组织/癌细胞全细胞裂解物组分中的一部分组分;全细胞裂解物组分可以分为水溶性组分和含有溶解剂的溶解液溶解的非水溶性组分;
    优选地,步骤(2)中,所述肿瘤抗原组分由一种或多种癌细胞和/或肿瘤组织的全细胞裂解得到,或者由一种或多种癌细胞和/或肿瘤组织的全细胞裂解后加工得到,或者由一种或多种癌细胞和/或肿瘤组织的全细胞加工后裂解得到,优选所述癌细胞或肿瘤组织中至少有一种与目标疾病类型相同;或者所述抗原组分由一种或多种癌细胞和/或肿瘤组织中的一部分组分组成,一部分组分中含有裂解液中的蛋白质/多肽组分和/或mRNA组分;
    优选地,步骤(1)中分选得到的T细胞为CD3+T细胞、CD3+CD8+T细胞、CD4+T细胞中的任一种或其组合。
  2. 如权利要求1所述的方法,其特征在于,负载有肿瘤抗原组分的纳米粒子和/或微米粒子可以与抗原提呈细胞和T细胞三者同时共孵育以激活癌细胞特异性T细胞;也可以负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞,然后将被激活的抗原提呈细胞再单独与T细胞二者一起共孵育以激活癌细胞特异性T细胞;负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞后,抗原提呈细胞可以不经过特殊处理再去与T细胞二者共孵育激活特异性T细胞,或者抗原提呈细胞可以经过固定、辐射、照射、修饰、灭活、矿化等处理后再与T细胞二者共孵育激活特异性T细胞。在分选癌细胞特异性T细胞时,可以使用一种标志物作为激活标志物进行分选,也可以使用多个标志物的组分作为激活标志物进行分选。
  3. 如权利要求1-2任一项所述的方法,其特征在于,抗原组分除了包含蛋白质和多肽组分还可以含有mRNA组分。
  4. 如权利要求1-3任一项所述的方法,其特征在于,所述步骤(1),分离得到外周血或者外周免疫系统的免疫细胞的同种同体或同种异体在分离提取上述细胞时可以不经任何处理,或者经过放疗、免疫治疗、化疗、粒子治疗、疫苗治疗处理。
  5. 如权利要求1-4任一项所述的方法,其特征在于,所述步骤(1)和步骤(2)中的分选方法为流式细胞术、磁珠法的任一种或其组合。
  6. 如权利要求1-5任一项所述的方法,其特征在于,所述肿瘤抗原组分为肿瘤组织和/或癌细胞的细胞裂解组分,包含有肿瘤组织和/或癌细胞细胞裂解后产生的水溶性组分和非水溶性组分的一种或两种,先分别收集水溶性组分和水不溶性组分并分别制备纳米或微米粒子,非水溶性组分使用含有溶解剂的溶解液溶解;或者也可以直接采用含有溶解剂的溶解液直接裂解癌细胞或肿瘤组织并溶解全细胞组分并制备纳米或微米粒子,所述非水溶性组分由含有溶解剂的溶解液溶解;
    优选地,抗原组分为肿瘤组织/癌细胞全细胞裂解物组分时其制备方法为:(1)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用;(2)使用含有溶解剂的溶解液裂解细胞,然后使用含有溶解剂的溶解液溶解裂解后的全细胞组分。
  7. 如权利要求1-6任一项所述的方法,其特征在于,所述肿瘤抗原组分为含有肿瘤组织和/或癌细胞的全细胞裂解组分中的一部分组分时,其制备方法为:(1)先制备肿瘤组织/癌细胞的裂解液,,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中分离提取的蛋白质和多肽组分与所有非水溶性组分一起作为抗原组分使用;(2)先制备肿瘤组织/癌细胞的裂解液,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从非水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将非水溶性组分中分离提取的蛋白质和多肽组分与所有水溶性组分一起作为抗原组分使用;(3) 先制备肿瘤组织/癌细胞的裂解液,,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分和非水溶性组分中使用适当方法分别分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中和非水溶性组分中分离提取的蛋白质和多肽组分一起作为抗原组分使用;(4)或者也可以直接采用含有溶解剂的溶解液直接裂解细胞或组织并溶解全细胞组分,然后通过适当方法制备其中的蛋白质/多肽组分,作为抗原组分使用。上述制备方法中还可以增加分离提取全细胞mRNA的步骤,并将全细胞mRNA作为抗原组分的一部分使用。所述适当处理方法包括但不限于盐析、加热和酶解等处理方法;所述非水溶性组分或者经过盐析、加热和酶解等处理后产生的沉淀使用含有溶解剂的溶解液溶解。
  8. 如权利要求1-7任一项所述的方法,其特征在于,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时,或者所述纳米粒子/微米粒子与抗原提呈细胞和T细胞同时共孵育时,纳米粒子/微米粒子浓度为2.5ng/mL到50mg/mL;共孵育时间为1-168小时。
