WO2024000725A1 - 一种癌细胞特异性t细胞疫苗、以及激活癌细胞特异性t细胞的方法 - Google Patents

一种癌细胞特异性t细胞疫苗、以及激活癌细胞特异性t细胞的方法 Download PDF

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WO2024000725A1
WO2024000725A1 PCT/CN2022/108966 CN2022108966W WO2024000725A1 WO 2024000725 A1 WO2024000725 A1 WO 2024000725A1 CN 2022108966 W CN2022108966 W CN 2022108966W WO 2024000725 A1 WO2024000725 A1 WO 2024000725A1
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cells
cell
antigen
nanoparticles
cancer
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French (fr)
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刘密
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苏州尔生生物医药有限公司
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Definitions

  • the present invention relates to the field of immunotherapy, and in particular to a cancer cell-specific T cell vaccine and a method for activating cancer cell-specific T cells.
  • T cells are the main cells in the body that specifically recognize and kill cancer cells.
  • Each clone of cancer cell-specific T cells can specifically recognize an antigenic epitope.
  • Cancer patients especially those who have undergone immunotherapy or radiotherapy, contain a certain number of cancer cell-specific T cells. However, the number of cancer cell-specific T cells in cancer patients is generally insufficient, which limits the ability of cancer cell-specific T cells to kill cancer cells. If the cancer cell-specific T cells in the body can be isolated, expanded and reinfused back to the patient , then the immune microenvironment can be regulated and cancer progression better controlled.
  • the present invention provides a nanoparticle and/or microparticle-assisted separation prepared by using antigen-presenting cells activated by nanoparticles (NP) or microparticles (MP) loaded with cancer cell whole cell antigens. and a method of amplifying killer (effector) cancer cell-specific T cells (T eff ), which uses nanoparticles or microparticles prepared from activated antigen-presenting cells to first activate broad-spectrum polyclonal cancer cell-specific T cells, and then use markers specifically expressed by activated killer cancer cell-specific T cells (T eff ) to separate and extract the above-mentioned cancer cell-specific T cells, and then infuse them back to the patient to prevent or treat cancer, effectively solving the problem.
  • NP nanoparticles
  • MP microparticles
  • the first object of the present invention is to provide a method for preparing a cancer T cell vaccine prepared from particle-activated antigen-presenting cells, which includes the following steps:
  • step S3 after sorting the activated cancer cell-specific T cells, a step of amplifying the cancer cell-specific T cells is also included.
  • the above-mentioned sorting is performed by using specific surface markers of cancer cell-specific T cells activated by cancer cell whole cell components.
  • Specific surface markers include but are not limited to CD69, PD-1, TIM-3, LAG-3, CD25, OX40 (CD134), TCF-1, CD137, CD44, CD39, CD103, CD56, CD279, CD278, CD244, CD27, CD154, CD28, etc.
  • Techniques for isolating cancer cell-specific T cells using surface markers include, but are not limited to, flow cytometry and magnetic bead sorting.
  • immune cells containing T cells can be derived from peripheral blood, peripheral immune organs or tumor-infiltrating lymphocytes. Before they are co-incubated with S2 products, the above immune cells can be sorted to sort out the T cells. Specifically, flow cytometry or magnetic bead sorting is used to collect samples from peripheral blood, peripheral immune tissue, and tumors. Sorting CD3 + cells, sorting CD45 + CD3 + cells, sorting CD3 + CD8 + cells, sorting CD45 + CD3 + CD8 + cells, sorting CD3 + CD4 + cells among infiltrating lymphocytes cells or sort out CD45 + CD3 + CD4 + cells.
  • the above-mentioned amplification is in vitro amplification, that is, the cancer cell-specific T cells are co-incubated with cytokines and/or antibodies.
  • the co-incubation system of the antigen-presenting cells and the first particles in step S1 and the co-incubation system of the nanovesicles and particles loaded with cell membrane components and immune cells in step S3 may contain cytokines and/or antibodies. .
  • the incubation system contains IL-2 and IL-7.
  • cytokines include, but are not limited to, interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 14 (IL-14), interleukin 4 (IL-4), interleukin 15 (IL-15), interleukin 21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 17 (IL-17), IL-12, interleukin 12 (IL-12), interleukin 6 (IL-6), Macrophage colony-stimulating factor (M-CSF), interleukin 33 (IL-33), gamma interferon (IFN- ⁇ ), TNF- ⁇ .
  • IL-2 interleukin 2
  • IL-7 interleukin 7
  • IL-14 interleukin 14
  • IL-4 interleukin 4
  • IL-15 interleukin 15
  • IL-21 interleukin 21
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL-17
  • antibodies include, but are not limited to, ⁇ CD-3 antibody, ⁇ CD-4 antibody, ⁇ CD-8 antibody, ⁇ CD-28 antibody, ⁇ CD-40 antibody, ⁇ OX-40 antibody, and ⁇ OX-40L antibody.
  • the co-incubation system may also contain unactivated antigen-presenting cells.
  • the first particle or the second particle can also be loaded with a bacterial lysis component and/or a bacterial outer vesicle lysis component, and the anti-bacterial lysis component and/or bacterial outer vesicle lysis component It is obtained by lysing bacteria or bacterial external vesicles through lysis solution containing lysis agent.
  • the lysis agent is urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerol, protein degrading enzyme, albumin, Aqueous solutions of lecithin, Triton, Tween, amino acids, glycosides, choline, etc.
  • Bacteria include but are not limited to Bacillus Calmette-Guérin, Escherichia coli, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Glactin coli, Lactobacillus reuteri, Lactobacillus rhamnosus, etc.
  • the first particle or the second particle is also loaded with an immune-enhancing adjuvant.
  • the immune-enhancing adjuvant includes but is not limited to pattern recognition receptor agonist, BCG, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl Dipeptides, Mycobacterium phlei, polyantigen, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, Imiquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium parvum vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycysteine Glycolic acid, manganese adjuvant, aluminum adjuvant, calcium adjuvant, cytokine, interleukin
  • the immune-enhancing adjuvant is two or more Toll-like receptor agonists, such as including (1) Poly(I:C) or Poly(ICLC); (2) CpG-ODN, wherein CpG-ODN is At least two of Class A CpG-ODN, Class B CpG-ODN and Class C CpG-ODN, and at least one of them is Class B CpG-ODN or Class C CpG-ODN.
  • Toll-like receptor agonists such as including (1) Poly(I:C) or Poly(ICLC); (2) CpG-ODN, wherein CpG-ODN is At least two of Class A CpG-ODN, Class B CpG-ODN and Class C CpG-ODN, and at least one of them is Class B CpG-ODN or Class C CpG-ODN.
  • type A CpG-ODN is selected from CpG-ODN 2216, CpG-ODN 1585 or CpG-ODN 2336
  • type B CpG-ODN is selected from CpG-ODN 1018, CpG-ODN 2006, CpG-ODN 1826, CpG-ODN 1668 , CpG-ODN 2007, CpG-ODN BW006 or CpG-ODN SL01
  • Class C CpG-ODN is selected from CpG-ODN 2395, CpG-ODN SL03 or CpG-ODN M362.
  • the first particle or the second particle is also loaded with positively charged polypeptides (such as KALA polypeptide, RALA polypeptide, melittin, etc.), arginine, polyarginine, lysine, and polylysine. , histidine, polyhistidine, NH 4 HCO 3 , protamine or histone, etc.
  • positively charged polypeptides such as KALA polypeptide, RALA polypeptide, melittin, etc.
  • arginine such as KALA polypeptide, RALA polypeptide, melittin, etc.
  • arginine such as KALA polypeptide, RALA polypeptide, melittin, etc.
  • arginine such as KALA polypeptide, RALA polypeptide, melittin, etc.
  • arginine such as KALA polypeptide, RALA polypeptide, melittin, etc.
  • arginine such as KALA polypeptide, RALA polypeptide, melittin, etc.
  • the first particle or the second particle is also loaded with a target that actively targets antigen-presenting cells.
  • the target may be mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, or CD11c antibody. , CD103 antibody, CD44 antibody, etc.
  • first particle or the second particle can be prepared from the following materials: organic synthetic polymer materials including but not limited to PLGA, PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acids, synthetic peptides, etc.; natural polymer materials include but are not limited to lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, peptides, etc.; inorganic materials include But it is not limited to ferric oxide, ferric oxide, carbonate, phosphate, etc.
