WO2023201787A1 - Cancer-specific t cell-based cell system, lymphocyte drug and use thereof - Google Patents

Cancer-specific t cell-based cell system, lymphocyte drug and use thereof Download PDF

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WO2023201787A1
WO2023201787A1 PCT/CN2022/092331 CN2022092331W WO2023201787A1 WO 2023201787 A1 WO2023201787 A1 WO 2023201787A1 CN 2022092331 W CN2022092331 W CN 2022092331W WO 2023201787 A1 WO2023201787 A1 WO 2023201787A1
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cells
cancer
water
allogeneic
nanoparticles
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PCT/CN2022/092331
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French (fr)
Chinese (zh)
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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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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

Definitions

  • the present invention relates to the technical field of immunotherapy, and in particular to a cell system based on cancer-specific T cells, lymphocyte drugs and their applications.
  • Immunity is a physiological function of the human body.
  • the human body relies on this function to identify "self” and “non-self” components, thereby destroying and removing abnormal substances (such as viruses, bacteria, etc.) in the human body, or damaged cells produced by the human body itself. and tumor cells, etc., to maintain human health.
  • Immunology technology has developed rapidly in recent years, especially in the field of cancer immunotherapy. With the continuous improvement of understanding of cancer, people have found that the human body's immune system and various immune cells play a key role in inhibiting the occurrence and development of cancer. By regulating the balance of the body's immune system, we are expected to influence and control the occurrence, development and treatment of cancer.
  • the body recognizes and kills cancer cells through various types of immune cells.
  • the human body's immune system consists of the natural immune system and the adaptive immune system. Cells of the natural immune system and cells of the adaptive immune system cooperate with each other to eliminate cancer cells.
  • the cells in the natural immune system that can kill cancer cells include NK cells, while the immune cells in the adaptive immune system that can kill cancer cells are mainly T cells, especially cancer-specific CD8 + T cells and CD4 + T cells.
  • the number and function of immune cells in the natural immune system are related to the status and function of the cancer patient. Generally, cancer patients with high levels of NK cells and ⁇ T cells in the natural immune system have a better prognosis.
  • Cancer-specific T cells of the adaptive immune system can generate a portion of pre-existing specific immunity stimulated by small amounts of water-soluble antigens released by tumor tissues.
  • an epitope can only activate one specific T cell. Due to the suppressive immune microenvironment of tumor tissue sites and the high heterogeneity of cancer cells, pre-existing cancer-specific T cells are far from broad-spectrum and diverse enough to effectively identify and kill cancer cells.
  • Cancer vaccines contain cancer-specific antigens, which can stimulate and activate a wider range of cancer-specific T cells in addition to the body's pre-existing specific immunity. Therefore, cancer vaccines are one of the methods to treat or prevent cancer.
  • cancer vaccines The basic function of cancer vaccines is to select appropriate cancer antigens to activate the human immune system to recognize abnormally mutated cancer cells. Cancer cells and tumor tissues are highly heterogeneous and have many mutations, so cancer cells or cancer tumor tissues themselves are the best sources of cancer antigens. The more types of antigens a vaccine contains and the more types of cancer-specific T cells it can activate, the better the efficacy of the vaccine will be. However, in clinical practice, many elderly patients or patients who have experienced radiotherapy and chemotherapy in the early stage have poor physical condition, physiological function and immune function. Therefore, there is an urgent need to find a new immunotherapy solution.
  • the present invention provides a cell system based on cancer-specific T cells.
  • the cancer-specific T cells in this cell system are activated by cancer vaccines and can more effectively activate cancer-specific immune responses.
  • This cell system provides an allogeneic lymphocyte drug. Since the effect of cancer-specific T cell activation is related to the individual's immune function, the present invention proposes a new strategy, that is, while the immune function is better, Inoculate the allogeneic cancer vaccine or inject dendritic cells (DC) cells activated in vitro, and then isolate and extract the highly activated cancer-specific immune cells and natural immune cells, and then infuse them back into the allogeneic allogeneic with poor immune function. , to solve the problem of poor immune function among the above-mentioned elderly cancer patients or cancer patients who have experienced radiotherapy or chemotherapy in the early stage.
  • DC dendritic cells
  • the first object of the present invention is to provide a cell system based on cancer-specific T cells, which cell system includes cancer-specific T cells activated by a cancer vaccine, wherein the cancer vaccine includes delivery particles and loaded cell components.
  • the delivery particles are nanoparticles or microparticles
  • the cellular components are water-soluble components and/or water-insoluble components derived from cancer cells and/or tumor tissues;
  • Cell components are obtained by lysing cancer cells and/or tumor tissues
  • Lysis is to lyse cancer cells or tumor tissue by adding water or an aqueous solution without a dissolving agent.
  • the supernatant obtained is a water-soluble component, and the part of the precipitate that is converted to soluble after being dissolved by a dissolving agent is a water-insoluble component;
  • lysis is to use a dissolving agent to lyse cancer cells or tumor tissues, and dissolve the lysed components to obtain a mixture containing both water-soluble components and water-insoluble components.
  • cancer cells or tumor tissues are frozen at -20°C to -273°C, and water or an aqueous solution without a dissolving agent is added, followed by repeated freezing and thawing until the cell membrane structure is destroyed.
  • cancer-specific T cells include CD4 + T cells and/or CD8 + T cells.
  • the cell system also includes natural immune cells, which do not need to be activated by cancer vaccines and can be autologous natural immune cells and/or allogeneic natural immune cells.
  • the anti-tumor product includes any of the following: (1) a preparation containing cancer-specific T cells activated by a cancer vaccine; (2) a preparation containing cancer-specific T cells activated by a cancer vaccine Preparations of cancer-specific T cells, and preparations containing natural immune cells (wherein, if there are more than one natural immune cells, the preparations containing natural immune cells can be one or more, and can be injected separately when used for patients) or mixed and injected together); (3) Preparations containing a mixture of cancer-specific T cells and natural immune cells activated by cancer vaccines.
  • natural immune cells include, but are not limited to, ⁇ T cells, natural killer cells (NK cells), neutrophils, and natural killer T cells (NKT cells).
  • the activation is injecting a cancer vaccine into the body to activate cancer-specific T cells, or injecting dendritic cells (DC) into the body after being stimulated by a cancer vaccine in vitro to activate cancer-specific T cells.
  • a cancer vaccine into the body to activate cancer-specific T cells
  • DC dendritic cells
  • the stimulation is to incubate DC cells with the cancer vaccine for a certain period of time.
  • the delivery particles loaded with cell components are engulfed by DC cells, they can be presented and activated by the DC cells for antigens, and can be reinfused into the body. Homing to lymph nodes and utilizing antigens loaded by DC cells to activate cancer-specific T cells.
  • the DC cells are incubated with the cancer vaccine for at least 4 hours, preferably 24-96 hours.
  • dendritic cells are derived from autologous dendritic cells, allogeneic dendritic cells, cell lines or stem cells.
  • DC cells can be derived from any cell from which isolated dendritic cells can be prepared, including but not limited to derived from stem cells, Cell lines, bone marrow cells, peripheral immune cells, etc.
  • an appropriate dissolution method is used to convert the non-water-soluble components from insoluble in pure water to soluble in a solution containing a dissolving agent.
  • the dissolving agent is selected from the group consisting of urea, guanidine hydrochloride, deoxycholate (such as sodium deoxycholate), dodecyl sulfate (such as sodium dodecyl sulfate, SDS), glycerol, and protein degrading enzymes.
  • the loading method is that water-soluble components and/or water-insoluble components are contained inside the delivery particles, and/or are loaded on the surface of the delivery particles, including but not limited to water-soluble components being loaded on the delivery particles at the same time.
  • 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 particle surface.
  • the water-soluble components are loaded in the particles, while the water-soluble components and the water-insoluble components are loaded on the particle surfaces at the same time.
  • the water-insoluble components are loaded in the particles and the water-soluble components are loaded on the particles.
  • Components and non-water-soluble components are loaded on the surface of the particles at the same time, water-soluble components and non-water-soluble components are loaded on the particles at the same time, and water-soluble components and non-water-soluble components are loaded on the surface of the particles at the same time.
  • the water-soluble components and the water-insoluble components are loaded inside the particles at the same time, and the water-soluble components and the water-insoluble components are loaded on the surface of the particles at the same time.
  • the ways in which water-soluble components and/or non-water-soluble components are loaded on the surface of delivery particles include adsorption, covalent attachment, charge interaction, hydrophobic interaction, one or more steps of solidification, and mineralization. and at least one of wrapping; the way in which the water-soluble component and/or the non-water-soluble component is loaded inside the delivery particle is any way that can load it inside the delivery particle, such as being loaded inside the delivery particle, through electrostatic It is loaded inside the delivery particles by adsorption and loaded inside the delivery particles through hydrophobic interaction.
  • the cell components are loaded on the delivery particles, they form one or more layers of structure.
  • the delivery particles are loaded with multiple layers of cell components, there are modifications between the layers.
  • the surface of the delivery particles is connected with a target that actively targets dendritic cells.
  • the target can be mannose, mannan, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody and other targets, leading the targeted delivery of cancer vaccines into dendritic cells.
  • the cancer vaccine also includes an immune-enhancing adjuvant, which is loaded on the delivery particles together with the cell components.
  • Immune-enhancing adjuvants include at least one of microbial-derived immune enhancers, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicine and other categories; Immune-enhancing adjuvants include, but are not limited to, pattern recognition receptor agonists, Bacillus Calmette-Guérin (BCG), manganese-related adjuvants, BCG cell wall scaffolds, BCG methanol extraction residues, BCG muramyl dipeptide, Mycobacterium phlei, polyanti-A Vitamins, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, miquimod, polysaccharides, cur
  • the delivery particles can also be co-loaded with components that assist particles or antigens in escaping from lysosomes and improve activation efficiency.
  • Substances that assist and increase the escape of particles or antigens from lysosomes include, but are not limited to, cell-penetrating peptides, membrane-penetrating peptides, substances with proton sponge effects, and substances with membrane fusion effects.
  • the surface of the delivery particles can be electrically neutral, negatively charged, or positively charged.
  • the delivery particles are nanoscale or micron-scale, which can ensure that the vaccine is engulfed by antigen-presenting cells.
  • the particle size In order to improve the phagocytosis efficiency, the particle size must be within an appropriate range.
  • the particle size of nanoparticles (NP) is 1nm-1000nm, preferably, the particle size is 30nm-1000nm, most preferably, the particle size is 100nm-600nm;
  • the particle size of microparticles (MP) is 1 ⁇ m-1000 ⁇ m, preferably,
  • the particle size is 1 ⁇ m-100 ⁇ m, more preferably, the particle size is 1 ⁇ m-10 ⁇ m, and most preferably, the particle size is 1 ⁇ m-5 ⁇ m.
  • the delivery particles are made of organic synthetic polymer materials, natural polymer materials or inorganic materials.
  • Organic synthetic polymer materials are biocompatible or degradable polymer materials, including but not limited to PLGA (polylactic acid-co-glycolic acid), PLA (polylactic acid), PGA (polyglycolic acid), PEG (polyethylene glycol) alcohol), PCL (polycaprolactone), Poloxamer (poloxamer), PVA (polyvinyl alcohol), PVP (polyvinylpyrrolidone), PEI (polyethyleneimine), PTMC (polytrimethylene carbonate) , polyanhydride, PDON (polydioxanone), PPDO (polydioxanone), PMMA (polymethylmethacrylate), PLGA-PEG, PLA-PEG, PGA-PEG, polyamino acid , synthetic peptides, synthetic lipids, etc.; natural polymer materials are biocompatible or degradable polymer materials
  • the cancer vaccine does not need to be modified during the preparation process, or appropriate modification technology can be used to improve the antigen loading capacity and/or immunogenicity of the nanovaccine or microvaccine to improve the dendritic vaccine.
  • Modification technologies include but are not limited to chemical modification and physical modification, such as biomineralization (such as silicification, calcification, magnesization), gelation, cross-linking, addition of charged substances, etc.
  • the shape of the cancer vaccine is any common shape, including but not limited to sphere, ellipsoid, barrel, polygon, rod, sheet, linear, worm-shaped, square, triangle, butterfly or disc.
  • cancer vaccines can be prepared using existing preparation methods, including but not limited to common solvent evaporation methods, dialysis methods, microfluidic methods, extrusion methods, and hot melt methods.
  • the cancer vaccine is prepared by the double emulsion method in the solvent evaporation method, as follows:
  • Step 1 Add a first predetermined volume of an aqueous phase solution containing a first predetermined concentration into a second predetermined volume of an organic phase containing a second predetermined concentration of medical material.
  • the aqueous phase solution contains cell components, which may or may not contain immune-enhancing adjuvants; the cell components are water-soluble components and/or original non-water-soluble components soluble in the dissolving agent, or water-soluble components and non-water-soluble components.
  • the first predetermined concentration requires the protein polypeptide concentration to be greater than 1ng/mL, preferably 1mg/mL-100mg/mL, to ensure that sufficient cancer antigens can be loaded to activate relevant immune responses.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL, preferably 0.01mg/mL-20mg/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 or modified PLGA or PLA
  • the organic solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc. , preferably methylene chloride.
  • the second predetermined concentration is 0.5 mg/mL-5000 mg/mL, preferably 100 mg/mL.
  • PLGA or modified PLGA or PLA is selected because this material is a biodegradable material and has been approved by the FDA for use as a pharmaceutical excipient. Studies have shown that PLGA or PLA both have certain immune-modulating functions and are therefore suitable as excipients in the preparation of nanoparticles or microparticles. In practical applications, appropriate materials can be selected according to actual conditions. In the embodiments of the present invention, some of the components for co-loading delivery particles contain polypeptides with lysosomal escape ability. In practical applications, any other substances that can increase the lysosomal escape of delivery particles or antigens can also be added.
  • 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 It 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.
  • Step 2 Subject the mixed solution obtained in Step 1 to ultrasonic treatment, stirring, homogenization treatment or microfluidic treatment.
  • the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm ⁇ 1500rpm, the stirring time is 0.1 hour ⁇ 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5W, and the time is greater than 0.1 seconds, such as 2 to 200 seconds; use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer for homogenization.
  • the pressure is greater than 5 psi, such as 20 psi to 100 psi, and use high shear.
  • the rotation speed should be greater than 100rpm, such as 1000rpm to 5000rpm; use microfluidic processing with a flow rate greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasound, stirring, homogenization treatment or microfluidic treatment are used for nanometerization and/or micronization. The length of ultrasonic time, stirring speed, homogenization pressure and time can control the size of the prepared micro-nano particles.
  • 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, stirring, homogenization treatment or microfluidic treatment.
  • 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 bring about 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 greater than 50 rpm and the stirring time is greater than 1 minute.
  • 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.
  • 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 fourth predetermined concentration is greater than 0.01 mg/mL, preferably 0.01-100 mg/mL. In some embodiments of the present invention, 5 mg/mL is selected.
  • 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. In the present invention, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. In the specific implementation process, the ratio of the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.
  • the predetermined stirring condition for this step is until the organic solvent is completely volatilized.
  • Step 5 After centrifuging the mixed solution that meets the predetermined stirring conditions in Step 4 at a rotation speed greater than 100 RPM for greater than 1 minute, remove the supernatant, and resuspend the precipitate in a fifth predetermined volume of a fifth predetermined concentration. in an aqueous solution containing a lyophilized protective agent or in a sixth predetermined volume of PBS (or physiological saline).
  • 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 conducting subsequent experiments related to the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles.
  • the freeze-drying protective agent is trehalose; the fifth predetermined concentration is 1-15% by mass, preferably 4%. The reason why it is set in this way is to not affect the freeze-drying effect during subsequent freeze-drying; or use Other lyoprotectants are used for freeze drying such as a mixed solution of sucrose and mannitol.
  • Step 6 After freeze-drying the suspension containing the lyoprotectant obtained in Step 5, the freeze-dried material is used for later use.
  • the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, preferably 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1.
  • natural immune cells include, but are not limited to, ⁇ T cells, natural killer cells (NK cells), natural killer T cells (NKT cells), neutrophils, etc.
  • This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components of melanoma tumor tissue to activate allogeneic cancer-specific T cells and prevent melanoma in mice.
  • B16F10 melanoma tumor tissue was first lysed to prepare water-soluble components and non-water-soluble components of the tumor tissue.
  • the nanovaccine and the blank nanoparticles used as controls were prepared by the double emulsion method in the solvent evaporation method.
  • nanovaccines 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 PLGA, the material used to prepare the nanoparticles, is 24KDa-38KDa.
  • the immune adjuvant used is poly(I:C) and the poly(I:C) is contained in the nanoparticles.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load cell components and adjuvants inside the nanoparticles.
  • Allogeneic cell mixtures are administered to cancer-affected mice to treat cancer.
  • the tumors of mice in both the PBS control group and the blank nanoparticle control group grew.
  • the tumor growth rate of mice treated with allogeneic cancer-specific T cells and natural immune cells activated by the nanovaccine was significantly slower, and some mice had tumors that disappeared and recovered.
  • the cell system of the present invention has good therapeutic effect on melanoma.
  • Example 3 Allogeneic cell system for prevention of melanoma lung metastasis
  • This example uses a mouse melanoma lung model to illustrate how to use an allogeneic cell system 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, a nanoparticle system loaded with water-soluble components and non-water-soluble components of the tumor tissue is prepared.
  • siliconization and adding charged substances were used to increase the loading capacity of the antigen, and only one round of mineralization was performed.
  • nanoparticles are used to first activate dendritic cells in vitro, and then the dendritic cells are injected to activate cancer-specific T cells.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the tumor grew to a volume of approximately 1000 mm 3
  • the mice were sacrificed and the tumor tissue was removed. Cut the tumor tissue into pieces and then grind it. Add collagenase and incubate in RPMI 1640 medium for 30 minutes. Then add an appropriate amount of pure water through a cell filter and freeze and thaw repeatedly 5 times. Ultrasound can be used to destroy the lysed cells.
  • 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. During the preparation process, the double emulsion method is first used to load the antigen and adjuvant inside the nanoparticles.
  • nanoparticles After loading the antigen inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and then the nanoparticles are resuspended using 7 ml PBS and mixed with 3 mL of PBS solution containing cell lysate (60 mg/mL) was mixed, centrifuged at 10,000 g for 20 min, and then reconstituted with 10 mL of silicate solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate, and 1.0 mM HCl, pH 3.0).
  • silicate solution containing 150 mM NaCl, 80 mM tetramethyl orthosilicate, and 1.0 mM HCl, pH 3.0.
  • the average particle size of the nanoparticles is about 350nm, and the surface potential of the nanoparticles is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 300 ⁇ g of protein or peptide components, and the poly(I:C) immune adjuvant used inside and outside each 1 mg of PLGA nanoparticles A total of about 0.02mg 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.
  • the particle size of the blank nanoparticles is about 300nm, and the surface potential is about -5mV.
  • pure water containing an equal amount of poly(I:C) or 5% SDS is used to replace the corresponding water-soluble components and non-containing components. Water-soluble 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
  • 6M guanidine hydrochloride was first used to lyse the whole cell fraction of B16F10 melanoma cancer cells. Then, the organic polymer material PLGA was used as the micron particle skeleton material, and CpG was used as the immune adjuvant to prepare a micron particle system loaded with whole cell components of cancer cells.
  • siliconization adding cationic substances and anionic substances were used to increase the loading capacity of the antigen, and two rounds of siliconization were performed. After micron particles activate allogeneic cancer-specific T cells, a cell mixture containing the above cells is injected into cancer-stricken mice to prevent cancer.
  • the cultured B16F10 melanoma cancer cell line was collected and centrifuged at 350g for 5 minutes. The supernatant was then discarded and washed twice with PBS. The cancer cells were then resuspended and lysed with 6M guanidine hydrochloride. The whole cell components were lysed and dissolved in 6M guanidine hydrochloride. That is the source of antigen raw materials for preparing micron particle systems.
  • the microparticles and the blank microparticles used as a control were prepared by the double emulsion method.
  • the double emulsion method was appropriately modified and improved.
  • two modification methods, low-temperature siliconization technology and addition of charged substances, were used to improve the antigenicity. load capacity.
  • 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.
  • 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. The 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. Use ultrapure water to centrifuge and wash. Resuspend in 3 mL of PBS containing polyaspartic acid (10 mg/mL) for 10 min.
  • micron particles loaded with lysate both inside and outside Before using the particles, resuspend them in 7 mL PBS and then add 3 mL of cancer tissue lysate component containing adjuvant (protein concentration 50 mg/mL) and incubate at room temperature for 10 min to obtain micron particles loaded with lysate both inside and outside.
  • the average particle size of the micron particles is about 2.45 ⁇ m, and the surface potential of the micron particles is about -3mV; each 1 mg of PLGA micron particles is loaded with approximately 160 ⁇ g of protein or peptide components, and each 1 mg of PLGA micron particle uses a total of 0.02 mg of CpG immune adjuvant, half of which is inside and outside. .
  • the particle size of the blank microparticles is about 2.43 ⁇ m, and the surface potential is about -3mV.
  • 6M guanidine hydrochloride containing an equal amount of CpG is used to replace the corresponding cell components.
  • T cells were isolated from mice on day 32 using flow cytometry. Total T cells contain ⁇ T cells and activated cancer-specific T cells.
  • the tumors of the mice in the control group all grew, while the tumor growth rate of the mice in the adoptively transferred T cell group slowed down significantly.
  • the cell system activated by micron particles modified with siliconization and addition of charged substances has a better preventive effect on melanoma than the cell system activated by micron particles not modified during the preparation process.
  • Example 5 Nanoparticles activate cancer-specific T cells for cancer prevention
  • an 8M urea aqueous solution (containing 500mM sodium chloride) was first used to lyse B16F10 melanoma tumor tissue and dissolve the tumor tissue lysate components. Then, a nanoparticle system loaded with whole cell components was prepared using PLGA as the nanoparticle skeleton material and Poly(I:C) and CpG as immune adjuvants. After nanoparticles are injected into mice to activate cancer-specific T cells, T cells and NK cells are isolated and extracted, and then the T cells and NK cells are given to allogeneic cancer-suffering mice for cancer prevention.
  • B16F10 cells 1.5 ⁇ 10 5 B16F10 cells were 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. Cut the tumor tissue into pieces and then grind it. Add an appropriate amount of 8M urea aqueous solution (containing 500mM sodium chloride) through a cell filter to lyse the cells and dissolve the cell lysate.
  • 8M urea aqueous solution containing 500mM sodium chloride
  • the nanoparticles and the blank nanoparticles used as a control were prepared by the solvent evaporation method.
  • the molecular weight of PLGA, the material used to prepare the nanoparticles, is 7KDa-17KDa.
  • the immune adjuvants used are poly(I:C) and CpG, and the lysate components and adjuvants are loaded inside the nanoparticles.
  • the preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysate components and adjuvants inside the nanoparticles.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 110 ⁇ g of protein or peptide components, and the poly(I:C) and CpG immune components used in each 1 mg of PLGA nanoparticles are Adjuvants are 0.02 mg each.
  • the particle size of the blank nanoparticles is about 250nm, and the surface potential is about -9mV.
  • 8M urea containing 500mM sodium chloride
  • poly(I:C) and CpG is used to replace the lysate component.
  • 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. On day 0, mice were injected subcutaneously with 100 ⁇ L of allogeneic cells containing 1 million T cells and 500,000 NK cells. At the same time, 1.5 ⁇ 10 5 B16F10 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.
  • mice in the control group all grew, while the tumor growth rate of mice transplanted with immune cells activated by antigen-loaded nanoparticles was significantly slower, and most of the mouse cancer cells were inoculated The tumor disappears.
  • Example 6 Nanoparticles are used to treat colon cancer after activating cancer-specific T 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 8M urea 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 source of raw materials for preparing nanoparticles.
  • the lysis method of BCG and the collection method of each component are the same as the lysis method and collection method of each component of cancer cells.
  • the water-soluble components and the dissolved water-insoluble components are mixed at a mass ratio of 1:1.
  • the nanovaccine and the blank nanoparticles used as controls were prepared using the solvent evaporation method.
  • the molecular weight of PLA, the material used to prepare the nanoparticles, is 40KDa.
  • the immune adjuvants used are CpG and BCG, and the adjuvants are distributed both inside and on the surface of the nanoparticles.
  • the preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysate mixture and adjuvant inside the nanoparticles.
  • nanoparticles After loading the lysate and adjuvant inside, 100 mg nanoparticles are centrifuged at 10000g for 20 minutes, and 10 mL containing Resuspend in 4% trehalose ultrapure water and freeze-dry for 48 h. Before use, resuspend 20 mg of nanoparticles in 0.9 mL of PBS, mix with 0.1 mL of sample containing lysate mixture (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 of the nanoparticles is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 120 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles contains 0.02 mg of CpG and BCG immune adjuvants.
  • the particle size of the blank nanoparticles is about 260 nm.
  • Example 5 Same as Example 5. However, total T cells, NK cells and NKT cells were separated and extracted.
  • the tumors of mice in both the PBS control group and the blank nanoparticle control group grew.
  • the tumor growth rate of mice in the immune cell transplantation group activated by nanoparticles was significantly slower, and some mice had tumors that disappeared and recovered.
  • the allogeneic immune cell treatment plan of the present invention has a good therapeutic effect on colon cancer.
  • Example 7 Immune cells activated by nanoparticles loaded with whole cell components of melanoma tumor tissue and lung cancer tumor tissue are used for the treatment of melanoma
  • This example uses melanoma as a cancer model to illustrate how to use nanoparticles loaded with whole cell components of melanoma and lung cancer tumor tissues to activate cancer-specific T cells, and use the cell vaccine to treat melanoma in allogeneic mice.
  • B16F10 melanoma tumor tissue and LLC lung cancer tumor tissue were first lysed to prepare a mixture of water-soluble components (mass ratio 3:1) and a mixture of non-water-soluble components (3:1) of the tumor tissue.
  • Use PLGA as the nanoparticle skeleton material, and use manganese particles and CpG as immune adjuvants to prepare nanoparticles loaded with the above mixture.
  • the nanoparticles are then used to activate cancer-specific T cells in mice, and the immune cells are isolated and extracted to treat allogeneic Melanoma in cancerous mice.
  • Each C57BL/6 mouse was subcutaneously inoculated with 1.5 ⁇ 10 5 B16F10 cells or 2 ⁇ 10 6 LLC lung cancer cells on the back.
  • the tumor grew to a volume of approximately 1000 mm 3
  • the mice were sacrificed and the tumor tissue was removed.
  • the methods for tumor lysis and collection of components are the same as in Example 1.
  • the nanoparticle system and the blank nanoparticles used as controls were prepared using the double emulsion 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 24KDa-38KDa, and the immune adjuvants used are manganese colloidal particles and CpG.
  • the manganese adjuvant is first prepared, and then the manganese adjuvant is mixed with the water-soluble component or the non-water-soluble component in the whole cell component and then used as the first aqueous phase to prepare nanoparticles internally loaded with antigen and adjuvant using the double emulsion method.
  • the manganese adjuvant particle size is approximately 13 nm.
  • the manganese adjuvant with the water-soluble component (60 mg/mL) or the non-water-soluble component (60 mg/mL) in the whole cell component at a volume ratio of 1:3, and then use the double emulsion method to dissolve the antigen. and manganese adjuvants were loaded into the interior of the nanoparticles. After loading the antigen (cleavage component) and adjuvant internally, 100 mg of the nanoparticles were centrifuged at 10,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours before use.
  • the average particle size of the nanoparticles is about 370nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 120 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles uses 0.01 mg of CpG adjuvant.
  • the particle size of the blank nanoparticles is about 330 nm.
  • pure water or 8M urea containing equal amounts of manganese adjuvant and CpG adjuvant are used to replace the corresponding water-soluble components and non-water-soluble components.
  • the tumors of mice in both the PBS control group and the blank nanoparticle control group grew.
  • the tumor growth rate of mice in the immune cell transplantation group activated by nanoparticles was significantly slower, and some mice had tumors that disappeared and recovered.
  • the allogeneic immune cell treatment plan of the present invention has a therapeutic effect on melanoma.
  • Example 8 Allogeneic immune cells activated by micron particles for the prevention of breast cancer
  • This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use 8M urea to dissolve whole cell components and prepare a micron particle system loaded with whole cell components, and use the micron particles to activate allogeneic cancer-specific T cells for breast cancer prevention.
  • breast cancer cells were first inactivated and denatured, the cancer cells were lysed with 8M urea, and then the whole cell components were dissolved. Then, a micron particle system loaded with whole cell components was prepared using PLGA as the micron particle skeleton material and CpG and Poly ICLC as immune adjuvants.
  • 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 appropriate amount of 8M urea was used to lyse the breast cancer cells and dissolve the lysate, which is the source of raw materials for preparing the particle system.
  • the double emulsion method was used to prepare the microparticle system and the blank microparticles used as controls.
  • the molecular weight of the microparticle skeleton material PLGA was 38KDa-54KDa.
  • the immune adjuvants used were CpG and Poly ICLC.
  • the double emulsion method is first used to prepare microparticles loaded with lysate components and adjuvants internally. After loading lysate and adjuvants internally, 100 mg micron particles are centrifuged at 9000g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose is used. Resuspend and dry for 48 hours before use.
  • the average particle size of the micron particle system is about 2.1 ⁇ m, and the surface potential of the micron particle system is about -5mV; each 1 mg of PLGA micron particles is loaded with approximately 110 ⁇ g of protein or peptide components, including 0.01 mg of CpG and Poly ICLC.
  • the particle size of blank microparticles is about 2.0 ⁇ m.
  • 8M urea containing equal amounts of CpG and Poly ICLC adjuvant is used to replace the corresponding cell components.
  • mice Select 6-8 week old female C57BL/6 mice and subcutaneously inject 100 ⁇ L of microparticles containing 2 mg PLGA on days 0, 4, 7, 14, 21, and 28.
  • total T cells and NK cells were isolated from mice using magnetic bead sorting.
  • Total T cells contain ⁇ T cells and activated cancer-specific T cells.
  • 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 of allogeneic immune cells containing 1 million T cells and 500,000 NK 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 of the immune cell treatment group activated by micron particles was significantly slower and the survival period of mice was significantly prolonged. It can be seen that the allogeneic activated immune cells of the present invention have a preventive effect on breast cancer.
  • Example 9 Nanoparticles activate allogeneic immune cells for the prevention of cancer metastasis
  • This example uses a mouse melanoma mouse lung metastasis cancer model to illustrate the use of nanoparticles to activate allogeneic immune cells and then transplant these immune cells to prevent cancer metastasis.
  • the specific dosage form, adjuvant, administration time, administration frequency, and dosage regimen can be adjusted according to the situation.
  • mouse melanoma tumor tissue and cancer cells were lysed and dissolved with 8M urea, and then the tumor tissue lysis component and the cancer cell lysis component were loaded into the nanoparticle system at a mass ratio of 1:2, and the particle system was used Activating cancer-specific T cells in allogeneic mice and preventing cancer metastasis in transplanted mice.
  • polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were used.
  • Nano The particles were used as control nanoparticles to analyze the efficacy of allogeneic immune cells activated by nanoparticles loaded with whole cell antigens and nanoparticles loaded with multiple peptide neoantigens in preventing cancer lung metastasis.
  • 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 components were miscible at a mass ratio of 1:2.
  • the nanoparticle system is prepared by a solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa.
  • the immune adjuvants used are CpG and Poly(I:C).
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the lysate components and adjuvants inside the nanoparticles. After loading the lysis components and adjuvants inside, 100 mg of nanoparticles are centrifuged at 10,000g for 20 minutes, and used Resuspend 10 mL of ultrapure water containing 4% trehalose and freeze-dry for 48 hours before use.
  • the average particle size of the nanoparticles is about 270nm; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components, including 0.02 mg of CpG and Poly(I:C).
  • the preparation method of control nanoparticles loaded with multiple antigen peptides is the same as above.
  • the particle size of the control nanoparticles is about 260 nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 100 ⁇ g of antigen peptides and an equal amount of adjuvant.
  • 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. On day 0, mice were intravenously injected with 100 ⁇ L of an allogeneic cell mixture containing 1 million T cells, 200,000 NK cells, and 200,000 NKT cells. 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.
  • immune cells activated by nanoparticles can effectively prevent cancer metastasis in allogeneic cells. Furthermore, immune cells activated by lysate- and adjuvant-loaded nanoparticles were better at preventing cancer metastasis in allogeneic compared with controls.
  • 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 activate mouse cancer-specific T cells, and then the nanoparticles containing Activated immune cells 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.
  • PLGA is used as the nanoparticle skeleton material
  • BCG is used as the adjuvant loaded on the nanoparticles
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • 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 and tumor tissues were harvested.
  • 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 water-insoluble components.
  • the BCG lysis method is the same as the tumor tissue lysis method.
  • the nanoparticles and the blank nanoparticles used as a control were prepared by the double emulsion method.
  • the molecular weight of PLGA, the material used to prepare the nanoparticles, is 7KDa-17KDa.
  • the immune adjuvant used is BCG, and BCG is loaded inside the nanoparticles.
  • the preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysate components and adjuvants inside the nanoparticles.
  • nanoparticles After loading the antigen lysis components and adjuvants inside, 100mg of the nanoparticles are centrifuged at 12000g for 20 minutes, and 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 nanoparticle injection, 20 mg of nanoparticles were dissolved in 0.9 mL of PBS, mixed with 0.1 mL of sample containing lysate (80 mg/mL) and GM-CSF (50 ng/mL), and incubated at room temperature for 10 min before use.
  • the average particle size of the nanoparticles is about 260nm, and the surface potential of the nanoparticles is about -4mV; each 1 mg of PLGA nanoparticles is loaded with approximately 130 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles uses 0.02 mg of BCG immune adjuvant.
  • the particle size of the blank nanoparticles is about 230 nm, and the blank nanoparticles are prepared with equal amounts of adjuvants.
  • immune cells activated by nanoparticles can effectively treat pancreatic cancer allogeneically. Furthermore, immune cells activated by lysate- and adjuvant-loaded nanoparticles treated cancer metastasis better allogeneically compared with controls.
  • Example 11 Target-modified nanoparticles activate allogeneic immune cells for cancer prevention
  • This example uses mannose as an active target to illustrate how targeted nanoparticles can activate allogeneic cancer-specific T cells and be used to prevent cancer.
  • the specific dosage form, adjuvant, administration time, administration frequency, and dosage regimen can be adjusted according to the situation.