  9. 如权利要求1-8任一项所述的方法,其特征在于,所述全细胞组分在裂解前或(和)裂解后既可经过灭活或(和)变性、固化、生物矿化、离子化、化学修饰、核酸酶处理等处理后再制备纳米粒子或微米粒子;也可细胞裂解前或(和)裂解后不经过任何灭活或(和)变性、固化、生物矿化、离子化、化学修饰、核酸酶处理直接制备。
  10. 如权利要求1-9任一项所述的方法,其特征在于,肿瘤组织细胞在裂解前经过了灭活或(和)变性处理,也可以在细胞裂解后做灭活或(和)变性处理,或者也可以细胞裂解前和裂解后均做灭活或(和)变性处理。
  11. 如权利要求1-10任一项所述的方法,其特征在于,细胞裂解前或(和)裂解后的灭活或(和)变性处理方法包括紫外照射、高温加热、放射线辐照、高压、固化、生物矿化、离子化、化学修饰、核酸酶处理、胶原酶处理、冷冻干燥中的任一种或其组合。
  12. 如权利要求1-11任一项所述的方法,其特征在于,所述步骤(2),所用抗原提呈细胞与T细胞数量比大于1:1;利用纳米粒子或微米粒子通过抗原提呈细胞的提呈后体外激活外周免疫细胞中预存的癌症特异性T细胞,所述纳米粒子或微米粒子选自粒径1nm-1000nm的纳米粒子或粒径1μm-1000μm的微米粒子。
  13. 如权利要求1-12任一项所述的方法,其特征在于,所述与T细胞和纳米粒子和/或微米粒子共孵育的抗原提呈细胞来源于自体、同种异体、细胞系、干细胞或以上的任意混合物;共孵育的抗原提呈细胞为B细胞、树突状细胞、巨噬细胞或者三者的任意混合物。
  14. 如权利要求1-13任一项所述的方法,其特征在于,所述抗原提呈细胞源于自体抗原提呈细胞、同种异体抗原提呈细胞、抗原提呈细胞系或者干细胞分化而来的抗原提呈细胞,优选为树突状细胞(DC)、B细胞、巨噬细胞中的任一种或其组合;更优选的使用多于一种抗原提呈细胞的组合。
  15. 如权利要求1-14任一项所述的方法,其特征在于,所述混合共孵育选自以下三种方式中的任一种:(a)三者直接混合共孵育一定时间;(b)微米粒子和/或纳米粒子与抗原提呈细胞先共孵育一段时间,再加入T细胞共孵育;(c)微米粒子和/或纳米粒子与抗原提呈细胞先共孵育一段时间,分选出孵育后的抗原提呈细胞后将抗原提呈细胞与T细胞二者共孵育。
  16. 如权利要求1-15任一项所述的方法,其特征在于,所述混合共孵育的培养条件为在30-38℃、1-10%CO2条件下共孵育1-168h。
  17. 如权利要求1-16任一项所述的方法,其特征在于,所述混合共孵育中可加入细胞因子;所加入的细胞因子包括但不限于白介素、肿瘤坏死因子、干扰素、生长因子;优选的,所加入的细胞因子中包含白介素7(IL-7)、白介素15(IL-15)。
  18. 如权利要求1-17任一项所述的方法,其特征在于,所述步骤(2),所述分选方法为使用带有荧光或者磁性或者特定配体的抗体与T细胞表面的特定细胞标志物结合后利用流式细胞术或者磁珠法等从细胞群中分离表达特定细胞标志物的细胞。
  19. 如权利要求1-18任一项所述的方法,其特征在于,所述步骤(3),所述分选得到的表达特定细胞标志物的T细胞为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中的任一种或其组合。
  20. 如权利要求1-19任一项所述的方法,其特征在于,步骤(3)中,所述细胞因子的浓度为1-6000ng/ml,优选为5-200ng/ml,更优选10-30ng/ml。
  21. 如权利要求1-20任一项所述的方法,其特征在于,所述步骤(3),所述细胞因子包括但不限于白介素、干扰素、肿瘤坏死因子。
  22. 如权利要求1-21任一项所述的方法,其特征在于,所述白介素包括但不限于白介素2(IL-2)、白介素7(IL-7)、白介素12(IL-12)、白介素15(IL-15)、白介素17(IL-17),白介素21(IL-21)。
  23. 如权利要求1-22任一项所述的方法,其特征在于,所述抗体的浓度为1-6000ng/ml,优选为5-100ng/ml,更优选10-30ng/ml。
  24. 如权利要求1-23任一项所述的方法,其特征在于,所述抗体包括但不限于αCD3抗体、αCD28抗体、αCD80抗体、αCD86抗体、αOX40抗体中的任一种或其组合。
  25. 如权利要求1-24任一项所述的方法,其特征在于,所述纳米粒子/微米粒子与抗原提呈细胞和T细胞混合物共孵育时间为至少1小时,优选为6-96小时。
  26. 如权利要求1-25任一项所述的方法,其特征在于,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时间为至少1小时,优选为6-96小时。
  27. 如权利要求1-26任一项所述的方法,其特征在于,所述激活的抗原提呈细胞和T细胞混合物共孵育时间为至少1小时,优选为6-96小时。
  28. 如权利要求1-27任一项所述的方法,其特征在于,所述扩增培养时间为至少1天,优选为4–72天。
  29. 如权利要求1-28任一项所述的方法,其特征在于,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时,或者所述纳米粒子/微米粒子与抗原提呈细胞和T细胞三者共孵育时,纳米粒子/微米粒子浓度为2.5ng/mL到50mg/mL。