  • the particle size of the first particle or the second particle is nanometer or micron, which can ensure that the particles are engulfed by the antigen-presenting cells.
  • the particle size should be within an appropriate range.
  • the particle size of nanoparticles is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, most preferably, the particle size is 50nm-600nm;
  • the particle size of microparticles is 1 ⁇ m-1000 ⁇ m, more preferably,
  • the particle size is 1 ⁇ m-100 ⁇ m, more preferably, the particle size is 1 ⁇ m-10 ⁇ m, and most preferably, the particle size is 1 ⁇ m-5 ⁇ m.
  • step S2 the activated antigen-presenting cells are subjected to mechanical destruction, membrane filtration or gradient centrifugation to prepare nanovesicles, or the activated antigen-presenting cells are subjected to mechanical destruction, membrane filtration or gradient centrifugation,
  • the product is combined with the second particle to obtain particles wrapped with cell membrane components.
  • the mechanical destruction method is selected from one or more of ultrasonic, homogenization, homogenization, high-speed stirring, high-pressure destruction, high-shear force destruction, swelling, chemical substances, and shrinkage.
  • the co-action mode is selected from one or more of co-incubation, co-extrusion, ultrasound, stirring, dialysis, ultrafiltration, homogenization and homogenization, and the antigen-presenting cell component is formed after co-action with nanoparticles or microparticles. New nanoparticles or microparticles are formed by covering the surface of the original nanoparticles or microparticles.
  • the tumor tissue and/or cancer cell whole cell components are prepared by the following steps: freezing the cancer cells and/or tumor tissue at -20°C to -273°C, adding water or a solution without a dissolving agent, and then repeating After freeze-thaw lysis, the supernatant obtained is a water-soluble component.
  • the soluble part of the precipitate is dissolved in a solution containing a dissolving agent and is converted into a non-water-soluble component.
  • the water-soluble component and the non-water-soluble component are combined.
  • the dissolving agent is selected from the group consisting of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as SDS), glycerin, protein degrading enzyme, albumin, lecithin, inorganic salts (0.1-2000mg/mL), Triton, and methane. At least one of temperature, amino acid, glycoside and choline.
  • the antigen-presenting cells include at least one of B cells, dendritic cells (DC) and macrophages, preferably two or more, and more preferably a combination of three types of cells.
  • B cells B cells
  • DC dendritic cells
  • macrophages preferably two or more, and more preferably a combination of three types of cells.
  • the obtained cancer cell-specific T cells include CD4 + T cells and/or CD8 + T cells, and preferably include both CD4 + T cells and CD8 + T cells.
  • nanoparticles and/or microparticles loaded with cancer cell whole cell antigens are used to specifically activate antigen-presenting cells, and then the antigen-presenting cells are prepared into nanoparticles or microparticles.
  • the prepared nanoparticles or micron particles are The particles are loaded with whole-cell antigen epitopes of cancer cells, and then nanoparticles or microparticles prepared from antigen-presenting cells are used to activate pre-existing activated lymphocytes in peripheral blood, peripheral immune tissue or tumor-infiltrating lymphocytes.
  • Cancer cell-specific T cells and then use the characteristics of activated cancer cell-specific T cells to secrete specific cytokines or highly express certain surface molecules, and use flow cytometry and other methods to isolate cancer cell-specific T cells. After amplification, it is infused back to the patient for use, which can isolate and amplify the most diverse and broad-spectrum cancer cell-specific T cells with the function of recognizing and killing cancer cells.
  • the second object of the present invention is to provide the use of the above-mentioned cancer T cell vaccine in the preparation of drugs for treating or preventing cancer.
  • T cells in peripheral blood, peripheral immune tissue or tumor-infiltrating lymphocytes are derived from autologous or allogeneic sources.
  • the antigen-presenting cells can be derived from the same type as the cancer cell-specific T cells, allogeneic, cell lines, or transformed from stem cells.
  • At least one of the cancer cells or tumor tissue used to prepare the antigen in the first particle or the second particle is the same as the target disease type treated by the above-mentioned drug.
  • the vaccine prepared by the invention is specific.
  • the third object of the present invention is to provide a method for activating cancer cell-specific T cells in vitro, which method includes the following steps:
  • the invention breaks through the limitations of existing activation methods and enables particles to be loaded with all antigens and activated antigen-presenting cell membranes, which can assist in the isolation of a wider spectrum and more diverse cancer cell-specific T cells, and are highly specific and useful in immunotherapy. The effect is better, thus providing more powerful drug candidates for cell therapy.
  • the present invention at least has the following advantages:
  • the present invention provides a technology for isolating cancer cell-specific T cells from immune cells using a nanoscale or micron-scale particle delivery system in vitro to assist in activation.
  • the isolated cancer cell-specific T cells are broad-spectrum and highly specific, including all
  • the cloned cells can specifically recognize and kill cancer cell effector (killer) cancer cell-specific T cells (T eff ).
  • T eff cancer cell effector cancer cell-specific T cells
  • the resulting cells can be used to prevent and treat cancer.
  • the activation process of antigen-presenting cells, the incubation process with T cells, and the loading materials of the first particle and the second particle were optimized to obtain a cancer vaccine with excellent therapeutic and preventive effects.
  • Figure 1 is a schematic diagram of the preparation process and application of the cell system of the present invention
  • a is a schematic diagram of collecting and preparing nanoparticles or microparticles for water-soluble antigens and water-insoluble antigens respectively
  • b is a lysis solution containing a dissolving agent to dissolve cancer cells
  • c is the use of nanoparticles and/or microparticles prepared in a or b to activate antigen-presenting cells, and use the particles prepared by the activated antigen-presenting cells to activate cancer cells
  • the characteristics of activated T cells are used to separate and extract cancer cell-specific T cells, and then the T cells are expanded and used to prevent or treat cancer.
  • Figures 2-14 are respectively the experimental results of mouse tumor growth rate and survival time when using isolated and amplified cancer cell-specific T cells to prevent or treat cancer in Examples 1-13; in Figure 2-14, a represents prevention or treatment The experimental results of tumor growth rate in cancer (n ⁇ 8); b is the experimental results of mouse survival period in preventing or treating cancer (n ⁇ 8), each data point is the mean ⁇ standard error (mean ⁇ SEM); In Figures 3, 4 and 13, c and d are the results of using flow cytometry to analyze the proportion of cancer cell-specific T cells activated by cancer cell whole cell antigens in the corresponding T cells; 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.
  • ⁇ represents the antigen presentation activated by nanoparticles/microparticles loaded with whole cell components of cancer cells and bacterial components lysed and dissolved by Tween 80 Compared with the T cell group prepared by particle-assisted isolation, p ⁇ 0.05, there is a significant difference; ⁇ represents the difference between nanoparticles/microparticles using two types of Class A CpG and Poly ICLC/Poly(I:C) as mixed adjuvants There is a significant difference at p ⁇ 0.05 between the T cell group prepared by activated antigen-presenting cells and nanoparticles/microparticles auxiliary isolation; ⁇ represents the difference between nanoparticles/micron using Class A CpG and Class B CpG as mixed adjuvants.
  • p ⁇ 0.01 there is a significant difference compared to the T cell group prepared by nanoparticles-assisted isolation of antigen-presenting cells activated by antigen-presenting cells; There is a significant difference at p ⁇ 0.01 between the T cell group assisted by nanoparticles prepared from antigen-presenting cells; 000 represents the difference compared with the use of nanoparticles that internally load cancer cell whole cell components but do not load any antigen-presenting cell membrane components on the surface.
  • p ⁇ 0.005 there is a significant difference compared with the T cell group assisted by nanoparticles/microparticles activated DC;
  • ⁇ represents p ⁇ 0.05 compared with the T cell group assisted by nanoparticles prepared from DC prepared by nanoparticles/microparticles activated.
  • Represents p ⁇ 0.005 there is a significant difference compared with the T cell group assisted by nanoparticles/microparticles prepared from antigen-presenting cells activated by nanoparticles/microparticles loaded with antigen without adjuvant
  • Represents p ⁇ 0.01 there is a significant difference compared with the T cell group assisted by nanoparticles/microparticles prepared by antigen-presenting cells activated by antigen-presenting cells loaded with antigen but not loaded with adjuvant
  • ⁇ represents the loaded cancer cells The whole cell component of nanoparticles/microparticles, DC cells and T cells were co-incubated to assist in the isolation of T cells.