  • the nanoparticle system can be taken up into dendritic cells through mannose receptors on the surface of dendritic cells, and then activate cancer needle-specific T cells.
  • 8M urea was used to lyse and dissolve the whole cell components of the cancer cells.
  • the nanoparticle system 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. For nanoparticles without a target, only PLGA is used.
  • the immune adjuvants used were Poly(I:C) and CpG. The preparation method is as described above. The lysate components and adjuvants are loaded into the nanoparticles using the double emulsion method.
  • nanoparticles are centrifuged at 10,000 g for 20 minutes and resuspended in 10 mL of ultrapure water containing 4% trehalose. Then freeze-dry for 48 hours before use.
  • the average particle size of nanoparticles with and without target is about 270nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide components, including 0.02 mg of Poly(I:C) and 0.02 mg of CpG.
  • the particle size of the control nanoparticles without adjuvant but with mannose target is also about 270nm. They are prepared using the same amount of cell components but do not contain any immune adjuvant.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide. components.
  • the tumor growth rate of mice treated with allogeneic activated immune cells was significantly slower.
  • the allogeneic cancer-specific T cells activated by the nanoparticles also have the effect of preventing cancer. Allogeneic cancer-specific T cells activated using adjuvanted nanoparticles are better than non-adjuvanted nanoparticles; immune cells activated using target-containing nanoparticles are better than non-target nanoparticles. of immune cells. This shows that the allogeneic immune cells of the present invention can prevent cancer, and the addition of active targeting targets and adjuvants helps nanoparticles activate allogeneic cancer-specific immune cells to play their role.
  • Example 12 Nanoparticles activate allogeneic immune cells to prevent liver cancer
  • Hepa1-6 liver cancer cells are first lysed, PLGA is used as the nanoparticle skeleton material, Poly(I:C) and BCG are used as immune adjuvants, and a nanoparticle system loaded with whole cell components of liver cancer cells is prepared using a solvent evaporation method. , and then use this particle system to activate allogeneic cancer-specific T cells, and isolate and extract allogeneic immune cells to prevent liver cancer.
  • 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 components of the cancer cells.
  • the cleavage method of BCG is the same as above.
  • the nanoparticle system is prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa, and the immune adjuvants used are BCG and Poly(I:C).
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the cleavage components and adjuvants inside the nanoparticles. After loading the cleavage components and adjuvants inside, 100mg nanoparticles are centrifuged at 10000g for 20 minutes, and 10 mL is used. Resuspend in ultrapure water containing 4% trehalose and freeze-dry for 48 hours before use.
  • the average particle size of the nanoparticles is about 270nm; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components, including 0.02 mg each of BCG and Poly(I:C).
  • 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. On day 32, CD4 + T cells, CD8 + T cells, ⁇ T cells and NK cells were isolated from mice using flow cytometry.
  • 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. On day 0, mice were injected with 100 ⁇ L of an allogeneic cell mixture containing 500,000 CD8 + T cells + 500,000 CD4 + T cells; or on day 0, mice were injected with 100 ⁇ L of an allogeneic cell mixture containing 500,000 CD8 + T cells + 500,000 CD8 + T cells An allogeneic cell mixture of CD4 + T cells + 500,000 NK cells + 500,000 ⁇ T cells. At the same time, each mouse was subcutaneously injected with 1.0 ⁇ 10 6 Hepa1-6 liver cancer cells on day 0. The tumor growth and mouse survival period were recorded in the same manner as in Example 1.
  • the tumor growth rate of mice treated with allogeneic activated immune cells was significantly slower.
  • the effect of using natural immune cells such as NK cells and NKT cells is better than using immune cells that do not contain natural immune system cells. This shows that the allogeneic immune cells of the present invention can prevent cancer, and the addition of natural immune cells helps to enhance the role of nanoparticles in activating cancer-specific immune cells in vitro.
  • Example 13 Calcified nanoparticle system activates allogeneic cancer-specific T cells for cancer prevention
  • This example illustrates that calcified nanoparticles activate allogeneic cancer-specific T cells.
  • other biomineralization technologies, cross-linking, gelation and other modified particles can also be used.
  • the specific dosage form, adjuvant, administration time, administration frequency, and dosage regimen can be adjusted according to the situation.
  • mouse melanoma tumor tissue and cancer cells were lysed and dissolved with 8M urea, and then the tumor tissue lysis component and the cancer cell lysis component were loaded into the nanoparticle system at a mass ratio of 1:1, and the particle system was used Activation of allogeneic cancer-specific T cells and transplantation of allogeneic immune cells for cancer prevention.
  • polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were used. Nano particles were used as control nanoparticles.
  • 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 components were miscible at a mass ratio of 1:1.
  • the nanoparticles are biocalcified after loading whole cell antigens inside and on the surface of the nanoparticles.
  • the nanoparticle system and the blank nanoparticles used as a control were prepared by a solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used was 7KDa-17KDa.
  • the immunoadjuvant CpG and Poly(I:C) used were loaded on the nanoparticles. inside the particle.
  • the preparation method is as follows. During the preparation process, the double emulsion method is first used to load the antigen inside the nanoparticles.
  • 100mg PLGA nanoparticles are centrifuged at 13000g for 20 minutes and resuspended in 18mL PBS. Then 2mL is added to dissolve in 8M urea tumor tissue and cancer cell lysate (60mg/mL), incubated at room temperature for 10 minutes, centrifuged at 12000g for 20 minutes and collected 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 140 ⁇ g of protein or polypeptide components, including 0.02 mg of CpG and Poly(I:C).
  • the preparation method of control nanoparticles loaded with multiple antigen peptides is the same as above.
  • the particle size of the control nanoparticles is about 290 nm.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 130 ⁇ g of antigen peptides and an equal amount of adjuvant.
  • Example 14 Nanoparticles activate allogeneic cancer-specific T cells for the treatment of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components of melanoma tumor tissue to activate allogeneic cancer-specific T cells and then reinfuse them together with natural immune cells. given to mice to treat 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 CpG were used as immune adjuvants.
  • GALA WEAALAEALAEALAEHLAEALAEALEALAA
  • a solvent evaporation method is used to prepare a nanoparticle system that co-loads adjuvants, lysosome escape substances and a mixture of water-soluble components or a mixture of non-water-soluble components, and then uses nanoparticles.
  • the particle system activates cancer-specific T cells allogeneically, then separates and extracts total T cells, NKT cells and NK cells, and injects the above cells into cancer-stricken mice to treat melanoma.
  • Substances that can promote lysosomal escape are added to the nanoparticle delivery carrier, such as the cell-penetrating peptide added in this embodiment, which can increase the lysosomal escape of the particles or particle-loaded antigens, thereby increasing the cross-presentation and cross-presentation of antigens. Increase the activation efficiency of CD8 + T cell immune response and better activate cancer-specific CD8 + T cell immune response.
  • the nanovaccine and the blank nanoparticles used as controls were 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 material used to prepare the nanoparticles, is 7KDa-17KDa.
  • the immune adjuvants used are poly(I:C) and CpG.
  • the lysosomal escape substance is GALA polypeptide, and the adjuvant and polypeptide are wrapped in the nanoparticles.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the lysis solution components and adjuvants inside the nanoparticles. After loading the lysis components inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL containing Resuspend in 4% trehalose ultrapure water and freeze-dry for 48 hours; resuspend it in 9 mL PBS before use, then add 1 mL of lysis buffer component (protein concentration 80 mg/mL) and incubate at room temperature for 10 min to obtain both internal and external load lysis. Nanoparticle systems of matter.
  • the average particle size of the nanoparticles is about 290nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 140 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C) and CpG immune Each adjuvant contains 0.02 mg, and the loaded GALA polypeptide is 0.05 mg.
  • the particle size of the blank nanoparticles is about 270nm.
  • the blank nanoparticles use equal amounts of adjuvant and GALA polypeptide in pure water or 2M arginine to replace the corresponding water-soluble components and non-water-soluble components.
  • the control groups in this study were the PBS group and the blank nanoparticles + free lysate control group.
  • Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On days 0, 7, 14, 21, 28 and 35, 100 ⁇ L of 1 mg PLGA nanoparticles loaded with water-soluble components and 100 ⁇ L of 1 mg PLGA loaded with original non-water-soluble components were subcutaneously injected, respectively. Nanoparticles.
  • the control group was injected with PBS or blank nanoparticles + free lysate loaded with an equal amount of immune adjuvant on corresponding days.
  • total T cells, NK cells, and NKT cells were isolated from mice using flow cytometry. Total T cells contain ⁇ T cells and activated cancer-specific T cells.
  • Allogeneic cell mixtures are administered to cancer-affected mice to treat cancer.
  • the tumors of mice in both the PBS control group and the blank nanoparticle control group grew.
  • the tumor growth rate of mice treated with allogeneic cancer-specific T cells and natural immune cells activated by the nanovaccine was significantly slower, and some mice had tumors that disappeared and recovered.
  • the cell system of the present invention has good therapeutic effect on melanoma.
  • Nanoparticles activate allogeneic cancer-specific T cells for the treatment of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components of melanoma tumor tissue to activate allogeneic cancer-specific T cells and then infuse them back into mice for treatment.
  • Melanoma B16F10 melanoma tumor tissue is first lysed, and then PLGA is used as the nanoparticle skeleton material, Poly(I:C) and CpG are used as immune adjuvants, and polylysine is used as a component to increase lysosomal escape.
  • the nanoparticle system is then used to activate cancer-specific T cells allogeneically, and then total CD4 + T cells and CD8 + T cells are isolated and extracted, and the above cells are injected into cancer-stricken mice to treat melanoma.
  • the nanovaccine and the blank nanoparticles used as a control were prepared by the double emulsion method and have the ability to target dendritic cells.
  • the nanoparticle preparation materials used are PLGA and mannan-modified PLGA, both of which have molecular weights of 24KDa-38KDa. When used, the mass ratio of unmodified PLGA and mannan-modified PLGA is 4:1.
  • the immune adjuvants used are poly(I:C) and CpG, the substance that increases lysosomal immune escape is polylysine, and the adjuvants and polylysine are encapsulated in nanoparticles.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the lysis solution components, adjuvants, and polylysine inside the nanoparticles. After loading the above components internally, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes. , and resuspended in 10 mL of ultrapure water containing 4% trehalose and then freeze-dried for 48 h.
  • the average particle size of the nanoparticles is about 280nm, and the surface potential of the nanoparticles is about -5mV; 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 poly(I:C) and CpG immune
  • the adjuvants are 0.02 mg each, and the polylysine is 0.05 mg.
  • the preparation materials and methods of the control nanoparticles are the same as above, except that polylysine is not loaded, the particle size is about 280nm, and equal amounts of adjuvants and cell lysis components are loaded.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On days 0, 7, 14, 21, 28 and 35, 100 ⁇ L of 2 mg PLGA nanoparticles were injected subcutaneously. CD3 + CD4 + T cells and CD3 + CD8 + T cells were isolated from mice on day 38 using flow cytometry.
  • Allogeneic cell mixtures are administered to cancer-affected mice to treat cancer.
  • the tumors in the PBS control group all grew.
  • the tumor growth rate of mice treated with allogeneic cancer-specific T cells activated by nanovaccines was significantly slower, and the T cells activated by adding nanoparticles that increase lysosomal escape substances were better than those that were not added Lysosomal escape of nanoparticle-activated T cells.
  • the cell system of the present invention has good therapeutic effect on melanoma.
  • Example 16 Allogeneic immune cells activated by micron particles for the prevention of breast cancer
  • This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use a micron particle system loaded with whole cell components and use the micron particles to activate allogeneic cancer-specific 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.
  • micron particle system loaded with whole cell components was prepared using PLGA as the micron particle skeleton material, CpG and Poly ICLC as immune adjuvants, and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) as a substance that enhances lysosome escape.
  • PLGA the micron particle skeleton material
  • CpG and Poly ICLC immune adjuvants
  • KALA polypeptide WEAKLAKALAKALAKHLAKALAKALKACEA
  • 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. water-soluble components. Dissolve the precipitate with 10% octylglucoside to obtain the dissolved original non-water-soluble components.
  • the double emulsion method was used to prepare the micron particle system and the control micron particles.
  • the molecular weight of the micron particle skeleton material PLGA is 38KDa-54KDa.
  • the immune adjuvants used are CpG and Poly ICLC.
  • the lysosomal escape increasing substance used is KALA polypeptide.
  • particles loaded with water-soluble components and particles loaded with non-water-soluble components are prepared separately and used together.
  • the double emulsion method is first used to prepare micron particles that are internally loaded with lysate components, adjuvants and KALA polypeptides.
  • micron particles After loading lysate and adjuvants internally, 100 mg of micron particles are centrifuged at 9000g for 20 minutes, and 10 mL containing 4% trehalose is used. Resuspend in ultrapure water and dry for 48 hours before use.
  • the average particle size of the micron particle system is about 3.1 ⁇ m, and the surface potential of the micron particle system is about -7mV; each 1 mg of PLGA micron particles is loaded with approximately 110 ⁇ g of protein or peptide components, including 0.01 mg of CpG and Poly ICLC, and 0.05 mg of KALA peptide. .
  • the particle size of the control microparticles is about 3.1 ⁇ m.
  • the preparation materials and preparation methods are the same as the microparticles described in this example, except that KALA polypeptide is not loaded, and equal amounts of CpG and Poly ICLC adjuvant cell lysate components are loaded.
  • mice Select 6-8 week old female BALB/c mice and subcutaneously inject 100 ⁇ L of 1 mg PLGA containing water-soluble components on days 0, 4, 7, 14, 21, and 28. particles and 100 ⁇ L of micron particles containing 1 mg of PLGA containing non-water-soluble components.
  • total T cells were isolated from mice using magnetic bead sorting. Total T cells contain ⁇ T cells and activated cancer-specific T cells.
  • the tumor growth rate in the immune cell treatment group activated by micron particles was significantly slower and the survival period of mice was significantly prolonged.
  • the effect of T cells activated by micron particles containing substances that increase lysosome escape function is better than that of T cells activated by micron particles that do not contain substances that increase lysosome escape function. It can be seen that the allogeneic activated immune cells of the present invention have a preventive effect on breast cancer.
  • Example 17 Allogeneic immune cells activated by micron particles for the prevention of breast cancer
  • This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use a micron particle system loaded with whole cell components and use the micron particles to activate allogeneic cancer-specific T cells for the prevention of breast cancer.
  • 8M urea solution was first used to lyse breast cancer cells and dissolve the lysis components.
  • PLA was used as the micron particle skeleton material
  • CpG and Poly ICLC were used as immune adjuvants
  • arginine and histidine were used as substances that enhance lysosomal escape to prepare a micron particle system loaded with whole cell components.
  • 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 (containing 500mM sodium chloride) was used to lyse the cancer cells and dissolve the lysate components, which are the antigen components for preparing the micron particle system.
  • the double emulsion method was used to prepare the microparticle system and the control microparticles.
  • the microparticle skeleton materials are unmodified PLA and mannose-modified PLA.
  • the molecular weights are both 40KDa.
  • the values of unmodified PLA and mannose-modified PLA are The ratio is 9:1.
  • the immune adjuvants used are CpG and Poly ICLC, and the lysosomal escape-increasing substances used are arginine and histidine.
  • the double emulsion method is first used to prepare micron particles internally loaded with lysate components, adjuvants, arginine and histidine.
  • micron particles are centrifuged at 9000g for 20 minutes, and 10 mL of ultrasonic acid containing 4% trehalose is used. Resuspend in pure water and dry for 48 hours before use.
  • the average particle size of the micron particle system is about 2.1 ⁇ m, and the surface potential of the micron particle system is about -7mV; each 1 mg of PLA micron particles is loaded with approximately 100 ⁇ g of protein or peptide components, including 0.01 mg each of CpG and Poly ICLC, and arginine and Histidine 0.05mg each.
  • the preparation materials and preparation methods of the control microparticles are the same as those of the microparticles described in this example.
  • the particle size is about 2.1 ⁇ m, the surface potential is about -7mV, and only arginine and histidine and an equal amount of cell lysate components are loaded. , without loading any adjuvants.
  • mice Female BALB/c mice aged 6 to 8 weeks were selected and subcutaneously injected with 100 ⁇ L of micron particles containing 2 mg PLA on days 0, 4, 7, 14, 21, and 28. On the 32nd day, the mice were sacrificed and their spleens were removed, mouse spleen single cell suspensions were prepared, and CD8 + T cells were sorted from mouse splenocytes using magnetic bead sorting.
  • the tumor growth rate of the immune cell treatment group activated by micron particles was significantly slower and the survival period of mice was significantly prolonged.
  • T cells activated by microparticles containing substances that increase lysosomal escape function and immune adjuvants are better than T cells activated by microparticles that only contain substances that increase lysosome escape function without adjuvants. It can be seen that the allogeneic activated immune cells of the present invention have a preventive effect on breast cancer.
  • Example 18 Nanoparticles activate allogeneic cancer-specific T cells for the treatment of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with melanoma cancer cells and whole cell components of tumor tissue to activate allogeneic cancer-specific T cells and then infuse them back into the mouse. mice to treat melanoma.
  • B16F10 melanoma cancer cells were first lysed to prepare water-soluble components and non-water-soluble components. Then, PLGA was used as the framework material, Poly(I:C) and CpG were used as immune adjuvants, and R8( RRRRRRRR) polypeptide is a substance that dissolves the ability to escape from lysosomes.
  • a nanoparticle system loaded with water-soluble components or non-water-soluble components is prepared.
  • the nanoparticle system is then co-incubated with dendritic cells in vitro and the dendritic cells are returned to the system. Allogeneic infusion of cancer-specific T cells is performed in vivo, and then CD8 + T cells are isolated and extracted for cancer treatment.
  • 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 through the cell filter and freeze and thaw repeatedly 5 times, and may be accompanied by ultrasound to destroy the lysed sample. Add nuclease for 10 minutes and then heat at 95°C for 5 minutes to inactivate the nuclease; collect the cultured B16F10 For cancer cell lines, first centrifuge to remove the culture medium, then wash twice with PBS and centrifuge to collect the cancer cells.
  • the water-soluble components of the tumor tissue and the water-soluble components of the cancer cells are mixed at a mass ratio of 1:1; the water-insoluble components of the tumor tissue and the non-water-soluble components of the cancer cells are mixed at a mass ratio of 1:1.
  • the above are the sources of antigen raw materials for preparing nanoparticle systems.
  • the nanoparticles were prepared using 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 material used to prepare nanoparticles, is 7KDa-17KDa.
  • the immune adjuvants used are poly(I:C) and CpG.
  • the R8 polypeptide is a substance that increases lysosomal escape, and the adjuvant and R8 polypeptide are loaded on the nanoparticles. within the particle.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the lysis solution components, adjuvants and R8 polypeptides inside the nanoparticles. After loading the lysis components inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes. And use 10mL of ultrapure water containing 4% trehalose to resuspend and freeze-dry for 48h; before use, resuspend it in 9mL PBS and then add 1mL of lysate component (protein concentration 80mg/mL) and incubate at room temperature for 10min to obtain the internal and external contents. Both lysate-loaded nanoparticle systems.
  • the average particle size of the nanoparticles is about 290nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 140 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C) and CpG immune Each adjuvant is 0.02mg, loading 0.1mg R8 polypeptide.
  • the particle size of the blank nanoparticles is about 270 nm, and the blank nanoparticles are loaded with equal amounts of adjuvant and R8 polypeptide pure water or 50% glycerin to replace the corresponding water-soluble components and non-water-soluble 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 RPMI1640 (10% FBS) medium, while adding recombinant mouse GM-CSF (20 ng/mL) 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).
  • BMDC Spread mouse BMDC into a cell culture plate, add 5 mL of RPMI 1640 (10% FBS) culture medium for every 100,000 BMDC cells, and then add 30 ⁇ g of PLGA nanoparticles loaded with water-soluble components and 30 ⁇ g of non-water-soluble loaded original
  • the component PLGA nanoparticles were incubated with BMDC for 48 h, and then the BMDC were collected and centrifuged at 300g for 5 minutes, washed twice with phosphate buffer saline (PBS) and resuspended in PBS for later use.
  • PBS phosphate buffer saline
  • blank nanoparticles + free lysate were added to co-incubate with BMDC cells.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected and 1 million BMDC cells were injected subcutaneously on days 0, 4, 7, 14, 21 and 28 respectively.
  • the injected BMDC cells in each group were activated by nanoparticles loaded with lysate or blank nanoparticles + free lysate.
  • CD8 + T cells were isolated from spleen single cell suspensions from mice on day 32 using flow cytometry.
  • 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. 100 ⁇ L of 800,000 CD8 + T cells were injected intravenously on days 4, 7, 10, 15, and 20 after melanoma inoculation. The method for monitoring tumor growth and survival in mice is the same as above.
  • the tumors of mice in both the PBS control group and the blank nanoparticle control group grew.
  • the tumor growth rate of mice treated with allogeneic cancer-specific T cells activated by the nanovaccine was significantly slower, and some mice had tumors that disappeared and recovered.
  • the cell system of the present invention has good therapeutic effect on melanoma.
  • This example uses mouse colon cancer as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components of colon cancer tumor tissue to activate allogeneic cancer-specific T cells and then infuse them back into mice for treatment.
  • Colon cancer 8M urea aqueous solution is first used to lyse colon cancer tumor tissue and dissolve the lysed components. Then, PLGA is used as the skeleton material, Poly(I:C) and CpG are used as immune adjuvants to prepare a nanoparticle system, and then used The nanoparticle system activates allogeneic cancer-specific T cells in vivo, and then CD8 + T cells are isolated and extracted for cancer treatment.
  • two different methods are used to activate cancer-specific T cells. One is to directly inject nanoparticles into the human body for activation, and the other is to activate dendritic cells in vitro and then inject them into the body to activate cancer-specific T cells. .
  • the nanoparticles were prepared using the double emulsion method.
  • the molecular weight of PLGA, the material used to prepare the nanoparticles, is 7KDa-17KDa.
  • the immune adjuvants used are poly(I:C) and CpG, and the adjuvants are loaded in the nanoparticles.
  • the preparation method is as described above. During the preparation process, the lysis solution components and adjuvants are first loaded inside the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 10,000 g for 20 minutes and resuspended in 10 mL of ultrapure water containing 4% trehalose. Freeze dry for 48 hours before use.
  • the average particle size of the nanoparticles is about 260nm, and the surface potential of the nanoparticles is about -7mV; each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C) and CpG immune Each adjuvant is 0.02mg.
  • BMDC Spread mouse BMDC into a cell culture plate, add 5 mL of RPMI 1640 (10% FBS) culture medium for every 100,000 BMDC cells, then add 50 ⁇ g of PLGA nanoparticles loaded with lysis components and incubate with the BMDC for 48 hours, and then collect After BMDC, centrifuge at 300g for 5 minutes, wash twice with phosphate buffer saline (PBS) and resuspend in PBS for later use.
  • RPMI 1640 50% FBS
  • PBS phosphate buffer saline
  • Allogeneic cells are given to cancer-stricken mice to treat cancer.
  • mice Select 6-8 week old female C57BL/6 as model mice to prepare colon cancer mice.
  • each mouse was subcutaneously inoculated with 2 ⁇ 10 6 MC38 cells on the lower right side of the back.
  • days 4, 7, 10, 15, 20 and 25 after inoculation of colon cancer cells 100 ⁇ L of 800,000 CD8 + T cells were injected intravenously.
  • the method for monitoring tumor growth and survival in mice is the same as above.
  • the PBS control group grew up quickly.
  • the tumor growth rate of mice treated with allogeneic cancer-specific T cells activated by the nanovaccine was significantly slower and the survival period was significantly prolonged, and the cancer-specific T cells activated by the direct injection of the nanovaccine had an effect Better than injecting dendritic cells to activate cancer-specific T cells.
  • the cell system of the present invention has good therapeutic effect on cancer.
  • Example 20 Nanoparticles activate allogeneic immune cells for the prevention of breast cancer
  • This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components and use the nanoparticles to activate allogeneic cancer-specific T cells for the prevention of breast cancer.
  • an 8M urea solution was first used to lyse breast cancer tumor tissue and dissolve the lysed components. Then, using CpG and Poly(I:C) as immune adjuvants, and arginine and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) as substances that enhance lysosomal escape, a PLGA nanoparticle system loaded with whole cell components was prepared.
  • mice that are 6-8 weeks old and subcutaneously inject 1 ⁇ 10 4T1 breast cancer cells into the back.
  • the mice are sacrificed and the mouse tumor tissue is removed. Cut into small pieces and pass through a cell sieve to prepare a single cell suspension. Wash twice with PBS and then use 8M urea aqueous solution (containing 500mM sodium chloride) to lyse the tumor tissue single cell suspension and dissolve the lysate components to prepare nanoparticles.
  • 8M urea aqueous solution containing 500mM sodium chloride
  • the nanoparticle system is prepared using the double emulsion method.
  • the nanoparticle skeleton materials are unmodified PLGA and mannan-modified PLGA.
  • the molecular weights are both 24-38KDa.
  • the ratio of unmodified PLGA to mannan-modified PLGA is is 4:1.
  • the immune adjuvants used were CpG and Poly(I:C), and the lysosomal escape-increasing substances used were arginine and KALA polypeptide.
  • the double emulsion method is first used to prepare nanoparticles internally loaded with lysate components, adjuvants, arginine and KALA polypeptides.
  • nanoparticles are centrifuged at 12,000g for 25 minutes, and 10 mL of ultrapure solution containing 4% trehalose is used. Resuspend in water and dry for 48 hours before use.
  • the average particle size of the nanoparticle system is about 250nm, and the surface potential of the nanoparticle system is about -8mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components, including 0.02 mg of CpG and Poly(I:C), and 0.02 mg of poly(I:C). 0.05mg each of amino acid and KALA.
  • mice Female BALC/c mice aged 6-8 weeks were selected and 100 ⁇ L of nanoparticles containing 2 mg PLGA were subcutaneously injected on days 0, 4, 7, 14, 21, and 28 respectively. On the 32nd day, the mice were sacrificed, peripheral blood was collected, and mouse peripheral blood mononuclear cells (PBMC) were isolated. CD3 + CD8 + T cells and ⁇ T cells were sorted from the PBMC using flow cytometry. The obtained CD3 + CD8 + T cells and ⁇ T cells were incubated with IL-2 (1000U/mL), IL-12 (1000U/mL) and ⁇ CD3/ ⁇ CD28 antibody (10ng/mL) respectively for 14 days to expand CD8 + T cells. and ⁇ T cells.
  • IL-2 1000U/mL
  • IL-12 1000U/mL
  • ⁇ CD3/ ⁇ CD28 antibody 10ng/mL
  • ⁇ T cells and NKT cells were isolated from PBMC, and the above two cells were co-incubated with IL-2 (1000U/mL), IL-12 (1000U/mL) and ⁇ CD3/ ⁇ CD28 antibody (10ng/mL) respectively. Incubate for 14 days to amplify and sort ⁇ T cells and NKT cells.
  • the tumor growth rate in the immune cell treatment group was significantly slower and the survival period of mice was significantly prolonged.
  • the mixed immune cells containing CD8 + T cells + ⁇ T cells are more effective than the mixed immune cells containing ⁇ T cells + NKT cells.
  • CD8 + T cells contain cancer-specific T cells of adaptive immunity, and ⁇ T cells and NKT cells all belong to the innate immune system. This shows that giving adaptive immune cells and innate immune cells at the same time is more effective than giving only innate immune cells. It can be seen that the allogeneic particle-activated immune cells of the present invention have a preventive effect on breast cancer.
  • Example 21 Allogeneic immune cells activated by micron particles for the treatment of breast cancer
  • This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use a micron particle system loaded with whole cell components and use the micron particles to activate allogeneic cancer-specific T cells for the treatment of breast cancer.
  • 8M urea solution was first used to lyse breast cancer cells and dissolve the lysis components. Then, using PLGA as the micron particle skeleton material, CpG and Poly ICLC as immune adjuvants, and arginine and lysine as substances that enhance lysosomal escape, a micron particle system loaded with whole cell components was prepared.
  • 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 (containing 500mM sodium chloride) was used to lyse the cancer cells and dissolve the lysate components, which are the antigen components for preparing the micron particle system.
  • the microparticle system is prepared using the double emulsion method.
  • the microparticle skeleton materials are unmodified PLGA and mannose-modified PLGA.
  • the molecular weights are both 24-38KDa.
  • the ratio of unmodified PLA to mannose-modified PLGA is 9. :1.
  • the immune adjuvants used are CpG and Poly ICLC, and the lysosomal escape-increasing substances used are arginine and lysine.
  • the double emulsion method is first used to prepare micron particles internally loaded with lysate components, adjuvants, arginine and lysine.
  • micron particles are centrifuged at 9000g for 20 minutes, and 10 mL of ultrasonic acid containing 4% trehalose is used. Resuspend in pure water and dry for 48 hours before use.
  • the average particle size of the micron particle system is about 2.1 ⁇ m, and the surface potential of the micron particle system is about -7mV; each 1 mg of PLGA micron particles is loaded with approximately 100 ⁇ g of protein or peptide components, including 0.01 mg each of CpG and Poly ICLC, and arginine and Lysine 0.05mg each.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected, and 100 ⁇ L of microparticles containing 2 mg PLGA were injected subcutaneously into the back on days 0, 4, 7, 14, 21, and 28. The mice were sacrificed on day 32, and the draining lymph nodes near the mice injected with micron particles were collected. The draining lymph nodes were minced and passed through a cell mesh to prepare a single-cell suspension. Incubate 2 mL of RPMI1640 complete medium containing 200,000 lymph node single cell suspension with the PLGA microparticles (30ug) prepared in step (2) for 48 hours, and then centrifuge at 400g for 5 minutes to collect the incubated single cell suspension.
  • CD3 + CD8 + T and CD3 + CD4 + T cells were sorted directly from the lymph node single cell suspension by flow cytometry, and compared with IL-2 (2000U/mL), IL-7 (500U/mL), and IL-15 (500U/mL) were incubated for 10 days to amplify the sorted CD8 + T cells and CD4 + T cells.
  • mice Female BALB/c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. At the same time, each mouse was subcutaneously injected with 1 ⁇ 10 6 4T1 cells on day 0. On days 5, 7, 10, 15, and 20, mice were subcutaneously injected with 100 ⁇ L containing 2 million cells. sorted and expanded T cells (containing 1.4 million CD8 + T cells and 600,000 CD4 + T cells), or subcutaneous injection of 100 ⁇ L containing 2 million cancer-specific T cells (containing 1.4 million CD8 + cancer-specific T cells and 600,000 CD4 + cancer-specific T cells). The size of the mouse tumor volume was recorded every 3 days starting from the 3rd day.
  • the tumor growth rate of the immune cell treatment group activated by micron particles was significantly slower and the survival period of mice was significantly prolonged.
  • the sorted cancer-specific T cells after expansion it is better to use the sorted cancer-specific T cells after expansion than directly sorted T cells after expansion.
  • the allogeneic cancer-specific T cells of the present invention have a good therapeutic effect on breast cancer.
  • the present invention uses the antigen-presenting cells (mainly DC cells and B cells) contained in the lymph node to assist the particles in activating cancer-specific T cells and then sorting the activated cancers.
  • the antigen-presenting cell auxiliary particle system from any other source can also be used to activate cancer-specific T cells, such as cell line-derived antigen-presenting cells, stem cell-derived antigen-presenting cells, and autologous sources. of antigen-presenting cells.

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Abstract

Provided are a cancer-specific T cell-based cell system, a lymphocyte drug and the use thereof. The cell system comprises cancer-specific T cells activated by a cancer vaccine, wherein the cancer vaccine comprises delivery particles and cell components loaded on the delivery particles, the delivery particles are nanoparticles or micron particles, and the cell components are water-soluble components and/or water-insoluble components in cells obtained by separating cancer cells and/or tumor tissues. Cancer-specific T cells can be activated by cancer vaccines or activated by injecting DC cells into the body after stimulation of DC cells in vitro by means of the cancer vaccines. An immune response is activated by means of an allogeneic body, innate immune cells and activated adaptive immune cells are transplanted into a receptor at the same time, and the clinical problem that a patient having poor immunocompetence cannot generate an effective immune response to a vaccine is solved.

Description

一种基于癌症特异性T细胞的细胞系统、淋巴细胞药物及其应用A cell system based on cancer-specific T cells, lymphocyte drugs and their applications 技术领域Technical field
本发明涉及免疫治疗技术领域，尤其涉及一种基于癌症特异性T细胞的细胞系统、淋巴细胞药物及其应用。The present invention relates to the technical field of immunotherapy, and in particular to a cell system based on cancer-specific T cells, lymphocyte drugs and their applications.
背景技术Background technique
免疫是人体的一种生理功能，人体依靠这种功能识别“自己”和“非己”成分，从而破坏和清除人体内的异常物质(如病毒、细菌等)，或人体本身所产生的损伤细胞和肿瘤细胞等，以维持人体的健康。近些年来免疫技术发展很快，尤其是癌症的免疫治疗领域。随着对癌症认识的不断提高，人们发现人体的免疫系统和各类免疫细胞在抑制癌症发生、发展的过程中扮演着关键角色。通过调节机体免疫系统的平衡，我们有望影响和控制癌症的发生、发展和治疗。Immunity is a physiological function of the human body. The human body relies on this function to identify "self" and "non-self" components, thereby destroying and removing abnormal substances (such as viruses, bacteria, etc.) in the human body, or damaged cells produced by the human body itself. and tumor cells, etc., to maintain human health. Immunology technology has developed rapidly in recent years, especially in the field of cancer immunotherapy. With the continuous improvement of understanding of cancer, people have found that the human body's immune system and various immune cells play a key role in inhibiting the occurrence and development of cancer. By regulating the balance of the body's immune system, we are expected to influence and control the occurrence, development and treatment of cancer.