  30. 如权利要求1-29任一项所述的方法,其特征在于,所述纳米粒子/微米粒子负载的抗原组分中的蛋白质和多肽组分含量高于10ng/mL。
  31. 如权利要求1-30任一项所述的方法,其特征在于,步骤(3)得到的体外扩增后得到的特异性T细胞,回输机体内发挥抗癌作用。
  32. 如权利要求1-31任一项所述的方法,其特征在于,所述纳米粒子和/或微米粒子所负载的癌症抗原为肿瘤组织和/或癌细胞的全细胞组分,含有肿瘤组织和/或癌细胞的水溶性组分和/或非水溶性组分。
  33. 如权利要求1-32任一项所述的方法,其特征在于,所述用于激活癌症特异性T细胞的纳米粒子和/或微米粒子,将一种和/或多种的肿瘤组织和/或癌细胞的组分负载到纳米粒子或微米粒子的负载方式为全细胞的水溶性成分和非水溶性成分分别或同时被包载于粒子内部,和/或分别或同时负载于粒子表面。
  34. 如权利要求1-33任一项所述的方法,其特征在于,所述用于激活癌症特异性T细胞的纳米粒子和/或微米粒子,其所负载的来源于肿瘤组织或癌细胞的全细胞组分中的原非水溶性部分采用适当增溶/溶解方法由在纯水中不溶变为在含增溶剂/溶解剂的水溶液中或有机溶剂中可溶;采用的溶解剂/增溶剂选自含有结构式1结构的化合物、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、多肽、氨基酸、糖苷和胆碱中的一种或多种;
    其中,结构式1如下:结构式1结构如下:
    ,R1为C、N、S或O,R2~R5独立地选自氢、烷基、氨基、羧基、取代或未取代胍基的至少一种;含有结构式1的化合物包括但不限于盐酸二甲双胍、硫酸二甲双胍、磺酸二甲双胍、二甲双胍盐、二甲双胍、盐酸聚六亚甲基胍、硫酸胍基丁胺、盐酸甲基胍、盐酸四甲基胍、尿素、盐酸胍、硫酸胍、磺酸胍、胍盐、其他含有胍基或脲的化合物、碳酸胍、精氨酸、胍基乙酸、胍基磷酸、氨基磺酸胍、胍基琥珀酸、盐酸氨基脲、氨基甲酰脲、乙酰脲、磺酰脲类化合物(格列本脲、格列齐特、格列喹酮、格列美脲等)、硫脲类化合物(硫氧嘧啶类、咪唑类等)、亚硝基脲类等含有结构式1结构的化合物。
  35. 如权利要求1-34任一项所述的方法,其特征在于,所述用于激活癌症特异性T细胞的纳米粒子和/或微米粒子的表面连接有主动靶向抗原提呈细胞的靶头。
  36. 如权利要求1-35任一项所述的方法,其特征在于,所述水溶性组分和/或非水溶性组分负载于所述癌症疫苗的表面的方式包括吸附、共价连接、电荷相互作用、疏水相互作用、一步或多步的固化、矿化和包裹中的至少一种。
  37. 如权利要求1-36任一项所述的方法,其特征在于,所述纳米粒子的粒径为1nm-1000nm;所述微米粒子的粒径为1μm-1000μm。
  38. 如权利要求1-37任一项所述的方法,其特征在于,所述的纳米尺寸粒子或微米尺寸粒子表面为电中性,带负电或者带正电。
  39. 如权利要求1-38任一项所述的方法,其特征在于,所述纳米疫苗和/或微米疫苗的制备材料为有机合成高分子材料、天然高分子材料或者无机材料。
  40. 如权利要求1-39任一项所述的方法,其特征在于,所述有机合成高分子材料为PLGA、PLA、PGA、PEG、PCL、Poloxamer、PVA、PVP、PEI、PTMC、聚酸酐、PDON、PPDO、PMMA、聚氨基酸、合成多肽;所述的天然高分子材料为卵磷脂、胆固醇、海藻酸钠、白蛋白、胶原蛋白、明胶、细胞膜成分、淀粉、糖类、多肽;所述的无机材料为三氧化二铁、四氧化三铁、碳酸钙、磷酸钙。
  41. 如权利要求1-40任一项所述的方法,其特征在于,所述的用于激活癌症特异性T细胞的纳米粒子和/或微米粒子,可将一种和/或多种的肿瘤组织和/或癌细胞的组分与免疫佐剂一起共负载于纳米粒子或微米粒子。
  42. 如权利要求1-41任一项所述的方法,其特征在于,所述水溶性部分和非水溶性部分都可以被含溶解剂/增溶剂的增溶水溶液或有机溶剂溶解。溶解剂/增溶剂为可以增加蛋白质或多肽在水溶液中溶解性的溶解剂/增溶剂中的至少一种;有机溶剂为可以溶解蛋白质或多肽的有机溶剂。
  43. 如权利要求1-42任一项所述的方法,其特征在于,所述的原非水溶性部分采用适当增溶/溶解方法由在纯水中不溶变为在含溶解剂/增溶剂的水溶液中或有机溶剂中可溶;采用的溶解剂/增溶剂选自含有结构式1结构的化合物、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、多肽、氨基酸、糖苷和胆碱中的一种或多种;其中,结构式1如下:结构式1结构如下:
    ,R1为C、N、S或O,R2~R5独立地选自氢、烷基、氨基、羧基、取代或未取代胍基的至少一种。含有结构式1的化合物包括但不限于盐酸二甲双胍、硫酸二甲双胍、磺酸二甲双胍、二甲双胍盐、二甲双胍、盐酸聚六亚甲基胍、硫酸胍基丁胺、盐酸甲基胍、盐酸四甲基胍、尿素、盐酸胍、硫酸胍、磺酸胍、胍盐、其他含有胍基或脲的化合物、碳酸胍、精氨酸、胍基乙酸、胍基磷酸、氨基磺酸胍、胍基琥珀酸、盐酸氨基脲、氨基甲酰脲、乙酰脲、磺酰脲类化合物(格列本脲、格列齐特、格列喹酮、格列美脲等)、硫脲类化合物(硫氧嘧啶类、咪唑类等)、亚硝基脲类含有结构式1结构的化合物。