  • ⁇ represents the preparation of antigen-presenting cells activated by nanoparticles/microparticles.
  • ⁇ represents the difference between the T cells prepared by activated nanoparticles/microparticles that do not load lysosomal escape substances Compared with the T cell group assisted by nanoparticles/microparticles, there is a significant difference at p ⁇ 0.05; Compared with the T cell group prepared by nanoparticles/micron particles assisted separation, p ⁇ 0.05, there is a significant difference; ⁇ represents nanoparticles or micron particles loaded with membrane cell components on the surface but not loaded with whole cell components of cancer cells inside.
  • ⁇ represents the antigen presentation activated by nanoparticles that are not loaded with adjuvants but loaded with whole cell components of cancer cells on the inside and loaded on the surface.
  • ⁇ represents the antigen presentation activated by nanoparticles/microparticles that only load two types of CpG as adjuvants.
  • ⁇ represents the difference compared with the T cell group prepared by using only antigen-presenting cells activated by nanoparticles/microparticles.
  • p ⁇ 0.05 there is a significant difference.
  • the T cell system for preventing or treating cancer includes cancer cell-specific T cells that have been specifically isolated and amplified from peripheral blood, peripheral immune tissue or tumor infiltrating lymphocytes.
  • cancer cell-specific T cells are first activated by nanoparticles and/or microparticles prepared from activated antigen-presenting cells, and then are isolated using specific molecules that are highly expressed after activation.
  • the cancer cell-specific T cells that are isolated and expanded can be of allogeneic or allogeneic origin.
  • the antigen-presenting cells used to prepare nanoparticles or microparticles are first activated by nanoparticles and/or microparticles loaded with tumor tissue and/or cancer cell whole cell antigens or mixtures thereof.
  • To prepare a T cell system for preventing or treating cancer its preparation process and application fields are shown in Figure 1.
  • cells or tissues When preparing nanoparticles or microparticles that activate antigen-presenting cells, cells or tissues can be lysed, water-soluble antigens and water-insoluble antigens can be collected separately and nano or microparticle systems can be prepared respectively; or dissolution containing a dissolving agent can also be directly used.
  • the liquid directly lyses cells or tissues and dissolves whole cell antigens of cancer cells to prepare nano or micro particle systems.
  • the whole cell antigen of the cancer cell of the present invention can undergo a process including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, protease endolysis or degradation before lysis or (and) after lysis.
  • Nanoparticles or microparticles are directly prepared by endolysis or degradation, or nuclease treatment.
  • tumor tissue cells undergo inactivation or (and) denaturation treatment before lysis.
  • inactivation or (and) denaturation treatment can also be performed after cell lysis, or 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.
  • Treatment methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, protease endolysis or degradation, collagenase treatment, freeze-drying and other treatment methods can also be used in the process.
  • Treatment methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, protease endolysis or degradation, collagenase treatment, freeze-drying and other treatment methods can also be used in the process.
  • Those skilled in the art can understand that during actual application, the skilled person can make appropriate adjustments according to specific circumstances.
  • the antigen-presenting cells When preparing activated antigen-presenting cells into nanoparticles or micron particles, the antigen-presenting cells are first mechanically destroyed, and then centrifuged and/or filtered with a filter membrane of a certain pore size. Optionally, the antigen-presenting cells are combined with the nanoparticles or micron particles. Particles work together.
  • Activated antigen-presenting cells contain a certain cell membrane structure after mechanical destruction.
  • the activated antigen-presenting cells After the activated antigen-presenting cells are mechanically destroyed, they interact with nanoparticles or microparticles to form new nanoparticles or microparticles.
  • the components of the antigen-presenting cells are located in the outer layer of the particles.
  • the assistance of antigen-presenting cells can also be added to the system.
  • the antigen-presenting cells prepared into nanoparticles or microparticles and the antigen-presenting cells used to incubate with T cells can be derived from autologous or allogeneic sources, or from cell lines or stem cells.
  • Antigen-presenting cells can be DC cells, B cells, macrophages, or any mixture of the above three, or other cells with antigen-presenting functions.
  • the system may contain cytokines and/or antibodies to improve activation efficiency.
  • the system can contain cytokines and/or antibodies to improve activation efficiency.
  • nanoparticles or microparticles loaded with cancer cell whole cell antigens are used to first activate antigen-presenting cells, and then the antigen-presenting cells are prepared into nanoparticles or microparticles, and the nanoparticles or microparticles prepared by the antigen-presenting cells are used.
  • the specific preparation method for micron particle-assisted isolation and expansion of cancer cell-specific T cells from peripheral blood, peripheral immune tissue or tumor-infiltrating lymphocytes is as follows:
  • Step 1 Add a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a second predetermined concentration of the raw material for preparing particles.
  • the aqueous solution may contain each component of the cancer cell/tumor tissue lysate and an immune-enhancing adjuvant; each component of the lysate is a water-soluble antigen or is dissolved in a solution containing urea during preparation. Or the original non-water-soluble antigen in the dissolution solution of guanidine hydrochloride and other dissolving agents.
  • the concentration of the water-soluble antigen or the original non-water-soluble antigen contained in the aqueous solution, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 1ng/mL, which can load enough cancer cell whole cell antigens to activate related cells.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the second predetermined concentration of raw materials for preparing particles ranges from 0.5 mg/mL to 5000 mg/mL, preferably 100 mg/mL.
  • the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase.
  • the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 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.
  • the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL ⁇ 100 mg/mL; when the aqueous phase solution is a lysate component/immune adjuvant solution, wherein The concentration of protein and polypeptide is greater than 1ng/mL, preferably 1mg/mL ⁇ 100mg/mL, and the concentration of immune adjuvant is greater than 0.01ng/mL, preferably 0.01mg/mL ⁇ 20mg/mL.
  • the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane;
  • the concentration of the organic phase is 0.5 mg/mL ⁇ 5000mg/mL, preferably 100mg/mL.
  • Step 2 subject the mixed solution obtained in Step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute.
  • the stirring is mechanical stirring or magnetic stirring
  • the stirring speed is greater than 50 rpm
  • the stirring time is greater than 1 minute.
  • the stirring speed is 50 rpm to 1500 rpm
  • the stirring time is 0.1 hour to 24 hours
  • the ultrasonic power is greater than 5W
  • the time Greater than 0.1 seconds such as 2 to 200 seconds
  • the pressure is greater than 5 psi, such as 20 psi to 100 psi.
  • the rotation speed of the shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; the flow rate of microfluidic processing is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used for nanonization and/or micronization.
  • the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared micro-nano particles. Too large or too small will cause to changes in particle size.
  • Step 3 Add the mixture obtained after step 2 to a third predetermined volume of aqueous solution containing a third predetermined concentration of emulsifier and perform ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or perform homogenization or microfluidic treatment. deal with.
  • the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be ultrasonically or stirred to form nanometers or micrometers.
  • 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 50rpm, and the stirring time is greater than 1 minute.
  • the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.5 to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W to 500W.
  • the time is greater than 0.1 seconds, such as 2 to 200 seconds; when homogenizing, use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer.
  • the pressure is greater than 20 psi, such as 20 psi to 100 psi.
  • the rotation speed is greater than 1000rpm, such as 1000rpm to 5000rpm; when using microfluidic processing, the flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used to nanonize or micronize the particles.
  • the length of ultrasonic time or stirring speed or homogenization process pressure and time can control the size of the prepared nano or micron particles. Too large or too small will cause Changes in particle size.
  • the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined volume is 5 mL
  • the third predetermined concentration is 20 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5.
  • the ratio of the second predetermined volume and the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles or microparticles of suitable size.
  • Step 4 Add the liquid obtained after the treatment in Step 3 to a fourth predetermined volume of the emulsifier aqueous solution with a fourth predetermined concentration, and stir until the predetermined stirring conditions are met.
  • the emulsifier aqueous solution is PVA solution or other solutions.
  • the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size.
  • the selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume.
  • the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10.
  • the ratio of the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.