机体通过各类免疫细胞识别和杀伤癌细胞。人体的免疫系统包含天然免疫系统和适应性免疫系统，天然免疫系统的细胞和适应性免疫系统的细胞相互配合，共同清除癌细胞。天然免疫系统中可以杀伤癌细胞的细胞包括NK细胞等，而适应性免疫系统中可以杀伤癌细胞的免疫细胞主要是T细胞，尤其是癌症特异性的CD8 +T细胞和CD4 +T细胞。天然免疫系统的免疫细胞的多少和功能强弱与癌症患者本身状态和机能有关，一般天然免疫系统的NK细胞和γδT细胞等含量高状态好的癌症患者预后较好。临床上很多年长的患者或者前期已经经历过放化疗的患者，其身体状态、生理机能和免疫功能很差，体内存在的先天免疫细胞较少或较弱，如果能补充一些同种异体年轻个体的先天免疫细胞，该部分患者的杀伤癌细胞的先天免疫系统能力会得到提升并进而更好的杀灭癌细胞。 The body recognizes and kills cancer cells through various types of immune cells. The human body's immune system consists of the natural immune system and the adaptive immune system. Cells of the natural immune system and cells of the adaptive immune system cooperate with each other to eliminate cancer cells. The cells in the natural immune system that can kill cancer cells include NK cells, while the immune cells in the adaptive immune system that can kill cancer cells are mainly T cells, especially cancer-specific CD8 + T cells and CD4 + T cells. The number and function of immune cells in the natural immune system are related to the status and function of the cancer patient. Generally, cancer patients with high levels of NK cells and γδ T cells in the natural immune system have a better prognosis. Clinically, many older patients or those who have experienced radiotherapy and chemotherapy in the early stage have poor physical condition, physiological function and immune function, and there are fewer or weaker innate immune cells in the body. If some allogeneic young individuals can be supplemented, By using innate immune cells, the ability of the innate immune system to kill cancer cells in these patients will be improved and thus better able to kill cancer cells.
适应性免疫系统的癌症特异性T细胞可以在肿瘤组织所释放的少量水溶性抗原刺激下产生一部分预存特异性免疫。但是，一种抗原表位只能激活一种特异性的T细胞。由于肿瘤组织部位抑制性的免疫微环境和癌细胞的高度异质性，预存的癌症特异性T细胞远远不够广谱和多样，因而识别和杀伤癌细胞效果有效。而癌症疫苗含有癌症特异性的抗原，可以刺激激活除机体预存特异性免疫以外的更广泛和多样的癌症特异性T细胞，因而癌症疫苗是治疗或预防癌症的方法之一。Cancer-specific T cells of the adaptive immune system can generate a portion of pre-existing specific immunity stimulated by small amounts of water-soluble antigens released by tumor tissues. However, an epitope can only activate one specific T cell. Due to the suppressive immune microenvironment of tumor tissue sites and the high heterogeneity of cancer cells, pre-existing cancer-specific T cells are far from broad-spectrum and diverse enough to effectively identify and kill cancer cells. Cancer vaccines contain cancer-specific antigens, which can stimulate and activate a wider range of cancer-specific T cells in addition to the body's pre-existing specific immunity. Therefore, cancer vaccines are one of the methods to treat or prevent cancer.
癌症疫苗的作用基础是选择合适的癌症抗原来激活人体免疫系统对异常突变的癌细胞的识别，癌细胞和肿瘤组织异质性很高，突变很多，所以癌症细胞或者癌症肿瘤组织本身是最好的癌症抗原的来源。疫苗所含抗原种类越多，所能激活的癌症特异性T细胞种类 越多，疫苗的疗效就会越好。但是，临床很多年长的患者或者前期已经经历过放化疗的患者，其身体状态、生理机能和免疫功能很差，因此，亟需寻找一种新的免疫治疗方案。The basic function of cancer vaccines is to select appropriate cancer antigens to activate the human immune system to recognize abnormally mutated cancer cells. Cancer cells and tumor tissues are highly heterogeneous and have many mutations, so cancer cells or cancer tumor tissues themselves are the best sources of cancer antigens. The more types of antigens a vaccine contains and the more types of cancer-specific T cells it can activate, the better the efficacy of the vaccine will be. However, in clinical practice, many elderly patients or patients who have experienced radiotherapy and chemotherapy in the early stage have poor physical condition, physiological function and immune function. Therefore, there is an urgent need to find a new immunotherapy solution.
发明内容Contents of the invention
为解决上述技术问题，本发明提供了一种基于癌症特异性T细胞的细胞系统，该细胞系统中的癌症特异性T细胞经癌症疫苗激活，能够更有效地激活癌症特异性免疫反应，并基于该细胞系统提供了一种同种异体淋巴细胞药物，由于癌症特异性T细胞激活时的效果与个体的免疫机能情况有关，因此本发明提出一种新的策略，即在免疫机能较好的同种异体上接种癌症疫苗或输入体外激活的树突状细胞(DC)细胞，然后分离提取被高效激活的癌症特异性免疫细胞和天然免疫细胞，再回输至免疫机能较差的同种异体上，解决上述年老的癌症患者或者前期经历过放疗或者化疗治疗的癌症患者中存在的免疫机能较差的问题。In order to solve the above technical problems, the present invention provides a cell system based on cancer-specific T cells. The cancer-specific T cells in this cell system are activated by cancer vaccines and can more effectively activate cancer-specific immune responses. This cell system provides an allogeneic lymphocyte drug. Since the effect of cancer-specific T cell activation is related to the individual's immune function, the present invention proposes a new strategy, that is, while the immune function is better, Inoculate the allogeneic cancer vaccine or inject dendritic cells (DC) cells activated in vitro, and then isolate and extract the highly activated cancer-specific immune cells and natural immune cells, and then infuse them back into the allogeneic allogeneic with poor immune function. , to solve the problem of poor immune function among the above-mentioned elderly cancer patients or cancer patients who have experienced radiotherapy or chemotherapy in the early stage.
本发明的第一个目的是提供一种基于癌症特异性T细胞的细胞系统，该细胞系统包括经癌症疫苗激活的癌症特异性T细胞，其中，癌症疫苗包括递送粒子及其负载的细胞组分，所述递送粒子为纳米粒子或微米粒子，所述细胞组分为来源于癌细胞和/或肿瘤组织的水溶性组分和/或非水溶性组分；The first object of the present invention is to provide a cell system based on cancer-specific T cells, which cell system includes cancer-specific T cells activated by a cancer vaccine, wherein the cancer vaccine includes delivery particles and loaded cell components. , the delivery particles are nanoparticles or microparticles, and the cellular components are water-soluble components and/or water-insoluble components derived from cancer cells and/or tumor tissues;
细胞组分经癌细胞和/或肿瘤组织裂解得到；Cell components are obtained by lysing cancer cells and/or tumor tissues;
裂解是将癌细胞或肿瘤组织加水或不含溶解剂的水溶液进行裂解，得到的上清液为水溶性组分，沉淀中经溶解剂溶解后转为可溶的部分为非水溶性组分；或，裂解是使用溶解剂裂解癌细胞或肿瘤组织，并溶解裂解后的组分，得到同时含有水溶性组分和非水溶性组分的混合物。Lysis is to lyse cancer cells or tumor tissue by adding water or an aqueous solution without a dissolving agent. The supernatant obtained is a water-soluble component, and the part of the precipitate that is converted to soluble after being dissolved by a dissolving agent is a water-insoluble component; Or, lysis is to use a dissolving agent to lyse cancer cells or tumor tissues, and dissolve the lysed components to obtain a mixture containing both water-soluble components and water-insoluble components.
进一步地，将癌细胞或肿瘤组织在-20℃～-273℃下冷冻，加水或不含溶解剂的水溶液后进行反复冻融裂解，直至细胞膜结构被破坏。Further, the cancer cells or tumor tissues are frozen at -20°C to -273°C, and water or an aqueous solution without a dissolving agent is added, followed by repeated freezing and thawing until the cell membrane structure is destroyed.
进一步地，所述癌症特异性T细胞包括CD4 +T细胞和/或CD8 +T细胞。 Further, the cancer-specific T cells include CD4 + T cells and/or CD8 + T cells.
进一步地，所述细胞系统还包括天然免疫细胞，该天然免疫细胞无需经癌症疫苗激活，可为自体天然免疫细胞和/或异体天然免疫细胞。Furthermore, the cell system also includes natural immune cells, which do not need to be activated by cancer vaccines and can be autologous natural immune cells and/or allogeneic natural immune cells.
将本发明的细胞系统制备成抗肿瘤制品时，该抗肿瘤制品包括以下任意一种：(1)含有经癌症疫苗激活后的癌症特异性T细胞的制剂；(2)含有经癌症疫苗激活后的癌症特异性T细胞的制剂，以及含有天然免疫细胞的制剂(其中，天然免疫细胞若多于一种，含有天然免疫细胞的制剂可为一种或多种，在给患者使用时可以分别注射或者混合后一起注射)；(3)含有经癌症疫苗激活后的癌症特异性T细胞和天然免疫细胞混合物的制剂。When the cell system of the present invention is prepared into an anti-tumor product, the anti-tumor product includes any of the following: (1) a preparation containing cancer-specific T cells activated by a cancer vaccine; (2) a preparation containing cancer-specific T cells activated by a cancer vaccine Preparations of cancer-specific T cells, and preparations containing natural immune cells (wherein, if there are more than one natural immune cells, the preparations containing natural immune cells can be one or more, and can be injected separately when used for patients) or mixed and injected together); (3) Preparations containing a mixture of cancer-specific T cells and natural immune cells activated by cancer vaccines.
进一步地，天然免疫细胞包括但不限于γδT细胞、自然杀伤细胞(NK细胞)、中性粒细胞和自然杀伤T细胞(NKT细胞)等。Further, natural immune cells include, but are not limited to, γδ T cells, natural killer cells (NK cells), neutrophils, and natural killer T cells (NKT cells).
进一步地，所述的激活为将癌症疫苗注射至体内激活癌症特异性T细胞，或将树突状 细胞(DC)在体外经癌症疫苗刺激后注射至体内激活癌症特异性T细胞。Further, the activation is injecting a cancer vaccine into the body to activate cancer-specific T cells, or injecting dendritic cells (DC) into the body after being stimulated by a cancer vaccine in vitro to activate cancer-specific T cells.
进一步地，所述的刺激是将DC细胞与癌症疫苗共孵育一定时间，负载细胞组分的递送粒子被DC细胞吞噬后，可被DC细胞进行抗原提呈和激活，回输至体内后即可归巢淋巴结并利用DC细胞负载的抗原激活癌症特异性T细胞。Further, the stimulation is to incubate DC cells with the cancer vaccine for a certain period of time. After the delivery particles loaded with cell components are engulfed by DC cells, they can be presented and activated by the DC cells for antigens, and can be reinfused into the body. Homing to lymph nodes and utilizing antigens loaded by DC cells to activate cancer-specific T cells.
进一步地，DC细胞与癌症疫苗共孵育至少4小时，优选为24-96小时。Further, the DC cells are incubated with the cancer vaccine for at least 4 hours, preferably 24-96 hours.
进一步地，树突状细胞为自体树突状细胞、异体树突状细胞、细胞系或干细胞来源，DC细胞可来源于任何可以制备分离树突状细胞的细胞，包括但不限于来源于干细胞、细胞系、骨髓细胞、外周免疫细胞等。Further, dendritic cells are derived from autologous dendritic cells, allogeneic dendritic cells, cell lines or stem cells. DC cells can be derived from any cell from which isolated dendritic cells can be prepared, including but not limited to derived from stem cells, Cell lines, bone marrow cells, peripheral immune cells, etc.
进一步地，癌症疫苗中，将非水溶性组分负载于递送粒子时，将非水溶性组分采用适当的溶解方法使其由在纯水中不溶变为在含溶解剂的溶液中可溶。采用溶解剂溶解时，溶解剂选自尿素、盐酸胍、脱氧胆酸盐(如脱氧胆酸钠)、十二烷基硫酸盐(如十二烷基硫酸钠，SDS)、甘油、蛋白质降解酶、白蛋白、卵磷脂、无机盐、Triton、吐温、DMSO(二甲基亚砜)、乙腈、乙醇、甲醇、DMF(N,N-二甲基甲酰胺)、丙醇、异丙醇、醋酸、胆固醇、氨基酸、糖苷和胆碱中的至少一种。Furthermore, in cancer vaccines, when loading non-water-soluble components on delivery particles, an appropriate dissolution method is used to convert the non-water-soluble components from insoluble in pure water to soluble in a solution containing a dissolving agent. When dissolving with a dissolving agent, the dissolving agent is selected from the group consisting of urea, guanidine hydrochloride, deoxycholate (such as sodium deoxycholate), dodecyl sulfate (such as sodium dodecyl sulfate, SDS), glycerol, and protein degrading enzymes. , albumin, lecithin, inorganic salts, Triton, Tween, DMSO (dimethyl sulfoxide), acetonitrile, ethanol, methanol, DMF (N,N-dimethylformamide), propanol, isopropyl alcohol, At least one of acetic acid, cholesterol, amino acids, glycosides and choline.
进一步地，癌症疫苗中，负载方式为水溶性组分和/或非水溶性组分被包载于递送粒子内部，和/或负载于递送粒子表面，包括但不限于水溶性组分同时负载于粒子中和负载于粒子表面，非水溶性组分同时负载于粒子中和负载于粒子表面，水溶性组分负载于粒子中而非水溶性组分负载于粒子表面，非水溶性组分负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负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负载于粒子中而只有水溶性组分负载于粒子表面，水溶性组分负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，非水溶性组分负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负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，水溶性组分和非水溶性组分同时负载于粒子内部，水溶性组分和非水溶性组分同时非水溶性组分同时负载于粒子表面。Further, in the cancer vaccine, the loading method is that water-soluble components and/or water-insoluble components are contained inside the delivery particles, and/or are loaded on the surface of the delivery particles, including but not limited to water-soluble components being loaded on the delivery particles at the same time. 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 particle surface. The water-soluble components are loaded in the particles, while the water-soluble components and the water-insoluble components are loaded on the particle surfaces at the same time. The water-insoluble components are loaded in the particles and the water-soluble components are loaded on the particles. Components and non-water-soluble components are loaded on the surface of the particles at the same time, water-soluble components and non-water-soluble components are loaded on the particles at the same time, and water-soluble components and non-water-soluble components are loaded on the surface of the particles at the same time. The water-soluble components and the water-insoluble components are loaded inside the particles at the same time, and the water-soluble components and the water-insoluble components are loaded on the surface of the particles at the same time.
进一步地，癌症疫苗中，水溶性组分和/或非水溶性组分负载于递送粒子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；水溶性组分和/或非水溶性组分负载于递送粒子内部的方式为任何可以将其负载于递送粒子内部的方式，如包载于递送粒子内部、通过静电吸附作用负载于递送粒子内部、通过疏水相互作用负载于递送粒子内部。Furthermore, in cancer vaccines, the ways in which water-soluble components and/or non-water-soluble components are loaded on the surface of delivery particles include adsorption, covalent attachment, charge interaction, hydrophobic interaction, one or more steps of solidification, and mineralization. and at least one of wrapping; the way in which the water-soluble component and/or the non-water-soluble component is loaded inside the delivery particle is any way that can load it inside the delivery particle, such as being loaded inside the delivery particle, through electrostatic It is loaded inside the delivery particles by adsorption and loaded inside the delivery particles through hydrophobic interaction.
进一步地，细胞组分负载于递送粒子后呈一层或多层结构，当递送粒子负载多层细胞组分时，层与层之间为修饰物。Furthermore, after the cell components are loaded on the delivery particles, they form one or more layers of structure. When the delivery particles are loaded with multiple layers of cell components, there are modifications between the layers.
进一步地，癌症疫苗中，递送粒子的表面连接有主动靶向树突状细胞的靶头。该靶头可为甘露糖、甘露聚糖、CD32抗体、CD11c抗体、CD103抗体、CD44抗体等靶头，带领 癌症疫苗靶向输送进树突状细胞。Furthermore, in cancer vaccines, the surface of the delivery particles is connected with a target that actively targets dendritic cells. The target can be mannose, mannan, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody and other targets, leading the targeted delivery of cancer vaccines into dendritic cells.
进一步地，癌症疫苗中，还包括免疫增强佐剂，其与细胞组分共同负载于递送粒子。免疫增强佐剂包括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、黄芪的有效成分等。将免疫增强佐剂与细胞组分共同负载于递送粒子上时，癌症疫苗被DC细胞吞噬后可以更好地激活癌症特异性T细胞。Furthermore, the cancer vaccine also includes an immune-enhancing adjuvant, which is loaded on the delivery particles together with the cell components. Immune-enhancing adjuvants include at least one of microbial-derived immune enhancers, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicine and other categories; Immune-enhancing adjuvants include, but are not limited to, pattern recognition receptor agonists, Bacillus Calmette-Guérin (BCG), manganese-related adjuvants, BCG cell wall scaffolds, BCG methanol extraction residues, BCG muramyl dipeptide, Mycobacterium phlei, polyanti-A Vitamins, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, miquimod, polysaccharides, curcumin, immune Adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium parvum vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvant, aluminum adjuvant, Calcium adjuvant, various cytokines, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, squalene, cytokines, vegetable oil, endotoxin, liposome adjuvant agent, MF59, double-stranded RNA, double-stranded DNA, aluminum-related adjuvants, CAF01, ginseng, active ingredients of astragalus, etc. When immune-enhancing adjuvants and cellular components are co-loaded on delivery particles, cancer vaccines can better activate cancer-specific T cells after being engulfed by DC cells.
进一步地，递送粒子还可共负载协助粒子或抗原逃离溶酶体和提高激活效率的成分。协助和增加粒子或抗原溶酶体逃逸的物质包括但不限于细胞穿透肽、膜穿透肽、具有质子海绵效应的物质、具有膜融合效应的物质。Furthermore, the delivery particles can also be co-loaded with components that assist particles or antigens in escaping from lysosomes and improve activation efficiency. Substances that assist and increase the escape of particles or antigens from lysosomes include, but are not limited to, cell-penetrating peptides, membrane-penetrating peptides, substances with proton sponge effects, and substances with membrane fusion effects.
进一步地，癌症疫苗中，递送粒子表面可为电中性、带负电或者带正电。Furthermore, in cancer vaccines, the surface of the delivery particles can be electrically neutral, negatively charged, or positively charged.
进一步地，癌症疫苗中，递送粒子纳米级或微米级，这样能保证疫苗被抗原提呈细胞吞噬，而为了提高吞噬效率，粒径大小要在适宜的范围内。纳米粒子(NP)的粒径为1nm-1000nm，优选地，粒径为30nm-1000nm，最优选地，粒径为100nm-600nm；微米粒子(MP)的粒径为1μm-1000μm，优选地，粒径为1μm-100μm，更优选地，粒径为1μm-10μm，最优选地，粒径为1μm-5μm。Furthermore, in cancer vaccines, the delivery particles are nanoscale or micron-scale, which can ensure that the vaccine is engulfed by antigen-presenting cells. In order to improve the phagocytosis efficiency, the particle size must be within an appropriate range. The particle size of nanoparticles (NP) is 1nm-1000nm, preferably, the particle size is 30nm-1000nm, most preferably, the particle size is 100nm-600nm; the particle size of microparticles (MP) is 1μm-1000μm, preferably, The particle size is 1 μm-100 μm, more preferably, the particle size is 1 μm-10 μm, and most preferably, the particle size is 1 μm-5 μm.
进一步地，癌症疫苗中，递送粒子由有机合成高分子材料、天然高分子材料或无机材料制得。有机合成高分子材料为生物相容或可降解的高分子材料，包括但不限于PLGA(聚乳酸-羟基乙酸共聚物)、PLA(聚乳酸)、PGA(聚乙醇酸)、PEG(聚乙二醇)、PCL(聚己内酯)、Poloxamer(泊洛沙姆)、PVA(聚乙烯醇)、PVP(聚乙烯吡咯烷酮)、PEI(聚乙烯亚胺)、PTMC(聚三亚甲基碳酸酯)、聚酸酐、PDON(聚对二氧六环酮)、PPDO(聚对二氧环己酮)、PMMA(聚甲基丙烯酸甲酯)、PLGA-PEG、PLA-PEG、PGA-PEG、聚氨基酸、合成多肽、合成脂质等；天然高分子材料为为生物相容或可降解的高分子材料，包括但不限于卵磷脂、胆固醇、海藻酸盐、白蛋白、胶原蛋白、明胶、细胞膜、淀粉、糖类、多肽等；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。Furthermore, in cancer vaccines, the delivery particles are made of organic synthetic polymer materials, natural polymer materials or inorganic materials. Organic synthetic polymer materials are biocompatible or degradable polymer materials, including but not limited to PLGA (polylactic acid-co-glycolic acid), PLA (polylactic acid), PGA (polyglycolic acid), PEG (polyethylene glycol) alcohol), PCL (polycaprolactone), Poloxamer (poloxamer), PVA (polyvinyl alcohol), PVP (polyvinylpyrrolidone), PEI (polyethyleneimine), PTMC (polytrimethylene carbonate) , polyanhydride, PDON (polydioxanone), PPDO (polydioxanone), PMMA (polymethylmethacrylate), PLGA-PEG, PLA-PEG, PGA-PEG, polyamino acid , synthetic peptides, synthetic lipids, etc.; natural polymer materials are biocompatible or degradable polymer materials, including but not limited to lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane, starch , sugars, peptides, etc.; inorganic materials are materials with no obvious biological toxicity, including but not limited to ferric oxide, ferric oxide, calcium carbonate, calcium phosphate, etc.
进一步地，癌症疫苗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疫苗(Nanovaccine)或微米疫苗(Microvaccine)的抗原负载量和/或免疫原性进而提高树突状细胞疫苗的疗效。修饰技术包括但不限于化学修饰和物理修饰，如生物矿化(如硅化、钙化、镁化)、凝胶化、交联、添加带电物质等。Furthermore, the cancer vaccine does not need to be modified during the preparation process, or appropriate modification technology can be used to improve the antigen loading capacity and/or immunogenicity of the nanovaccine or microvaccine to improve the dendritic vaccine. Efficacy of cell-based vaccines. Modification technologies include but are not limited to chemical modification and physical modification, such as biomineralization (such as silicification, calcification, magnesization), gelation, cross-linking, addition of charged substances, etc.
进一步地，癌症疫苗的形状为常见的任意形状，包括但不限于球形、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。Furthermore, the shape of the cancer vaccine is any common shape, including but not limited to sphere, ellipsoid, barrel, polygon, rod, sheet, linear, worm-shaped, square, triangle, butterfly or disc.
进一步地，癌症疫苗可以采用已有的制备方法制备，包括但不仅限于常见的溶剂挥发法、透析法、微流控法、挤出法、热熔法。在本发明的一些实施方案中，癌症疫苗采用溶剂挥发法中的复乳法制备得到，具体如下：Furthermore, cancer vaccines can be prepared using existing preparation methods, including but not limited to common solvent evaporation methods, dialysis methods, microfluidic methods, extrusion methods, and hot melt methods. In some embodiments of the present invention, the cancer vaccine is prepared by the double emulsion method in the solvent evaporation method, as follows:
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的医用材料的有机相中。Step 1: Add a first predetermined volume of an aqueous phase solution containing a first predetermined concentration into a second predetermined volume of an organic phase containing a second predetermined concentration of medical material.
其中，水相溶液含有细胞组分，可含有或不含有免疫增强佐剂；细胞组分为水溶性组分和/或溶于溶解剂的原非水溶性组分，或者水溶性组分和非水溶性组分同时溶于溶解剂中的混合物。第一预定浓度要求蛋白质多肽浓度大于1ng/mL，优选1mg/mL-100mg/mL，以确保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，优选0.01mg/mL-20mg/mL；Wherein, the aqueous phase solution contains cell components, which may or may not contain immune-enhancing adjuvants; the cell components are water-soluble components and/or original non-water-soluble components soluble in the dissolving agent, or water-soluble components and non-water-soluble components. A mixture of water-soluble components simultaneously dissolved in a dissolving agent. The first predetermined concentration requires the protein polypeptide concentration to be greater than 1ng/mL, preferably 1mg/mL-100mg/mL, to ensure that sufficient cancer antigens can be loaded to activate relevant immune responses. The concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL, preferably 0.01mg/mL-20mg/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或经修饰的PLGA或PLA，有机溶剂为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。第二预定浓度为0.5mg/mL-5000mg/mL，优选为100mg/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. In some embodiments, the medical polymer material is PLGA or modified PLGA or PLA, and the organic solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc. , preferably methylene chloride. The second predetermined concentration is 0.5 mg/mL-5000 mg/mL, preferably 100 mg/mL.
在本发明实施例中，之所以选择PLGA或经修饰的PLGA或PLA，是由于该材料为生物可降解材料且已被FDA批准用作药物辅料。研究表明PLGA或PLA都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。在实际应用中可根据实际情况选择合适的材料。在本发明的实施例中，部分实施递送粒子共负载的组分中含有具有溶酶体逃逸能力的多肽，在实际应用中也可以加入任何其他可增加递送粒子或者抗原溶酶体逃逸的物质。In the embodiment of the present invention, PLGA or modified PLGA or PLA is selected because this material is a biodegradable material and has been approved by the FDA for use as a pharmaceutical excipient. Studies have shown that PLGA or PLA both have certain immune-modulating functions and are therefore suitable as excipients in the preparation of nanoparticles or microparticles. In practical applications, appropriate materials can be selected according to actual conditions. In the embodiments of the present invention, some of the components for co-loading delivery particles contain polypeptides with lysosomal escape ability. In practical applications, any other substances that can increase the lysosomal escape of delivery particles or antigens can also be added.
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，优选为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子或微米粒子的尺寸大小。In practice, the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase. In the present invention, the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is It is 1:1.1-1:5000, preferably 1:10. During the specific implementation process, the first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.
步骤2，将步骤1得到的混合液进行超声处理、搅拌、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，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、搅拌、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、搅拌速度、均质处理压力及时间能控制制备的微纳粒子大小。Step 2: Subject the mixed solution obtained in Step 1 to ultrasonic treatment, stirring, homogenization treatment or microfluidic treatment. Preferably, the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm ~ 1500rpm, the stirring time is 0.1 hour ~ 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5W, and the time is greater than 0.1 seconds, such as 2 to 200 seconds; use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer for homogenization. When using a high-pressure/ultra-high-pressure homogenizer, the pressure is greater than 5 psi, such as 20 psi to 100 psi, and use high shear. When switching to a homogenizer, the rotation speed should be greater than 100rpm, such as 1000rpm to 5000rpm; use microfluidic processing with a flow rate greater than 0.01mL/min, such as 0.1mL/min-100mL/min. Ultrasound, stirring, homogenization treatment or microfluidic treatment are used for nanometerization and/or micronization. The length of ultrasonic time, stirring speed, homogenization pressure and time can control the size of the prepared micro-nano particles.
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超声处理、搅拌、均质处理或微流控处理。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, stirring, homogenization treatment or microfluidic treatment.
该步骤是为了继续纳米化或微米化，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。This step is to continue 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 bring about changes in particle size. For this reason, it is necessary to choose the appropriate ultrasound time. In the present invention, 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, and the stirring time is greater than 1 minute, such as 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute. For example, the stirring speed is 50 rpm ~ 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. When using a high-pressure/ultra-high-pressure homogenizer, the pressure is greater than 20 psi, such as 20 psi to 100 psi. When using a high-shear homogenizer, 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.
在本发明中，乳化剂水溶液为聚乙烯醇(PVA)水溶液，第三预定浓度大于1mg/mL，优选1-100mg/mL，本发明的一些实施例中选择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，优选为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。In the present invention, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, and the third predetermined concentration is greater than 1 mg/mL, preferably 1-100 mg/mL, and 20 mg/mL is selected in some embodiments of the present invention. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present invention, the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5. In order to control the size of nanoparticles or microparticles during specific implementation, the ratio of the second predetermined volume and the third predetermined volume can be adjusted. Similarly, 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.
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。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.
本步骤中，乳化剂水溶液依然为PVA。In this step, the emulsifier aqueous solution is still PVA.
第四预定浓度大于0.01mg/mL，优选为0.01-100mg/mL，本发明的一些实施例中选择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，优选为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。The fourth predetermined concentration is greater than 0.01 mg/mL, preferably 0.01-100 mg/mL. In some embodiments of the present invention, 5 mg/mL is selected. 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. In the present invention, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. In the specific implementation process, the ratio of the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.
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。In the present invention, the predetermined stirring condition for this step is until the organic solvent is completely volatilized.
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，并将沉淀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。Step 5: After centrifuging the mixed solution that meets the predetermined stirring conditions in Step 4 at a rotation speed greater than 100 RPM for greater than 1 minute, remove the supernatant, and resuspend the precipitate in a fifth predetermined volume of a fifth predetermined concentration. in an aqueous solution containing a lyophilized protective agent or in a sixth predetermined volume of PBS (or physiological saline).
其中，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；或直接使用进行注射或者与细胞共孵育。Among them, 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 subsequent experiments related to the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles can be directly performed; or it can be used directly Inject or incubate with cells.
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。冻干保护剂选用海藻糖(Trehalose)；第五预定浓度为质量百分比1-15％，优选为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；或使用其他冻干保护剂进行冷冻干燥处理如蔗糖和甘露醇的混合溶液。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 conducting subsequent experiments related to the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles. The freeze-drying protective agent is trehalose; the fifth predetermined concentration is 1-15% by mass, preferably 4%. The reason why it is set in this way is to not affect the freeze-drying effect during subsequent freeze-drying; or use Other lyoprotectants are used for freeze drying such as a mixed solution of sucrose and mannitol.
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。Step 6: After freeze-drying the suspension containing the lyoprotectant obtained in Step 5, the freeze-dried material is used for later use.
步骤7，将步骤5中得到的重悬于PBS(或生理盐水)中的含纳米粒或微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和/或溶解的原非水溶性组分混合后使用，即表面负载细胞组分的癌症疫苗。Step 7: Resuspend the nanoparticle- or microparticle-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 frozen suspension obtained in Step 6. The dried freeze-dried material containing nanoparticles or microparticles and a freeze-drying protective agent is used directly; or the above sample is mixed with a seventh predetermined volume of water-soluble components and/or dissolved original non-water-soluble components and used, that is, Cancer vaccines with surface-loaded cellular components.
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，优选为1:100到100:1，最优体积比为1:30到30:1。In the present invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, preferably 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1.
在一些实施例中，所述重悬的纳米粒子和/或微米粒子混悬液体积为9mL时，水溶性组分和/或溶解的原非水溶性组分的体积为1mL。在实际使用时可将二者体积和比例根据需要进行调整。In some embodiments, when the volume of the resuspended nanoparticle and/or microparticle suspension is 9 mL, the volume of the water-soluble component and/or the dissolved original non-water-soluble component is 1 mL. In actual use, the volume and proportion of the two can be adjusted as needed.
进一步地，细胞系统用于治疗或预防癌症时：(1)将步骤5制备的纳米疫苗和/或微米疫苗注射给供体(如同种异体供体)；或者将步骤5制备的纳米疫苗和/或微米疫苗与树突状细胞混合孵育一定时间，然后将树突状细胞注射给供体；(2)从供体中收集步骤(1)激活的供体体内的癌症特异性T细胞；或者收集步骤(1)激活的供体体内的癌症特异性T细胞以及NK细胞，NKT细胞以及γδT细胞。将收集的上述细胞回输到患者体内预防或治疗癌症。Further, when the cell system is used to treat or prevent cancer: (1) inject the nanovaccine and/or micron vaccine prepared in step 5 into a donor (such as an allogeneic donor); or inject the nanovaccine and/or micron vaccine prepared in step 5 Or micron vaccine and dendritic cells are mixed and incubated for a certain period of time, and then the dendritic cells are injected into the donor; (2) collecting from the donor the cancer-specific T cells activated in the donor in step (1); or collecting Step (1) activates cancer-specific T cells and NK cells, NKT cells and γδ T cells in the donor. The collected cells are reinfused into the patient's body to prevent or treat cancer.
上述方法中，若需在递送粒子表面负载多层细胞组分或对癌症疫苗进行修饰，可在上述步骤4之后进行以下操作：In the above method, if you need to load multiple layers of cellular components on the surface of the delivery particles or modify the cancer vaccine, you can perform the following operations after the above step 4:
S1，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，并将沉淀重新混悬于第八预定体积的第八预定浓度的含有水溶性组分和/或非水溶性组分的溶液中(该溶液中可含有或不含有佐剂)。S1, after centrifuging the mixed liquid that meets the predetermined stirring conditions in Step 4 at a rotation speed of greater than 100 RPM for greater than 1 minute, remove the supernatant, and resuspend the precipitate in an eighth predetermined volume of an eighth predetermined concentration of in a solution of water-soluble components and/or non-water-soluble components (the solution may or may not contain adjuvants).
S2，将S1得到的混合液进行离心，去除上清液，并将沉淀重新混悬于第九预定体积的修饰处理试剂中(如固化处理试剂或矿化处理试剂),作用一定时间后离心洗涤，和/或将沉淀重悬于第十预定体积的含有带正电或者带负电的物质并作用一定时间。S2, centrifuge the mixed solution obtained in S1, remove the supernatant, and resuspend the precipitate in the ninth predetermined volume of modification treatment reagent (such as solidification treatment reagent or mineralization treatment reagent), and then centrifuge and wash after a certain period of time. , and/or resuspend the precipitate in a tenth predetermined volume containing positively or negatively charged substances and act for a certain period of time.
本发明的S2中，沉淀重新混悬于第十预定体积的带电物质后可不进行冻干，对混合液进行离心，所得沉淀重悬于PBS(或生理盐水)中，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或者直使用进行注射或者与细胞进行孵育。In S2 of the present invention, the precipitate is resuspended in the tenth predetermined volume of charged substance without freeze-drying. The mixed solution is centrifuged, and the resulting precipitate is resuspended in PBS (or physiological saline) and directly processed for subsequent nanoparticle or micron Relevant experiments in which cancer cells/tissue lysates are loaded on the particle surface are either directly injected or incubated with cells.
或，将S2所得混合液进行离心，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。Alternatively, the mixture obtained in S2 is centrifuged, and the precipitate is resuspended in an aqueous solution containing a drying protective agent and then vacuum dried at room temperature or freeze vacuum dried. After drying, the subsequent nanoparticles or microparticles are adsorbed to the surface of cancer cell lysates. Related experiments.
S3，将S2中得到的重悬于PBS(或生理盐水)中的含纳米粒或微米粒的混悬液或者采用PBS(或生理盐水)重悬S2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。S3: Resuspend the suspension containing nanoparticles or microparticles obtained in S2 in PBS (or physiological saline) or resuspend the dried suspension containing nanoparticles or microparticles obtained in S2 with PBS (or physiological saline). The dried substance with the 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.
进一步地，S1-S3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。Furthermore, the S1-S3 modification and antigen loading steps can be repeated multiple times to increase the loading capacity of the antigen. Moreover, when adding positively or negatively charged substances, substances with the same charge can be added multiple times or substances with different charges can be added alternately.