  44. 如权利要求1-43任一项所述的方法,其特征在于,用于激活癌症特异性T细胞的纳米粒子或微米粒子所负载的细胞组分来源于一种或多种癌细胞和/或一种或多种肿瘤组织全细胞中得到的组分,将非水溶性组分负载到递送粒子上,使该纳米或微米系统中含有更多的抗原,更优选地,将水溶性组分和非水溶性组分同时负载到递送粒子上,使递送粒子上负载了全细胞组分抗原。
  45. 如权利要求1-44任一项所述的方法,其特征在于,用于激活癌症特异性T细胞的纳米粒子和/或微米粒子负载的细胞组分或其混合物,混合物包括但不限于水溶性组分互相混合,或者非水溶性组分互相混合,或者全部或部分水溶性组分与全部或部分水溶性组分互相混合。
  46. 如权利要求1-45任一项所述的方法,其特征在于,在纳米粒子和/或微米粒子中,细胞组分或其混合物被负载于纳米粒子或微米粒子内部和/或表面,具体的,所述负载方式为细胞的水溶性组分和非水溶性组分分别或同时被包载于粒子内部,和/或分别或同时负载于粒子表面,包括但不限于水溶性组分同时装载于粒子中和负载于粒子表面,非水溶性组分同时装载于粒子中和负载于粒子表面,水溶性组分装载于粒子中而非水溶性组分负载于粒子表面,非水溶性组分装载于粒子中而水溶性组分负载于粒子表面,水溶性组分和非水溶性组分装载于粒子中而只有非水溶性组分负载于粒子表面,水溶性组分和非水溶性组分装载于粒子中而只有水溶性组分负载于粒子表面,水溶性组分装载于粒子中而水溶性组分和非水溶性组分同时负载于粒子表面,非水溶性组分装载于粒子中而水溶性组分和非水溶性组分同时负载于粒子表面,水溶性组分和非水溶性组分同时装载于粒子中而且水溶性组分和非水溶性组分同时负载于粒子表面。
  47. 如权利要求1-46任一项所述的方法,其特征在于,用于激活癌症特异性T细胞的纳米粒子或微米粒子的内部和/或表面还可包括免疫增强佐剂,免疫增强佐剂包括但不限于微生物来源的免疫增强剂、人或动物免疫系统的产物、固有免疫激动剂、适应性免疫激动剂、化学合成药物、真菌多糖类、中药及其他类中的至少一类;免疫增强佐剂包括但不限于模式识别受体激动剂、卡介苗(BCG)、锰相关佐剂、卡介苗细胞壁骨架、卡介苗甲醇提取残余物、卡介苗胞壁酰二肽、草分枝杆菌、多抗甲素、矿物油、病毒样颗粒、免疫增强的再造流感病毒小体、霍乱肠毒素、皂苷及其衍生物、Resiquimod、胸腺素、新生牛肝活性肽、米喹莫特、多糖、姜黄素、免疫佐剂CpG、免疫佐剂poly(I:C)、免疫佐剂poly ICLC、短小棒状杆菌苗、溶血性链球菌制剂、辅酶Q10、左旋咪唑、聚胞苷酸、锰佐剂、铝佐剂、钙佐剂、各种细胞因子、白细胞介素、干扰素、聚肌苷酸、聚腺苷酸、明矾、磷酸铝、羊毛脂、角鲨烯、细胞因子、植物油、内毒素、脂质体佐剂、MF59、双链RNA、双链DNA、铝相关佐剂、CAF01、人参、黄芪的有效成分中的至少一种。本领域技术人员可以理解,此处为列举并非穷举,免疫增强佐剂也可采用其他可使免疫反应增强的物质。
  48. 如权利要求1-47任一项所述的方法,其特征在于,免疫增强佐剂优选Toll样受体激动剂。
  49. 如权利要求1-48任一项所述的方法,其特征在于,免疫增强佐剂优选两种以上Toll样受体激动剂联用。
  50. 如权利要求1-49任一项所述的方法,其特征在于,将免疫佐剂与细胞组分共负载于纳米粒子或微米粒子中,在纳米粒子或微米粒子被抗原提呈细胞吞噬后可以更好的癌症特异性T细胞。
  51. 如权利要求1-50任一项所述的方法,其特征在于,微米粒子或纳米粒子中负载增加纳米粒子和/或微米粒子或其负载的抗原从溶酶体中逃逸至细胞质的物质;所述增加溶酶体逃逸的物质包括氨基酸、多肽、糖类、脂类、可以产生质子海绵效应的无机盐,优选地,所述增加溶酶体逃逸的物质中的氨基酸包含带正电的氨基酸,优选地,所述增加溶酶体逃逸的多肽种包含带正电的氨基酸。
  52. 如权利要求1-51任一项所述的方法,其特征在于,纳米粒子或微米粒子的表面可以不连接具有主动靶向功能的靶头,或者连接有主动靶向功能的靶头,
    优选地,主动靶向靶头可为甘露糖、CD19抗体、CD20抗体、BCMA抗体、CD32抗体、CD11c抗体、CD103抗体、CD44抗体等常用的靶头,带领粒子系统靶向输送到抗原提呈细胞。
  53. 如权利要求1-52任一项所述的方法,其特征在于,所述纳米粒子和/或微米粒子的表面连接有主动靶向抗原提呈细胞的靶头。
  54. 如权利要求1-53任一项所述的方法,其特征在于,纳米粒子或微米粒子在制备过程中可以不做修饰处理,也可以采用适当的修饰技术以提高纳米粒子或微米粒子的抗原负载量。修饰技术包括但不限于生物矿化(如硅化、钙化、镁化)、凝胶化、交联、化学修饰、添加带电物质。
  55. 如权利要求1-54任一项所述的方法,其特征在于,细胞组分或其混合物被负载于纳米粒子或微米粒子内部的形式为任何可以将细胞组分或其混合物负载于纳米粒子或微米粒子内部的方式。