  • the predetermined stirring condition of this step is until the volatilization of the organic solvent is completed, that is, the volatilization of methylene chloride in step 1 is completed.
  • Step 5 After centrifuging the mixed liquid that meets the predetermined stirring conditions in Step 4 at a rotation speed of greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in a fifth predetermined volume of Five predetermined concentrations of an aqueous solution containing a lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).
  • Step 6 After freeze-drying the suspension containing the lyoprotectant obtained in Step 5, the freeze-dried material is used for later use.
  • Step 7 Resuspend a sixth predetermined volume of the nanoparticle-containing suspension obtained in Step 5 in PBS (or physiological saline) or use a sixth predetermined volume of PBS (or physiological saline) to resuspend the nanoparticle-containing suspension obtained in Step 6
  • PBS or physiological saline
  • the freeze-dried substance containing nanoparticles or microparticles and a lyoprotectant is used directly; or the above sample is mixed with a seventh predetermined volume of water-soluble antigen or the dissolved original non-water-soluble antigen and used.
  • the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferred volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1 .
  • Step 8 Incubate the antigen-presenting cells with the nanoparticles and/or microparticles prepared above for a certain period of time.
  • the tumor tissue and/or cancer cells and antigen-presenting cells used to prepare nanoparticles and/or microparticles can be from autologous or allogeneic sources.
  • Step 9 Collect the co-incubated cells and perform mechanical damage such as sonication, homogenization, and mechanical stirring.
  • Step 10 Centrifuge the ultrasonicated sample and/or filter it with a filter membrane of a certain pore size, and/or interact with nanoparticles and/or microparticles loaded with whole cell components of cancer cells to prepare a sample based on antigen-presenting cells. Nanoparticles or microparticles.
  • Step 11 Obtain peripheral blood, peripheral immune tissue or tumor tissue, and collect T cells or immune cells containing T cells in the above tissues.
  • the above peripheral blood, peripheral immune tissue or tumor tissue can be from autologous or allogeneic.
  • Step 12 Mix the nanoparticles and/or microparticles prepared in step 10 with the immune cells containing T cells obtained in step 11 and incubate them together for a certain period of time.
  • Step 13 Use flow cytometry, magnetic bead sorting, etc. to isolate T cells activated by the antigen.
  • step 14 the isolated T cells activated by the cancer cell whole cell antigen are expanded in vitro.
  • Step 15 Inject the expanded cancer cell-specific T cells back into the patient's body to prevent or treat cancer.
  • Example 1 Isolation and expansion of cancer cell-specific T cells for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticles prepared by nanoparticle-activated antigen-presenting cells to assist in the isolation and expansion of cancer cell-specific T cells from tumor-infiltrating lymphocytes for the prevention of melanoma.
  • B16F10 melanoma tumor tissue was lysed to prepare water-soluble antigen and water-insoluble antigen of the tumor tissue.
  • the organic polymer material PLGA was used as the nanoparticle skeleton material, and Polyinosinic-polycytidylic acid (poly(I:C )) is an immune adjuvant that uses a solvent evaporation method to prepare a nanoparticle system loaded with water-soluble antigens and non-water-soluble antigens of tumor tissue.
  • the nanoparticles are then used to activate antigen-presenting cells, and the antigen-presenting cells are mechanically destroyed and centrifuged to prepare the system.
  • Nanoparticles are used to assist in the isolation of cancer cell-specific T cells from tumor-infiltrating lymphocytes, and after amplification, they are injected into the body to prevent melanoma.
  • the nanoparticle 1 is prepared by the double emulsion method in the solvent evaporation method. During preparation, nanoparticles loaded with water-soluble antigens in whole cell antigens of cancer cells and nanoparticles loaded with non-water-soluble antigens in whole cell antigens of cancer cells are prepared separately, and then used together.
  • 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 only distributed inside 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, and then 100 mg of nanoparticles are centrifuged at 10,000 g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose is used. Resuspend and freeze-dry for 48 hours.
  • the average particle size of the nanoparticles 1 is about 280nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components.
  • the poly(I:C) immune adjuvant used per 1 mg of PLGA nanoparticles is 0.02 mg.
  • This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare BMDC.
  • 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.
  • 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.
  • RPMI 1640 (10% FBS) medium to stop lysis, centrifuge at 400 g for 3 min, and discard the supernatant.
  • the cells were placed in a 10 mm culture dish and cultured in RPMI 1640 (10% FBS) medium with recombinant mouse GM-CSF (20 ng/mL) added at 37 degrees Celsius and 5% CO 2 for 7 days. On the third day, shake the culture bottle gently and add the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20ng/mL).
  • Nanoparticles loaded with whole cell components of cancer cells derived from tumor tissue 250 ⁇ g nanoparticles loaded with water-soluble components + 250 ⁇ g nanoparticles loaded with non-water-soluble components were completely cultured with BMDC (10 million) in 15 mL RPMI1640.
  • the base was incubated for a total of 48 hours (37°C, 5% CO 2 ); the incubation system contained cytokine combination 1: granulocyte-macrophage colony-stimulating factor (GM-CSF, 500U/mL), IL-2 (500U/mL) ), IL-7 (500U/mL), IL-12 (500U/mL) or containing cytokine component 2: GM-CSF (500U/mL), IL-4 (500U/mL), tumor necrosis factor ⁇ ( TNF- ⁇ , 500U/mL), IL10 (500U/mL).
  • cytokine combination 1 granulocyte-macrophage colony-stimulating factor (GM-CSF, 500U/mL), IL-2 (500U/mL) ), IL-7 (500U/mL), IL-12 (500U/mL) or containing cytokine component 2: GM-CSF (500U/mL), IL-4 (
  • step (3) collect the DCs (10 million) prepared in step (3) without any nanoparticle or microparticle activation, then wash the cells twice with physiological saline, resuspend the cells in physiological saline and incubate at 4°C and 7.5W. Sonicate for 20 minutes to disrupt cells and prepare samples containing cell membrane components. The sample was then centrifuged at 2000g for 20 minutes and the supernatant was collected. The supernatant was centrifuged at 7000g for 20 minutes and the supernatant was collected. The supernatant was mixed with 40 mg of nanoparticles loaded with cancer cell whole cell components prepared in step (2).
  • Particle 1 (20 mg of nanoparticles loaded with water-soluble components + 20 mg of nanoparticles loaded with non-water-soluble components) was incubated for 10 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane. The extrudate was centrifuged at 15,000g for 120 minutes. Collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain nanoparticle 3 with a particle size of 300 nm.
  • the incubated DCs (10 million) with the addition of cytokine component 2 by centrifugation at 400 g for 5 minutes, then wash the cells twice with physiological saline, resuspend the cells in physiological saline, and incubate at 4°C and 7.5W. Sonicate for 20 minutes to disrupt the cells and prepare samples containing cell membrane components. The sample was then centrifuged at 2000g for 20 minutes and the supernatant was collected. The supernatant was centrifuged at 7000g for 20 minutes and the supernatant was collected. The supernatant was mixed with 40 mg of nanoparticles loaded with cancer cell whole cell components prepared in step (2).
  • Particle 1 (20 mg of nanoparticles loaded with water-soluble components + 20 mg of nanoparticles loaded with non-water-soluble components) was incubated for 10 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane. The extrudate was centrifuged at 15,000g for 120 minutes. Collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain nanoparticles. Among them, nanoparticles 4 were obtained by incubating the nanoparticles 1 with the membrane components, with a particle size of 300 nm.
  • Particle 1 (20 mg of nanoparticles loaded with water-soluble components + 20 mg of nanoparticles loaded with non-water-soluble components) was incubated for 10 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter membrane. The extrudate was centrifuged at 15,000g for 120 minutes. Collect and discard the supernatant, collect the precipitate, and resuspend the precipitate in PBS to obtain nanoparticle 5 with a particle size of 300 nm.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the mice were sacrificed and the mouse tumor tissue and spleen cells were harvested.
  • the mouse tumor tissue was cut into small pieces and digested with collagenase for 15 minutes, then a single cell suspension was prepared through a cell mesh, centrifuged and washed with PBS, and then flow cytometry was used to separate viable cells from the tumor tissue single cell suspension.
  • CD3 + T cells are labeled with live-dead cell dye to remove dead cells).