本发明的癌症疫苗中，细胞组分来源于至少一种癌细胞和/或至少一种肿瘤组织中的细胞，且所述至少有一种癌细胞或肿瘤组织与预防或治疗的疾病相同。将非水溶性组分负载到递送粒子上，使该疫苗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，使递送粒子负载全部抗原，然后注射至供体体内以激活癌症特异性T细胞或在体外与DC细胞共孵育，再回输至供体体内以激活癌症特异性T细胞。In the cancer vaccine of the present invention, the cell component is derived from at least one cancer cell and/or cells in at least one tumor tissue, and the at least one cancer cell or tumor tissue is the same as the disease to be prevented or treated. The non-water-soluble components are loaded onto the delivery particles so that the vaccine system contains more antigens. More preferably, the water-soluble components and the non-water-soluble components are loaded onto the delivery particles at the same time so that the delivery particles are loaded with all the antigens. The antigen is then injected into the donor to activate cancer-specific T cells or incubated with DC cells in vitro, and then reinfused back into the donor to activate cancer-specific T cells.
本发明要求保护上述基于癌症特异性T细胞的细胞系统在制备预防或治疗癌症产品中的应用。The present invention claims the use of the above-mentioned cancer-specific T cell-based cell system in the preparation of products for the prevention or treatment of cancer.
进一步地，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，以激活机体免疫系统，从而延缓癌症的进展、治疗癌症或者预防癌症的复发、转移。Furthermore, it is administered multiple times before the occurrence of cancer, after the occurrence of cancer, or after surgical removal of tumor tissue to activate the body's immune system, thereby delaying the progression of cancer, treating cancer, or preventing recurrence and metastasis of cancer.
本发明的第二个目的是提供一种同种异体淋巴细胞药物，该同种异体淋巴细胞药物包括经癌症疫苗激活的癌症特异性T细胞，所述癌症特异性T细胞来源于同种异体的个体，其中，癌症疫苗包括递送粒子及其负载的细胞组分，所述递送粒子为纳米粒子或微米粒子，所述细胞组分为来源于癌细胞和/或肿瘤组织的水溶性组分和/或非水溶性组分；The second object of the present invention is to provide an allogeneic lymphocyte drug, which includes cancer-specific T cells activated by a cancer vaccine, and the cancer-specific T cells are derived from allogeneic Individual, wherein the cancer vaccine includes delivery particles and loaded cellular components, the delivery particles are nanoparticles or microparticles, and the cellular component is a water-soluble component derived from cancer cells and/or tumor tissue and/ or water-insoluble components;
细胞组分经癌细胞和/或肿瘤组织裂解得到；Cell components are obtained by lysing cancer cells and/or tumor tissues;
裂解是将癌细胞或肿瘤组织在-20℃～-273℃下冷冻，加水或不含溶解剂的溶液后进行反复冻融裂解，上清液为水溶性组分，沉淀中经溶解剂溶解后转为可溶的部分为非水溶性组分；或裂解是使用溶解剂裂解癌细胞或肿瘤组织，并溶解裂解后的组分，得到同时含有水溶性组分和非水溶性组分的混合物。Lysis is to freeze cancer cells or tumor tissues at -20°C to -273°C, add water or a solution without a dissolving agent, and then freeze and thaw repeatedly. The supernatant is a water-soluble component, and the precipitate is dissolved by a dissolving agent. Convert the soluble part into water-insoluble components; or lysis is to use a dissolving agent to lyse cancer cells or tumor tissues and dissolve the lysed components to obtain a mixture containing both water-soluble components and non-water-soluble components.
进一步地，所述同种异体的个体可为一个或多个。Furthermore, the number of allogeneic individuals may be one or more.
进一步地，所述癌症特异性T细胞包括CD4 +T细胞和/或CD8 +T细胞。 Further, the cancer-specific T cells include CD4 + T cells and/or CD8 + T cells.
进一步地，所述同种异体淋巴细胞药物还包括来源于同种异体的天然免疫细胞，该天然免疫细胞无需经癌症疫苗激活，经癌症疫苗激活的癌症特异性T细胞从同种异体供体的体内分离后，与天然免疫细胞混合，即得所述同种异体淋巴细胞药物，将该药物注射入同种异体受体的体内用于相关疾病的治疗。Further, the allogeneic lymphocyte medicine also includes natural immune cells derived from allogeneic donors. The natural immune cells do not need to be activated by cancer vaccines. The cancer-specific T cells activated by cancer vaccines are obtained from allogeneic donors. After being separated in the body and mixed with natural immune cells, the allogeneic lymphocyte drug is obtained, and the drug is injected into the body of the allogeneic recipient for the treatment of related diseases.
进一步地，天然免疫细胞包括但不限于γδT细胞、自然杀伤细胞(NK细胞)、自然杀伤T细胞(NKT细胞)、中性粒细胞等。Further, natural immune cells include, but are not limited to, γδ T cells, natural killer cells (NK cells), natural killer T cells (NKT cells), neutrophils, etc.
进一步地，天然免疫细胞或经癌症疫苗激活的癌症特异性T细胞通过流式细胞术、磁珠分选等细胞分选技术从同种异体供体中分离得到。Further, natural immune cells or cancer-specific T cells activated by cancer vaccines are isolated from allogeneic donors through cell sorting technologies such as flow cytometry and magnetic bead sorting.
进一步地，所述的激活为将癌症疫苗注射至同种异体供体的体内，激活癌症特异性T细胞，或将DC细胞在体外经癌症疫苗刺激后注射至同种异体供体的体内激活癌症特异性T细胞。Further, the activation is to inject a cancer vaccine into the body of an allogeneic donor to activate cancer-specific T cells, or to inject DC cells into the body of an allogeneic donor after being stimulated by a cancer vaccine in vitro to activate cancer. specific T cells.
进一步地，DC细胞为自体DC细胞和/或同种异体DC细胞、细胞系或干细胞来源的DC细胞。Further, the DC cells are autologous DC cells and/or allogeneic DC cells, DC cells derived from cell lines or stem cells.
进一步地，所述的刺激是将DC细胞与癌症疫苗进行共孵育至少4小时，负载细胞组分的递送粒子被DC细胞吞噬后，可被DC细胞进行抗原提呈和激活，回输至体内后即可归巢淋巴结并利用DC细胞负载的抗原激活癌症特异性T细胞。Further, the stimulation is to incubate DC cells with the cancer vaccine for at least 4 hours. After the delivery particles loaded with cell components are engulfed by DC cells, they can be presented and activated by the DC cells for antigen, and then infused back into the body. It can then home to lymph nodes and activate cancer-specific T cells using the antigens loaded by DC cells.
进一步地，溶解剂选自尿素、盐酸胍、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、无机盐、Triton、吐温、二甲基亚砜、乙腈、乙醇、甲醇、N,N-二甲基甲酰胺、丙醇、异丙醇、醋酸、胆固醇、氨基酸、糖苷和胆碱中的至少一种。除以上限定，其余对淋巴细胞药物中癌症疫苗的处理同上述细胞系统中的癌症疫苗。Further, the dissolving agent is selected from the group consisting of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, inorganic salts, Triton, Tween, dimethyl sulfoxide, At least one of acetonitrile, ethanol, methanol, N,N-dimethylformamide, propanol, isopropyl alcohol, acetic acid, cholesterol, amino acids, glycosides and choline. Except for the above limitations, other treatments for cancer vaccines in lymphocyte drugs are the same as those for cancer vaccines in the above cell systems.
本发明要求保护上述同种异体淋巴细胞药物在制备预防或治疗癌症产品中的应用。The present invention claims the use of the above-mentioned allogeneic lymphocyte medicine in the preparation of products for preventing or treating cancer.
进一步地，本发明所述的淋巴细胞药物输至同种异体体内使用时，将经过分选的细胞进行体外扩增。其中，经过分选的细胞指通过流式细胞术、磁珠分选等细胞分选技术从同种异体供体中分离得到的含有目标T细胞的混合物；体外扩增的方法包括但不限于与细胞因子和/或抗体共孵育。Furthermore, when the lymphocyte drug of the present invention is infused into an allogeneic body for use, the sorted cells are expanded in vitro. Among them, sorted cells refer to a mixture containing target T cells separated from allogeneic donors through cell sorting technologies such as flow cytometry, magnetic bead sorting, etc.; in vitro amplification methods include but are not limited to Cytokine and/or antibody co-incubation.
进一步地，与淋巴细胞药物共孵育的细胞因子包括但不限于白介素2(IL-2)、白介素7(IL-7)、白介素15(IL-15)、白介素21(IL-21)、白介素17(IL-17)、白介素12(IL-12)、白介素6(IL-6)、白介素33(IL-33)、γ干扰素(IFN-γ)、TNF-α。Further, cytokines co-incubated with lymphocyte drugs include but are not limited to interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 21 (IL-21), interleukin 17 (IL-17), interleukin 12 (IL-12), interleukin 6 (IL-6), interleukin 33 (IL-33), gamma interferon (IFN-γ), TNF-α.
进一步地，与淋巴细胞药物共孵育的抗体包括但不限于αCD-3抗体、αCD-4抗体、αCD-8抗体、αCD-28抗体、αCD-40抗体、αOX-40抗体、αOX-40L抗体。Further, antibodies co-incubated with lymphocyte drugs include, but are not limited to, αCD-3 antibodies, αCD-4 antibodies, αCD-8 antibodies, αCD-28 antibodies, αCD-40 antibodies, αOX-40 antibodies, and αOX-40L antibodies.
将本发明的同种异体淋巴细胞药物制备成抗肿瘤制品时，该抗肿瘤制品包括以下任意 一种：(1)含有经癌症疫苗激活的来源于同种异体的癌症特异性T细胞的制剂；(2)含有经癌症疫苗激活的来源于同种异体的癌症特异性T细胞的制剂，以及含有来源于同种异体的天然免疫细胞的制剂(其中，天然免疫细胞若多于一种，含有天然免疫细胞的制剂可为一种或多种，在给患者使用时可以分别注射或者混合后一起注射)；(3)含有经癌症疫苗激活的癌症特异性T细胞和天然免疫细胞混合物的制剂，所述癌症特异性T细胞和天然免疫细胞均来源于同种异体的个体。When the allogeneic lymphocyte drug of the present invention is prepared into an anti-tumor product, the anti-tumor product includes any of the following: (1) a preparation containing allogeneic cancer-specific T cells activated by a cancer vaccine; (2) Preparations containing cancer-specific T cells derived from allogeneic cells activated by cancer vaccines, and preparations containing natural immune cells derived from allogeneic cells (wherein, if there is more than one type of natural immune cells, natural immune cells containing The preparation of immune cells can be one or more, which can be injected separately or mixed and injected together when used for patients); (3) preparations containing a mixture of cancer-specific T cells activated by cancer vaccines and natural immune cells, so The cancer-specific T cells and innate immune cells are derived from allogeneic individuals.
进一步地，所述的应用为将同种异体供体体内分离得到的淋巴细胞药物回输至同种异体受体体内，回输方法包括但不限于静脉注射、肌肉注射、皮下注射、皮内注射、腹腔注射、瘤内注射。Further, the application is to reinfuse lymphocyte drugs isolated from the body of the allogeneic donor into the body of the allogeneic recipient. The reinfusion method includes but is not limited to intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection. , intraperitoneal injection, intratumoral injection.
进一步地，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。Further, the drug may be administered multiple times before the occurrence of cancer, after the occurrence of cancer, or after surgical removal of tumor tissue.
借由上述方案，本发明至少具有以下优点：Through the above solutions, the present invention at least has the following advantages:
本发明提供了一种基于癌症特异性T细胞的细胞药物，并将该细胞药物应用于同种异体癌症的预防和治疗，使用癌症疫苗激活免疫机能较好的同种异体的癌症特异性免疫细胞，然后分离提取被激活的癌症特异性免疫细胞和天然免疫细胞，应用于癌症的治疗或者预防。癌症疫苗在同种异体内激活的癌症特异性T细胞，在过继转移到患者体内后能特异性的识别和杀伤癌细胞；如果同时将同种异体体内分离的天然免疫系统的γδT细胞、NK细胞和NKT细胞过继转移至患者体内，这些天然免疫细胞也可以通过各自的作用机制协同杀伤癌细胞。本发明提供的同种异体细胞药物，在应用于疾病的预防和治疗时，远远优于现有技术中直接分离得到或采用其他方式激活得到的同种异体T细胞或天然免疫细胞的效果。The present invention provides a cell drug based on cancer-specific T cells, applies the cell drug to the prevention and treatment of allogeneic cancer, and uses a cancer vaccine to activate allogeneic cancer-specific immune cells with better immune function. , and then isolate and extract the activated cancer-specific immune cells and natural immune cells, and apply them to the treatment or prevention of cancer. The cancer-specific T cells activated by the cancer vaccine in the allogeneic body can specifically recognize and kill cancer cells after adoptive transfer into the patient; if the γδ T cells and NK cells of the natural immune system isolated in the allogeneic body are also combined Adoptively transferred with NKT cells into the patient's body, these natural immune cells can also coordinately kill cancer cells through their respective mechanisms of action. When applied to the prevention and treatment of diseases, the allogeneic cell medicine provided by the present invention is far superior to the effect of allogeneic T cells or natural immune cells directly isolated or activated in other ways in the prior art.
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。The above description is only an overview of the technical solutions of the present invention. In order to have a clearer understanding of the technical means of the present invention and implement them according to the contents of the description, the preferred embodiments of the present invention are described below with detailed drawings.
附图说明Description of the drawings
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明。In order to make the content of the present invention easier to understand clearly, the present invention will be described in further detail below based on specific embodiments of the present invention and in conjunction with the accompanying drawings.
图1为本发明疫苗系统的制备过程及细胞系统应用示意图；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为采用含有溶解剂的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中制备的上述粒子激活同种异体体内的癌症特异性T细胞后分离提取含有癌症特异性T细胞的免疫细胞，并用该类细胞预防或治疗癌症的示意图；Figure 1 is a schematic diagram of the preparation process of the vaccine system of the present invention and the application of the cell system; wherein a is a schematic diagram of collecting and preparing nanoparticles or microparticles from water-soluble components and non-water-soluble components respectively; b is a schematic diagram using a solution containing a dissolving agent Schematic diagram of dissolving whole cell components and preparing nanoparticles or microparticles; c is the use of the above particles prepared in a or b to activate cancer-specific T cells in allogeneic bodies and then separate and extract immune cells containing cancer-specific T cells, Schematic diagram of using this type of cells to prevent or treat cancer;
图2分别为实施例1中用纳米粒激活癌症特异性T细胞结果以及使用同种异体免疫细胞预防黑色素瘤时小鼠肿瘤生长速度和生存期实验结果；a,流式细胞术分析注射纳米粒的小鼠脾细胞与抗原共孵育后可以被激活并分泌IFN-γ的CD8 +T细胞占脾细胞中CD8 +T细胞的比例；b,流式细胞术分析注射纳米粒的小鼠脾细胞与抗原共孵育后可以被激活并分 泌IFN-γ的CD4 +T细胞占脾细胞中CD4 +T细胞的比例；c,预防癌症时的肿瘤生长速度实验结果(n＝8)；d,预防癌症时的小鼠生存期实验结果(n＝8)，每个数据点为平均值±标准误差(mean±SEM)；a和b图中显著性差异分析采用t检验；c图中肿瘤生长抑制实验的显著性差异采用ANOVA法分析；d图中显著性差异采用Kaplan-Meier和log-rank test分析；***表示与PBS空白对照组相比p＜0.005，有显著性差异；###标示与含免疫佐剂的空白纳米粒+游离裂解液激活的细胞对照组相比p＜0.005，有显著性差异；&&&表示与PBS刺激的细胞对照组相比p＜0.005，有显著性差异； Figure 2 shows the results of using nanoparticles to activate cancer-specific T cells in Example 1 and the experimental results of mouse tumor growth rate and survival time when using allogeneic immune cells to prevent melanoma; a, flow cytometry analysis of injected nanoparticles The ratio of CD8 + T cells that can be activated and secrete IFN-γ to CD8 + T cells in spleen cells after co-incubation of mouse splenocytes with antigens; b, flow cytometry analysis of splenocytes of mice injected with nanoparticles and The ratio of CD4 + T cells that can be activated and secrete IFN-γ to CD4 + T cells in spleen cells after co-incubation with antigens; c, experimental results of tumor growth rate when preventing cancer (n=8); d, when preventing cancer The mouse survival experiment results (n=8), each data point is the mean ± standard error (mean ± SEM); the significant difference analysis in pictures a and b uses t test; the tumor growth inhibition experiment in picture c Significant differences were analyzed using ANOVA; significant differences in Figure d were analyzed using Kaplan-Meier and log-rank tests; *** indicates p < 0.005 compared with the PBS blank control group, and there is a significant difference; ### indicates the difference with Compared with the control group of cells activated by blank nanoparticles + free lysate containing immune adjuvant, there is a significant difference at p<0.005;&&& means there is a significant difference at p<0.005 compared with the control group of cells stimulated by PBS;
图3-20分别为实施例2-19中用本发明所述细胞系统预防或治疗癌症时小鼠肿瘤生长速度和生存期实验结果；a,预防或治疗癌症时的肿瘤生长速度实验结果(n≥8)；b,预防或治疗癌症时的小鼠生存期实验结果(n≥8)，每个数据点为平均值±标准误差(mean±SEM)；a图中肿瘤生长抑制实验的显著性差异采用ANOVA法分析，b图中显著性差异采用Kaplan-Meier和log-rank test分析；***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＜0.01，有显著性差异；###标示与含免疫佐剂的空白纳米粒+游离裂解液激活的细胞对照组相比p＜0.005，有显著性差异；&&&表示与PBS刺激的细胞对照组相比p＜0.005，有显著性差异；$代表与多肽纳米粒激活的细胞组相比p＜0.05，有显著性差异；★代表与未修饰的的纳米粒或微米粒激活的细胞组相比p＜0.05，有显著性差异；θθθ代表与无靶头有佐剂的纳米粒激活的细胞组相比p＜0.05，有显著性差异；δδδ代表与不使用佐剂的纳米粒激活的细胞组相比p＜0.05，有显著性差异；λ代表与只使用纳米粒激活的癌症特异性T细胞组相比p＜0.05，有显著性差异；ηηη代表与不使用佐剂只使用增加溶酶体逃逸物质的纳米粒激活的癌症特异性T细胞组相比p＜0.005，有显著性差异；π代表与使用含有佐剂但是不含增加溶酶体逃逸物质的纳米粒激活的癌症特异性T细胞组相比p＜0.05，有显著性差异；ω代表与使用纳米粒先激活DC,再注射DC到同种异体体内激活的癌症特异性T细胞组相比p＜0.05，有显著性差异；′Ω′Ω′Ω代表与只使用γδT细胞+NKT细胞组相比p＜0.005，有显著性差异；
Figure PCTCN2022092331-appb-000001
代表与使用扩增后的CD4 +T细胞和CD8 +T细胞组相比p＜0.05，有显著性差异。 Figures 3-20 are respectively the experimental results of mouse tumor growth rate and survival time when using the cell system of the present invention to prevent or treat cancer in Examples 2-19; a, the experimental results of tumor growth rate when preventing or treating cancer (n ≥8); b, mouse survival experimental results when preventing or treating cancer (n≥8), each data point is the mean ± standard error (mean ± SEM); the significance of the tumor growth inhibition experiment in figure a Differences were analyzed using ANOVA, and significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests; *** 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 compared with the PBS blank control group. p < 0.01, there is a significant difference between groups; ### indicates p < 0.005, there is a significant difference compared with the control group of cells activated by blank nanoparticles containing immune adjuvant + free lysate; &&& indicates stimulation with PBS Compared with the cell control group, p < 0.005, there is a significant difference; $ represents a significant difference compared with the cell group activated by peptide nanoparticles, p <0.05; ★ represents compared with the cell group activated by unmodified nanoparticles or microparticles Compared with the cell group, p < 0.05, there is a significant difference; θθθ represents the p < 0.05, there is a significant difference compared with the cell group activated by nanoparticles without target and adjuvant; δδδ represents the difference from the nanoparticles without adjuvant. p<0.05, there is a significant difference compared to the activated cell group; λ represents p<0.05, there is a significant difference compared to the cancer-specific T cell group activated with nanoparticles only; ηηη represents the difference compared with the cancer-specific T cell group activated only without adjuvant Compared with the cancer-specific T cell group activated by nanoparticles that increase lysosomal escape substances, p<0.005, there is a significant difference; π represents the cancer-specific T cells activated by nanoparticles containing adjuvants but not containing substances that increase lysosomal escape. Compared with the specific T cell group, p < 0.05, there is a significant difference; ω represents p < 0.05, compared with the cancer-specific T cell group that uses nanoparticles to activate DC first, and then injects DC into the allogeneic body to activate the cancer-specific T cells. Sexual difference; ′Ω′Ω′Ω represents p<0.005, which means there is a significant difference compared with the group using only γδT cells + NKT cells;
Figure PCTCN2022092331-appb-000001
It means there is a significant difference compared with the group using expanded CD4 + T cells and CD8 + T cells at p < 0.05.
具体实施方式Detailed ways
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。The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.
本发明所述的用于预防或治疗癌症的细胞系统，其包含来自于同种异体的被纳米疫苗和/或微米疫苗激活的癌症特异性T细胞。纳米疫苗和/或微米疫苗负载全细胞组分或其混合物，可直接注射到同种异体体内激活癌症特异性T细胞或者用于体外激活树突状细胞后再注射到同种异体体内激活癌症特异性T细胞。然后分离提取同种异体体内被激活的含有癌症特异性T细胞的细胞系统，即可制备预防或治疗癌症的细胞系统，其制备过程及应用 领域如图1所示。The cell system for preventing or treating cancer according to the present invention includes cancer-specific T cells derived from allogeneic cells and activated by nano-vaccines and/or micro-vaccines. Nano-vaccines and/or micro-vaccines are loaded with whole cell components or mixtures thereof, which can be directly injected into allogeneic bodies to activate cancer-specific T cells or used to activate dendritic cells in vitro and then injected into allogeneic bodies to activate cancer-specific T cells. Sexual T cells. Then, the cell system containing cancer-specific T cells activated in the allogeneic body is isolated and extracted, and a cell system for preventing or treating cancer can be prepared. The preparation process and application fields are shown in Figure 1.
在制备纳米疫苗或微米疫苗时，可裂解细胞或组织后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系统；或者也可以直接采用含有溶解剂的溶液直接裂解细胞或组织并溶解全细胞组分并制备纳米或微米粒子系统。本发明所述全细胞组分在裂解前或(和)裂解后既可经过包括但不限于灭活或(和)变性、固化、生物矿化、离子化、化学修饰、交联化、核酸酶处理等处理后再制备纳米疫苗或微米疫苗；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、交联化、核酸酶处理直接制备纳米疫苗或微米疫苗。本发明部分实施例中，肿瘤组织细胞在裂解前经过了灭活或(和)变性处理，在实际使用过程中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；本发明部分实施例中细胞裂解前或(和)裂解后的灭活或(和)变性处理方法为紫外照射和高温加热，在实际使用过程中也可以采用包括但不限于放射线辐照、高压、固化、生物矿化、离子化、化学修饰、交联化、核酸酶处理、胶原酶处理、冷冻干燥等处理方法。本领域技术人员可以理解，在实际应用过程中技术人员可根据具体情况进行适当调整。When preparing nano-vaccines or micro-vaccines, cells or tissues can be lysed and water-soluble components and water-insoluble components can be collected separately to prepare nano- or micro-particle systems respectively; or a solution containing a dissolving agent can be used to directly lyse cells or Organize and solubilize whole-cell fractions and prepare nano- or microparticle systems. The whole cell components of the present invention can undergo a process including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, cross-linking, nuclease, etc. before lysis or (and) after lysis. Nano-vaccines or micro-vaccines can be prepared after other treatments; it can also be done without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, cross-linking, etc. before or after cell lysis. Nuclease treatment directly prepares nano vaccines or micro vaccines. In some embodiments of the present invention, tumor tissue cells are inactivated or/and denatured before lysis. In actual use, they can also be inactivated or/and denatured after cell lysis, or the cells can also be lysed. Inactivation or (and) denaturation treatment is performed before and after lysis; in some embodiments of the present invention, the inactivation or (and) denaturation treatment method before or (and) after cell lysis is ultraviolet irradiation and high-temperature heating. In actual use Treatment methods including, but not limited to, radiation irradiation, high pressure, curing, biomineralization, ionization, chemical modification, cross-linking, nuclease treatment, collagenase treatment, freeze-drying, etc. may also be used during the process. Those skilled in the art can understand that during actual application, the skilled person can make appropriate adjustments according to specific circumstances.
在使用癌症疫苗体外激活DC然后再给同种异体注射被体外激活的DC时，DC可以来源于自体或者同种异体，也可以来自于细胞系或者干细胞。When using cancer vaccines to activate DCs in vitro and then injecting allogeneic DCs activated in vitro, the DCs can be derived from autologous or allogeneic sources, or from cell lines or stem cells.
同种异体体内的癌症特异性T细胞被激活后，可以从外周血提取含有被激活的癌症特异性T细胞的细胞系统，也可以从任何其他含有癌症特异性T细胞的组织提取分离。分离提取含有被激活的癌症特异性T细胞的细胞系统时可以采用流式细胞术或者磁珠分选，或者其他任何可以提取分离该类细胞系统的方法。After allogeneic cancer-specific T cells are activated, the cell system containing the activated cancer-specific T cells can be extracted from peripheral blood or isolated from any other tissue containing cancer-specific T cells. Flow cytometry or magnetic bead sorting can be used to isolate and extract cell systems containing activated cancer-specific T cells, or any other method that can extract and isolate such cell systems.
实施例1纳米粒子激活同种异体的癌症特异性T细胞用于黑色素瘤的预防Example 1 Nanoparticles activate allogeneic cancer-specific T cells for melanoma prevention
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用负载有黑色素瘤肿瘤组织的全细胞组分的纳米粒子系统激活同种异体体内的癌症特异性T细胞后预防小鼠的黑色素瘤。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，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系统，然后将纳米粒子系统注射到同种异体体内，从同种异体体内分离提取含有癌症特异性T细胞的细胞后，将所提取的细胞注射到小鼠体内预防黑色素瘤。This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components of melanoma tumor tissue to activate allogeneic cancer-specific T cells and prevent melanoma in mice. In this example, B16F10 melanoma tumor tissue was first lysed to prepare water-soluble components and non-water-soluble components of the tumor tissue. Then, the organic polymer material PLGA was used as the nanoparticle skeleton material, and Polyinosinic-polycytidylic acid (poly( I:C)) uses a solvent evaporation method to prepare a nanoparticle system loaded with water-soluble components and non-water-soluble components of tumor tissue as an immune adjuvant, and then injects the nanoparticle system into the allogeneic body. After cells containing cancer-specific T cells are isolated and extracted in vivo, the extracted cells are injected into mice to prevent melanoma.
(1)肿瘤组织的裂解及各组分的收集(1) Lysis of tumor tissue and collection of components
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞，在肿瘤长到体积分别为约1000mm 3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，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系统的抗原原料来源。 1.5 × 10 5 B16F10 cells were 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 pieces and then ground. An appropriate amount of pure water was added through a cell strainer and frozen and thawed 5 times repeatedly, accompanied by ultrasound to destroy the lysed 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 8M urea to the resulting precipitate to dissolve the precipitate and remove the insoluble component from the pure water. The water-insoluble components of water are converted into soluble in 8M urea aqueous solution. The above are the sources of antigen raw materials for preparing nanoparticle systems.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中纳米疫苗及作为对照的空白纳米粒采用溶剂挥发法中的复乳法制备。在制备时负载水溶性组分的纳米疫苗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24KDa-38KDa，所采用的免疫佐剂为poly(I:C)且poly(I:C)包载于纳米粒子内。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细胞组分和佐剂，在内部负载水溶性组分或非水溶性组分后，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280nm左右，纳米粒子表面电位为-9mV左右；每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分，每1mgPLGA纳米粒所使用的poly(I:C)免疫佐剂为0.02mg。空白纳米粒粒径为260nm左右，表面电位为-8mV左右空白纳米粒制备时分别采用等量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。In this example, the nanovaccine and the blank nanoparticles used as controls were prepared by the double emulsion method in the solvent evaporation method. During preparation, nanovaccines 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 PLGA, the material used to prepare the nanoparticles, is 24KDa-38KDa. The immune adjuvant used is poly(I:C) and the poly(I:C) is contained in the nanoparticles. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load cell components and adjuvants inside the nanoparticles. After loading water-soluble components or non-water-soluble components inside, 100 mg of nanoparticles are centrifuged at 10,000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h. The average particle size of the nanoparticles is about 280nm, and the surface potential of the nanoparticles is about -9mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein or peptide components, and the poly(I:C) immune adjuvant used for each 1 mg of PLGA nanoparticles is 0.02mg. The particle size of the blank nanoparticles is about 260nm, and the surface potential is about -8mV. When preparing the blank nanoparticles, an equal amount of poly(I:C) pure water or 8M urea is used to replace the corresponding water-soluble components and non-water-soluble components. .
(3)含有癌症特异性T细胞的细胞系统的制备(3) Preparation of cell systems containing cancer-specific T cells
选取6-8周的雌性C57BL/6为模型小鼠。在第0天，第3天，第7天，第14天，第21天，第28天和第35天分别给小鼠皮下注射100μL含水溶性组分的1mgPLGA纳米粒子和100μL含溶解后的非水溶性组分的1mgPLGA纳米粒子。PBS对照组和空白纳米粒+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38天处死各组小鼠，分别收集各组小鼠的脾脏，制备小鼠脾脏细胞单细胞悬液，并进行红细胞裂解处理以去除单细胞悬液中的红细胞。然后采用磁珠分选法分选得到CD8 +T细胞和CD4 +T细胞。将所分离得到的上述细胞按下述方法过继移植给同种异体小鼠预防癌症。 Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On days 0, 3, 7, 14, 21, 28 and 35, mice were subcutaneously injected with 100 μL of 1 mg PLGA nanoparticles containing water-soluble components and 100 μL of dissolved non- 1 mg of PLGA nanoparticles in water-soluble component. The PBS control group and the blank nanoparticle + free lysate control group were injected with the same dose at the corresponding time as a control. On the 38th day, the mice in each group were sacrificed, the spleens of the mice in each group were collected, a single cell suspension of mouse spleen cells was prepared, and the red blood cells were lysed to remove the red blood cells in the single cell suspension. Then, CD8 + T cells and CD4 + T cells were sorted using magnetic bead sorting method. The isolated cells were adoptively transplanted into allogeneic mice according to the following method to prevent cancer.
使用之前，将小鼠脾细胞中的CD8 +T细胞和CD4 +T细胞与含有各类黑色素瘤抗原的肿瘤组织裂解液共孵育24小时，然后采用流式细胞术分析小鼠脾细胞中可以被裂解液中的抗原激活的CD8 +T细胞和CD4 +T细胞的比例。所得结果即为脾细胞中被纳米疫苗激活的黑色素瘤特异性T细胞的比例。如图2a和图2b所示，纳米疫苗可有效激活黑色素瘤特异性的CD8 +T细胞和CD4 +T细胞。 Before use, CD8 + T cells and CD4 + T cells in mouse splenocytes were incubated with tumor tissue lysate containing various melanoma antigens for 24 hours, and then flow cytometry was used to analyze whether the mouse splenocytes could be Ratio of antigen-activated CD8 + T cells to CD4 + T cells in lysate. The result is the proportion of melanoma-specific T cells in splenocytes activated by the nanovaccine. As shown in Figure 2a and Figure 2b, the nanovaccine can effectively activate melanoma-specific CD8 + T cells and CD4 + T cells.
(4)同种异体的癌症特异性T细胞用于癌症的预防(4) Allogeneic cancer-specific T cells for cancer prevention
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)制备得到的100万个CD4 +T细胞(60万个)和CD8 +T细胞(40万个)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1.5×10 5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×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma 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 the recipient mice. immune cells in mice. Then, 1 million CD4 + T cells (600,000 cells) and CD8 + T cells (400,000 cells) prepared in step (3) were intravenously injected into the recipient mice. The next day, 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. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图2c和2d所示，接受PBS对照组和空白纳米粒对照组小鼠T细胞的受体小鼠的肿瘤都长大。与对照组相比，接收纳米粒免疫激活的T细胞的受体小鼠体内的肿瘤生长速度明显变慢，而且部分小鼠肿瘤消失痊愈。综上所述，本发明同种异体的癌症特异性T细胞对黑色素瘤具有良好的预防效果。As shown in Figures 2c and 2d, the tumors of the recipient mice that received T cells from mice in the PBS control group and the blank nanoparticle control group grew. Compared with the control group, the tumor growth rate in the recipient mice that received immune-activated T cells from the nanoparticles was significantly slower, and some mice had tumors that disappeared and recovered. In summary, the allogeneic cancer-specific T cells of the present invention have a good preventive effect on melanoma.
实施例2纳米粒子激活同种异体的癌症特异性T细胞后用于黑色素瘤的治疗Example 2 Nanoparticles activate allogeneic cancer-specific T cells for the treatment of melanoma
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用负载有黑色素瘤癌细胞和肿瘤组织的全细胞组分的纳米粒子系统激活同种异体体内的癌症特异性T细胞后，与天然免疫细胞一同回输给小鼠以治疗黑色素瘤。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)和CpG为免疫佐剂采用溶剂挥发法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，然后使用纳米粒子系统在同种异体体内激活癌症特异性T细胞，然后分离提取总T细胞、NKT细胞和NK细胞，将上述细胞注射给患癌小鼠治疗黑色素瘤。This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with melanoma cancer cells and whole cell components of tumor tissue to activate allogeneic cancer-specific T cells in vivo and interact with natural immune cells. Together they were infused back into mice to treat melanoma. In this example, 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 framework material, Poly(I:C) and CpG as immune adjuvants, a nanoparticle system loaded with a mixture of water-soluble components and a mixture of non-water-soluble components was prepared using a solvent evaporation method, and then the nanoparticles were used The system activates cancer-specific T cells allogeneically, then separates and extracts total T cells, NKT cells and NK cells, and injects the above cells into cancer-stricken mice to treat melanoma.