  56. 如权利要求1-55任一项所述的方法,其特征在于,细胞组分或其混合物被负载于纳米粒子或微米粒子表面的方式包括但不限于吸附、共价连接、电荷相互作用(如添加带正电的物质、添加带负电的物质)、疏水相互作用、一步或多步的固化、矿化、包裹等。
  57. 如权利要求1-56任一项所述的方法,其特征在于,负载于纳米粒子或微米粒子表面的水溶性组分和/或非水溶性组分负载后为一层或多层,疫苗表面负载多层水溶性组分和/或非水溶性组分时,层与层之间为修饰物。
  58. 如权利要求1-57任一项所述的方法,其特征在于,所诉纳米粒子的粒径大小为1nm-1000nm,更优选地,粒径大小为30nm-1000nm,最优选地,粒径大小为100nm-600nm。
  59. 如权利要求1-58任一项所述的方法,其特征在于,微米粒子的粒径大小为1μm-1000μm,更优选地,粒径大小为1μm-100μm,更优选地,粒径大小为1μm-10μm,最优选地,粒径大小为1μm-5μm。
  60. 如权利要求1-59任一项所述的方法,其特征在于,所述纳米粒子或微米粒子的形状包括球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形或圆盘形中的任一种。
  61. 如权利要求1-60任一项所述的方法,其特征在于,所述水溶性组分和/或非水溶性组分负载于所述癌症疫苗的表面的方式包括吸附、共价连接、电荷相互作用、疏水相互作用、一步或多步的固化、矿化和包裹中的至少一种。
  62. 如权利要求1-61任一项所述的方法,其特征在于,所述纳米疫苗和/或微米疫苗的制备材料为有机合成高分子材料、天然高分子材料或者无机材料。
  63. 如权利要求1-62任一项所述的方法,其特征在于,所述有机合成高分子材料为生物相容或可降解的高分子材料,包括PLGA、PLA、PGA、PLGA-PEG、PLA-PEG、PGA-PEG、PEG、PCL、Poloxamer、PVA、PVP、PEI、PTMC、聚酸酐、PDON、PPDO、PMMA、聚氨基酸、合成多肽、合成脂质中的任一种或其组合。
  64. 如权利要求1-63任一项所述的方法,其特征在于,所述天然高分子材料为生物相容或可降解的高分子材料,包括为卵磷脂、胆固醇、海藻酸钠、白蛋白、胶原蛋白、明胶、细胞膜成分、淀粉、糖类、多肽中的任一种或其组合。
  65. 如权利要求1-64任一项所述的方法,其特征在于,所述无机材料为无明显生物毒性的材料,包括但不限于三氧化二铁、四氧化三铁、碳酸钙、磷酸钙等。
  66. 如权利要求1所述的一种来源于自体或同种异体的用于预防或治疗癌症的癌症特异性T细胞的制备方法,具体包括如下步骤:
    (1)从外周血或外周免疫器官的免疫细胞中分离单个核细胞PBMC;优选地,所述PBMC经过第一步分选,获得以下至少一种效应性细胞:CD3+CD8+T细胞、CD19+B细胞、CD3+T细胞、CD8+T细胞、CD4+T细胞、B220+B细胞、CD69-的PBMC细胞、CD25-的PBMC细胞、CD3+CD69+T细胞、CD11c+DC细胞;
    (2)制备负载有肿瘤抗原组分的纳米粒子和/或微米粒子;
    优选地,所述肿瘤抗原组分的制备为先分离得到肿瘤细胞或组织,裂解所述肿瘤细胞或组织获得水溶性组分、非水溶性组分、全组分的任一种或其组合;
    优选地,负载有肿瘤抗原组分的纳米粒子和/或微米粒子利用复乳法制备;
    (3)在培养基中加入负载有肿瘤抗原组分的纳米粒子和/或微米粒子,与(1)所述的PBMC细胞或T细胞共孵育,得细胞培养物,从细胞培养物中进一步分选出特异性T细胞,所述特异性T细胞为包括但不限于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+等T细胞的任一种或其组合;
    优选地,共孵育过程还加入5-5000万个/ml的抗原提呈细胞,所述抗原提呈细胞为B细胞、DC细胞、巨噬细胞中的任一种或其组合;
    优选地,负载有肿瘤抗原组分的纳米粒子和/或微米粒子粒子可以与抗原提呈细胞和T细胞三者同时共孵育以激活癌细胞特异性T细胞;也可以负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞,然后将被激活的抗原提呈细胞再单独与T细胞二者一起共孵育以激活癌细胞特异性T细胞;负载有肿瘤抗原组分的纳米粒子和/或微米粒子先与抗原提呈细胞共孵育激活抗原提呈细胞后,抗原提呈细胞可以不经过特殊处理再去与T细胞二者共孵育激活特异性T细胞,或者抗原提呈细胞可以经过固定、辐射、照射、修饰、灭活、矿化等处理后再与T细胞二者共孵育激活特异性T细胞;
    优选地,在培养基中加入2.5ng-50mg/ml的负载有肿瘤抗原组分的纳米粒子和/或微米粒子,与1-5000万个/ml的PBMC细胞或分选细胞,在30-38℃、1-5%CO2条件下共孵育4-96h,得细胞培养物;
    优选地,共孵育过程还加入10-500ng/ml的白介素,所述白介素为IL-2、IL-7、IL-12、IL-15、IL-17、IL-21中的任一种或其组合;
    所述培养基为DMEM高糖完全培养基、RPM1640培养基、AIMV无血清培养基中的任一种;