  • Nanoparticle 2 (100 ⁇ g) or nanoparticle 3 (100 ⁇ g) or nanoparticle 4 (100 ⁇ g) or nanoparticle 5 (100 ⁇ g) and T cells (500,000) derived from tumor-infiltrating lymphocytes were coagulated in 3 mL of RPMI 1649 complete medium. Incubate for 96 hours (37°C, 5% CO2 ). Then flow cytometry is used to sort the CD3 + CD8 + CD69 + T cells in the incubated cells, which are cancer cell-specific CD8 + T cells.
  • the cancer cell-specific T cells obtained by the above sorting were combined with IL-2 (2000U/mL), IL-7 (2000U/mL), IL-12 (200U/mL), IL-15 (200U/mL) and ⁇ CD -3 antibody (10ng/mL) was incubated for 10 days (the medium was changed every two days) to amplify and sort to obtain cancer cell-specific T cells, which are T cell vaccines.
  • the cancer cell-specific T cell vaccine obtained by assisted sorting and amplification of nanoparticles 2 is T cell vaccine 1
  • the cancer cell-specific T cell vaccine obtained by assisted sorting and amplification of nanoparticles 3 is T cell vaccine.
  • the cancer cell-specific T cell vaccine obtained by assisted sorting and amplification of nanoparticles 4 is T cell vaccine 3
  • the cancer cell-specific T cell vaccine obtained by assisted sorting and amplification of nanoparticles 5 is T cell vaccine 4.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma tumor-bearing mice.
  • the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate the recipient mice. immune cells in mice.
  • 4 million cancer cell-specific T cells (T cell vaccine 1, or T cell vaccine 2, or T cell vaccine 3, or T cell vaccine 4) prepared in step (6) or 100 ⁇ L of PBS were intravenously injected into the subject. body mice.
  • each recipient mouse was inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 cells on the lower right side of the back. Monitor mouse tumor growth rate and mouse survival time.
  • T cell vaccine 4 has the best effect, and T cell vaccine 4 is more effective than T cell vaccine 1, T cell vaccine 2 and T cell vaccine 3. This shows that the effect of adding cytokine combination 1 during the activation of antigen-presenting cells is better than that of cytokine component 2; the surface of the nanoparticles that assist in the isolation of T cells is loaded with antigen-presenting cell membrane components, and the inside is a solid nanoparticle loaded with cell components.
  • the effect of particles is better than that of nanovesicle structures that only load membrane components on the surface; the effect of nanoparticles prepared by antigen-presenting cells activated by nanoparticles loaded with whole cell components of cancer cells is much better than that of unactivated antigen-presenting cells.
  • the cancer cell-specific T cells of the present invention have a good preventive effect on melanoma.
  • Antigen-presenting cells activated by nanoparticles loaded with cancer cell whole cell components will degrade and present the whole cell antigens in the cancer cell whole cell components loaded with the engulfed nanoparticles, which will be presented to the cell membrane surface by the antigen-presenting cells.
  • Cancer cell epitopes have bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the cell membrane components of the antigen-presenting cells contain antigen epitopes that bind to MHC.
  • the cell membrane components in the above antigen-presenting cells will form nanoparticles or microparticles, and be loaded with MHC molecules and degraded. It can directly activate cancer cell-specific T cells without the assistance of antigen-presenting cells.
  • Example 2 Isolation and expansion of cancer cell-specific T cells for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticle-activated antigen-presenting cells to prepare nanoparticles to assist in the isolation and expansion of cancer cell-specific T cells for the prevention of melanoma.
  • B16F10 melanoma tumor tissue was lysed to prepare water-soluble antigen and water-insoluble antigen of the tumor tissue.
  • PLGA was used as the nanoparticle framework material
  • poly(I:C) and CpG1018 were used as immune adjuvants and solvents were used.
  • the volatilization method is used to prepare a nanoparticle system loaded with water-soluble antigens and non-water-soluble antigens of tumor tissue, and then the nanoparticles are used to activate antigen-presenting cells, and the activated antigen-presenting cells are prepared into nanoparticles to assist in the isolation of tumor-infiltrating lymphocytes.
  • the cancer cell-specific T cells in the tumor are expanded and injected into the body to prevent melanoma.
  • the nanoparticles and the blank nanoparticles and peptide nanoparticles used as controls were prepared by the solvent evaporation method.
  • the molecular weight of PLGA, the preparation material for nanoparticles 1 loaded with whole cell components, is 7Da-17KDa.
  • the immune adjuvants used are poly(I:C) and CpG1018, and the adjuvants are contained inside the nanoparticles.
  • the preparation method is as described above. During the preparation process, the double emulsion method is first used to load the antigen and adjuvant inside the nanoparticles.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein and peptide components, and each 1 mg of PLGA nanoparticles uses 0.02 mg of poly(I:C) and CpG1018 immune adjuvants.
  • polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were loaded with equal mass.
  • Peptide nanoparticle 2 is used as a control nanoparticle. Its preparation materials and preparation methods are the same as nanoparticle 1.
  • the particle size of control nanoparticle 2 is about 280 nm, and it is loaded with 100 ⁇ g of peptide component and an equal amount of adjuvant.
  • the preparation materials and methods of blank nanoparticle 3 are the same as those of nanoparticle 1.
  • the particle size is about 280 nm. It only carries the same amount of immune adjuvant but does not load any antigen component.
  • Bone marrow-derived dendritic cells (BMDC) and B cells were used as antigen-presenting cells.
  • the preparation of BMDC is the same as in Example 1.
  • the B cell extraction process is as follows: kill the mouse and remove the mouse spleen, then prepare a mouse splenocyte single cell suspension, and then use magnetic bead sorting to sort CD19 + B cells from the splenocyte single cell suspension. Mix BMDC and B cells at a ratio of 1:1 and use them as mixed antigen-presenting cells.
  • nanoparticle 1 500 ⁇ g or peptide nanoparticle 2 (500 ⁇ g) or blank nanoparticle 3 (500 ⁇ g) + free lysate with 20 million mixed antigen-presenting cells (10 million BMDC + 10 million B cells) in 15 mL Incubate in RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contains a combination of cytokines: IL-15 (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL) mL), IL-12 (1000U/mL).
  • IL-15 500U/mL
  • IL-2 500U/mL
  • IL-7 500U/mL
  • IL-12 1000U/mL
  • nanoparticle 1 and 20 million BMDCs were incubated in 15mL RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ); the incubation system contained a combination of cytokines: IL-15 (500U/mL), IL-2 ( 500U/mL), IL-7 (500U/mL), IL-12 (1000U/mL).
  • IL-15 500U/mL
  • IL-2 500U/mL
  • IL-7 500U/mL
  • IL-12 1000U/mL
  • Particle 1 50 mg or peptide nanoparticle 2 (50 mg) or blank nanoparticle 3 (50 mg) was incubated for 10 minutes, and then repeatedly co-extruded using a 0.45 ⁇ m filter. The extruded liquid was centrifuged at 15000g for 60 minutes and discarded. After removing the supernatant, resuspend in physiological saline and the resulting precipitate is nanoparticles.
  • the nanoparticles prepared by using the mixed antigen-presenting cell membrane components activated by blank nanoparticles 3 and the blank nanoparticles 3 are nanoparticles 4, with a particle size of 300 nm;
  • the mixed antigen-presenting cell membrane components activated by peptide nanoparticles 2 are
  • the nanoparticles prepared by the interaction between the cell membrane components and the polypeptide nanoparticles 2 are nanoparticles 5 with a particle size of 300 nm;
  • the obtained nanoparticles were nanoparticle 6, with a particle size of 300 nm.
  • step (2) Collect the filtrate and incubate it with the nanoparticle 1 (50 mg) prepared in step (2) for 10 minutes, and then Repeated co-extrusion using a 0.45 ⁇ m filter membrane, centrifuge the extrudate at 15,000 g for 60 minutes, discard the supernatant and resuspend in physiological saline, and the resulting precipitate is Nanoparticle 7, with a particle size of 300 nm.