(1)肿瘤组织和癌细胞的裂解及各组分的收集(1) Lysis of tumor tissue and cancer cells and collection of components
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞，在肿瘤长到体积分别为约1000mm 3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，并可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系统的抗原原料来源。 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 through a cell strainer and freeze and thaw repeatedly 5 times, and can be 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. After the tumor tissue or cancer cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant to obtain 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 are converted into soluble in 8M urea aqueous solution. The water-soluble components of the tumor tissue and the water-soluble components of the cancer cells are mixed at a mass ratio of 1:1; the water-insoluble components of the tumor tissue and the non-water-soluble components of the cancer cells are mixed at a mass ratio of 1:1. The above are the sources of antigen raw materials for preparing nanoparticle systems.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中纳米疫苗及作为对照的空白纳米粒采用复乳法制备。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CpG且佐剂只分布于纳米粒子内部。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液组分和佐剂，在内部负载裂解组分后，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；在使用前将其用9mLPBS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，得到内外都负载裂解物的纳米粒子系统。该纳米粒子平均粒径为290nm左右，纳米粒子表面电位为-5mV左右；每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分，每1mg PLGA纳米粒所负载的poly(I:C)和CpG免疫佐剂各0.02mg。空白纳米粒粒径为270nm左右，表面电位为-6mV左右，空白纳米粒分别采用等量poly(I:C)和CpG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。In this example, the nanovaccine and the blank nanoparticles used as controls were prepared by the double emulsion method. During preparation, 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 material used to prepare the nanoparticles, is 7KDa-17KDa. The immune adjuvants used are poly(I:C) and CpG, and the adjuvants are only distributed inside the nanoparticles. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysis solution components and adjuvants inside the nanoparticles. After loading the lysis components inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL containing Resuspend 4% trehalose in ultrapure water and freeze-dry for 48 hours; resuspend it in 9 mL PBS before use, then add 1 mL of lysate component (protein concentration 80 mg/mL) and incubate at room temperature for 10 min to obtain a lysate loaded both internally and externally. of nanoparticle systems. The average particle size of the nanoparticles is about 290nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 140 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C) and CpG immune Each adjuvant is 0.02mg. The particle size of the blank nanoparticles is about 270nm, and the surface potential is about -6mV. The blank nanoparticles use equal amounts of poly(I:C) and CpG pure water or 8M urea to replace the corresponding water-soluble components and non-water-soluble components. point.
(3)纳米粒子激活癌症特异性T细胞(3) Nanoparticles activate cancer-specific T cells
本研究对照组分别是PBS组、空白纳米粒+游离裂解液对照组。选取6-8周的雌性C57BL/6为模型小鼠。在第0天、第7天、第14天、第21天、第28天和第35天分别皮下注射100μL负载水溶性组分的1mg PLGA纳米粒和100μL负载原非水溶性组分的1mg PLGA纳米粒。对照组在相应天数分别注射PBS或者负载等量免疫佐剂的空白纳米粒+游离裂解物。第38天从小鼠中利用流式细胞术分离总T细胞、NK细胞和NKT细胞。总T细胞中含有γδT细胞和被激活的癌症特异性T细胞。The control groups in this study were the PBS group and the blank nanoparticles + free lysate control group. Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On days 0, 7, 14, 21, 28 and 35, 100 μL of 1 mg PLGA nanoparticles loaded with water-soluble components and 100 μL of 1 mg PLGA loaded with original non-water-soluble components were subcutaneously injected, respectively. Nanoparticles. The control group was injected with PBS or blank nanoparticles + free lysate loaded with an equal amount of immune adjuvant on corresponding days. On day 38, total T cells, NK cells, and NKT cells were isolated from mice using flow cytometry. Total T cells contain γδ T cells and activated cancer-specific T cells.
(4)同种异体的细胞混合物给与患癌小鼠治疗癌症(4) Allogeneic cell mixtures are administered to cancer-affected mice to treat cancer.
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1.5×10 5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100μL含50万个T细胞、20万个NK细胞和20万个NKT细胞的同种异体的细胞混合物。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×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 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. On days 4, 7, 10, 15, and 20 after melanoma inoculation, 100 μL of allogeneic cells containing 500,000 T cells, 200,000 NK cells, and 200,000 NKT cells were injected intravenously, respectively. of cell mixture. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图3所示，PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大。与对照组相比，纳米疫苗激活的来自同种异体的癌症特异性T细胞和天然免疫细胞处理的小鼠肿瘤生长速度明显变慢，而且部分小鼠肿瘤消失痊愈。综上所述，本发明的细胞系统对黑色素瘤具有良好的治疗效果。As shown in Figure 3, the tumors of mice in both the PBS control group and the blank nanoparticle control group grew. Compared with the control group, the tumor growth rate of mice treated with allogeneic cancer-specific T cells and natural immune cells activated by the nanovaccine was significantly slower, and some mice had tumors that disappeared and recovered. In summary, the cell system of the present invention has good therapeutic effect on melanoma.
实施例3来自同种异体的细胞系统用于黑色素瘤肺转移的预防Example 3 Allogeneic cell system for prevention of melanoma lung metastasis
本实施例以小鼠黑色素瘤肺模型来说明如何使用同种异体的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系统。在本实施例中采用了硅化和添加带电物质的方法来增加抗原的负载量，且只进行了一轮矿化处理。本实施例中，使用纳米粒子先在体外激活树突状细胞，再注射树突状细胞激活癌症特异性 T细胞。This example uses a mouse melanoma lung model to illustrate how to use an allogeneic cell system to prevent cancer metastasis. In this example, B16F10 melanoma tumor tissue is first lysed to prepare water-soluble components and non-water-soluble components of the tumor tissue; then, a nanoparticle system loaded with water-soluble components and non-water-soluble components of the tumor tissue is prepared. . In this embodiment, siliconization and adding charged substances were used to increase the loading capacity of the antigen, and only one round of mineralization was performed. In this embodiment, nanoparticles are used to first activate dendritic cells in vitro, and then the dendritic cells are injected to activate cancer-specific T cells.
(1)肿瘤组织的裂解及各组分的收集(1) Lysis of tumor tissue and collection of components
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞，在肿瘤长到体积约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨，加入胶原酶在RPMI 1640培养基中孵育30min,然后通过细胞过滤网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可溶于纯水的水溶性组分；在所得沉淀部分中加入％5的SDS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％5的SDS水溶液中可溶。以上即为制备粒子的抗原原料来源。 1.5×10 5 B16F10 cells were 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. Cut the tumor tissue into pieces and then grind it. Add collagenase and incubate in RPMI 1640 medium for 30 minutes. Then add an appropriate amount of pure water through a cell filter and freeze and thaw repeatedly 5 times. Ultrasound can be used to destroy the lysed cells. After the cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant, which is the water-soluble component soluble in pure water; add %5 SDS aqueous solution to the resulting precipitate to dissolve the precipitate. The non-water-soluble components that are insoluble in pure water are converted into soluble in %5 SDS aqueous solution. The above are the sources of antigen raw materials for preparing particles.
(2)纳米粒子的制备(2) Preparation of nanoparticles
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中的复乳法制备，对复乳法进行了适当的修饰改进，在纳米粒子制备过程中采用低温硅化技术和添加带电物质两种修饰方法提高抗原的负载量。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抗原和佐剂，在内部负载抗原后，将100mg纳米粒子在10000g离心20分钟，然后使用7mLPBS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系统。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纳米粒内外所使用的poly(I:C)免疫佐剂共约为0.02mg且内外各半。In this example, the nanoparticles and the blank nanoparticles used as a control were prepared by the double emulsion method in the solvent evaporation method. The double emulsion method was appropriately modified and improved. In the preparation process of the nanoparticles, both low-temperature siliconization technology and the addition of charged substances were used. This modification method increases the loading capacity of the antigen. 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 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. During the preparation process, the double emulsion method is first used to load the antigen and adjuvant inside the nanoparticles. After loading the antigen inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and then the nanoparticles are resuspended using 7 ml PBS and mixed with 3 mL of PBS solution containing cell lysate (60 mg/mL) was mixed, centrifuged at 10,000 g for 20 min, and then reconstituted with 10 mL of silicate solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate, and 1.0 mM HCl, pH 3.0). Suspend and fix at room temperature for 10 min, then fix at -80°C for 24 h, centrifuge and wash with ultrapure water, resuspend and react with 3 mL of PBS containing protamine (5 mg/mL) and polylysine (10 mg/mL). 10 min, then centrifuge at 10,000g for 20 min, wash, resuspend in 10 mL of PBS solution containing cell lysate (50 mg/mL) and incubate for 10 min, then centrifuge at 10,000 g for 20 min, resuspend in 10 mL of ultrapure water containing 4% trehalose and freeze Dry for 48 hours; resuspend the particles in 7 mL of PBS before use, then add 3 mL of adjuvanted cancer tissue lysate component (protein concentration 50 mg/mL) and incubate at room temperature for 10 min to obtain frozen siliconized and lysate loaded lysate inside and outside. Modified nanoparticle systems with the addition of cationic species. The average particle size of the nanoparticles is about 350nm, and the surface potential of the nanoparticles is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 300 μg of protein or peptide components, and the poly(I:C) immune adjuvant used inside and outside each 1 mg of PLGA nanoparticles A total of about 0.02mg and half inside and outside.
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7mLPBS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-4mV左 右；每1mg PLGA纳米粒子约负载150μg蛋白质或多肽组分，每1mgPLGA纳米粒内外所使用的poly(I:C)免疫佐剂共约为0.02mg且内外各半。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. During the preparation process, 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. , before using the particles, resuspend them in 7 mL PBS and then add 3 mL of cancer tissue lysate component containing adjuvant (protein concentration 50 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 320nm, and the surface potential of the nanoparticles is about -4mV; each 1 mg of PLGA nanoparticles is loaded with approximately 150 μg of protein or peptide components, and the poly(I:C) immune adjuvant used inside and outside each 1 mg of PLGA nanoparticles A total of about 0.02mg and half inside and outside.
空白纳米粒粒径为300nm左右，表面电位为-5mV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5％的SDS代替相对应的水溶性组分和非水溶性组分。The particle size of the blank nanoparticles is about 300nm, and the surface potential is about -5mV. When preparing the blank nanoparticles, pure water containing an equal amount of poly(I:C) or 5% SDS is used to replace the corresponding water-soluble components and non-containing components. Water-soluble components.
(3)树突状细胞的制备(3) Preparation of dendritic cells
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RPMI1640(10％FBS)培养基，同时加入重组小鼠GM-CSF(20ng/mL)，37度，5％CO 2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。 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). First, a 6-8 week old C57 mouse was sacrificed by cervical dislocation. The tibia and femur of the hind legs were surgically removed and placed in PBS. The muscle tissue around the bones was removed with scissors and tweezers. Use scissors to cut off both ends of the bone, and then use a syringe to draw the PBS solution. The needles are inserted into the bone marrow cavity from both ends of the bone, and the bone marrow is repeatedly flushed into the culture dish. Collect the bone marrow solution, centrifuge at 400g for 3 minutes, and then add 1 mL of red blood cell lysis solution to lyse the red blood. Add 3 mL of 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 RPMI1640 (10% FBS) medium, while adding recombinant mouse GM-CSF (20 ng/mL) 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). On the 6th day, half of the culture medium was replaced. On day 7, collect a small amount of suspended and semi-adherent cells. Through flow cytometry, when the proportion of CD86 + CD80 + cells in CD11c + cells is between 15-20%, the induced cultured BMDC can be used for the following One step experiment.
(4)树突状细胞的激活(4) Activation of dendritic cells
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负载水溶性组分的PLGA纳米粒子和30μg负载非水溶性组分的PLGA纳米粒子与BMDC共孵育48h，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。对照组加入空白纳米粒+游离裂解液与BMDC细胞共孵育。Spread mouse BMDC into a cell culture plate, add 5 mL of RPMI 1640 (10% FBS) culture medium for every 100,000 BMDC cells, and then add 30 μg of PLGA nanoparticles loaded with water-soluble components and 30 μg of non-water-soluble components. The separated PLGA nanoparticles were incubated with BMDC for 48 h, and then the BMDC were collected and centrifuged at 300g for 5 minutes, washed twice with phosphate buffer saline (PBS) and resuspended in PBS for later use. In the control group, blank nanoparticles + free lysate were added to co-incubate with BMDC cells.
(5)癌症特异性T细胞的激活(5) Activation of cancer-specific T cells
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100万个BMDC细胞。各组注射的BMDC细胞分别被负载裂解液的纳米粒子或者空白纳米粒子+游离裂解液激活过。第32天从小鼠中利用流式细胞术从脾脏单细胞悬液中分离总T细胞。总T细胞中含有γδT细胞和被激活的癌症特异性T细胞。Female C57BL/6 mice aged 6-8 weeks were selected and 1 million BMDC cells were injected subcutaneously on days 0, 4, 7, 14, 21 and 28 respectively. The injected BMDC cells in each group were activated by nanoparticles loaded with lysate or blank nanoparticles + free lysate. Total T cells were isolated from spleen single cell suspensions from mice on day 32 using flow cytometry. Total T cells contain γδ T cells and activated cancer-specific T cells.
(6)同种异体细胞系统用于癌症转移的预防(6) Allogeneic cell systems for the prevention of cancer metastasis
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50万个同种异体的T细胞。同时在第1天给每只小鼠静脉注射接种0.5×10 5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。 Melanoma tumor-bearing 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. On day 0, mice were intravenously injected with 100 μL of 500,000 allogeneic T cells. 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.
(7)实验结果(7)Experimental results
如图4所示，对照组小鼠的癌灶较多都长大，而经本发明所述细胞系统预处理的小鼠几乎没有癌灶。而且，采用硅化和添加带电物质修饰处理的纳米粒子激活的细胞系统对黑色素瘤肺转移的预防效果优于制备过程中未做修饰处理的纳米粒子激活的细胞系统。As shown in Figure 4, the mice in the control group had more cancer foci and grew up, while the mice pretreated with the cell system of the present invention had almost no cancer foci. Moreover, the cell system activated by nanoparticles modified by siliconization and addition of charged substances has a better preventive effect on melanoma lung metastasis than the cell system activated by nanoparticles that are not modified during the preparation process.
实施例4微米粒子激活的同种异体细胞用于癌症的预防Example 4 Micron particle-activated allogeneic cells for cancer prevention
本实施例中，首先使用6M盐酸胍裂解B16F10黑色素瘤癌细胞全细胞组分。然后，以有机高分子材料PLGA为微米粒骨架材料，以CpG为免疫佐剂制备负载有癌细胞的全细胞组分的微米粒子系统。在本实施例中采用了硅化、添加阳离子物质和阴离子物质的方法增加抗原的负载量，而且进行了两轮硅化处理。微米粒子激活同种异体体内的癌症特异性T细胞后，将包含上述细胞的细胞混合物注射给患癌小鼠预防癌症。In this example, 6M guanidine hydrochloride was first used to lyse the whole cell fraction of B16F10 melanoma cancer cells. Then, the organic polymer material PLGA was used as the micron particle skeleton material, and CpG was used as the immune adjuvant to prepare a micron particle system loaded with whole cell components of cancer cells. In this embodiment, siliconization, adding cationic substances and anionic substances were used to increase the loading capacity of the antigen, and two rounds of siliconization were performed. After micron particles activate allogeneic cancer-specific T cells, a cell mixture containing the above cells is injected into cancer-stricken mice to prevent cancer.
(1)癌细胞的裂解(1) Lysis of cancer cells
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，全细胞组分裂解并溶于6M盐酸胍后即为制备微米粒子系统的抗原原料来源。The cultured B16F10 melanoma cancer cell line was collected and centrifuged at 350g for 5 minutes. The supernatant was then discarded and washed twice with PBS. The cancer cells were then resuspended and lysed with 6M guanidine hydrochloride. The whole cell components were lysed and dissolved in 6M guanidine hydrochloride. That is the source of antigen raw materials for preparing micron particle systems.
(2)微米粒子系统的制备(2) Preparation of micron particle system
本实施例中微米粒子及作为对照的空白微米粒采用复乳法制备，对复乳法进行了适当的修饰改进，在微米粒子制备过程中采用低温硅化技术和添加带电物质两种修饰方法提高抗原的负载量。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7mLPBS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2.50μm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微米粒使用CpG 免疫佐剂共0.02mg且内外各半。In this example, the microparticles and the blank microparticles used as a control were prepared by the double emulsion method. The double emulsion method was appropriately modified and improved. During the preparation process of the micron particles, two modification methods, low-temperature siliconization technology and addition of charged substances, were used to improve the antigenicity. load capacity. 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. During the preparation process, 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. The 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. Use ultrapure water to centrifuge and wash. Resuspend in 3 mL of PBS containing polyaspartic acid (10 mg/mL) for 10 min. Then centrifuge at 10000 g for 15 min to wash. Use 10 mL of PBS containing cells. 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. minutes, and resuspended in 10 mL of ultrapure water containing 4% trehalose and then freeze-dried for 48 h; before use, resuspend the particles in 7 mL of PBS and then add 3 mL of cancer cell lysate containing adjuvant (protein concentration 50 mg/mL ) and reacted at room temperature for 10 minutes to obtain modified micron particles loaded with lysate both inside and outside, which were modified by two rounds of freezing silicification, adding cationic substances and anionic substances. The average particle size of the micron particles is about 2.50 μm, and the surface potential of the micron particles is about -2mV; each 1 mg of PLGA micron particles is loaded with approximately 340 μg of protein or peptide components, and each 1 mg of PLGA micron particles uses a total of 0.02 mg of CpG immune adjuvant, half of which is inside and outside. .
未经修饰处理的微米粒子制备方法步骤基本与修饰处理的微米粒子的制备相同，只是未经过硅化、添加阳离子物质和阴离子物质处理这些步骤。在制备过程中首先采用复乳法在微米粒子内部负载细胞组分，在内部负载细胞组分后在10000g离心15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7mLPBS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微米粒子。该微米粒子平均粒径为2.45μm左右，微米粒子表面电位为-3mV左右；每1mg PLGA微米粒子约负载160μg蛋白质或多肽组分，每1mgPLGA微米粒使用CpG免疫佐剂共0.02mg且内外各半。The steps for preparing unmodified micron particles are basically the same as those for preparing modified micron particles, except that the steps of siliconization, addition of cationic substances and anionic substances are not carried out. During the preparation process, the double emulsion method is first used to load cell components inside the micron particles. After loading the cell components inside, they are centrifuged at 10,000g for 15 minutes, then resuspended in 10 mL of ultrapure water containing 4% trehalose and then freeze-dried for 48 hours. Before using the particles, resuspend them in 7 mL PBS and then add 3 mL of cancer tissue lysate component containing adjuvant (protein concentration 50 mg/mL) and incubate at room temperature for 10 min to obtain micron particles loaded with lysate both inside and outside. The average particle size of the micron particles is about 2.45 μm, and the surface potential of the micron particles is about -3mV; each 1 mg of PLGA micron particles is loaded with approximately 160 μg of protein or peptide components, and each 1 mg of PLGA micron particle uses a total of 0.02 mg of CpG immune adjuvant, half of which is inside and outside. .
空白微米粒粒径为2.43μm左右，表面电位为-3mV左右，空白微米粒制备时采用含有等量CpG的6M盐酸胍代替相对应的细胞组分。The particle size of the blank microparticles is about 2.43 μm, and the surface potential is about -3mV. When preparing the blank microparticles, 6M guanidine hydrochloride containing an equal amount of CpG is used to replace the corresponding cell components.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
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。第32天从小鼠中利用流式细胞术分离总T细胞。总T细胞中含有γδT细胞和被激活的癌症特异性T细胞。Select 6-8 week old female C57BL/6 mice and subcutaneously inject 100 μL of microparticles containing 2 mg PLGA on days 0, 4, 7, 14, 21, and 28. Total T cells were isolated from mice on day 32 using flow cytometry. Total T cells contain γδ T cells and activated cancer-specific T cells.
(4)同种异体细胞系统用于癌症的预防(4) Allogeneic cell systems for cancer prevention
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天给小鼠皮下注射100μL含100万个T细胞的同种异体的细胞。同时在第0天给每只小鼠皮下注射接种1.5×10 5个B16F10细胞，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×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 Melanoma tumor-bearing 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. On day 0, mice were injected subcutaneously with 100 μL of allogeneic cells containing 1 million T cells. At the same time, 1.5×10 5 B16F10 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. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图5所示，对照组小鼠的肿瘤都长大，而使用过继转移的T细胞组的小鼠肿瘤生长速度都明显变慢。而且，采用硅化和添加带电物质修饰处理的微米粒子激活的细胞系统对黑色素瘤预防效果优于制备过程中未做修饰处理的微米粒子激活的细胞系统。As shown in Figure 5, the tumors of the mice in the control group all grew, while the tumor growth rate of the mice in the adoptively transferred T cell group slowed down significantly. Moreover, the cell system activated by micron particles modified with siliconization and addition of charged substances has a better preventive effect on melanoma than the cell system activated by micron particles not modified during the preparation process.
实施例5纳米粒子激活癌症特异性T细胞用于癌症的预防Example 5 Nanoparticles activate cancer-specific T cells for cancer prevention
本实施例中，首先使用8M尿素水溶液((含500mM氯化钠))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PLGA为纳米粒骨架材料，以Poly(I:C)和CpG为免疫佐剂制备负载有全细胞组分的纳米粒子系统。纳米粒子注射到小鼠体内激活癌症特异性T细胞后，分离提取T细胞和NK细胞，然后将T细胞和NK细胞给与同种异体的患癌小鼠用于预防癌症。In this example, an 8M urea aqueous solution (containing 500mM sodium chloride) was first used to lyse B16F10 melanoma tumor tissue and dissolve the tumor tissue lysate components. Then, a nanoparticle system loaded with whole cell components was prepared using PLGA as the nanoparticle skeleton material and Poly(I:C) and CpG as immune adjuvants. After nanoparticles are injected into mice to activate cancer-specific T cells, T cells and NK cells are isolated and extracted, and then the T cells and NK cells are given to allogeneic cancer-suffering mice for cancer prevention.
(1)肿瘤组织的收集及裂解(1) Collection and lysis of tumor tissue
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞，在肿瘤长到体积约1000mm 3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8M尿素水溶液(含500mM氯化钠)裂解细胞，并溶解细胞裂解物。以上即为制备纳米粒子系统的抗原原料来源。 1.5×10 5 B16F10 cells were 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. Cut the tumor tissue into pieces and then grind it. Add an appropriate amount of 8M urea aqueous solution (containing 500mM sodium chloride) through a cell filter to lyse the cells and dissolve the cell lysate. The above are the sources of antigen raw materials for preparing nanoparticle systems.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
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。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CpG,且裂解物组分和佐剂负载于纳米粒子内部。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物组分和佐剂，在内部负载抗原裂解组分和佐剂后，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。该纳米粒子平均粒径为270nm左右，纳米粒子表面电位为-8mV左右；每1mg PLGA纳米粒子约负载110μg蛋白质或多肽组分，每1mg PLGA纳米粒所使用的poly(I:C)和CpG免疫佐剂各为0.02mg。空白纳米粒粒径为250nm左右，表面电位为-9mV左右，空白纳米粒制备时采用含等量poly(I:C)和CpG的8M尿素(含500mM氯化钠)代替裂解物组分。In this example, the nanoparticles and the blank nanoparticles used as a control were prepared by the solvent evaporation method. The molecular weight of PLGA, the material used to prepare the nanoparticles, is 7KDa-17KDa. The immune adjuvants used are poly(I:C) and CpG, and the lysate components and adjuvants are loaded inside the nanoparticles. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysate components and adjuvants inside the nanoparticles. After loading the antigen lysis components and adjuvants inside, 100mg of the nanoparticles are centrifuged at 12000g for 20 minutes, and 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. The average particle size of the nanoparticles is about 270nm, and the surface potential of the nanoparticles is about -8mV; each 1 mg of PLGA nanoparticles is loaded with approximately 110 μg of protein or peptide components, and the poly(I:C) and CpG immune components used in each 1 mg of PLGA nanoparticles are Adjuvants are 0.02 mg each. The particle size of the blank nanoparticles is about 250nm, and the surface potential is about -9mV. When preparing the blank nanoparticles, 8M urea (containing 500mM sodium chloride) containing equal amounts of poly(I:C) and CpG is used to replace the lysate component.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
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。第32天从小鼠中利用流式细胞术分离总T细胞和NK细胞。总T细胞中含有γδT细胞和被激活的癌症特异性T细胞。Female C57BL/6 mice aged 6-8 weeks were selected and subcutaneously injected with 100 μL of nanoparticles containing 2 mg PLGA on days 0, 4, 7, 14, 21, and 28. Total T cells and NK cells were isolated from mice on day 32 using flow cytometry. Total T cells contain γδ T cells and activated cancer-specific T cells.
(4)同种异体细胞系统用于癌症的预防(4) Allogeneic cell systems for cancer prevention
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天给小鼠皮下注射100μL含100万个T细胞和50万个NK细胞的同种异体的细胞。同时在第0天给每只小鼠皮下注射接种1.5×10 5个B16F10细胞，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×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 Melanoma tumor-bearing 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. On day 0, mice were injected subcutaneously with 100 μL of allogeneic cells containing 1 million T cells and 500,000 NK cells. At the same time, 1.5×10 5 B16F10 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. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图6所示，对照组小鼠的肿瘤都长大，而经负载抗原的纳米粒子激活过的免疫细胞进行移植的小鼠肿瘤生长速度都明显变慢，且大部分小鼠癌细胞接种后肿瘤消失。As shown in Figure 6, the tumors of mice in the control group all grew, while the tumor growth rate of mice transplanted with immune cells activated by antigen-loaded nanoparticles was significantly slower, and most of the mouse cancer cells were inoculated The tumor disappears.
实施例6纳米粒子激活癌症特异性T细胞后用于治疗结肠癌Example 6 Nanoparticles are used to treat colon cancer after activating cancer-specific T cells
本实施例以MC38小鼠结肠癌为癌症模型来说明如何使用同种异体的免疫细胞治疗结肠癌。首先裂解结肠癌肿瘤组织和肺癌癌细胞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PLA为纳米粒骨架材料，以CpG和卡介苗(BCG)为免疫佐剂制备纳米粒子，并用该纳米粒子激活癌症特异性T细胞，然后分离提取含有癌症特异性T细胞的免疫细胞用于治疗同种异体的结肠癌。This example uses MC38 mouse colon cancer as a cancer model to illustrate how to use allogeneic immune cells to treat colon cancer. First, colon cancer tumor tissue and lung cancer cells are 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 are The component mixture is mixed in a mass ratio of 1:1. Then, the organic polymer material PLA is used as the nanoparticle skeleton material, and CpG and Bacillus Calmette-Guérin (BCG) are used as immune adjuvants to prepare nanoparticles. The nanoparticles are used to activate cancer-specific T cells, and then the cells containing cancer-specific T cells are isolated and extracted. Immune cells for treatment of allogeneic colon cancer.
(1)肿瘤组织和癌细胞的裂解及各组分的收集(1) Lysis of tumor tissue and cancer cells and collection of components
在每只C57BL/6小鼠背部皮下接种2×10 6个MC38细胞在肿瘤长到体积分别为约1000mm 3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，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。 2 × 10 6 MC38 cells were 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 pieces and then ground. An appropriate amount of pure water was added through a cell strainer and frozen and thawed 5 times repeatedly, accompanied by ultrasound to destroy the lysed cells. After the cells are lysed, centrifuge the lysate at a speed greater than 5000g for 5 minutes and take the supernatant to obtain the water-soluble components that are soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate and remove the insoluble components. The water-insoluble components of pure water are converted into soluble in 8M urea aqueous solution.
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，并可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。Collect the cultured LLC lung cancer cell lines and centrifuge them at 350g for 5 minutes. Then discard the supernatant and wash twice with PBS. Then resuspend the cells in ultrapure water and freeze and thaw repeatedly 5 times. Ultrasound can be used to destroy the lysed cells. . After the cells are lysed, centrifuge the lysate at 3000g for 6 minutes and take the supernatant to obtain the water-soluble components that are soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate and remove the insoluble components in pure water. The water-insoluble components of water are converted into soluble in 8M urea aqueous solution.
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8M尿素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照质量比1:1混合，该混合物为制备纳米粒子的原料来源。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 8M urea 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 source of raw materials for preparing nanoparticles.
(2)BCG的裂解和各组分的收集(2) BCG lysis and collection of components
BCG的裂解方法和各组分的收集方法同癌细胞的裂解方法和各组分的收集方法，水溶性组分和溶解的非水溶性组分按质量比1:1混合。The lysis method of BCG and the collection method of each component are the same as the lysis method and collection method of each component of cancer cells. The water-soluble components and the dissolved water-insoluble components are mixed at a mass ratio of 1:1.
(3)纳米粒子的制备(3) Preparation of nanoparticles
本实施例中纳米疫苗及作为对照的空白纳米粒采用溶剂挥发法制备。所采用的纳米粒子制备材料PLA分子量为40KDa，所采用的免疫佐剂为CpG和BCG，且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裂解物混合物和佐剂，在内部负载裂解物和佐剂后，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。使用前将20mg纳米粒重悬于0.9mLPBS中，并于0.1mL含有裂解物混合物(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，纳米粒子表面电位为-3mV左右；每1mg PLGA纳米粒子约负载120μg蛋白质或多肽组分，每1mgPLGA纳米粒含有CpG和BCG免疫佐剂各0.02mg。空白纳米粒粒径为260nm左右，空白纳米粒制备时分别采用含有等量佐剂尿素溶液代替相对应裂解物组分。In this example, the nanovaccine and the blank nanoparticles used as controls were prepared using the solvent evaporation method. The molecular weight of PLA, the material used to prepare the nanoparticles, is 40KDa. The immune adjuvants used are CpG and BCG, and the adjuvants are distributed both inside and on the surface of the nanoparticles. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysate mixture and adjuvant inside the nanoparticles. After loading the lysate and adjuvant inside, 100 mg nanoparticles are centrifuged at 10000g for 20 minutes, and 10 mL containing Resuspend in 4% trehalose ultrapure water and freeze-dry for 48 h. Before use, resuspend 20 mg of nanoparticles in 0.9 mL of PBS, mix with 0.1 mL of sample containing lysate mixture (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 of the nanoparticles is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 120 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles contains 0.02 mg of CpG and BCG immune adjuvants. The particle size of the blank nanoparticles is about 260 nm. When preparing the blank nanoparticles, a solution containing an equal amount of adjuvant urea was used instead of the corresponding lysate component.
(4)癌症特异性T细胞的激活(4) Activation of cancer-specific T cells
同实施例5。但是分离提取总T细胞、NK细胞和NKT细胞。Same as Example 5. However, total T cells, NK cells and NKT cells were separated and extracted.
(5)同种异体细胞系统用于癌症的治疗(5) Allogeneic cell systems for cancer treatment
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2×10 6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60万个总T细胞、20万个NK细胞和20万个NKT细胞的同种异体免疫细胞。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×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 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 μL containing 600,000 total T cells. cells, allogeneic immune cells of 200,000 NK cells and 200,000 NKT cells. The size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(6)实验结果(6)Experimental results
如图7所示，PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大。与对照组相比纳米粒子激活的免疫细胞移植组小鼠肿瘤生长速度明显变慢，而且部分小鼠肿瘤消失痊愈。综上所述，本发明同种异体免疫细胞治疗方案对结肠癌具有良好的治疗效果。As shown in Figure 7, the tumors of mice in both the PBS control group and the blank nanoparticle control group grew. Compared with the control group, the tumor growth rate of mice in the immune cell transplantation group activated by nanoparticles was significantly slower, and some mice had tumors that disappeared and recovered. In summary, the allogeneic immune cell treatment plan of the present invention has a good therapeutic effect on colon cancer.
实施例7被负载黑色素瘤肿瘤组织和肺癌肿瘤组织全细胞组分的纳米粒子激活的免疫细胞用于黑色素瘤的治疗Example 7 Immune cells activated by nanoparticles loaded with whole cell components of melanoma tumor tissue and lung cancer tumor tissue are used for the treatment of melanoma
本实施例以黑色素瘤为癌症模型来说明如何使用负载黑色素瘤和肺癌肿瘤组织的全细胞组分的纳米粒子激活癌症特异性T细胞，并用该细胞疫苗治疗同种异体小鼠的黑色素瘤。本实施例中，首先裂解B16F10黑色素瘤肿瘤组织和LLC肺癌肿瘤组织以制备肿瘤组织的水溶性组分混合物(质量比3:1)和非水溶性组分混合物(3:1)。以PLGA为纳米粒骨架材料，以锰颗粒和CpG为免疫佐剂制备负载上述混合物的纳米粒子，然后采用该纳米粒子激活小鼠体内的癌症特异性T细胞，并分离提取免疫细胞治疗同种异体患癌小鼠的黑色素瘤。This example uses melanoma as a cancer model to illustrate how to use nanoparticles loaded with whole cell components of melanoma and lung cancer tumor tissues to activate cancer-specific T cells, and use the cell vaccine to treat melanoma in allogeneic mice. In this example, B16F10 melanoma tumor tissue and LLC lung cancer tumor tissue were first lysed to prepare a mixture of water-soluble components (mass ratio 3:1) and a mixture of non-water-soluble components (3:1) of the tumor tissue. Use PLGA as the nanoparticle skeleton material, and use manganese particles and CpG as immune adjuvants to prepare nanoparticles loaded with the above mixture. The nanoparticles are then used to activate cancer-specific T cells in mice, and the immune cells are isolated and extracted to treat allogeneic Melanoma in cancerous mice.
(1)肿瘤组织的裂解及各组分的收集(1) Lysis of tumor tissue and collection of components
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞或者2×10 6个LLC肺癌细胞，在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。肿瘤的裂解和各组分收集方法同实施例1。将来自黑色素瘤肿瘤组织的和来自肺癌肿瘤组织的水溶性组分和溶解于8M尿素中的原非水溶性组分分别按照3:1的比例混合即为制备纳米粒子的抗原来源。 Each C57BL/6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells or 2 × 10 6 LLC lung cancer cells on the back. When the tumor grew to a volume of approximately 1000 mm 3 , the mice were sacrificed and the tumor tissue was removed. The methods for tumor lysis and collection of components are the same as in Example 1. Mix the water-soluble components from melanoma tumor tissue and lung cancer tumor tissue with the original non-water-soluble components dissolved in 8M urea at a ratio of 3:1 respectively to prepare the antigen source of the nanoparticles.