    所述的共孵育是在30-38℃条件下孵育1-168h,优选为4-96h,更优选为6-72h;
    所述特异性T细胞活率大于60%,优选大于70%,更优选大于80%;
    (4)扩增(3)获得的特异性T细胞。
  67. 如权利要求66所述的制备方法,其特征在于,所述扩增步骤为,在扩增培养基中,加入10-50万个细胞/ml的步骤(2)分选得到的特异性T细胞,在30-38℃、1-5%CO2条件下共孵育,每2-3天使用扩增培养基换液,共孵育4-72天后,获得扩增后的特异性T细胞。
  68. 如权利要求66-67任一项所述的制备方法,其特征在于,优选地,在扩增培养基中扩增,所述扩增培养基为DMEM高糖完全培养基、RPM1640培养基中的任一种;
    优选地,扩增培养的条件为30-38℃培养5-30天后;
    优选地,所述扩增培养基还包含200-1000U/ml的白介素、10-200ng/ml的抗体和/或1-100ng/mL粒细胞-巨噬细胞集落刺激因子(GM-CSF);
    优选地,所述白介素为IL-2、IL-7、IL-12、IL-15、IL-17、TNF-α、IL-21中的任一种或其组合;
    优选地,所述抗体为αCD3抗体、αCD28抗体中的任一种或其组合。
  69. 如权利要求66-68任一项所述的制备方法,其特征在于,所述步骤(1),所述细胞选自患肿瘤疾病个体的任何部位,优选为脾脏细胞、淋巴细胞。
  70. 如权利要求66-69任一项所述的制备方法,其特征在于,所述扩增后的特异性T细胞活率大于60%,优选大于75%,更优选大于80%。
  71. 如权利要求66-70任一项所述的制备方法,其特征在于,所述步骤(2),所述分选方法为使用带有荧光或者磁性或者特定配体的抗体与T细胞表面的特定细胞标志物结合后利用流式细胞术或者磁珠法等从细胞群中分离表达特定细胞标志物的细胞。
  72. 如权利要求66-71任一项所述的制备方法,其特征在于,所述混合共孵育中可加入细胞因子;所加入的细胞因子包括但不限于白介素、肿瘤坏死因子、干扰素、生长因子;优选的,所加入的细胞因子中包含白介素7(IL-7)、白介素15(IL-15)。
  73. 如权利要求66-72任一项所述的制备方法,其特征在于,步骤(3)中,所述细胞因子的浓度为1-6000ng/ml,优选为5-100ng/ml,更优选10-30ng/ml。
  74. 如权利要求66-73任一项所述的制备方法,其特征在于,所述步骤(3),所述细胞因子包括但不限于白介素、干扰素、肿瘤坏死因子。
  75. 如权利要求66-74任一项所述的制备方法,其特征在于,所述白介素包括但不限于白介素2(IL-2)、白介素7(IL-7)、白介素12(IL-12)、白介素15(IL-15)、白介素17(IL-17),白介素21(IL-21)。
  76. 如权利要求66-75任一项所述的制备方法,其特征在于,所述抗体的浓度为1-6000ng/ml,优选为5-100ng/ml,更优选10-30ng/ml。
  77. 如权利要求66-76任一项所述的制备方法,其特征在于,所述抗体包括但不限于αCD3抗体、αCD28抗体、αCD80抗体、αCD86抗体、αOX40抗体中的任一种或其组合。
  78. 如权利要求66-77任一项所述的制备方法,其特征在于,所述纳米粒子/微米粒子与抗原提呈细胞和T细胞混合物共孵育时间为至少1小时,优选为6-96小时。
  79. 如权利要求66-78任一项所述的制备方法,其特征在于,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时间为至少1小时,优选为6-96小时。
  80. 如权利要求66-79任一项所述的制备方法,其特征在于,所述激活的抗原提呈细胞和T细胞混合物共孵育时间为至少1小时,优选为6-96小时。
  81. 如权利要求66-80任一项所述的制备方法,其特征在于,所述扩增培养时间为至少1天,优选为4–36天。
  82. 如权利要求66-81任一项所述的制备方法,其特征在于,所述纳米粒子/微米粒子单独与抗原提呈细胞共孵育时,或者所述纳米粒子/微米粒子与抗原提呈细胞和T细胞三者共孵育时,纳米粒子/微米粒子浓度为2.5ng/mL到50mg/mL。
  83. 如权利要求66-82任一项所述的制备方法,其特征在于,所述纳米粒子/微米粒子负载的抗原组分中的蛋白质和多肽组分含量高于10ng/mL。
  84. 如权利要求66-83任一项所述的制备方法,其特征在于,肿瘤抗原组分为全细胞组分时的制备方法为:(1)先裂解癌细胞/肿瘤组织,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用;(2)使用含有溶解剂的溶解液裂解细胞,然后使用含有溶解剂的溶解液溶解裂解后的全细胞组分。