  • mice On day 0, 5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse. On days 7, 14, 21 and 28, the mice were subcutaneously injected with 100 ⁇ L of cancer cell-loaded serum. Cellular component of 1 mg PLGA nanoparticles. The mice were sacrificed on day 32, and the spleen and tumor tissues of the mice were collected. The mouse tumor tissue was cut into small pieces and passed through a cell sieve to prepare a single cell suspension. After centrifugation and washing with PBS, flow cytometry was used to separate living cells from the single cell suspension of the tumor tissue (live and dead cell dyes were used to mark dead cells). cells to remove dead cells) CD3 + T cells.
  • a splenocyte single cell suspension was prepared by passing the mouse spleen through a cell screen and lysing red blood cells, and flow cytometry was used to sort live cells from the splenocyte single cell suspension (live and dead cell dyes were used to mark dead cells). cells to remove dead cells) CD19 + B cells.
  • T cells (400,000) were incubated in 5 mL RPMI1640 complete medium for a total of 48 hours (37°C, 5% CO 2 ), and then flow cytometry was used to sort the incubated CD3 + CD134 + T cells, which were cancerous Cancer cell-specific T cells activated by whole cell antigens; the cancer cell-specific T cells obtained using nanoparticles 4 to assist sorting are T cell vaccine 1; the cancer cell-specific T cells obtained using nanoparticles 5 to assist sorting The cells are T cell vaccine 2; the cancer cell-specific T cells obtained using nanoparticles 6 to assist sorting are T cell vaccine 3; the cancer cell-specific T cells obtained using nanoparticles 7 to assist sorting are T cell vaccine 4 .
  • step (5) Or incubate 100 ⁇ g of the antigen-presenting cell-based nanoparticles 6 prepared in step (5) with T cells (400,000) from tumor-infiltrating lymphocytes in 5 mL of RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ), and then use flow cytometry to sort the incubated CD3 + CD134 + T cells, which are cancer cell-specific T cells activated by cancer cell whole cell antigens and are T cell vaccines 5.
  • anti-mouse CD3 antibody, anti-mouse CD4 antibody, anti-mouse CD8 antibody and anti-mouse CD134 antibody were used to label the incubated T cells, and then flow cytometry was used to analyze the interaction between different nanoparticles and T cells.
  • the cancer cell whole cell antigens loaded by the nanoparticles can be degraded into antigen epitopes after being phagocytosed by the antigen presenting cells and presented to the surface of the antigen presenting cells. Specific T cells that can recognize the cancer cell whole cell antigens can recognize them.
  • Cancer cells are activated after whole-cell antigen epitopes and express specific surface markers.
  • T cells that are activated and can be sorted can be identified. and the number of cancer cell-specific T cells with killing efficacy.
  • the T cell vaccine obtained by the above sorting was incubated with IL-2 (2000U/mL) and ⁇ CD-3 antibody (20ng/mL) respectively for 14 days (the medium was changed every two days) to amplify the sorted cancer cells.
  • Specific T cell vaccines were incubated with IL-2 (2000U/mL) and ⁇ CD-3 antibody (20ng/mL) respectively for 14 days (the medium was changed every two days) to amplify the sorted cancer cells.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare melanoma tumor-bearing mice.
  • the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate the recipient mice. immune cells in mice.
  • 1 million cancer cell specific T cell vaccines (T cell vaccine 1, or T cell vaccine 2, or T cell vaccine 3, or T cell vaccine 4, or T cell vaccine 5) prepared in step (6) Or 100 ⁇ L PBS was injected intravenously into recipient mice.
  • each recipient mouse was inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 cells on the lower right side of the back. Monitor mouse tumor growth rate and mouse survival time.
  • T cell vaccine 3 and T cell vaccine 5 are the most effective. T cell vaccine 3 and T cell vaccine 5 are both significantly better than T cell vaccine 2 and T cell vaccine 4; and T cell vaccine 3 and T cell vaccine 5 are equally effective.
  • T cell vaccine 5 shows that the method of assisting the isolation of broad-spectrum cancer cell-specific T cells according to the present invention does not rely on antigen-presenting cells.
  • the same effect as adding antigen-presenting cells to assist in isolating cancer cell-specific T cells can be obtained without adding antigen-presenting cells, which is also the purpose of the present invention.
  • One advantage of nanoparticles or microparticles prepared from activated antigen-presenting cells is to assist in the sorting and expansion of cancer cell-specific T cells.
  • Nanoparticles loaded with whole-cell antigens of cancer cells activated by antigen-presenting cells are more effective in preventing cancer than nanoparticles loaded with four antigen peptides activated by antigen-presenting cells.
  • Cell-prepared nanoparticles assist in the isolation and expansion of cancer cell-specific T cells. This shows that the types of cancer cell-specific T cells prepared by nanoparticle-assisted isolation of antigen-presenting cells activated by nanoparticles loaded with four neoantigen peptides are limited, so the expanded T cell system contains a small number of T cell clones. , fewer cancer cells can be identified and killed.
  • Nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with cancer cell whole cell antigens can assist in the isolation of a wider spectrum of cancer cell-specific T cells, so the number of T cell clones that can be obtained after expansion is wider. The higher the spectrum, the more cancer cells can be identified and killed, and the better the effect of treating or preventing cancer.
  • Nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole cell components can activate CD8 +
  • the proportions of CD134 + T cells and CD4 + CD134 + T cells in CD8 + T cells and CD4 + T cells were significantly higher than those in the control group. It can be seen that the nanoparticles prepared by the antigen-presenting cells activated by the nanoparticles loaded with whole cell components according to the present invention can better assist in the isolation of cancer cell-specific T cells with the ability to recognize and kill cancer cells.
  • Example 3 Sorting and amplifying cancer cell-specific T cells for use in the treatment of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to use nanoparticles prepared by nanoparticle-activated antigen-presenting cells to assist in the isolation and expansion of cancer cell-specific T cells in mouse peripheral immune cells and then use them to treat melanoma.
  • B16F10 melanoma tumor tissue and cancer cells were first lysed to prepare a water-soluble antigen mixture (mass ratio 1:1) and a water-insoluble antigen mixture (mass ratio 1:1) of tumor tissue and cancer cells, and then The water-soluble antigen mixture and the water-insoluble antigen mixture are mixed at a mass ratio of 1:1.
  • PLGA was used as the nanoparticle skeleton material
  • Poly(I:C) and CpG2006 were used as adjuvants to prepare nanoparticles loaded with lysate components, and then the nanoparticles were incubated with antigen-presenting cells for a period of time to activate antigen presentation. cells, and prepare antigen-presenting cells into nanoparticles to assist in isolating and activating cancer cell-specific T cells, and amplify them for the treatment of melanoma.
  • 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 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.
  • centrifuge Bifidobacterium longum at 5000g for 30 minutes, then discard the precipitate and collect the supernatant. Filter the supernatant with a 1 ⁇ m filter, ultrasonicate at 4°C for 5 minutes at 20W, and then centrifuge at 16000g for 90 minutes. , resuspend the pellet in PBS to form the collected bacterial outer vesicle membrane components, and then use Tween 80 aqueous solution to lyse and dissolve the bacterial membrane components.
  • nanoparticle 1 is prepared by the double emulsion method.
  • the molecular weight of the preparation material PLGA is 7KDa-17KDa.
  • the immune adjuvants used are poly(I:C) and CpG2006 and the adjuvants are encapsulated 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. Then 100 mg of nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of ultrapure solution containing 4% trehalose is used. Resuspend in water and freeze-dry for 48 hours before use.
  • the average particle size of nanoparticles 1 is about 250nm.
  • Each 1 mg of PLGA nanoparticles 1 is loaded with approximately 130 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA nanoparticles 1 carries 0.02 mg of poly(I:C) and CpG2006 immune adjuvant.
  • the preparation materials and preparation method of nanoparticle 2 are the same as nanoparticle 1.
  • the nanoparticle 2 is internally loaded with the antigen component prepared in step (1) and the 8M urea-dissolved bacterial outer vesicle membrane component prepared in step (2) at the same time, and the mass ratio of the two is 1:1.
  • the immune adjuvants used were poly(I:C) and CpG2006, and the adjuvants were encapsulated in nanoparticles.
  • the double emulsion method was first used to load the tumor tissue inside the nanoparticles with lysate components, bacterial external vesicle components and adjuvants.
  • nanoparticles were centrifuged at 10,000g for 20 minutes, and 10 mL containing 4% trehalose was used. Resuspend in ultrapure water and freeze-dry for 48 hours before use.