(2)纳米粒子的制备(2) Preparation of nanoparticles
本实施例中纳米粒子系统及作为对照的空白纳米粒采用复乳法制备。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锰胶体颗粒和CpG。先制备锰佐剂，然后将锰佐剂与全细胞组分中的水溶性组分或非水溶性组分混合后作为第一水相采用复乳法制备内部负载抗原和佐剂的纳米粒。在制备锰佐剂时，先将1mL 0.3M的Na 3PO 4溶液加入到9mL生理盐水中，后加入2mL  0.3M的MnCl 2溶液，放置过夜后，即得到Mn 2OHPO 4胶体锰佐剂，锰佐剂粒径约为13nm。然后将锰佐剂与全细胞组分全细胞组分中的水溶性组分(60mg/mL)或非水溶性组分(60mg/mL)按1:3体积比混合后采用复乳法将抗原和锰佐剂负载到纳米粒内部。在内部负载抗原(裂解组分)和佐剂后，将100mg纳米粒子在10000g离心20分钟，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370nm左右，纳米粒子表面电位为-5mV左右；每1mg PLGA纳米粒子约负载120μg蛋白质或多肽组分，每1mg PLGA纳米粒使用CpG佐剂为0.01mg。 In this example, the nanoparticle system and the blank nanoparticles used as controls were prepared using the double emulsion 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 adjuvants used are manganese colloidal particles and CpG. The manganese adjuvant is first prepared, and then the manganese adjuvant is mixed with the water-soluble component or the non-water-soluble component in the whole cell component and then used as the first aqueous phase to prepare nanoparticles internally loaded with antigen and adjuvant using the double emulsion method. When preparing manganese adjuvant, first add 1mL of 0.3M Na 3 PO 4 solution to 9mL of physiological saline, then add 2mL of 0.3M MnCl 2 solution, and leave it overnight to obtain Mn 2 OHPO 4 colloidal manganese adjuvant. The manganese adjuvant particle size is approximately 13 nm. Then, mix the manganese adjuvant with the water-soluble component (60 mg/mL) or the non-water-soluble component (60 mg/mL) in the whole cell component at a volume ratio of 1:3, and then use the double emulsion method to dissolve the antigen. and manganese adjuvants were loaded into the interior of the nanoparticles. After loading the antigen (cleavage component) and adjuvant internally, 100 mg of the nanoparticles were centrifuged at 10,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours before use. The average particle size of the nanoparticles is about 370nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 120 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles uses 0.01 mg of CpG adjuvant.
空白纳米粒粒径为330nm左右，空白纳米粒制备时分别采用含有等量锰佐剂和CpG佐剂的纯水或8M尿素代替相对应的水溶性组分和非水溶性组分。The particle size of the blank nanoparticles is about 330 nm. When preparing the blank nanoparticles, pure water or 8M urea containing equal amounts of manganese adjuvant and CpG adjuvant are used to replace the corresponding water-soluble components and non-water-soluble components.
(3)树突状细胞的制备(3) Preparation of dendritic cells
同实施例3。Same as Example 3.
(4)树突状细胞的激活(4) Activation of dendritic cells
同实施例3。Same as Example 3.
(5)癌症特异性T细胞的激活(5) Activation of cancer-specific T cells
同实施例3。Same as Example 3.
(6)同种异体细胞系统用于癌症的治疗(6) Allogeneic cell systems for cancer treatment
同实施例2。Same as Example 2.
(7)实验结果(7)Experimental results
如图8所示，PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大。与对照组相比纳米粒子激活的免疫细胞移植组小鼠肿瘤生长速度明显变慢，而且部分小鼠肿瘤消失痊愈。综上所述，本发明同种异体免疫细胞治疗方案对黑色素瘤具有治疗效果。As shown in Figure 8, the tumors of mice in both the PBS control group and the blank nanoparticle control group grew. Compared with the control group, the tumor growth rate of mice in the immune cell transplantation group activated by nanoparticles was significantly slower, and some mice had tumors that disappeared and recovered. In summary, the allogeneic immune cell treatment plan of the present invention has a therapeutic effect on melanoma.
实施例8被微米粒子激活同种异体免疫细胞用于乳腺癌的预防Example 8 Allogeneic immune cells activated by micron particles for the prevention of breast cancer
本实施例以4T1小鼠三阴性乳腺癌为癌症模型来说明如何采用8M尿素溶解全细胞组分并制备负载有全细胞组分的微米粒子系统，并以该微米粒子激活同种异体的癌症特异性T细胞，用于预防乳腺癌。本实施例中，首先对乳腺癌细胞进行灭活和变性处理并以8M尿素裂解癌细胞后溶解全细胞组分。然后，以PLGA为微米粒子骨架材料，以CpG和Poly ICLC为免疫佐剂制备负载有全细胞组分的微米粒子系统。This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use 8M urea to dissolve whole cell components and prepare a micron particle system loaded with whole cell components, and use the micron particles to activate allogeneic cancer-specific T cells for breast cancer prevention. In this example, breast cancer cells were first inactivated and denatured, the cancer cells were lysed with 8M urea, and then the whole cell components were dissolved. Then, a micron particle system loaded with whole cell components was prepared using PLGA as the micron particle skeleton material and CpG and Poly ICLC as immune adjuvants.
(1)癌细胞的裂解(1) Lysis of cancer cells
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系统的原料来源。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 appropriate amount of 8M urea was used to lyse the breast cancer cells and dissolve the lysate, which is the source of raw materials for preparing the particle system.
(2)微米粒子系统的制备(2) Preparation of micron particle system
本实施例中制备微米粒子系统及作为对照的空白微米粒子采用复乳法，微米粒子骨架 材料PLGA分子量为38KDa-54KDa，所采用的免疫佐剂为CpG和Poly ICLC。制备时先采用复乳法制备内部负载裂解物组分和佐剂的微米粒子,在内部负载裂解物和佐剂后，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，微米粒子系统表面电位为-5mV左右；每1mg PLGA微米粒子约负载110μg蛋白质或多肽组分，含CpG和Poly ICLC各0.01mg。空白微米粒粒径为2.0μm左右，空白微米粒制备时采用含有等量CpG和Poly ICLC佐剂的8M尿素代替相对应的细胞组分。In this example, the double emulsion method was used to prepare the microparticle system and the blank microparticles used as controls. The molecular weight of the microparticle skeleton material PLGA was 38KDa-54KDa. The immune adjuvants used were CpG and Poly ICLC. During preparation, the double emulsion method is first used to prepare microparticles loaded with lysate components and adjuvants internally. After loading lysate and adjuvants internally, 100 mg micron particles are centrifuged at 9000g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose is used. Resuspend and dry for 48 hours before use. The average particle size of the micron particle system is about 2.1 μm, and the surface potential of the micron particle system is about -5mV; each 1 mg of PLGA micron particles is loaded with approximately 110 μg of protein or peptide components, including 0.01 mg of CpG and Poly ICLC. The particle size of blank microparticles is about 2.0 μm. When preparing blank microparticles, 8M urea containing equal amounts of CpG and Poly ICLC adjuvant is used to replace the corresponding cell components.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
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。第32天从小鼠中利用磁珠分选法分离总T细胞和NK细胞。总T细胞中含有γδT细胞和被激活的癌症特异性T细胞。Select 6-8 week old female C57BL/6 mice and subcutaneously inject 100 μL of microparticles containing 2 mg PLGA on days 0, 4, 7, 14, 21, and 28. On day 32, total T cells and NK cells were isolated from mice using magnetic bead sorting. Total T cells contain γδ T cells and activated cancer-specific T cells.
(4)同种异体细胞系统用于癌症的预防(4) Allogeneic cell systems for cancer prevention
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100万个T细胞和50万个NK细胞的同种异体免疫细胞。同时在第0天给每只小鼠皮下注射接种1×10 6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。肿瘤体积采用公式v＝0.52×a×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 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 μL of allogeneic immune cells containing 1 million T cells and 500,000 NK 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. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图9所示，与对照组相比，被微米粒激活的免疫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，本发明所述的来自同种异体的激活免疫细胞对乳腺癌具有预防效果。As shown in Figure 9, compared with the control group, the tumor growth rate of the immune cell treatment group activated by micron particles was significantly slower and the survival period of mice was significantly prolonged. It can be seen that the allogeneic activated immune cells of the present invention have a preventive effect on breast cancer.
实施例9纳米粒子激活同种异体免疫细胞用于癌症转移的预防Example 9 Nanoparticles activate allogeneic immune cells for the prevention of cancer metastasis
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纳米粒子激活同种异体的免疫细胞后移植该类免疫细胞预防癌症转移。在实际应用时具体剂型，佐剂，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以8M尿素裂解后溶解，然后肿瘤组织裂解组分和癌细胞裂解组分按质量比1:2负载于纳米粒子系统，并用该粒子系统激活同种异体小鼠体内的癌症特异性T细胞，预防移植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激活的同种异体的免疫细胞预防癌症肺转移中的功效。This example uses a mouse melanoma mouse lung metastasis cancer model to illustrate the use of nanoparticles to activate allogeneic immune cells and then transplant these immune cells to prevent cancer metastasis. In actual application, the specific dosage form, adjuvant, administration time, administration frequency, and dosage regimen can be adjusted according to the situation. In this example, mouse melanoma tumor tissue and cancer cells were lysed and dissolved with 8M urea, and then the tumor tissue lysis component and the cancer cell lysis component were loaded into the nanoparticle system at a mass ratio of 1:2, and the particle system was used Activating cancer-specific T cells in allogeneic mice and preventing cancer metastasis in transplanted mice. In this example, four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were used. Nano The particles were used as control nanoparticles to analyze the efficacy of allogeneic immune cells activated by nanoparticles loaded with whole cell antigens and nanoparticles loaded with multiple peptide neoantigens in preventing cancer lung metastasis.
(1)肿瘤组织和癌细胞的裂解(1) Lysis of tumor tissue and cancer cells
收集小鼠B16F10黑色素瘤肿瘤组织和培养的癌细胞后采用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组分按质量比1:2混溶。After collecting mouse B16F10 melanoma tumor tissue and cultured cancer cells, 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 components were miscible at a mass ratio of 1:2.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中纳米粒子系统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CpG和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裂解物组分和佐剂，在内部负载裂解组分和佐剂后，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。该纳米粒子平均粒径为270nm左右；每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分，含CpG和Poly(I:C)各0.02mg。负载多种抗原多肽的对照纳米粒子制备方法同上，对照纳米粒子粒径为260nm左右，每1mg PLGA纳米粒子约负载100μg抗原多肽和等量佐剂。In this example, the nanoparticle system is prepared by a solvent evaporation method. The molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa. The immune adjuvants used are CpG and Poly(I:C). The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysate components and adjuvants inside the nanoparticles. After loading the lysis components and adjuvants inside, 100 mg of nanoparticles are centrifuged at 10,000g for 20 minutes, and used Resuspend 10 mL of ultrapure water containing 4% trehalose and freeze-dry for 48 hours before use. The average particle size of the nanoparticles is about 270nm; each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein or peptide components, including 0.02 mg of CpG and Poly(I:C). The preparation method of control nanoparticles loaded with multiple antigen peptides is the same as above. The particle size of the control nanoparticles is about 260 nm. Each 1 mg PLGA nanoparticle is loaded with approximately 100 μg of antigen peptides and an equal amount of adjuvant.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
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。第32天从小鼠中利用流式细胞术分离总T细胞、NK细胞和NKT细胞。总T细胞中含有γδT细胞和被激活的癌症特异性T细胞。Select 6-8 week old female C57BL/6 mice and subcutaneously inject 200 μL of 2 mg PLGA nanoparticles on days 0, 4, 7, 14, 21 and 28 respectively. On day 32, total T cells, NK cells and NKT cells were isolated from mice using flow cytometry. Total T cells contain γδ T cells and activated cancer-specific T cells.
(4)同种异体细胞系统用于癌症转移的预防(4) Allogeneic cell systems for the prevention of cancer metastasis
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含100万个T细胞、20万个NK细胞和20万个NKT细胞的同种异体的细胞混合物。同时在第1天给每只小鼠静脉注射接种0.5×10 5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。 Melanoma tumor-bearing 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. On day 0, mice were intravenously injected with 100 μL of an allogeneic cell mixture containing 1 million T cells, 200,000 NK cells, and 200,000 NKT cells. 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.
(5)实验结果(5)Experimental results
如图10所示，被纳米粒激活的免疫细胞可在同种异体中有效预防癌症转移。而且，与对照组相比，被负载裂解物和佐剂的纳米粒激活的免疫细胞在同种异体中预防癌症转移的效果更好。As shown in Figure 10, immune cells activated by nanoparticles can effectively prevent cancer metastasis in allogeneic cells. Furthermore, immune cells activated by lysate- and adjuvant-loaded nanoparticles were better at preventing cancer metastasis in allogeneic compared with controls.
实施例10纳米粒子激活同种异体中的癌症特异性T细胞用于胰腺癌的治疗Example 10 Nanoparticle activation of allogeneic cancer-specific T cells for the treatment of pancreatic cancer
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，并使用该纳米粒子激活小鼠癌症特异性T细胞，然后使用含有被激活细胞的免疫细胞治疗胰腺癌。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为负载于纳米粒子的佐剂，注射纳米粒子时混合粒细胞-巨噬细胞集落刺激因子(GM-CSF)作为佐剂。In this example, 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 activate mouse cancer-specific T cells, and then the nanoparticles containing Activated immune cells treat pancreatic cancer. In the experiment, 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. When preparing particles, 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. A 3:1 mixture of non-water-soluble components of cancerous tumor tissue. PLGA is used as the nanoparticle skeleton material, BCG is used as the adjuvant loaded on the nanoparticles, and granulocyte-macrophage colony-stimulating factor (GM-CSF) is mixed as the adjuvant when the nanoparticles are injected.
(1)肿瘤组织的裂解及各组分的收集(1) Lysis of tumor tissue and collection of components
在每只C57BL/6小鼠腋下皮下接种2×10 6个MC38结肠癌细胞或接种1×10 6个Pan02胰腺癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000mm 3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。BCG的裂解方法同肿瘤组织裂解方法。 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. When the inoculated tumors in each mouse grew to a volume of approximately 1000 mm 3 The mice were sacrificed and tumor tissues were harvested. 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 water-insoluble components. The BCG lysis method is the same as the tumor tissue lysis method.
(2)纳米粒子的制备(2) Preparation of nanoparticles
本实施例中纳米粒子及作为对照的空白纳米粒采用复乳法制备。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,且BCG负载于纳米粒子内部。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物组分和佐剂，在内部负载抗原裂解组分和佐剂后，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。在纳米粒子注射前将20mg纳米粒子溶于0.9mL PBS中，与0.1mL含裂解物(80mg/mL)和GM-CSF(50ng/mL)的样品混合并在室温作用10min后使用。该纳米粒子平均粒径为260nm左右，纳米粒子表面电位为-4mV左右；每1mg PLGA纳米粒子约负载130μg蛋白质或多肽组分，每1mg PLGA纳米粒使用BCG免疫佐剂0.02mg。空白纳米粒粒径为230nm左右，空白纳米粒制备时采用含等量佐剂。In this example, the nanoparticles and the blank nanoparticles used as a control were prepared by the double emulsion method. The molecular weight of PLGA, the material used to prepare the nanoparticles, is 7KDa-17KDa. The immune adjuvant used is BCG, and BCG is loaded inside the nanoparticles. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysate components and adjuvants inside the nanoparticles. After loading the antigen lysis components and adjuvants inside, 100mg of the nanoparticles are centrifuged at 12000g for 20 minutes, and 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 nanoparticle injection, 20 mg of nanoparticles were dissolved in 0.9 mL of PBS, mixed with 0.1 mL of sample containing lysate (80 mg/mL) and GM-CSF (50 ng/mL), and incubated at room temperature for 10 min before use. The average particle size of the nanoparticles is about 260nm, and the surface potential of the nanoparticles is about -4mV; each 1 mg of PLGA nanoparticles is loaded with approximately 130 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles uses 0.02 mg of BCG immune adjuvant. The particle size of the blank nanoparticles is about 230 nm, and the blank nanoparticles are prepared with equal amounts of adjuvants.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
同实施例6。Same as Example 6.
(4)疫苗用于癌症的治疗(4) Vaccines are used to treat cancer
同实施例6。Same as Example 6.
(5)实验结果(5)Experimental results
如图11所示，被纳米粒激活的免疫细胞可在同种异体中有效治疗胰腺癌。而且，与对照组相比，被负载裂解物和佐剂的纳米粒激活的免疫细胞在同种异体中治疗癌症转移的效果更好。As shown in Figure 11, immune cells activated by nanoparticles can effectively treat pancreatic cancer allogeneically. Furthermore, immune cells activated by lysate- and adjuvant-loaded nanoparticles treated cancer metastasis better allogeneically compared with controls.
实施例11靶头修饰纳米粒激活同种异体免疫细胞用于癌症的预防Example 11 Target-modified nanoparticles activate allogeneic immune cells for cancer prevention
本实施例以甘露糖为主动靶向的靶头为例说明如何靶向纳米粒激活同种异体的癌症特异性T细胞并用于预防癌症。在实际应用时具体剂型，佐剂，给药时间、给药次数、给药方案可根据情况调整。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，并进而激活癌针特异性T细胞。This example uses mannose as an active target to illustrate how targeted nanoparticles can activate allogeneic cancer-specific T cells and be used to prevent cancer. In actual application, the specific dosage form, adjuvant, administration time, administration frequency, and dosage regimen can be adjusted according to the situation. The nanoparticle system can be taken up into dendritic cells through mannose receptors on the surface of dendritic cells, and then activate cancer needle-specific T cells.
(1)癌细胞的裂解(1) Lysis of cancer cells
收集培养的B16F10癌细胞后采用8M尿素裂解和溶解癌细胞全细胞组分。After collecting the cultured B16F10 cancer cells, 8M urea was used to lyse and dissolve the whole cell components of the cancer cells.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中纳米粒子系统使用复乳法制备。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。不带靶头的纳米粒只使用PLGA。所采用的免疫佐剂为Poly(I:C)和CpG。制备方法如前所述，采用复乳法将裂解物组分和佐剂共负载于纳米粒子内部，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。带有靶头和不带靶头的纳米粒子的平均粒径均为270nm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CpG各0.02mg。不负载佐剂的但带有甘露糖靶头的对照纳米粒粒径也为270nm左右，制备时采用等量细胞组分但是不含任何免疫佐剂，每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分。In this example, the nanoparticle system 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. For nanoparticles without a target, only PLGA is used. The immune adjuvants used were Poly(I:C) and CpG. The preparation method is as described above. The lysate components and adjuvants are loaded into the nanoparticles using the double emulsion method. Then 100 mg of the nanoparticles are centrifuged at 10,000 g for 20 minutes and resuspended in 10 mL of ultrapure water containing 4% trehalose. Then freeze-dry for 48 hours before use. The average particle size of nanoparticles with and without target is about 270nm. Each 1 mg of PLGA nanoparticles is loaded with approximately 80 μg of protein or peptide components, including 0.02 mg of Poly(I:C) and 0.02 mg of CpG. The particle size of the control nanoparticles without adjuvant but with mannose target is also about 270nm. They are prepared using the same amount of cell components but do not contain any immune adjuvant. Each 1 mg of PLGA nanoparticles is loaded with approximately 80 μg of protein or peptide. components.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
同实施例1。Same as Example 1.
(4)疫苗用于癌症的预防(4) Vaccine for cancer prevention
同实施例1。Same as Example 1.
(5)实验结果(5)Experimental results
如图12所示，与对照组相比，使用同种异体被激活免疫细胞处理的小鼠肿瘤生长速度明显变慢。而且，纳米粒子只负载裂解物组分不负载佐剂时，纳米粒子激活的同种异体的癌症特异性T细胞也有预防癌症的效果。使用含有佐剂的纳米粒子激活的同种异体的癌症特异性T细胞效果好于不含佐剂的纳米粒子；使用含有靶头的纳米粒子所激活的免疫细胞好于不含靶头纳米粒子激活的免疫细胞。这说明本发明所述的来自同种异体的免疫细胞可以预防癌症，而且主动靶向靶头和佐剂的加入有助于纳米粒子激活同种异体的癌症特异性免疫细胞从而发挥作用。As shown in Figure 12, compared with the control group, the tumor growth rate of mice treated with allogeneic activated immune cells was significantly slower. Moreover, when the nanoparticles only load the lysate component and not the adjuvant, the allogeneic cancer-specific T cells activated by the nanoparticles also have the effect of preventing cancer. Allogeneic cancer-specific T cells activated using adjuvanted nanoparticles are better than non-adjuvanted nanoparticles; immune cells activated using target-containing nanoparticles are better than non-target nanoparticles. of immune cells. This shows that the allogeneic immune cells of the present invention can prevent cancer, and the addition of active targeting targets and adjuvants helps nanoparticles activate allogeneic cancer-specific immune cells to play their role.
实施例12纳米粒子激活同种异体的免疫细胞预防肝癌Example 12 Nanoparticles activate allogeneic immune cells to prevent liver cancer
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BCG为免疫佐剂采用溶剂挥发法制备负载肝癌细胞全细胞组分的纳米粒子系统，然后以该粒子系统激活同种异体的癌症特异性T细胞，分离提取同种异体的免疫细胞预防肝癌。In this example, Hepa1-6 liver cancer cells are first lysed, PLGA is used as the nanoparticle skeleton material, Poly(I:C) and BCG are used as immune adjuvants, and a nanoparticle system loaded with whole cell components of liver cancer cells is prepared using a solvent evaporation method. , and then use this particle system to activate allogeneic cancer-specific T cells, and isolate and extract allogeneic immune cells to prevent liver cancer.
(1)癌细胞的裂解及各组分的收集(1) Lysis of cancer cells and collection of components
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。BCG的裂解方法同上。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 components of the cancer cells. The cleavage method of BCG is the same as above.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中纳米粒子系统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24KDa-38KDa，所采用的免疫佐剂为BCG和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裂解组分和佐剂，在内部负载裂解组分和佐剂后，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。该纳米粒子平均粒径为270nm左右；每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分，含有BCG和Poly(I:C)各0.02mg。In this embodiment, the nanoparticle system is prepared by the double emulsion method in the solvent evaporation method. The molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa, and the immune adjuvants used are BCG and Poly(I:C). The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the cleavage components and adjuvants inside the nanoparticles. After loading the cleavage components and adjuvants inside, 100mg nanoparticles are centrifuged at 10000g for 20 minutes, and 10 mL is used. Resuspend in ultrapure water containing 4% trehalose and freeze-dry for 48 hours before use. The average particle size of the nanoparticles is about 270nm; each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein or peptide components, including 0.02 mg each of BCG and Poly(I:C).
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
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。第32天从小鼠中利用流式细胞术分离CD4 +T细胞、CD8 +T细胞，γδT细胞和NK细胞。 Female C57BL/6 mice aged 6-8 weeks were selected and 200 μL of 2 mg PLGA nanoparticles were subcutaneously injected on days 0, 4, 7, 14, 21, and 28 respectively. On day 32, CD4 + T cells, CD8 + T cells, γδ T cells and NK cells were isolated from mice using flow cytometry.
(4)同种异体细胞系统用于癌症转移的预防(4) Allogeneic cell systems for the prevention of cancer metastasis
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100μL含50万个CD8 +T细胞+50万个CD4 +T细胞的同种异体的细胞混合物；或者第0天注射100μL含50万个CD8 +T细胞+50万个CD4 +T细胞+50万个NK细胞+50万个γδT细胞的同种异体的细胞混合物。同时在第0天给每只小鼠皮下注射接种1.0×10 6个Hepa1-6肝癌细胞，肿瘤生长和小鼠生存期记录方式同实施例1。 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. On day 0, mice were injected with 100 μL of an allogeneic cell mixture containing 500,000 CD8 + T cells + 500,000 CD4 + T cells; or on day 0, mice were injected with 100 μL of an allogeneic cell mixture containing 500,000 CD8 + T cells + 500,000 CD8 + T cells An allogeneic cell mixture of CD4 + T cells + 500,000 NK cells + 500,000 γδ T cells. At the same time, each mouse was subcutaneously injected with 1.0×10 6 Hepa1-6 liver cancer cells on day 0. The tumor growth and mouse survival period were recorded in the same manner as in Example 1.
(5)实验结果(5)Experimental results
如图13所示，与对照组相比，使用同种异体被激活免疫细胞处理的小鼠肿瘤生长速度明显变慢。而且，使用含有NK细胞和NKT细胞等天然免疫细胞的效果好于不含天然免疫系统免疫细胞。这说明本发明所述的来自同种异体的免疫细胞可以预防癌症，而且天然免疫细胞的加入有助于增强纳米粒子体外激活癌症特异性免疫细胞发挥作用。As shown in Figure 13, compared with the control group, the tumor growth rate of mice treated with allogeneic activated immune cells was significantly slower. Moreover, the effect of using natural immune cells such as NK cells and NKT cells is better than using immune cells that do not contain natural immune system cells. This shows that the allogeneic immune cells of the present invention can prevent cancer, and the addition of natural immune cells helps to enhance the role of nanoparticles in activating cancer-specific immune cells in vitro.
实施例13钙化的纳米粒子系统激活同种异体癌症特异性T细胞用于癌症的预防Example 13 Calcified nanoparticle system activates allogeneic cancer-specific T cells for cancer prevention
本实施例说明钙化的纳米粒子激活同种异体的癌症特异性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，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以8M尿素裂解后溶解，然后肿瘤组织裂解组分和癌细胞裂解组分按质量比1:1负载于纳米粒子系统，并用该粒子系统激活同种异体的癌症特异性T细胞，并将同种异体的免疫细胞移植用于癌症的预防。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。This example illustrates that calcified nanoparticles activate allogeneic cancer-specific T cells. In actual use, other biomineralization technologies, cross-linking, gelation and other modified particles can also be used. In actual application, the specific dosage form, adjuvant, administration time, administration frequency, and dosage regimen can be adjusted according to the situation. In this example, mouse melanoma tumor tissue and cancer cells were lysed and dissolved with 8M urea, and then the tumor tissue lysis component and the cancer cell lysis component were loaded into the nanoparticle system at a mass ratio of 1:1, and the particle system was used Activation of allogeneic cancer-specific T cells and transplantation of allogeneic immune cells for cancer prevention. In this example, four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were used. Nano particles were used as control nanoparticles.
(1)肿瘤组织和癌细胞的裂解(1) Lysis of tumor tissue and cancer cells
收集小鼠B16F10黑色素瘤肿瘤组织和培养的癌细胞后采用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组分按质量比1:1混溶。After collecting mouse B16F10 melanoma tumor tissue and cultured cancer cells, 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 components were miscible at a mass ratio of 1:1.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
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系统及作为对照的空白纳米粒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7KDa-17KDa，所采用免疫佐剂CpG和Poly(I:C)负载于纳米粒子内部。制备方法如下所述，在制备过程中首先采用复乳法在纳米粒子内部负载抗原，在内部负载裂解组分后，将100mg PLGA纳米粒子在13000g离心20min后使用18mLPBS重悬，然后加入2mL溶解于8M尿素的肿瘤组织和癌细胞裂解液(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20mLDMEM培养基中，然后加入200μL of CaCl 2(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140μg蛋白质或多肽组分，CpG和Poly(I:C)各0.02mg。负载多种抗原多肽的对照纳米粒子制备方法同上，对照纳米粒子粒径为290nm左右，每1mg PLGA纳米粒子约负载130μg抗原多肽和等量佐剂。 In this embodiment, the nanoparticles are biocalcified after loading whole cell antigens inside and on the surface of the nanoparticles. In this example, the nanoparticle system and the blank nanoparticles used as a control were prepared by a solvent evaporation method. The molecular weight of the nanoparticle preparation material PLGA used was 7KDa-17KDa. The immunoadjuvant CpG and Poly(I:C) used were loaded on the nanoparticles. inside the particle. The preparation method is as follows. During the preparation process, the double emulsion method is first used to load the antigen inside the nanoparticles. After loading the cleavage components inside, 100mg PLGA nanoparticles are centrifuged at 13000g for 20 minutes and resuspended in 18mL PBS. Then 2mL is added to dissolve in 8M urea tumor tissue and cancer cell lysate (60mg/mL), incubated at room temperature for 10 minutes, centrifuged at 12000g for 20 minutes and collected the precipitate. The 100 mg PLGA nanoparticles were then resuspended in 20 mL DMEM medium, and then 200 μL of CaCl 2 (1 mM) was added and reacted at 37°C for two hours. Then, collect the precipitate after centrifugation at 10,000 g for 20 minutes, resuspend in ultrapure water, and centrifuge and wash twice. The average particle size of the nanoparticles is about 290nm; each 1 mg of PLGA nanoparticles is loaded with approximately 140 μg of protein or polypeptide components, including 0.02 mg of CpG and Poly(I:C). The preparation method of control nanoparticles loaded with multiple antigen peptides is the same as above. The particle size of the control nanoparticles is about 290 nm. Each 1 mg PLGA nanoparticle is loaded with approximately 130 μg of antigen peptides and an equal amount of adjuvant.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
同实施例1。Same as Example 1.
(4)疫苗用于癌症的预防(4) Vaccine for cancer prevention
同实施例1。Same as Example 1.
(5)实验结果(5)Experimental results
如图14所示，与对照组相比，钙化纳米粒激活的同种异体的免疫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激活的免疫细胞效果好于使用负载几种抗原多肽的纳米粒子。As shown in Figure 14, compared with the control group, allogeneic immune cells activated by calcified nanoparticles can prolong the survival of mice and effectively prevent cancer. Moreover, nanoparticles loaded with whole cell components activate immune cells better than nanoparticles loaded with several antigen peptides.
实施例14纳米粒子激活同种异体的癌症特异性T细胞后用于黑色素瘤的治疗Example 14 Nanoparticles activate allogeneic cancer-specific T cells for the treatment of melanoma
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用负载有黑色素瘤肿瘤组织的全细胞组分的纳米粒子系统激活同种异体体内的癌症特异性T细胞后，与天然免疫细胞一同回输给小鼠以治疗黑色素瘤。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CpG为免疫佐剂，以GALA(WEAALAEALAEALAEHLAEALAEALEALAA)为促进溶酶体逃逸组分，采用溶剂挥发法制备共负载佐剂、溶酶体逃逸物质和水溶性组分混合物或非水溶性组分混合物的纳米粒子系统，然后使用纳米粒子系统在同种异体体内激活癌症特异性T细胞，然后分离提取总T细胞、NKT细胞和NK细胞，将上述细胞注射给患癌小鼠治疗黑色素瘤。在纳米粒子递送载体中加入了可以促进溶酶体逃逸的物质，比如本实施例所加的细胞穿透肽， 可以增加粒子或粒子负载的抗原溶酶体逃逸，进而增加抗原的交叉提呈和增加CD8 +T细胞免疫反应的激活效能，更好的激活癌症特异性CD8 +T细胞免疫反应。 This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components of melanoma tumor tissue to activate allogeneic cancer-specific T cells and then reinfuse them together with natural immune cells. given to mice to treat melanoma. In this example, 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 CpG were used as immune adjuvants. Using GALA (WEAALAEALAEALAEHLAEALAEALEALAA) as a component that promotes lysosome escape, a solvent evaporation method is used to prepare a nanoparticle system that co-loads adjuvants, lysosome escape substances and a mixture of water-soluble components or a mixture of non-water-soluble components, and then uses nanoparticles. The particle system activates cancer-specific T cells allogeneically, then separates and extracts total T cells, NKT cells and NK cells, and injects the above cells into cancer-stricken mice to treat melanoma. Substances that can promote lysosomal escape are added to the nanoparticle delivery carrier, such as the cell-penetrating peptide added in this embodiment, which can increase the lysosomal escape of the particles or particle-loaded antigens, thereby increasing the cross-presentation and cross-presentation of antigens. Increase the activation efficiency of CD8 + T cell immune response and better activate cancer-specific CD8 + T cell immune response.
(1)肿瘤组织和癌细胞的裂解及各组分的收集(1) Lysis of tumor tissue and cancer cells and collection of components
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞，在肿瘤长到体积分别为约1000mm 3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，并可伴有超声以破坏裂解所得样品。待肿瘤组织或癌细胞裂解后，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2.0M的精氨酸氯化钠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原料来源。 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 through a cell strainer and freeze and thaw repeatedly 5 times, and may be accompanied by ultrasound to destroy the lysed sample. After the tumor tissue or cancer cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant to obtain the water-soluble component that is soluble in pure water; add 2.0M arginine chloride to the resulting precipitate. The sodium chloride solution dissolves the precipitated part to convert the non-water-soluble components that are insoluble in pure water into soluble in the aqueous solution. The above are the sources of antigen raw materials for preparing nanoparticle systems.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中纳米疫苗及作为对照的空白纳米粒采用复乳法制备。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CpG，溶酶体逃逸物质为GALA多肽，且佐剂和多肽包裹于纳米粒子内。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液组分和佐剂，在内部负载裂解组分后，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；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，得到内外都负载裂解物的纳米粒子系统。该纳米粒子平均粒径为290nm左右，纳米粒子表面电位为-5mV左右；每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分，每1mg PLGA纳米粒所负载的poly(I:C)和CpG免疫佐剂各0.02mg，负载GALA多肽0.05mg。空白纳米粒粒径为270nm左右，空白纳米粒分别采用等量佐剂和GALA多肽的纯水或2M精氨酸代替相对应的水溶性组分和非水溶性组分。In this example, the nanovaccine and the blank nanoparticles used as controls were prepared by the double emulsion method. During preparation, 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 material used to prepare the nanoparticles, is 7KDa-17KDa. The immune adjuvants used are poly(I:C) and CpG. The lysosomal escape substance is GALA polypeptide, and the adjuvant and polypeptide are wrapped in the nanoparticles. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysis solution components and adjuvants inside the nanoparticles. After loading the lysis components inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL containing Resuspend in 4% trehalose ultrapure water and freeze-dry for 48 hours; resuspend it in 9 mL PBS before use, then add 1 mL of lysis buffer component (protein concentration 80 mg/mL) and incubate at room temperature for 10 min to obtain both internal and external load lysis. Nanoparticle systems of matter. The average particle size of the nanoparticles is about 290nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 140 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C) and CpG immune Each adjuvant contains 0.02 mg, and the loaded GALA polypeptide is 0.05 mg. The particle size of the blank nanoparticles is about 270nm. The blank nanoparticles use equal amounts of adjuvant and GALA polypeptide in pure water or 2M arginine to replace the corresponding water-soluble components and non-water-soluble components.