  85. 如权利要求66-84任一项所述的制备方法,所述肿瘤抗原组分为全细胞组分中一部分组分时,其制备方法为:(1)先制备肿瘤组织/癌细胞的裂解液,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中分离提取的蛋白质和多肽组分与所有非水溶性组分一起作为抗原组分使用;(2)先制备肿瘤组织/癌细胞的裂解液,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从非水溶性组分中使用适当方法分离提取水溶性组分中的蛋白质和多肽组分,然后将非水溶性组分中分离提取的蛋白质和多肽组分与所有水溶性组分一起作为抗原组分使用;(3)先制备肿瘤组织/癌细胞的裂解液,然后分别制备水溶性组分和非水溶性组分,然后将非水溶性组分使用特定含有溶解液的溶解剂溶解后使用,然后从水溶性组分和非水溶性组分中使用适当方法分别分离提取水溶性组分中的蛋白质和多肽组分,然后将水溶性组分中和非水溶性组分中分离提取的蛋白质和多肽组分一起作为抗原组分使用;(4)或者也可以直接采用含有溶解剂的溶解液直接裂解细胞或组织并溶解全细胞组分,然后通过适当方法制备其中的蛋白质/多肽组分,作为抗原组分使用;
    优选地,所述适当处理方法包括但不限于盐析、加热和酶解等处理方法;所述非水溶性组分或者经过盐析、加热和酶解等处理后产生的沉淀使用含有溶解剂的溶解液溶解;
    优选地,上述制备方法中还可以增加分离提取全细胞mRNA的步骤,并将全细胞mRNA作为抗原组分的一部分使用。
  86. 如权利要求66-85任一项所述的制备方法,其特征在于,步骤(2)中,所述肿瘤抗原组分的制备为先分离得到肿瘤细胞或组织,裂解所述肿瘤细胞或组织获得水溶性组分、非水溶性组分、全组分的任一种或其组合。
  87. 如权利要求66-86任一项所述的制备方法,其特征在于,步骤(2)中,所述肿瘤抗原组分经过盐析、加热、酶解处理。
  88. 如权利要求66-87任一项所述的制备方法,其特征在于,所述水溶性部分和非水溶性部分都可以被含溶解剂的增溶水溶液或有机溶剂溶解。
  89. 如权利要求66-88任一项所述的制备方法,其特征在于,所述溶解剂/增溶剂为可以增加蛋白质或多肽在水溶液中溶解性的溶解剂/增溶剂中的至少一种;有机溶剂为可以溶解蛋白质或多肽的有机溶剂。
  90. 如权利要求66-89任一项所述的制备方法,其特征在于,所述的非水溶性部分采用适当增溶方法由在纯水中不溶变为在含溶解剂的水溶液中或有机溶剂中可溶;采用的溶解剂选自含有结构式1结构的化合物、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、多肽、氨基酸、糖苷和胆碱中的一种或多种;其中,结构式1如下:结构式1结构如下:
    R1为C、N、S或O,R2~R5独立地选自氢、烷基、氨基、羧基、取代或未取代胍基中的至少一种。含有结构式1结构的化合物包括但不限于盐酸二甲双胍、硫酸二甲双胍、磺酸二甲双胍、二甲双胍盐、二甲双胍、尿素、盐酸胍、硫酸胍、磺酸胍、胍盐、其他含有胍基的化合物、碳酸胍、精氨酸、胍基乙酸、胍基磷酸、氨基磺酸胍、胍基琥珀酸、盐酸氨基脲、氨基甲酰脲、乙酰脲、磺酰脲类化合物(格列本脲、格列齐特、格列喹酮、格列美脲等)、硫脲类化合物(硫氧嘧啶类、咪唑类等)、亚硝基脲类等。
  91. 如权利要求66-90任一项所述的制备方法,其特征在于,将免疫佐剂与细胞组分共负载于纳米粒子或微米粒子中,在纳米粒子或微米粒子被抗原提呈细胞吞噬后可以更好的癌症特异性T细胞。
  92. 如权利要求66-91任一项所述的制备方法,其特征在于,所述水溶性组分的制备方法为:将肿瘤组织或癌细胞切块后研磨,过滤制得单细胞悬液,加水反复冻融1-5次后,超声裂解,裂解物以5000-10000g的转速离心5-10分钟后,取上清液即为水溶性组分,沉淀部分为非水溶性组分。
  93. 如权利要求66-92任一项所述的制备方法,其特征在于,所述抗原组分为非水溶性组分中加入溶解剂溶解后得到的可溶组分,所述溶解剂为含有结构式1结构的化合物、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、多肽、氨基酸、糖苷和胆碱中的一种或多种;其中,结构式1如下:结构式1结构如下:
    R1为C、N、S或O,R2~R5独立地选自氢、烷基、氨基、羧基、取代或未取代胍基中的至少一种。含有结构式1结构的化合物包括但不限于盐酸二甲双胍、硫酸二甲双胍、磺酸二甲双胍、二甲双胍盐、二甲双胍、尿素、盐酸胍、硫酸胍、磺酸胍、胍盐、其他含有胍基的化合物、碳酸胍、精氨酸、胍基乙酸、胍基磷酸、氨基磺酸胍、胍基琥珀酸、盐酸氨基脲、氨基甲酰脲、乙酰脲、磺酰脲类化合物(格列本脲、格列齐特、格列喹酮、格列美脲等)、硫脲类化合物(硫氧嘧啶类、咪唑类等)、。
  94. 如权利要求66-93任一项所述的制备方法,其特征在于,所述肿瘤组织或癌细胞提前进行紫外高温加热进行灭活和变性处理、加入核酸酶灭活处理中的任一种或其组合。
  95. 如权利要求66-94任一项所述的制备方法,其特征在于,所述抗原组分为水溶性组分经盐析和加热沉淀后得到的组分。
  96. 如权利要求66-95任一项所述的制备方法,其特征在于,所述抗原组分为肿瘤组织裂解离心后的沉淀部分即非水溶性组分中加入溶解剂溶解后得到的沉淀物中,再加入增溶剂进行二次溶解得到的可溶组分,所述增溶剂为吐温80、硫酸胍中的任一种或其组合。