  • the average particle size of nanoparticles 2 is about 250nm.
  • Each 1 mg of PLGA nanoparticles 2 is loaded with approximately 130 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA nanoparticles 2 carries 0.02 mg of poly(I:C) and CpG2006 immune adjuvant each.
  • the preparation materials and preparation method of nanoparticle 3 are the same as nanoparticle 1.
  • the nanoparticle 3 is simultaneously loaded with the antigen component prepared in step (1) and the bacterial outer vesicle membrane component dissolved in Tween 80 prepared in step (2), and the mass ratio of the two is 1:1.
  • the immune adjuvants used were poly(I:C) and CpG2006, and the adjuvants were encapsulated in nanoparticles.
  • the double emulsion method was first used to load the tumor tissue inside the nanoparticles with lysate components, bacterial external vesicle components and adjuvants.
  • nanoparticles 3 100 mg were centrifuged at 10,000g for 20 minutes, and 10 mL containing 4% trehalose was used. Resuspend in ultrapure water and freeze-dry for 48 hours before use.
  • the average particle size of nanoparticles 3 is about 250nm.
  • Each 1 mg of PLGA nanoparticles 3 is loaded with approximately 130 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA nanoparticles 3 is loaded with 0.02 mg of poly(I:C) and CpG2006 immune adjuvant.
  • blank nanoparticle 4 The preparation materials and methods of blank nanoparticle 4 are the same as nanoparticle 1, but blank nanoparticle 4 only carries the same amount of adjuvant and does not load any tumor tissue lysate components.
  • the particle size of the nanoparticles 4 is approximately 250 nm.
  • mice After the C57BL/6 mice were sacrificed, the spleens of the mice were removed, a single cell suspension of mouse splenocytes was prepared, and CD19 + B cells in the splenocytes were isolated using magnetic bead sorting.
  • 500 ⁇ g of nanoparticle 1, or 500 ⁇ g of nanoparticle 2, or 500 ⁇ g of nanoparticle 3, or 500 ⁇ g of nanoparticle 4 were incubated with B cells (10 million cells) in 15 mL of RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ), the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), albumin (50ng /mL) and CD80 antibody (10ng/mL).
  • the incubated B cells were collected by centrifugation at 400g for 5 minutes, then washed with PBS three times, resuspended in PBS water and sonicated at low power (10W) for 15 minutes. Then centrifuge the sample at 500g for 5 minutes and collect the supernatant. The supernatant is filtered through membranes with pore sizes of 30um, 10um, 5um, 0.45um, and 0.22um in sequence. The resulting filtrate sample is centrifuged at 18000g for 60 minutes and then discarded. The supernatant and the pellet were resuspended in PBS to obtain nanoparticles.
  • the nanoparticles prepared by using antigen-presenting cells activated by nanoparticle 1 are nanoparticles 5, with a particle size of 110 nanometers;
  • the nanoparticles prepared by using antigen-presenting cells activated by nanoparticles 2 are nanoparticles 6, with a particle size of 110 nanometers.
  • the nanoparticles prepared using antigen-presenting cells activated by nanoparticles 3 are nanoparticles 7, with a particle size of 110 nanometers;
  • the nanoparticles prepared using antigen-presenting cells activated with nanoparticles 4 are nanoparticles 8, with a particle size of 110 nanometers nanometer.
  • mice Inoculate 5 ⁇ 10 5 B16F10 cells subcutaneously on the back of each C57BL/6 mouse on day 0, and subcutaneously inject 0.5 mg of PLGA prepared in step (3) into the mice on day 10, 17 and 24 respectively. Nanoparticles1.
  • the mice were sacrificed on day 31, the mouse spleens were removed and mouse spleen single cell suspension was prepared, and magnetic bead sorting method was used to sort the live cells in the spleen cells (use live-dead cell dye to mark dead cells to remove dead cells) of CD45 + CD3 + T cells.
  • step (6) Put the isolated T cells (4 million) and 100 ⁇ g of nanoparticles prepared in step (6) (nanoparticle 5, or nanoparticle 6, or nanoparticle 7, or nanoparticle 8) in 40 mL of high-glucose DMEM complete medium
  • the CCP was incubated for 72 hours (37°C, 5% CO 2 ), and then flow cytometry was used to sort the incubated CD3 + CD134 + T cells, which are cancer cell-specific T cells specifically activated by cancer cell whole cell antigens. .
  • the cancer cell-specific T cells obtained by using nanoparticles 5 to assist sorting are T cell vaccine 1; the cancer cell-specific T cells obtained using nanoparticles 6 to assist sorting are T cell vaccines 2; using nanoparticles 7 to assist sorting
  • the selected cancer cell-specific T cells are T cell vaccine 3; the cancer cell-specific T cells obtained using nanoparticles 8-assisted sorting are T cell vaccine 4.
  • the T cells in the splenocytes without nanoparticle-assisted sorting were incubated with different nanoparticles prepared in step (6) in DMEM high-glucose complete medium for 48 hours, and then the incubated cells were collected and used with The IFN- ⁇ antibody of the fluorescent probe labels the incubated T cells, and then flow cytometry is used to analyze the proportion of IFN- ⁇ + T cells in the T cells.
  • the cancer cell whole cell antigens loaded by the nanoparticles can be degraded into antigen epitopes after being phagocytosed by the antigen-presenting cells and presented to the surface of the antigen-presenting cell membrane.
  • the nanoparticles prepared by the antigen-presenting cells are loaded with the above-mentioned degraded and presented products.
  • the antigenic epitope can be recognized by cancer cell-specific T cells and activate cancer cell-specific T cells. After activation, they secrete killer cytokines.
  • IFN- ⁇ is the most important cytokine secreted by antigen-specific T cells that are activated after recognizing antigens.
  • CD3 + IFN- ⁇ + T cells analyzed using flow cytometry are cancer cell-specific T cells that can recognize and kill cancer cells.
  • the cancer cell-specific T cell vaccines obtained by the above sorting were incubated with IL-2 (2000U/mL) and IL-7 (2000U/mL) in DMEM high-glucose complete medium for 7 days (37°C, 5% CO 2 , change the medium every two days) to amplify the cancer cell-specific T cell vaccine obtained by sorting.
  • the nanoparticles prepared by PBS control group and blank nanoparticle-activated antigen presenting cells assisted in the isolation and expansion of T cell vaccine 4.
  • the mice in the control group had very fast tumor growth and a very short survival period. short.
  • the tumor growth rate of mice treated with T cell vaccine 1, T cell vaccine 2 and T cell vaccine 3 was significantly slower, and some mice had tumors disappearing and cured.
  • the effect of T cell vaccine 2 is better than that of T cell vaccine 1 and T cell vaccine 3, which shows that the antigen presenting cells activated after loading the bacterial outer vesicle components lysed and dissolved into nanoparticles using appropriate methods are beneficial to the auxiliary Isolation of cancer cell-specific T cells.
  • the cell system of the present invention has excellent therapeutic effect on cancer.
  • the T cell vaccine activates cancer cell-specific T cells in vitro.
  • the proportion of cancer cell-specific T cells that can be activated when using different particles to assist sorting is the same as in Figures a and b. related to the efficacy.
  • the proportion of activated cancer cell-specific T cells sorted using CD134 as a surface marker of activation is consistent with the proportion of T cells that can release the killer cytokine IFN- ⁇ upon encountering cancer cell antigens, indicating that , T cells sorted using CD134 as an activated surface marker are cancer cell-specific T cells that can specifically recognize and kill cancer cells. It can be seen that the separation method of the present invention can effectively sort cancer cell-specific T cells in tumor tissues that have the ability to recognize and kill cancer cells.
  • Example 4 Nanoparticle-assisted isolation and expansion of cancer cell-specific T cells for cancer prevention
  • 6M guanidine hydrochloride was first used to cleave the whole cell antigen of B16F10 melanoma cancer cells. Then, a micron particle system loaded with cancer cell whole cell antigens was prepared using PLGA as the micron particle skeleton material and CpG BW006 (Class B), CPG2216 (Class A) and Poly ICLC as immune adjuvants. After using micron particles to activate antigen-presenting cells, the antigen-presenting cells are prepared into nanoparticles to assist in the isolation of cancer cell-specific T cells and amplified for cancer prevention.