(3)纳米粒子激活癌症特异性T细胞(3) Nanoparticles activate cancer-specific T cells
本研究对照组分别是PBS组、空白纳米粒+游离裂解液对照组。选取6-8周的雌性C57BL/6为模型小鼠。在第0天、第7天、第14天、第21天、第28天和第35天分别皮下注射100μL负载水溶性组分的1mg PLGA纳米粒和100μL负载原非水溶性组分的1mg PLGA纳米粒。对照组在相应天数分别注射PBS或者负载等量免疫佐剂的空白纳米粒+游离裂解物。第38天从小鼠中利用流式细胞术分离总T细胞、NK细胞和NKT细胞。总T细胞中含有γδT细胞和被激活的癌症特异性T细胞。The control groups in this study were the PBS group and the blank nanoparticles + free lysate control group. Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On days 0, 7, 14, 21, 28 and 35, 100 μL of 1 mg PLGA nanoparticles loaded with water-soluble components and 100 μL of 1 mg PLGA loaded with original non-water-soluble components were subcutaneously injected, respectively. Nanoparticles. The control group was injected with PBS or blank nanoparticles + free lysate loaded with an equal amount of immune adjuvant on corresponding days. On day 38, total T cells, NK cells, and NKT cells were isolated from mice using flow cytometry. Total T cells contain γδ T cells and activated cancer-specific T cells.
(4)同种异体的细胞混合物给与患癌小鼠治疗癌症(4) Allogeneic cell mixtures are administered to cancer-affected mice to treat cancer.
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1.5×10 5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100μL含80万个T细胞、10万个NK细胞和10万个NKT细胞的同种 异体的细胞混合物。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×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 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. On days 4, 7, 10, 15, and 20 after melanoma inoculation, 100 μL of allogeneic cells containing 800,000 T cells, 100,000 NK cells, and 100,000 NKT cells were injected intravenously, respectively. of cell mixture. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图15所示，PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大。与对照组相比，纳米疫苗激活的来自同种异体的癌症特异性T细胞和天然免疫细胞处理的小鼠肿瘤生长速度明显变慢，而且部分小鼠肿瘤消失痊愈。综上所述，本发明的细胞系统对黑色素瘤具有良好的治疗效果。As shown in Figure 15, the tumors of mice in both the PBS control group and the blank nanoparticle control group grew. Compared with the control group, the tumor growth rate of mice treated with allogeneic cancer-specific T cells and natural immune cells activated by the nanovaccine was significantly slower, and some mice had tumors that disappeared and recovered. In summary, the cell system of the present invention has good therapeutic effect on melanoma.
实施例15纳米粒子激活同种异体的癌症特异性T细胞后用于黑色素瘤的治疗Example 15 Nanoparticles activate allogeneic cancer-specific T cells for the treatment of melanoma
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用负载有黑色素瘤肿瘤组织的全细胞组分的纳米粒子系统激活同种异体体内的癌症特异性T细胞后，回输给小鼠以治疗黑色素瘤。本实施例中，首先裂解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CpG为免疫佐剂，以聚赖氨酸为增加溶酶体逃逸组分制备纳米粒子系统，然后使用纳米粒子系统在同种异体体内激活癌症特异性T细胞，然后分离提取总CD4 +T细胞和CD8 +T细胞，将上述细胞注射给患癌小鼠治疗黑色素瘤。 This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components of melanoma tumor tissue to activate allogeneic cancer-specific T cells and then infuse them back into mice for treatment. Melanoma. In this example, B16F10 melanoma tumor tissue is first lysed, and then PLGA is used as the nanoparticle skeleton material, Poly(I:C) and CpG are used as immune adjuvants, and polylysine is used as a component to increase lysosomal escape. The nanoparticle system is then used to activate cancer-specific T cells allogeneically, and then total CD4 + T cells and CD8 + T cells are isolated and extracted, and the above cells are injected into cancer-stricken mice to treat melanoma.
(1)肿瘤组织和癌细胞的裂解及各组分的收集(1) Lysis of tumor tissue and cancer cells and collection of components
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞，在肿瘤长到体积分别为约1000mm 3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单细胞悬液，然后使用5％脱氧胆酸钠水溶液裂解细胞并溶解裂解物组分。以上即为制备纳米粒子系统的抗原原料来源。 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 and pass through a cell strainer to prepare a single cell suspension, then use 5% sodium deoxycholate aqueous solution to lyse the cells and dissolve the lysate components. The above are the sources of antigen raw materials for preparing nanoparticle systems.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中纳米疫苗及作为对照的空白纳米粒采用复乳法制备，具有靶向树突状细胞的能力。所采用的纳米粒子制备材料为PLGA和甘露聚糖修饰的PLGA，二者分子量都为24KDa-38KDa，使用时未修饰PLGA和甘露聚糖修饰PLGA的质量比为4:1。所采用的免疫佐剂为poly(I:C)和CpG，增加溶酶体免疫逃逸的物质为聚赖氨酸，且佐剂和聚赖氨酸包载于纳米粒子内。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液组分和佐剂、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100μg蛋白质或多肽组分，每1mg PLGA纳米粒所负载的poly(I:C)和CpG免疫佐剂各0.02mg,聚赖氨酸0.05mg。对照纳米粒制备材料和方法同上，只是不负载聚赖氨酸，其粒径为280nm左右，负载等量佐剂和细胞裂解组分。In this example, the nanovaccine and the blank nanoparticles used as a control were prepared by the double emulsion method and have the ability to target dendritic cells. The nanoparticle preparation materials used are PLGA and mannan-modified PLGA, both of which have molecular weights of 24KDa-38KDa. When used, the mass ratio of unmodified PLGA and mannan-modified PLGA is 4:1. The immune adjuvants used are poly(I:C) and CpG, the substance that increases lysosomal immune escape is polylysine, and the adjuvants and polylysine are encapsulated in nanoparticles. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysis solution components, adjuvants, and polylysine inside the nanoparticles. After loading the above components internally, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes. , and resuspended in 10 mL of ultrapure water containing 4% trehalose and then freeze-dried for 48 h. The average particle size of the nanoparticles is about 280nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C) and CpG immune The adjuvants are 0.02 mg each, and the polylysine is 0.05 mg. The preparation materials and methods of the control nanoparticles are the same as above, except that polylysine is not loaded, the particle size is about 280nm, and equal amounts of adjuvants and cell lysis components are loaded.
(3)纳米粒子激活癌症特异性T细胞(3) Nanoparticles activate cancer-specific T cells
本研究对照组分别是PBS组、对照纳米粒子处理组。选取6-8周的雌性C57BL/6为模型小鼠。在第0天、第7天、第14天、第21天、第28天和第35天分别皮下注射100μL的2mg PLGA纳米粒。第38天从小鼠中利用流式细胞术分离CD3 +CD4 +T细胞和CD3 +CD8 +T细胞。 The control groups in this study were the PBS group and the control nanoparticle treatment group. Female C57BL/6 mice aged 6-8 weeks were selected as model mice. On days 0, 7, 14, 21, 28 and 35, 100 μL of 2 mg PLGA nanoparticles were injected subcutaneously. CD3 + CD4 + T cells and CD3 + CD8 + T cells were isolated from mice on day 38 using flow cytometry.
(4)同种异体的细胞混合物给与患癌小鼠治疗癌症(4) Allogeneic cell mixtures are administered to cancer-affected mice to treat cancer.
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1.5×10 5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100μL含60万个CD8 +T细胞和40万个CD4 +T细胞的细胞混合物。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×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 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. On days 4, 7, 10, 15 and 20 after melanoma inoculation, 100 μL of cell mixture containing 600,000 CD8 + T cells and 400,000 CD4 + T cells was injected intravenously. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图16所示，PBS对照组的肿瘤都长大。与对照组相比，纳米疫苗激活的来自同种异体的癌症特异性T细胞处理的小鼠肿瘤生长速度明显变慢，而且加入增加溶酶体逃逸物质的纳米粒子激活的T细胞好于未加入溶酶体逃逸的纳米粒子激活的T细胞。综上所述，本发明的细胞系统对黑色素瘤具有良好的治疗效果。As shown in Figure 16, the tumors in the PBS control group all grew. Compared with the control group, the tumor growth rate of mice treated with allogeneic cancer-specific T cells activated by nanovaccines was significantly slower, and the T cells activated by adding nanoparticles that increase lysosomal escape substances were better than those that were not added Lysosomal escape of nanoparticle-activated T cells. In summary, the cell system of the present invention has good therapeutic effect on melanoma.
实施例16被微米粒子激活同种异体免疫细胞用于乳腺癌的预防Example 16 Allogeneic immune cells activated by micron particles for the prevention of breast cancer
本实施例以4T1小鼠三阴性乳腺癌为癌症模型来说明如何采用负载全细胞组分的微米粒子系统，并以该微米粒子激活同种异体的癌症特异性T细胞，用于预防乳腺癌。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CpG和Poly ICLC为免疫佐剂，以KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)为增强溶酶体逃逸的物质，制备负载有全细胞组分的微米粒子系统。This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use a micron particle system loaded with whole cell components and use the micron particles to activate allogeneic cancer-specific T cells for the prevention of breast cancer. In this embodiment, 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, a micron particle system loaded with whole cell components was prepared using PLGA as the micron particle skeleton material, CpG and Poly ICLC as immune adjuvants, and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) as a substance that enhances lysosome escape.
(1)癌细胞的裂解(1) Lysis of cancer cells
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。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. water-soluble components. Dissolve the precipitate with 10% octylglucoside to obtain the dissolved original non-water-soluble components.
(2)微米粒子系统的制备(2) Preparation of micron particle system
本实施例中制备微米粒子系统及作为对照微米粒子采用复乳法，微米粒子骨架材料PLGA分子量为38KDa-54KDa，所采用的免疫佐剂为CpG和Poly ICLC，所采用的溶酶体逃逸增加物质为KALA多肽。制备时，负载水溶性组分的粒子和负载非水溶性组分的粒子分别制备，使用时一起使用。制备时先采用复乳法制备内部负载裂解物组分、佐剂和KALA 多肽的微米粒子,在内部负载裂解物和佐剂后，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3.1μm左右，微米粒子系统表面电位为-7mV左右；每1mg PLGA微米粒子约负载110μg蛋白质或多肽组分，含CpG和Poly ICLC各0.01mg，含KALA多肽0.05mg。对照微米粒粒径为3.1μm左右，制备材料和制备方法与本实施例所述微米粒子相同，只是不负载KALA多肽，负载等量CpG和Poly ICLC佐剂细胞裂解物组分。In this example, the double emulsion method was used to prepare the micron particle system and the control micron particles. The molecular weight of the micron particle skeleton material PLGA is 38KDa-54KDa. The immune adjuvants used are CpG and Poly ICLC. The lysosomal escape increasing substance used is KALA polypeptide. During preparation, particles loaded with water-soluble components and particles loaded with non-water-soluble components are prepared separately and used together. During preparation, the double emulsion method is first used to prepare micron particles that are internally loaded with lysate components, adjuvants and KALA polypeptides. After loading lysate and adjuvants internally, 100 mg of micron particles are centrifuged at 9000g for 20 minutes, and 10 mL containing 4% trehalose is used. Resuspend in ultrapure water and dry for 48 hours before use. The average particle size of the micron particle system is about 3.1 μm, and the surface potential of the micron particle system is about -7mV; each 1 mg of PLGA micron particles is loaded with approximately 110 μg of protein or peptide components, including 0.01 mg of CpG and Poly ICLC, and 0.05 mg of KALA peptide. . The particle size of the control microparticles is about 3.1 μm. The preparation materials and preparation methods are the same as the microparticles described in this example, except that KALA polypeptide is not loaded, and equal amounts of CpG and Poly ICLC adjuvant cell lysate components are loaded.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
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含水溶性组分的1mg PLGA的微米粒子和100μL含非水溶性组分的1mg PLGA的微米粒子。第32天从小鼠中利用磁珠分选法分离总T细胞。总T细胞中含有γδT细胞和被激活的癌症特异性T细胞。Select 6-8 week old female BALB/c mice and subcutaneously inject 100 μL of 1 mg PLGA containing water-soluble components on days 0, 4, 7, 14, 21, and 28. particles and 100 μL of micron particles containing 1 mg of PLGA containing non-water-soluble components. On day 32, total T cells were isolated from mice using magnetic bead sorting. Total T cells contain γδ T cells and activated cancer-specific T cells.
(4)同种异体细胞系统用于癌症的预防(4) Allogeneic cell systems for cancer prevention
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100万个T细胞。同时在第0天给每只小鼠皮下注射接种1×10 6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。肿瘤体积采用公式v＝0.52×a×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 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 μL containing 1 million 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. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图17所示，与对照组相比，被微米粒激活的免疫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，含有增加溶酶体逃逸功能的物质的微米粒子所激活的T细胞效果好于不含有溶酶体逃逸功能的物质的微米粒子所激活的T细胞。由此可见，本发明所述的来自同种异体的激活免疫细胞对乳腺癌具有预防效果。As shown in Figure 17, compared with the control group, the tumor growth rate in the immune cell treatment group activated by micron particles was significantly slower and the survival period of mice was significantly prolonged. Moreover, the effect of T cells activated by micron particles containing substances that increase lysosome escape function is better than that of T cells activated by micron particles that do not contain substances that increase lysosome escape function. It can be seen that the allogeneic activated immune cells of the present invention have a preventive effect on breast cancer.
实施例17被微米粒子激活同种异体免疫细胞用于乳腺癌的预防Example 17 Allogeneic immune cells activated by micron particles for the prevention of breast cancer
本实施例以4T1小鼠三阴性乳腺癌为癌症模型来说明如何采用负载全细胞组分的微米粒子系统，并以该微米粒子激活同种异体的癌症特异性T细胞，用于预防乳腺癌。本实施例中，首先使用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CpG和Poly ICLC为免疫佐剂，以精氨酸和组氨酸为增强溶酶体逃逸的物质，制备负载有全细胞组分的微米粒子系统。This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use a micron particle system loaded with whole cell components and use the micron particles to activate allogeneic cancer-specific T cells for the prevention of breast cancer. In this example, 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, CpG and Poly ICLC were used as immune adjuvants, and arginine and histidine were used as substances that enhance lysosomal escape to prepare a micron particle system loaded with whole cell components.
(1)癌细胞的裂解(1) Lysis of cancer cells
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(含500mM氯化钠)裂解癌细胞并溶解裂解物组分，即为制备微米粒子系统的抗原组分。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 (containing 500mM sodium chloride) was used to lyse the cancer cells and dissolve the lysate components, which are the antigen components for preparing the micron particle system.
(2)微米粒子系统的制备(2) Preparation of micron particle system
本实施例中制备微米粒子系统及作为对照微米粒子采用复乳法，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CpG和Poly ICLC，所采用的溶酶体逃逸增加物质为精氨酸和组氨酸。制备时先采用复乳法制备内部负载裂解物组分、佐剂、精氨酸和组氨酸的微米粒子,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。该微米粒子系统平均粒径为2.1μm左右，微米粒子系统表面电位为-7mV左右；每1mg PLA微米粒子约负载100μg蛋白质或多肽组分，含CpG和Poly ICLC各0.01mg，含精氨酸和组氨酸各0.05mg。对照微米粒制备材料和制备方法与本实施例所述微米粒子相同，粒径为2.1μm左右，表面电位为-7mV左右，只负载精氨酸和组氨酸和等量的细胞裂解物组分，而不负载任何佐剂。In this example, the double emulsion method was used to prepare the microparticle system and the control microparticles. The microparticle skeleton materials are unmodified PLA and mannose-modified PLA. The molecular weights are both 40KDa. The values of unmodified PLA and mannose-modified PLA are The ratio is 9:1. The immune adjuvants used are CpG and Poly ICLC, and the lysosomal escape-increasing substances used are arginine and histidine. During preparation, the double emulsion method is first used to prepare micron particles internally loaded with lysate components, adjuvants, arginine and histidine. Then, 100 mg of micron particles are centrifuged at 9000g for 20 minutes, and 10 mL of ultrasonic acid containing 4% trehalose is used. Resuspend in pure water and dry for 48 hours before use. The average particle size of the micron particle system is about 2.1 μm, and the surface potential of the micron particle system is about -7mV; each 1 mg of PLA micron particles is loaded with approximately 100 μg of protein or peptide components, including 0.01 mg each of CpG and Poly ICLC, and arginine and Histidine 0.05mg each. The preparation materials and preparation methods of the control microparticles are the same as those of the microparticles described in this example. The particle size is about 2.1 μm, the surface potential is about -7mV, and only arginine and histidine and an equal amount of cell lysate components are loaded. , without loading any adjuvants.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
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 PLA的微米粒子。第32天处死小鼠后摘取小鼠脾脏，制备小鼠脾脏单细胞悬液，并从小鼠脾细胞中利用磁珠分选法分选得到CD8 +T细胞。 Female BALB/c mice aged 6 to 8 weeks were selected and subcutaneously injected with 100 μL of micron particles containing 2 mg PLA on days 0, 4, 7, 14, 21, and 28. On the 32nd day, the mice were sacrificed and their spleens were removed, mouse spleen single cell suspensions were prepared, and CD8 + T cells were sorted from mouse splenocytes using magnetic bead sorting.
(4)同种异体细胞系统用于癌症的预防(4) Allogeneic cell systems for cancer prevention
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100万个CD8 +T细胞。同时在第0天给每只小鼠皮下注射接种1×10 6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。肿瘤体积采用公式v＝0.52×a×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 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 subcutaneously on day 0 with 100 μL containing 1 million CD8 + 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. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图18所示，与对照组相比，被微米粒激活的免疫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，含有增加溶酶体逃逸功能的物质和免疫佐剂的微米粒子所激活的T细胞效果好于只含有溶酶体逃逸功能的物质而不含有佐剂的微米粒子所激活的T细胞。由此可见，本发明所述的来自同种异体的激活免疫细胞对乳腺癌具有预防效果。As shown in Figure 18, compared with the control group, the tumor growth rate of the immune cell treatment group activated by micron particles was significantly slower and the survival period of mice was significantly prolonged. Moreover, T cells activated by microparticles containing substances that increase lysosomal escape function and immune adjuvants are better than T cells activated by microparticles that only contain substances that increase lysosome escape function without adjuvants. It can be seen that the allogeneic activated immune cells of the present invention have a preventive effect on breast cancer.
实施例18纳米粒子激活同种异体的癌症特异性T细胞后用于黑色素瘤的治疗Example 18 Nanoparticles activate allogeneic cancer-specific T cells for the treatment of melanoma
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用负载有黑色素瘤癌细胞和肿瘤组织的全细胞组分的纳米粒子系统激活同种异体体内的癌症特异性T细胞后，回输给小鼠以治疗黑色素瘤。本实施例中，首先裂解B16F10黑色素瘤癌细胞以制备水溶性组分和非水溶性组分，然后，以PLGA为骨架材料，以Poly(I:C)和CpG为免疫佐剂，以R8(RRRRRRRR)多肽为溶解溶酶体逃逸能力的物质，制备负载水溶性组分或非水溶性组分的纳米粒子系统， 然后将纳米粒子系统与树突状细胞体外共孵育后将树突状细胞回输同种异体体内激活癌症特异性T细胞，然后分离提取CD8 +T细胞用于癌症治疗。 This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with melanoma cancer cells and whole cell components of tumor tissue to activate allogeneic cancer-specific T cells and then infuse them back into the mouse. mice to treat melanoma. In this example, B16F10 melanoma cancer cells were first lysed to prepare water-soluble components and non-water-soluble components. Then, PLGA was used as the framework material, Poly(I:C) and CpG were used as immune adjuvants, and R8( RRRRRRRR) polypeptide is a substance that dissolves the ability to escape from lysosomes. A nanoparticle system loaded with water-soluble components or non-water-soluble components is prepared. The nanoparticle system is then co-incubated with dendritic cells in vitro and the dendritic cells are returned to the system. Allogeneic infusion of cancer-specific T cells is performed in vivo, and then CD8 + T cells are isolated and extracted for cancer treatment.
(1)肿瘤组织和癌细胞的裂解及各组分的收集(1) Lysis of tumor tissue and cancer cells and collection of components
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞，在肿瘤长到体积分别为约1000mm 3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，并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5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。待肿瘤组织或癌细胞酶作用处理后，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系统的抗原原料来源。 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 through the cell filter and freeze and thaw repeatedly 5 times, and may be accompanied by ultrasound to destroy the lysed sample. Add nuclease for 10 minutes and then heat at 95°C for 5 minutes to inactivate the nuclease; collect the cultured B16F10 For cancer cell lines, first centrifuge to remove the culture medium, then wash twice with PBS and centrifuge to collect the cancer cells. Resuspend the cancer cells in ultrapure water, freeze and thaw repeatedly three times, and destroy the cancer cells with ultrasonic destruction. Add nuclease to the sample and react for 10 minutes, then heat at 95°C for 5 minutes to inactivate the nuclease. After the tumor tissue or cancer cells are treated with enzymes, centrifuge the lysate at 5000g for 5 minutes and take the supernatant, which is the water-soluble component soluble in pure water; add 50% glycerol to the resulting precipitate to dissolve the precipitate. Partially converted into soluble. The water-soluble components of the tumor tissue and the water-soluble components of the cancer cells are mixed at a mass ratio of 1:1; the water-insoluble components of the tumor tissue and the non-water-soluble components of the cancer cells are mixed at a mass ratio of 1:1. The above are the sources of antigen raw materials for preparing nanoparticle systems.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中纳米粒子采用复乳法制备。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CpG，R8多肽为增加溶酶体逃逸的物质，且佐剂和R8多肽负载于纳米粒子内。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液组分、佐剂和R8多肽，在内部负载裂解组分后，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；在使用前将其用9mLPBS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，得到内外都负载裂解物的纳米粒子系统。该纳米粒子平均粒径为290nm左右，纳米粒子表面电位为-5mV左右；每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分，每1mg PLGA纳米粒所负载的poly(I:C)和CpG免疫佐剂各0.02mg，负载0.1mg R8多肽。空白纳米粒粒径为270nm左右，空白纳米粒分别负载等量佐剂和R8多肽的纯水或50％甘油代替相应的水溶性组分和非水溶性组分。In this example, the nanoparticles were prepared using the double emulsion method. During preparation, 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 material used to prepare nanoparticles, is 7KDa-17KDa. The immune adjuvants used are poly(I:C) and CpG. The R8 polypeptide is a substance that increases lysosomal escape, and the adjuvant and R8 polypeptide are loaded on the nanoparticles. within the particle. The preparation method is as mentioned above. During the preparation process, the double emulsion method is first used to load the lysis solution components, adjuvants and R8 polypeptides inside the nanoparticles. After loading the lysis components inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes. And use 10mL of ultrapure water containing 4% trehalose to resuspend and freeze-dry for 48h; before use, resuspend it in 9mL PBS and then add 1mL of lysate component (protein concentration 80mg/mL) and incubate at room temperature for 10min to obtain the internal and external contents. Both lysate-loaded nanoparticle systems. The average particle size of the nanoparticles is about 290nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 140 μg of protein or peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C) and CpG immune Each adjuvant is 0.02mg, loading 0.1mg R8 polypeptide. The particle size of the blank nanoparticles is about 270 nm, and the blank nanoparticles are loaded with equal amounts of adjuvant and R8 polypeptide pure water or 50% glycerin to replace the corresponding water-soluble components and non-water-soluble components.
(3)树突状细胞的制备(3) Preparation of dendritic cells
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RPMI1640(10％FBS)培养基，同时加入重组小鼠GM-CSF(20ng/mL)，37度，5％CO 2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。 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). First, a 6-8 week old C57 mouse was sacrificed by cervical dislocation. The tibia and femur of the hind legs were surgically removed and placed in PBS. The muscle tissue around the bones was removed with scissors and tweezers. Use scissors to cut off both ends of the bone, and then use a syringe to draw the PBS solution. The needles are inserted into the bone marrow cavity from both ends of the bone, and the bone marrow is repeatedly flushed into the culture dish. Collect the bone marrow solution, centrifuge at 400g for 3 minutes, and then add 1 mL of red blood cell lysis solution to lyse the red blood. Add 3 mL of 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 RPMI1640 (10% FBS) medium, while adding recombinant mouse GM-CSF (20 ng/mL) 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). On the 6th day, half of the culture medium was replaced. On day 7, collect a small amount of suspended and semi-adherent cells. Through flow cytometry, when the proportion of CD86 + CD80 + cells in CD11c + cells is between 15-20%, the induced cultured BMDC can be used for the following One step experiment.
(4)树突状细胞的激活(4) Activation of dendritic cells
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负载水溶性组分的PLGA纳米粒子和30μg负载原非水溶性组分的PLGA纳米粒子与BMDC共孵育48h，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。对照组加入空白纳米粒+游离裂解液与BMDC细胞共孵育。Spread mouse BMDC into a cell culture plate, add 5 mL of RPMI 1640 (10% FBS) culture medium for every 100,000 BMDC cells, and then add 30 μg of PLGA nanoparticles loaded with water-soluble components and 30 μg of non-water-soluble loaded original The component PLGA nanoparticles were incubated with BMDC for 48 h, and then the BMDC were collected and centrifuged at 300g for 5 minutes, washed twice with phosphate buffer saline (PBS) and resuspended in PBS for later use. In the control group, blank nanoparticles + free lysate were added to co-incubate with BMDC cells.
(5)癌症特异性T细胞的激活(5) Activation of cancer-specific T cells
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100万个BMDC细胞。各组注射的BMDC细胞分别被负载裂解液的纳米粒子或者空白纳米粒子+游离裂解液激活过。第32天从小鼠中利用流式细胞术从脾脏单细胞悬液中分离CD8 +T细胞。 Female C57BL/6 mice aged 6-8 weeks were selected and 1 million BMDC cells were injected subcutaneously on days 0, 4, 7, 14, 21 and 28 respectively. The injected BMDC cells in each group were activated by nanoparticles loaded with lysate or blank nanoparticles + free lysate. CD8 + T cells were isolated from spleen single cell suspensions from mice on day 32 using flow cytometry.
(6)同种异体的细胞混合物给与患癌小鼠治疗癌症(6) Allogeneic cell mixtures are given to cancer-stricken mice to treat cancer.
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1.5×10 5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100μL的80万个CD8 +T细胞。小鼠肿瘤生长和生存期监测方法同上。 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. 100 μL of 800,000 CD8 + T cells were injected intravenously on days 4, 7, 10, 15, and 20 after melanoma inoculation. The method for monitoring tumor growth and survival in mice is the same as above.
(7)实验结果(7)Experimental results
如图19所示，PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大。与对照组相比，纳米疫苗激活的来自同种异体的癌症特异性T细胞处理的小鼠肿瘤生长速度明显变慢，而且部分小鼠肿瘤消失痊愈。综上所述，本发明的细胞系统对黑色素瘤具有良好的治疗效果。As shown in Figure 19, the tumors of mice in both the PBS control group and the blank nanoparticle control group grew. Compared with the control group, the tumor growth rate of mice treated with allogeneic cancer-specific T cells activated by the nanovaccine was significantly slower, and some mice had tumors that disappeared and recovered. In summary, the cell system of the present invention has good therapeutic effect on melanoma.
实施例19同种异体的癌症特异性T细胞用于结肠癌的治疗Example 19 Allogeneic cancer-specific T cells for the treatment of colon cancer
本实施例以小鼠结肠癌为癌症模型来说明如何使用负载有结肠癌肿瘤组织的全细胞组分的纳米粒子系统激活同种异体体内的癌症特异性T细胞后，回输给小鼠以治疗结肠癌。本实施例中，首先使用8M尿素水溶液裂解结肠癌肿瘤组织并溶解裂解组分，然后，以PLGA为骨架材料，以Poly(I:C)和CpG为免疫佐剂，制备纳米粒子系统，然后使用纳米粒子系统激活同种异体体内的癌症特异性T细胞，然后分离提取CD8 +T细胞用于癌症治疗。本实施例中，激活癌症特异性T细胞使用两种不同的方法，一种为直接将纳米粒子注射进入人体 激活，一种为先体外激活树突状细胞再注射进入体内激活癌症特异性T细胞。 This example uses mouse colon cancer as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components of colon cancer tumor tissue to activate allogeneic cancer-specific T cells and then infuse them back into mice for treatment. Colon cancer. In this example, 8M urea aqueous solution is first used to lyse colon cancer tumor tissue and dissolve the lysed components. Then, PLGA is used as the skeleton material, Poly(I:C) and CpG are used as immune adjuvants to prepare a nanoparticle system, and then used The nanoparticle system activates allogeneic cancer-specific T cells in vivo, and then CD8 + T cells are isolated and extracted for cancer treatment. In this embodiment, two different methods are used to activate cancer-specific T cells. One is to directly inject nanoparticles into the human body for activation, and the other is to activate dendritic cells in vitro and then inject them into the body to activate cancer-specific T cells. .
(1)肿瘤组织和癌细胞的裂解及各组分的收集(1) Lysis of tumor tissue and cancer cells and collection of components
收集肿瘤组织时先在每只C57BL/6小鼠背部皮下接种2×10 6个MC38结肠癌细胞，在肿瘤长到体积分别为约1000mm 3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系统的抗原原料来源。 When collecting tumor tissue, 2 × 10 MC38 colon cancer cells were subcutaneously inoculated on the back of each C57BL/6 mouse . When the tumor grew to a volume of approximately 1000 mm, the mice were sacrificed and the tumor tissue was removed and cut into sections. After the block was ground, an 8M urea aqueous solution was added through a cell strainer to digest the tumor tissue and dissolve the lysed components. The above are the sources of antigen raw materials for preparing nanoparticle systems.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中纳米粒子采用复乳法制备。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CpG，且佐剂负载于纳米粒子内。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后备用。该纳米粒子平均粒径为260nm左右，纳米粒子表面电位为-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纳米粒所负载的poly(I:C)和CpG免疫佐剂各0.02mg。In this example, the nanoparticles were prepared using the double emulsion method. The molecular weight of PLGA, the material used to prepare the nanoparticles, is 7KDa-17KDa. The immune adjuvants used are poly(I:C) and CpG, and the adjuvants are loaded in the nanoparticles. The preparation method is as described above. During the preparation process, the lysis solution components and adjuvants are first loaded inside the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 10,000 g for 20 minutes and resuspended in 10 mL of ultrapure water containing 4% trehalose. Freeze dry for 48 hours before use. The average particle size of the nanoparticles is about 260nm, and the surface potential of the nanoparticles is about -7mV; each 1 mg of PLGA nanoparticles is loaded with approximately 90 μg of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I:C) and CpG immune Each adjuvant is 0.02mg.
(3)树突状细胞的制备(3) Preparation of dendritic cells
同实施例18。Same as Example 18.
(4)树突状细胞的激活(4) Activation of dendritic cells
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50μg负载裂解组分的PLGA纳米粒子与BMDC共孵育48h，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。Spread mouse BMDC into a cell culture plate, add 5 mL of RPMI 1640 (10% FBS) culture medium for every 100,000 BMDC cells, then add 50 μg of PLGA nanoparticles loaded with lysis components and incubate with the BMDC for 48 hours, and then collect After BMDC, centrifuge at 300g for 5 minutes, wash twice with phosphate buffer saline (PBS) and resuspend in PBS for later use.
(5)癌症特异性T细胞的激活(5) Activation of cancer-specific T cells
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100万个被预激活的BMDC细胞或者注射2mg的PLGA纳米疫苗。注射的BMDC细胞被负载裂解组分和佐剂的纳米粒子预激活过。第32天从小鼠中利用流式细胞术从脾脏单细胞悬液中分离CD8 +T细胞。 Select 6-8 week old female C57BL/6 mice and subcutaneously inject 1 million preactivated BMDC cells on days 0, 4, 7, 14, 21 and 28 respectively. Inject 2 mg of PLGA nanovaccine. The injected BMDC cells were preactivated with nanoparticles loaded with lytic components and adjuvants. CD8 + T cells were isolated from spleen single cell suspensions from mice on day 32 using flow cytometry.
(6)同种异体的细胞给与患癌小鼠治疗癌症(6) Allogeneic cells are given to cancer-stricken mice to treat cancer.
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2×10 6个MC38细胞。在接种结肠癌细胞后第4天、第7天、第10天、第15天、第20天和第25天分别静脉注射100μL的80万个CD8 +T细胞。小鼠肿瘤生长和生存期监测方法同上。 Select 6-8 week old female C57BL/6 as model mice to prepare colon cancer mice. On day 0, each mouse was subcutaneously inoculated with 2 × 10 6 MC38 cells on the lower right side of the back. On days 4, 7, 10, 15, 20 and 25 after inoculation of colon cancer cells, 100 μL of 800,000 CD8 + T cells were injected intravenously. The method for monitoring tumor growth and survival in mice is the same as above.
(7)实验结果(7)Experimental results
如图20所示，PBS对照组都很快长大。与对照组相比，纳米疫苗激活的来自同种异体的癌症特异性T细胞处理的小鼠肿瘤生长速度明显变慢且生存期明显延长，而且被直接注 射纳米疫苗激活的癌症特异性T细胞效果好于注射树突状细胞激活的癌症特异性T细胞。综上所述，本发明的细胞系统对癌症具有良好的治疗效果。As shown in Figure 20, the PBS control group grew up quickly. Compared with the control group, the tumor growth rate of mice treated with allogeneic cancer-specific T cells activated by the nanovaccine was significantly slower and the survival period was significantly prolonged, and the cancer-specific T cells activated by the direct injection of the nanovaccine had an effect Better than injecting dendritic cells to activate cancer-specific T cells. In summary, the cell system of the present invention has good therapeutic effect on cancer.
实施例20纳米粒子激活同种异体免疫细胞用于乳腺癌的预防Example 20 Nanoparticles activate allogeneic immune cells for the prevention of breast cancer
本实施例以4T1小鼠三阴性乳腺癌为癌症模型来说明如何采用负载全细胞组分的纳米粒子系统，并以该纳米粒子激活同种异体的癌症特异性T细胞，用于预防乳腺癌。本实施例中，首先使用8M尿素溶液对乳腺癌肿瘤组织进行裂解并溶解裂解组分。然后，以CpG和Poly(I:C)为免疫佐剂，以精氨酸和KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)为增强溶酶体逃逸的物质，制备负载有全细胞组分的PLGA纳米粒子系统。This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components and use the nanoparticles to activate allogeneic cancer-specific T cells for the prevention of breast cancer. In this example, an 8M urea solution was first used to lyse breast cancer tumor tissue and dissolve the lysed components. Then, using CpG and Poly(I:C) as immune adjuvants, and arginine and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) as substances that enhance lysosomal escape, a PLGA nanoparticle system loaded with whole cell components was prepared.