  97. 如权利要求1-96所述的用于预防或治疗癌症的癌症特异性T细胞的制备方法制备得到的特异性T细胞。
  98. 一种来源于自体或同种异体的用于预防或治疗癌症的癌症特异性T细胞,所述特异性T细胞为包括但不限于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+T细胞的任一种或其组合;所述特异性T细胞活率大于60%,优选大于70%,更优选大于80%;
    优选地,所述特异性T细胞由负载有肿瘤抗原组分的纳米粒子和/或微米粒子,与从外周血或外周免疫器官的免疫细胞中分离的单个核细胞(PBMC)共孵育获得。
  99. 如权利要求98所述的特异性T细胞,其特征在于,所述PBMC细胞经过分选后,再与负载有肿瘤抗原组分的纳米粒子和/或微米粒子共孵育,
    优选地,与负载有肿瘤抗原组分的纳米粒子和/或微米粒子共孵育后的细胞再次经过分选,获得特异性T细胞。
    优选地,所述特异性T细胞还可经过体外扩增的步骤,该步骤可以在前述分选步骤之前,也可在前述分选步骤之后,优选在分选步骤之后进行。
    优选地,所述的PBMC细胞与所述的纳米/微米粒子共孵育时,还存在抗原递呈细胞(APC),
    优选地,所述各种类型的T细胞可以单一使用,也可以根据患者需要组合使用。
  100. 含如权利要求97-99任一项所述的特异性T细胞的药物组合物,其制备方法,包括下述步骤,在将癌症特异性T细胞回输给患者前可以在癌症特异性T细胞中添加具有增强天然免疫系统的物质,如白蛋白、NK细胞、中性粒细胞、γδT细胞、NK T细胞,
    优选地,所述特异性T细胞的细胞浓度为(0.01-100)×107个/ml,优选为(0.1-8)×107个/ml,
    优选地,所述药物组合物中还包含羟乙基淀粉、糖、盐中的任一种或其组合。
  101. 如权利要求100所述的药物组合物的制备方法,包括下述步骤,在将癌症特异性T细胞回输给患者前可以在癌症特异性T细胞中添加具有增强天然免疫系统的物质,如白蛋白、NK细胞、中性粒细胞、γδT细胞、NK T细胞,
    优选地,所述特异性T细胞的细胞浓度为(0.01-100)×107个/ml,优选为(0.1-8)×107个/ml,
    优选地,所述药物组合物中还包含羟乙基淀粉、糖、盐中的任一种或其组合。
  102. 如权利要求97-99任一项所述的特异性T细胞在制备癌症治疗或预防药物中的应用。
  103. 如权利要求102所述的应用,所述特异性T细胞在癌症发生前、癌症发生后或手术切除肿瘤组织后多次给药。
  104. 如权利要求97-99任一项所述的特异性T细胞在制备预防癌症复发或预防癌症转移的药物中的应用。
  105. 如权利要求97-99任一项所述的特异性T细胞用于制备抗肿瘤免疫治疗的制品中的应用。
  106. 如权利要求105所述的应用,所述肿瘤选自实体瘤、血液瘤和淋巴瘤。包括但不限于肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液瘤如白血病、脑肿瘤、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤、骨肉瘤、骨癌、胰腺癌、皮肤癌、前列腺癌、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、霍奇金病、非霍奇金淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症、转移癌、循环肿瘤细胞的任一种或其组合。
    优选地,所述肿瘤选自黑色素瘤、结肠癌、三阴性乳腺癌、胰腺癌、转移癌、肝癌、结肠癌、淋巴瘤、食管癌、非小细胞肺癌中的任一种或其组合。
  107. 如权利要求105所述的应用,所述免疫治疗选自抗肿瘤治疗中的免疫治疗或根治术后免疫治疗的任一种或其组合,优选地,所述免疫治疗选自原发性肝细胞癌根治术后免疫治疗。
  108. 如权利要求102-107任一项所述的应用,所述药物用于成年患者或儿童患者。
  109. 如权利要求97-99任一项所述的特异性T细胞用于免疫治疗的应用。
  110. 如权利要求109所述的应用,所述免疫治疗的给药方式为静脉注射、皮下注射、瘤内注射、腹腔注射、肌肉注射、皮内注射中的任一种或其组合。
  111. 如权利要求97-99任一项所述的特异性T细胞与放疗、化疗、靶向治疗、手术治疗或免疫治疗的任一种联用于抗肿瘤或肿瘤免疫治疗中的应用。
  112. 如权利要求97-99任一项所述的特异性T细胞用于制备增强抗病毒能力的药物中的应用。
  113. 如权利要求97-99任一项所述的特异性T细胞用于制备增强治疗自身免疫性疾病的药物中的应用。
  114. 如权利要求113所述的应用,所述自身免疫性疾病选自系统性红斑狼疮、类风湿性关节炎、硬皮病、甲状腺机能亢进、青少年糖尿病、原发性血小板紫癜、自身免疫性溶血性贫血、溃疡性结肠炎、皮肤病的任一种或其并发症。
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