(1)肿瘤组织的裂解(1) Lysis of tumor tissue
选取6-8周的雌性BALB/c小鼠，背部皮下注射1×10 6个4T1乳腺癌细胞，待肿瘤体积长到1000mm 3左右时，处死小鼠，摘取小鼠肿瘤组织，将肿瘤组织切成小块后通过细胞筛网制备单细胞悬液，用PBS洗涤两遍后使用8M尿素水溶液(含500mM氯化钠)裂解肿瘤组织单细胞悬液并溶解裂解物组分，即为制备纳米粒子系统的抗原组分。 Select female BALB/c mice that are 6-8 weeks old and subcutaneously inject 1× 10 4T1 breast cancer cells into the back. When the tumor volume grows to about 1000 mm, the mice are sacrificed and the mouse tumor tissue is removed. Cut into small pieces and pass through a cell sieve to prepare a single cell suspension. Wash twice with PBS and then use 8M urea aqueous solution (containing 500mM sodium chloride) to lyse the tumor tissue single cell suspension and dissolve the lysate components to prepare nanoparticles. Antigenic components of particle systems.
(2)纳米粒子系统的制备(2) Preparation of nanoparticle system
本实施例中制备纳米粒子系统采用复乳法，纳米粒子骨架材料为未修饰的PLGA和甘露聚糖修饰的PLGA，分子量都为24-38KDa，未修饰的PLGA和甘露聚糖修饰的PLGA的比例为4:1。所采用的免疫佐剂为CpG和Poly(I:C)，所采用的溶酶体逃逸增加物质为精氨酸和KALA多肽。制备时先采用复乳法制备内部负载裂解物组分、佐剂、精氨酸和KALA多肽的纳米粒子，尔后，将100mg纳米粒子在12000g离心25分钟，使用10mL含4％海藻糖的超纯水重悬后干燥48h后备用。该纳米粒子系统平均粒径为250nm左右，纳米粒子系统表面电位为-8mV左右；每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分，含CpG和Poly(I:C)各0.02mg，含精氨酸和KALA各0.05mg。In this example, the nanoparticle system is prepared using the double emulsion method. The nanoparticle skeleton materials are unmodified PLGA and mannan-modified PLGA. The molecular weights are both 24-38KDa. The ratio of unmodified PLGA to mannan-modified PLGA is is 4:1. The immune adjuvants used were CpG and Poly(I:C), and the lysosomal escape-increasing substances used were arginine and KALA polypeptide. During preparation, the double emulsion method is first used to prepare nanoparticles internally loaded with lysate components, adjuvants, arginine and KALA polypeptides. Then, 100 mg of nanoparticles are centrifuged at 12,000g for 25 minutes, and 10 mL of ultrapure solution containing 4% trehalose is used. Resuspend in water and dry for 48 hours before use. The average particle size of the nanoparticle system is about 250nm, and the surface potential of the nanoparticle system is about -8mV; each 1 mg of PLGA nanoparticles is loaded with approximately 100 μg of protein or peptide components, including 0.02 mg of CpG and Poly(I:C), and 0.02 mg of poly(I:C). 0.05mg each of amino acid and KALA.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
选取6-8周的雌性BALC/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2mgPLGA的纳米粒子。第32天处死小鼠，收集小鼠外周血，并分离得到小鼠外周血单个核细胞(PBMC)，从PBMC中利用流式细胞术分选CD3 +CD8 +T细胞和γδT细胞，将分选得到CD3 +CD8 +T细胞和γδT细胞分别与IL-2(1000U/mL)、IL-12(1000U/mL)和αCD3/αCD28抗体(10ng/mL)共孵育14天以扩增CD8 +T细胞和γδT细胞。对照组小鼠从PBMC中分离γδT细胞和NKT细胞，并将上述两种细胞分别于IL-2(1000U/mL)、IL-12(1000U/mL)和αCD3/αCD28抗体(10ng/mL)共孵育14天以扩增分选得到γδT细胞和NKT细胞。 Female BALC/c mice aged 6-8 weeks were selected and 100 μL of nanoparticles containing 2 mg PLGA were subcutaneously injected on days 0, 4, 7, 14, 21, and 28 respectively. On the 32nd day, the mice were sacrificed, peripheral blood was collected, and mouse peripheral blood mononuclear cells (PBMC) were isolated. CD3 + CD8 + T cells and γδ T cells were sorted from the PBMC using flow cytometry. The obtained CD3 + CD8 + T cells and γδ T cells were incubated with IL-2 (1000U/mL), IL-12 (1000U/mL) and αCD3/αCD28 antibody (10ng/mL) respectively for 14 days to expand CD8 + T cells. and γδ T cells. In the control group, γδT cells and NKT cells were isolated from PBMC, and the above two cells were co-incubated with IL-2 (1000U/mL), IL-12 (1000U/mL) and αCD3/αCD28 antibody (10ng/mL) respectively. Incubate for 14 days to amplify and sort γδ T cells and NKT cells.
(4)同种异体细胞系统用于癌症的预防+(4) Allogeneic cell system for cancer prevention+
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。在第0天给小鼠皮下注射100万个CD8 +T细胞+50万个γδT细胞；或者注射75万个γδT细胞+75万个NKT细胞。同时在第0天给每只小鼠皮下注射接种1×10 6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。肿瘤体积采用公式v＝0.52×a×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 Female BALB/c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. On day 0, mice were injected subcutaneously with 1 million CD8 + T cells + 500,000 γδ T cells; or with 750,000 γδ T cells + 750,000 NKT 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. Tumor volume was calculated using the formula v = 0.52 × a × b 2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图21所示，与对照组相比，免疫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，含有CD8 +T细胞+γδT细胞的混合免疫细胞效果好于γδT细胞+NKT细胞的混合免疫细胞。CD8 +T细胞中含有适应性免疫的癌症特异性T细胞，γδT细胞和NKT细胞都属于天然免疫系统。这说明同时给予适应性免疫细胞和天然免疫细胞效果好于只给与天然免疫细胞。由此可见，本发明所述的来自同种异体的被粒子激活的免疫细胞对乳腺癌具有预防效果。 As shown in Figure 21, compared with the control group, the tumor growth rate in the immune cell treatment group was significantly slower and the survival period of mice was significantly prolonged. Moreover, the mixed immune cells containing CD8 + T cells + γδ T cells are more effective than the mixed immune cells containing γδ T cells + NKT cells. CD8 + T cells contain cancer-specific T cells of adaptive immunity, and γδ T cells and NKT cells all belong to the innate immune system. This shows that giving adaptive immune cells and innate immune cells at the same time is more effective than giving only innate immune cells. It can be seen that the allogeneic particle-activated immune cells of the present invention have a preventive effect on breast cancer.
实施例21被微米粒子激活同种异体免疫细胞用于乳腺癌的治疗Example 21 Allogeneic immune cells activated by micron particles for the treatment of breast cancer
本实施例以4T1小鼠三阴性乳腺癌为癌症模型来说明如何采用负载全细胞组分的微米粒子系统，并以该微米粒子激活同种异体的癌症特异性T细胞，用于治疗乳腺癌。本实施例中，首先使用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PLGA为微米粒子骨架材料，以CpG和Poly ICLC为免疫佐剂，以精氨酸和赖氨酸为增强溶酶体逃逸的物质，制备负载有全细胞组分的微米粒子系统。This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use a micron particle system loaded with whole cell components and use the micron particles to activate allogeneic cancer-specific T cells for the treatment of breast cancer. In this example, 8M urea solution was first used to lyse breast cancer cells and dissolve the lysis components. Then, using PLGA as the micron particle skeleton material, CpG and Poly ICLC as immune adjuvants, and arginine and lysine as substances that enhance lysosomal escape, a micron particle system loaded with whole cell components was prepared.
(1)癌细胞的裂解(1) Lysis of cancer cells
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(含500mM氯化钠)裂解癌细胞并溶解裂解物组分，即为制备微米粒子系统的抗原组分。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 (containing 500mM sodium chloride) was used to lyse the cancer cells and dissolve the lysate components, which are the antigen components for preparing the micron particle system.
(2)微米粒子系统的制备(2) Preparation of micron particle system
本实施例中制备微米粒子系统采用复乳法，微米粒子骨架材料为未修饰的PLGA和甘露糖修饰的PLGA，分子量都为24-38KDa，未修饰的PLA和甘露糖修饰的PLGA的比例为9:1。所采用的免疫佐剂为CpG和Poly ICLC，所采用的溶酶体逃逸增加物质为精氨酸和赖氨酸。制备时先采用复乳法制备内部负载裂解物组分、佐剂、精氨酸和来氨酸的微米粒子,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。该微米粒子系统平均粒径为2.1μm左右，微米粒子系统表面电位为-7mV左右；每1mg PLGA微米粒子约负载100μg蛋白质或多肽组分，含CpG和Poly ICLC各0.01mg，含精氨酸和赖氨酸各0.05mg。In this example, the microparticle system is prepared using the double emulsion method. The microparticle skeleton materials are unmodified PLGA and mannose-modified PLGA. The molecular weights are both 24-38KDa. The ratio of unmodified PLA to mannose-modified PLGA is 9. :1. The immune adjuvants used are CpG and Poly ICLC, and the lysosomal escape-increasing substances used are arginine and lysine. During preparation, the double emulsion method is first used to prepare micron particles internally loaded with lysate components, adjuvants, arginine and lysine. Then, 100 mg of micron particles are centrifuged at 9000g for 20 minutes, and 10 mL of ultrasonic acid containing 4% trehalose is used. Resuspend in pure water and dry for 48 hours before use. The average particle size of the micron particle system is about 2.1 μm, and the surface potential of the micron particle system is about -7mV; each 1 mg of PLGA micron particles is loaded with approximately 100 μg of protein or peptide components, including 0.01 mg each of CpG and Poly ICLC, and arginine and Lysine 0.05mg each.
(3)癌症特异性T细胞的激活(3) Activation of cancer-specific T cells
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天分别后背皮下注射100μL含2mg PLGA的微米粒子。第32天处死小鼠，收集小鼠注射微米粒子附近的引流淋巴结，将引流淋巴结切碎后通过细胞筛网制备单细胞悬液。将2mL含有20万个淋巴结单细胞悬液的RPMI1640完全培养基与步骤(2)所制备的PLGA微米粒子(30ug)共孵育48小时，然后在400g离心5分钟收集孵育后的单细胞悬液。从孵育后的淋巴结细胞中利用流式细胞术分选CD3 +CD8 +CD69 +T细胞和CD3 +CD4 +CD69 +T细胞，即为识别癌症特异性抗原表位后被癌症抗原表位激活的癌症特异性T细胞，将分选得到的CD3 +CD8 +CD69 +和CD3 +CD4 +CD69 +癌症特异性T细胞与IL-2(2000U/mL)、IL-7(500U/mL)、IL-15(500U/mL)共孵育10天以扩增分选得到癌细胞特异性CD8 +T细胞和CD4 +T细胞。对照组小鼠的淋巴结单细胞悬液不与微米粒子共孵育，而是直接从淋巴结单细胞悬液中利用流式细胞术分选得到CD3 +CD8 +T和CD3 +CD4 +T细胞，并与IL-2(2000U/mL)、IL-7(500U/mL)、IL-15(500U/mL)共孵育10天以扩增分选得到的CD8 +T细胞和CD4 +T细胞。 Female C57BL/6 mice aged 6-8 weeks were selected, and 100 μL of microparticles containing 2 mg PLGA were injected subcutaneously into the back on days 0, 4, 7, 14, 21, and 28. The mice were sacrificed on day 32, and the draining lymph nodes near the mice injected with micron particles were collected. The draining lymph nodes were minced and passed through a cell mesh to prepare a single-cell suspension. Incubate 2 mL of RPMI1640 complete medium containing 200,000 lymph node single cell suspension with the PLGA microparticles (30ug) prepared in step (2) for 48 hours, and then centrifuge at 400g for 5 minutes to collect the incubated single cell suspension. Use flow cytometry to sort CD3 + CD8 + CD69 + T cells and CD3 + CD4 + CD69 + T cells from incubated lymph node cells, which are cancers that are activated by cancer antigen epitopes after recognizing cancer-specific antigen epitopes. Specific T cells, combine the sorted CD3 + CD8 + CD69 + and CD3 + CD4 + CD69 + cancer-specific T cells with IL-2 (2000U/mL), IL-7 (500U/mL), IL-15 (500U/mL) for 10 days to amplify and sort to obtain cancer cell-specific CD8 + T cells and CD4 + T cells. The lymph node single cell suspension of mice in the control group was not incubated with micron particles. Instead, CD3 + CD8 + T and CD3 + CD4 + T cells were sorted directly from the lymph node single cell suspension by flow cytometry, and compared with IL-2 (2000U/mL), IL-7 (500U/mL), and IL-15 (500U/mL) were incubated for 10 days to amplify the sorted CD8 + T cells and CD4 + T cells.
(4)同种异体细胞系统用于癌症的治疗(4) Allogeneic cell systems for cancer treatment
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。同时在第0天给每只小鼠皮下注射接种1×10 6个4T1细胞，第5天、第7天、第10天和第15天、第20天分别给小鼠皮下注射100μL含200万个分选扩增后的T细胞(含140万个CD8 +T细胞和60万个CD4 +T细胞)，或者皮下注射100μL含200万个癌症特异性T细胞(含140万个CD8 +癌症特异性T细胞和60万个CD4 +癌症特异性T细胞)。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×b 2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，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 Female BALB/c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice. At the same time, each mouse was subcutaneously injected with 1 × 10 6 4T1 cells on day 0. On days 5, 7, 10, 15, and 20, mice were subcutaneously injected with 100 μL containing 2 million cells. sorted and expanded T cells (containing 1.4 million CD8 + T cells and 600,000 CD4 + T cells), or subcutaneous injection of 100 μL containing 2 million cancer-specific T cells (containing 1.4 million CD8 + cancer-specific T cells and 600,000 CD4 + cancer-specific T cells). The size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v=0.52×a× b2 , where v is the tumor volume, a is the tumor length, and b is the tumor width. Due to the ethics of animal experimentation, when the mouse tumor volume exceeds 2000mm3 in the mouse survival test, the mouse is deemed dead and the mouse is euthanized.
(5)实验结果(5)Experimental results
如图22所示，与对照组相比，被微米粒激活的免疫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，分选得到的癌症特异性T细胞经扩增后使用好于直接分选的T细胞经扩增后使用。由此可见，本发明所述的来自同种异体的癌症特异性T细胞对乳腺癌具有良好的治疗效果。本发明在淋巴结单细胞悬液与粒子系统体外共孵育时，使用淋巴结中所含有的抗原提呈细胞(主要是DC细胞和B细胞)辅助粒子激活癌症特异性T细胞后分选被激活的癌症特异性T细胞，在实际使用时也可以使用任何其他来源的抗原提呈细胞辅助粒子系统激活癌症特异性T细胞，比如细胞系来源的抗原提呈细胞、干细胞来源的抗原提呈细胞、自体来源的抗原提呈细胞。As shown in Figure 22, compared with the control group, the tumor growth rate of the immune cell treatment group activated by micron particles was significantly slower and the survival period of mice was significantly prolonged. Moreover, it is better to use the sorted cancer-specific T cells after expansion than directly sorted T cells after expansion. It can be seen that the allogeneic cancer-specific T cells of the present invention have a good therapeutic effect on breast cancer. When the lymph node single cell suspension and the particle system are co-incubated in vitro, the present invention uses the antigen-presenting cells (mainly DC cells and B cells) contained in the lymph node to assist the particles in activating cancer-specific T cells and then sorting the activated cancers. For specific T cells, in actual use, the antigen-presenting cell auxiliary particle system from any other source can also be used to activate cancer-specific T cells, such as cell line-derived antigen-presenting cells, stem cell-derived antigen-presenting cells, and autologous sources. of antigen-presenting cells.
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。Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or modifications may be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (37)

  1. 一种基于癌症特异性T细胞的细胞系统，其特征在于：所述细胞系统包括经癌症疫苗激活的癌症特异性T细胞，其中，癌症疫苗包括递送粒子及其负载的细胞组分，所述递送粒子为纳米粒子或微米粒子，所述细胞组分为来源于癌细胞和/或肿瘤组织的水溶性组分和/或非水溶性组分；A cell system based on cancer-specific T cells, characterized in that: the cell system includes cancer-specific T cells activated by a cancer vaccine, wherein the cancer vaccine includes delivery particles and their loaded cell components, and the delivery The particles are nanoparticles or microparticles, and the cellular components are water-soluble components and/or water-insoluble components derived from cancer cells and/or tumor tissues;
    所述细胞组分经癌细胞和/或肿瘤组织裂解得到；The cellular components are obtained by lysing cancer cells and/or tumor tissues;
    所述的裂解是将癌细胞或肿瘤组织加水或不含溶解剂的水溶液进行裂解，得到的上清液为所述水溶性组分，沉淀中经溶解剂溶解后转为可溶的部分为所述非水溶性组分；或所述的裂解是使用溶解剂裂解癌细胞或肿瘤组织，并溶解裂解后的组分，得到同时含有所述水溶性组分和非水溶性组分的混合物。The lysis is to lyse cancer cells or tumor tissues by adding water or an aqueous solution without a dissolving agent. The supernatant obtained is the water-soluble component, and the soluble part of the precipitate after being dissolved by the dissolving agent is the solubilized component. The water-insoluble component; or the lysis is to use a dissolving agent to lyse cancer cells or tumor tissue, and dissolve the lysed components to obtain a mixture containing both the water-soluble component and the water-insoluble component.
  2. 根据权利要求1所述的细胞系统，其特征在于：所述癌症特异性T细胞包括CD4 +T细胞和/或CD8 +T细胞。 The cell system according to claim 1, wherein the cancer-specific T cells include CD4 + T cells and/or CD8 + T cells.
  3. 根据权利要求1所述的细胞系统，其特征在于：所述细胞系统还包括天然免疫细胞。The cell system according to claim 1, characterized in that: the cell system further includes innate immune cells.
  4. 根据权利要求3所述的细胞系统，其特征在于：所述天然免疫细胞选自γδT细胞、自然杀伤细胞、中性粒细胞和自然杀伤T细胞中的至少一种。The cell system according to claim 3, wherein the natural immune cells are selected from at least one of γδ T cells, natural killer cells, neutrophils and natural killer T cells.
  5. 根据权利要求1所述的细胞系统，其特征在于：所述的激活是将癌症疫苗注射至体内激活癌症特异性T细胞，或将树突状细胞经癌症疫苗刺激后注射至体内激活癌症特异性T细胞。The cell system according to claim 1, wherein the activation is to inject a cancer vaccine into the body to activate cancer-specific T cells, or to inject dendritic cells into the body after being stimulated by the cancer vaccine to activate cancer-specific T cells. T cells.
  6. 根据权利要求5所述的细胞系统，其特征在于：所述的刺激是将树突状细胞与癌症疫苗在体外共孵育，直至癌症疫苗中负载的细胞组分被树突状细胞进行抗原提呈和激活。The cell system according to claim 5, characterized in that: the stimulation is to co-incubate dendritic cells and cancer vaccine in vitro until the cellular components loaded in the cancer vaccine are antigen-presented by dendritic cells. and activation.
  7. 根据权利要求1所述的细胞系统，其特征在于：所述溶解剂选自尿素、盐酸胍、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、无机盐、Triton、吐温、二甲基亚砜、乙腈、乙醇、甲醇、N,N-二甲基甲酰胺、丙醇、异丙醇、醋酸、胆固醇、氨基酸、糖苷和胆碱中的至少一种。The cell system according to claim 1, wherein the dissolving agent is selected from the group consisting of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, inorganic At least one of salt, Triton, Tween, dimethyl sulfoxide, acetonitrile, ethanol, methanol, N,N-dimethylformamide, propanol, isopropanol, acetic acid, cholesterol, amino acids, glycosides and choline kind.
  8. 根据权利要求1所述的细胞系统，其特征在于：所述水溶性组分和/或非水溶性组分负载于递送粒子内部，和/或负载于递送粒子表面。The cell system according to claim 1, wherein the water-soluble component and/or the water-insoluble component is loaded inside the delivery particle, and/or is loaded on the surface of the delivery particle.
  9. 根据权利要求1所述的细胞系统，其特征在于：所述水溶性组分和/或非水溶性组分负载于递送粒子表面的方式为吸附、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。The cell system according to claim 1, characterized in that: the water-soluble component and/or the water-insoluble component is loaded on the surface of the delivery particles in a manner such as adsorption, covalent connection, charge interaction, hydrophobic interaction, At least one of one or more steps of solidification, mineralization and encapsulation.
  10. 根据权利要求1所述的细胞系统，其特征在于：所述递送粒子的表面连接有主动靶向树突状细胞的靶头。The cell system according to claim 1, wherein the surface of the delivery particles is connected with a target head that actively targets dendritic cells.
  11. 根据权利要求10所述的细胞系统，其特征在于：所述靶头选自甘露糖、CD32抗体、CD11c抗体、CD103抗体和CD44抗体中的至少一种。The cell system according to claim 10, wherein the target is selected from at least one of mannose, CD32 antibody, CD11c antibody, CD103 antibody and CD44 antibody.
  12. 根据权利要求1所述的细胞系统，其特征在于：所述递送粒子上还负载免疫增强佐剂。The cell system according to claim 1, wherein the delivery particles are also loaded with an immune-enhancing adjuvant.
  13. 根据权利要求1所述的细胞系统，其特征在于：所述递送粒子上还负载有协助递送粒子或抗原逃离溶酶体的物质。The cell system according to claim 1, wherein the delivery particles are also loaded with substances that assist the delivery particles or antigens in escaping from lysosomes.
  14. 根据权利要求1所述的细胞系统，其特征在于：所述递送粒子由有机合成高分子材料、天然高分子材料或无机材料制得；The cell system according to claim 1, characterized in that: the delivery particles are made of organic synthetic polymer materials, natural polymer materials or inorganic materials;
    所述有机合成高分子材料选自聚乳酸-羟基乙酸共聚物、聚乳酸、聚乙醇酸、聚乙二醇、聚己内酯、泊洛沙姆、聚乙烯醇、聚乙烯吡咯烷酮、聚乙烯亚胺、聚三亚甲基碳酸酯、聚酸酐、聚对二氧六环酮、聚对二氧环己酮、聚甲基丙烯酸甲酯、PLGA-PEG、PLA-PEG、PGA-PEG、聚氨基酸、合成多肽和合成脂质中的至少一种；所述天然高分子材料选自卵磷脂、胆固醇、海藻酸盐、白蛋白、胶原蛋白、明胶、细胞膜、淀粉、糖类和多肽中的至少一种；所述无机材料选自三氧化二铁、四氧化三铁、碳酸钙和磷酸钙中的至少一种。The organic synthetic polymer material is selected from polylactic acid-glycolic acid copolymer, polylactic acid, polyglycolic acid, polyethylene glycol, polycaprolactone, poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene oxide Amine, polytrimethylene carbonate, polyanhydride, polydioxanone, polydioxanone, polymethyl methacrylate, PLGA-PEG, PLA-PEG, PGA-PEG, polyamino acid, At least one of synthetic polypeptides and synthetic lipids; the natural polymer material is selected from at least one of lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane, starch, sugars and polypeptides ; The inorganic material is selected from at least one of ferric oxide, ferric tetroxide, calcium carbonate and calcium phosphate.
  15. 根据权利要求1所述的细胞系统，其特征在于：所述癌症疫苗经过表面修饰，所述表面修饰的方式为化学修饰或物理修饰。The cell system according to claim 1, wherein the cancer vaccine is surface modified, and the surface modification is chemical modification or physical modification.
  16. 权利要求1-15任一项所述的基于癌症特异性T细胞的细胞系统在制备预防或治疗癌症产品中的应用。Application of the cancer-specific T cell-based cell system according to any one of claims 1 to 15 in the preparation of products for the prevention or treatment of cancer.
  17. 一种同种异体淋巴细胞药物，其特征在于：所述同种异体淋巴细胞药物包括经癌症疫苗激活的癌症特异性T细胞，所述癌症特异性T细胞来源于同种异体的个体，其中，癌症疫苗包括递送粒子及其负载的细胞组分，所述递送粒子为纳米粒子或微米粒子，所述细胞组分为来源于癌细胞和/或肿瘤组织的水溶性组分和/或非水溶性组分；An allogeneic lymphocyte drug, characterized in that: the allogeneic lymphocyte drug includes cancer-specific T cells activated by a cancer vaccine, and the cancer-specific T cells are derived from an allogeneic individual, wherein, Cancer vaccines include delivery particles and loaded cellular components. The delivery particles are nanoparticles or microparticles. The cellular components are water-soluble components and/or non-water-soluble components derived from cancer cells and/or tumor tissues. components;
    所述细胞组分经癌细胞和/或肿瘤组织裂解得到；The cellular components are obtained by lysing cancer cells and/or tumor tissues;
    所述的裂解是将癌细胞或肿瘤组织在-20℃～-273℃下冷冻，加水或不含溶解剂的溶液后进行反复冻融裂解，上清液为所述水溶性组分，沉淀中经溶解剂溶解后转为可溶的部分为所述非水溶性组分；或所述的裂解是使用溶解剂裂解癌细胞或肿瘤组织，并溶解裂解后的组分，得到同时含有所述水溶性组分和非水溶性组分的混合物。The lysis is to freeze cancer cells or tumor tissues at -20°C to -273°C, add water or a solution without a dissolving agent, and perform repeated freeze-thaw lysis. The supernatant is the water-soluble component, and the precipitated The part that becomes soluble after being dissolved by a dissolving agent is the water-insoluble component; or the lysis is to use a dissolving agent to lyse cancer cells or tumor tissues, and dissolve the cleaved components to obtain the water-soluble component that also contains the water-soluble component. A mixture of water-insoluble and water-insoluble components.
  18. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述同种异体的个体为一个或多个。The allogeneic lymphocyte medicine according to claim 17, wherein the number of allogeneic individuals is one or more.
  19. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述癌症特异性T细胞包括CD4 +T细胞和/或CD8 +T细胞。 The allogeneic lymphocyte drug according to claim 17, wherein the cancer-specific T cells include CD4 + T cells and/or CD8 + T cells.
  20. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述同种异体淋巴细胞药物还包括来源于同种异体的天然免疫细胞。The allogeneic lymphocyte drug according to claim 17, wherein the allogeneic lymphocyte drug further includes natural immune cells derived from allogeneic cells.
  21. 根据权利要求20所述的同种异体淋巴细胞药物，其特征在于：所述天然免疫细胞选自γδT细胞、自然杀伤细胞、中性粒细胞和自然杀伤T细胞中的至少一种。The allogeneic lymphocyte medicine according to claim 20, wherein the natural immune cells are selected from at least one of γδ T cells, natural killer cells, neutrophils and natural killer T cells.
  22. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述的激活为将癌症疫苗注射至同种异体体内激活癌症特异性T细胞，或将树突状细胞经癌症疫苗刺激后注射至同种异体体内激活癌症特异性T细胞。The allogeneic lymphocyte medicine according to claim 17, characterized in that: the activation is to inject a cancer vaccine into the allogeneic body to activate cancer-specific T cells, or to activate dendritic cells after being stimulated by the cancer vaccine. Injection into allogeneic cells activates cancer-specific T cells.
  23. 根据权利要求22所述的同种异体淋巴细胞药物，其特征在于：所述树突状细胞为自体树突状细胞、同种异体的树突状细胞、细胞系或干细胞来源。The allogeneic lymphocyte medicine according to claim 22, wherein the dendritic cells are derived from autologous dendritic cells, allogeneic dendritic cells, cell lines or stem cells.
  24. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述的刺激是将树突状细胞与癌症疫苗在体外共孵育，直至癌症疫苗中负载的细胞组分被树突状细胞进行抗原提呈和激活。The allogeneic lymphocyte medicine according to claim 17, characterized in that: the stimulation is to co-incubate dendritic cells with the cancer vaccine in vitro until the cellular components loaded in the cancer vaccine are absorbed by the dendritic cells. Perform antigen presentation and activation.
  25. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述溶解剂选自尿素、盐酸胍、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、无机盐、Triton、吐温、二甲基亚砜、乙腈、乙醇、甲醇、N,N-二甲基甲酰胺、丙醇、异丙醇、醋酸、胆固醇、氨基酸、糖苷和胆碱中的至少一种。The allogeneic lymphocyte medicine according to claim 17, characterized in that: the dissolving agent is selected from the group consisting of urea, guanidine hydrochloride, deoxycholate, lauryl sulfate, glycerol, protein degrading enzyme, albumin, Lecithin, inorganic salts, Triton, Tween, dimethyl sulfoxide, acetonitrile, ethanol, methanol, N,N-dimethylformamide, propanol, isopropanol, acetic acid, cholesterol, amino acids, glycosides and choline at least one of them.
  26. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述水溶性组分和/或非水溶性组分分别或同时负载于递送粒子内部，或分别或同时负载于递送粒子表面。The allogeneic lymphocyte drug according to claim 17, characterized in that: the water-soluble component and/or the water-insoluble component are loaded inside the delivery particles respectively or simultaneously, or are loaded separately or simultaneously on the surface of the delivery particles. .
  27. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述水溶性组分和/或非水溶性组分负载于递送粒子表面的方式为吸附、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。The allogeneic lymphocyte drug according to claim 17, characterized in that: the water-soluble component and/or the water-insoluble component is loaded on the surface of the delivery particle by adsorption, covalent connection, charge interaction, At least one of hydrophobic interaction, one or more steps of solidification, mineralization and encapsulation.
  28. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述递送粒子的表面连接有主动靶向树突状细胞的靶头。The allogeneic lymphocyte drug according to claim 17, wherein the surface of the delivery particles is connected with a target head that actively targets dendritic cells.
  29. 根据权利要求28所述的同种异体淋巴细胞药物，其特征在于：所述靶头选自甘露糖、甘露聚糖、CD32抗体、CD11c抗体、CD103抗体和CD44抗体中的至少一种。The allogeneic lymphocyte drug according to claim 28, wherein the target is selected from at least one of mannose, mannan, CD32 antibody, CD11c antibody, CD103 antibody and CD44 antibody.
  30. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述递送粒子上还负载免疫增强佐剂。The allogeneic lymphocyte drug according to claim 17, wherein the delivery particles are also loaded with an immune-enhancing adjuvant.
  31. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述递送粒子上还负载有协助递送粒子或抗原逃离溶酶体的物质。The allogeneic lymphocyte drug according to claim 17, wherein the delivery particles are also loaded with substances that assist the delivery particles or antigens in escaping from lysosomes.
  32. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：制备所述递送粒子的材料为有机合成高分子材料、天然高分子材料或无机材料；The allogeneic lymphocyte drug according to claim 17, characterized in that: the material for preparing the delivery particles is an organic synthetic polymer material, a natural polymer material or an inorganic material;
    所述有机合成高分子材料选自聚乳酸-羟基乙酸共聚物、聚乳酸、聚乙醇酸、聚乙二醇、聚己内酯、泊洛沙姆、聚乙烯醇、聚乙烯吡咯烷酮、聚乙烯亚胺、聚三亚甲基碳酸酯、聚酸酐、聚对二氧六环酮、聚对二氧环己酮、聚甲基丙烯酸甲酯、PLGA-PEG、PLA-PEG、PGA-PEG、聚氨基酸、合成多肽和合成脂质中的至少一种；所述天然高分子材料选自卵磷 脂、胆固醇、海藻酸盐、白蛋白、胶原蛋白、明胶、细胞膜、淀粉、糖类和多肽中的至少一种；所述无机材料选自三氧化二铁、四氧化三铁、碳酸钙和磷酸钙中的至少一种。The organic synthetic polymer material is selected from polylactic acid-glycolic acid copolymer, polylactic acid, polyglycolic acid, polyethylene glycol, polycaprolactone, poloxamer, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene oxide Amine, polytrimethylene carbonate, polyanhydride, polydioxanone, polydioxanone, polymethylmethacrylate, PLGA-PEG, PLA-PEG, PGA-PEG, polyamino acid, At least one of synthetic polypeptides and synthetic lipids; the natural polymer material is selected from at least one of lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane, starch, sugars and polypeptides ; The inorganic material is selected from at least one of ferric oxide, ferric tetroxide, calcium carbonate and calcium phosphate.
  33. 根据权利要求17所述的同种异体淋巴细胞药物，其特征在于：所述癌症疫苗经过表面修饰，所述表面修饰的方式为化学修饰或物理修饰。The allogeneic lymphocyte drug according to claim 17, characterized in that: the cancer vaccine is surface modified, and the surface modification method is chemical modification or physical modification.
  34. 权利要求17-33任一项所述的同种异体淋巴细胞药物在制备预防或治疗癌症产品中的应用。Application of the allogeneic lymphocyte drug according to any one of claims 17 to 33 in the preparation of products for the prevention or treatment of cancer.
  35. 根据权利要求34所述的应用，其特征在于：所述的应用为将同种异体供体体内分离得到的淋巴细胞药物输至同种异体受体体内。The application according to claim 34, characterized in that the application is to transport the lymphocyte drug separated from the body of the allogeneic donor into the body of the allogeneic recipient.
  36. 根据权利要求35所述的应用，其特征在于：将淋巴细胞药物输至同种异体受体体内前，还包括对所述淋巴细胞药物进行体外扩增的步骤。The application according to claim 35, characterized in that: before infusing the lymphocyte drug into the allogeneic recipient, it further includes the step of in vitro amplification of the lymphocyte drug.
  37. 一种抗肿瘤制品，其特征在于，所述抗肿瘤制品包括以下任意一种：(1)含有经癌症疫苗激活的来源于同种异体的癌症特异性T细胞的制剂；(2)含有经癌症疫苗激活的来源于同种异体的癌症特异性T细胞的制剂，以及含有来源于同种异体的天然免疫细胞的制剂；(3)含有经癌症疫苗激活的癌症特异性T细胞和天然免疫细胞混合物的制剂，所述癌症特异性T细胞和天然免疫细胞均来源于同种异体的个体。An anti-tumor product, characterized in that the anti-tumor product includes any one of the following: (1) a preparation containing allogeneic cancer-specific T cells activated by a cancer vaccine; (2) a preparation containing cancer-specific T cells activated by a cancer vaccine Vaccine-activated preparations of allogeneic cancer-specific T cells, and preparations containing allogeneic-derived natural immune cells; (3) mixtures containing cancer vaccine-activated cancer-specific T cells and natural immune cells A preparation in which the cancer-specific T cells and natural immune cells are derived from allogeneic individuals.
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