  • the cultured B16F10 melanoma cancer cell line was collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, and then the cancer cells were resuspended and lysed with 6M guanidine hydrochloride.
  • the whole cell antigen of the cancer cells was lysed and dissolved in 6M Guanidine hydrochloride is the source of antigen raw materials for preparing micron particle systems.
  • the micron particles are prepared by the double emulsion method.
  • the molecular weight of PLGA, the material used to prepare micron particles 1, is 38KDa-54KDa, and the immune adjuvants used are CpG BW006, CPG2216 and Poly ICLC.
  • Poly ICLC is a toll-like receptor 3 agonist, while various CpGs are Toll-like receptor 9 agonists, and both Toll-like receptor 3 and Toll-like receptor 9 are located in the endosome membrane structure within the cell.
  • the lysate components and immune adjuvant were loaded into the micron particles, and then centrifuged at 10,000g for 15 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 hours; before use, the particles were Resuspend in 7 mL of PBS and then add 3 mL of cancer cell lysate component (protein concentration 50 mg/mL) and incubate at room temperature for 10 min to obtain micron particles 1 loaded with lysate both inside and outside.
  • cancer cell lysate component protein concentration 50 mg/mL
  • the average particle size of the microparticles is about 2.50 ⁇ m, and the surface potential is about -2mV; each 1 mg of PLGA microparticles is loaded with approximately 140 ⁇ g of protein or peptide components, and the loaded CpG BW006 (Class B), CPG2216 (Class A) and Poly ICLC 0.02mg each.
  • control microparticle 2 The preparation materials and preparation methods of control microparticle 2 are the same, and the loaded immune adjuvants are CpG2336 (Class A), CPG2216 (Class A) and Poly ICLC.
  • the diameter of the control micron particles 2 is about 2.50 ⁇ m, and the surface potential is about -2mV.
  • Each 1 mg PLGA micron particle is loaded with approximately 140 ⁇ g of protein or peptide components.
  • Each 1 mg PLGA micron particle is loaded with CpG2336 (Class A) and CPG2216 (Class A). and Poly ICLC immune adjuvant are 0.02mg each.
  • control microparticle 3 The preparation materials and preparation methods of control microparticle 3 are the same, and the loaded immune adjuvants are CpG BW006 (category B) and CPG2216 (category A).
  • the adjuvant used in control microparticle 3 is 0.02 mg per 1 mg PLGA microparticle, the particle size is about 2.50 ⁇ m, and the surface potential is about -2mV.
  • Each 1 mg PLGA microparticle is loaded with approximately 140 ⁇ g of protein or peptide components.
  • Each 1 mg PLGA microparticle contains The loaded CpG BW006 (Category B) and CPG2216 (Category A) are 0.03mg each.
  • mice After the mice were killed, mouse lymph nodes and spleens were collected. The mouse lymph nodes or spleens were minced and ground, and filtered through a cell mesh to prepare a single cell suspension. After mixing the lymph node single cell suspension and the spleen single cell suspension, flow cytometry was used. CD19 + B cells and CD11c + DC were sorted by cytometry.
  • Micron particles (500 ⁇ g) loaded with whole cell components of cancer cells were incubated with prepared DCs (10 million) and B cells (10 million) in 20 mL high-glucose DMEM complete medium for 72 hours (37°C, 5% CO2 ), the incubation system contains granulocyte-macrophage colony-stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL ) and CD86 antibody (10ng/mL).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL-2 500U/mL
  • IL-7 200U/mL
  • IL-12 200U/mL
  • CD86 antibody 10ng/mL
  • the incubated DC and B cells were collected by centrifugation at 400g for 5 minutes, and then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors.
  • PBS 4°C phosphate buffer solution
  • the cells were resuspended in PBS water and incubated at 4°C at low power (PBS). 22.5W) Ultrasonic for 1 minute. Then centrifuge the sample at 3000g for 15 minutes and collect the supernatant. Centrifuge the supernatant at 8000g for 15 minutes and collect the supernatant. Then collect the supernatant after centrifugation at 16000g for 90 minutes. Discard the supernatant and collect the pellet. Place the pellet in PBS. After resuspension, nanoparticles are obtained, and the diameter of the nanoparticles is 110 nanometers.
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • mice were treated with radiation irradiation at the tumor site. The mice were sacrificed on the 25th day, and the tumor tissues of mice in each group were collected. The tumor tissues of the mice were cut into small pieces and passed through a cell sieve to prepare a single cell suspension. Then, magnetic bead sorting method was used to sort the tumor tissue single cells.
  • CD3 + T cells among live cells in a cell suspension dead cells are removed using live-dead cell dye to label them).
  • the sorted T cells (5 million) and the nanoparticles (100 ⁇ g) prepared in step (5) were incubated in 2 mL RPMI1640 complete medium for 24 hours (37°C, 5% CO 2 ), and then magnetic beads were used for sorting CD69 + T cells among T cells are sorted by this method, which are cancer cell-specific T cells activated by cancer cell whole cell antigens.
  • the cancer cell-specific T cells sorted above were incubated with IL-2 (2000U/mL), ⁇ CD-3 antibody (20ng/mL) and ⁇ CD-28 antibody (20ng/mL) in RPMI1640 complete medium for 7 days. (Change the medium once every two days) to amplify and sort the cancer cell-specific T cells.
  • nanoparticles prepared from antigen-presenting cells activated by microparticles loaded with CpG adjuvant and Poly ICLC mixed adjuvant assist in the isolation and expansion of T cells in preventing melanoma, which is better than microparticles loaded with two CpG mixed adjuvants.
  • Nanoparticles prepared from activated antigen-presenting cells assist in the isolation of expanded T cells.
  • Nanoparticles prepared by activated antigen-presenting cells using microparticles loaded with one type B CpG, one type A CpG and Poly ICLC mixed adjuvant were better than using micron particles loaded with two type A CpG and Poly ICLC mixed adjuvant.
  • Nanoparticles prepared from particle-activated antigen-presenting cells This shows that the nanoparticles activated by antigen-presenting cells loaded with mixed adjuvants of two different toll-like receptors can produce better nanoparticles, and that nanoparticles containing class B CpG and Toll-like receptor 3 agonists are used as mixed adjuvants. Nanoparticles prepared by activating antigen-presenting cells with micron particles are more effective.
  • 8M urea was first used to lyse B16F10 melanoma tumor tissue and dissolve the tumor tissue lysate components. Then, PLGA was used as the nanoparticle skeleton material, and Poly(I:C), CpG2006 (B type) and CpGSL01 (B type) were used as immune adjuvants to prepare nanoparticles loaded with cancer cell whole cell antigens, and the nanoparticles were used to activate the antigens. Nanoparticles are prepared after presenting cells, and then assist in isolating cancer cell-specific T cells from tumor-infiltrating lymphocytes, and then amplify the above cells for cancer prevention.
  • nanoparticles were prepared by solvent evaporation method.
  • the molecular weight of PLGA, the material used to prepare nanoparticle 1, is 7KDa-17KDa.
  • the immune adjuvants used are Poly(I:C), CpG2006 and CpGSL01, and the lysate components and adjuvants are encapsulated inside the nanoparticles.
  • the preparation method is as described above.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles uses Poly(I:C), CpG2006 and CpGSL01 is 0.02 mg each.
  • the preparation materials and preparation method of control nanoparticle 2 are the same as above.
  • the particle size is about 270nm. It is loaded with an equal amount of lysate components.
  • the loaded immune adjuvant is Poly(I:C). Each 1mg of PLGA is loaded with 0.06mg of Poly(I:C). .
  • control nanoparticle 3 The particle size of control nanoparticle 3 is about 270nm, and it is loaded with an equal amount of lysate components.
  • the loaded immune adjuvants are Poly(I:C), CpG1585 (Class A) and CpG2216 (Class A). Each 1 mg of PLGA is loaded with Poly(I :C), CpG1585 (Category A) and CpG2216 (Category A) 0.02mg each.
  • mouse lymph nodes were removed to prepare mouse lymph node single cell suspension, and then flow cytometry was used to sort CD11c + DC and CD19 + B cells from the lymph node cell single cell suspension.
  • Nanoparticles 1 (500 ⁇ g), nanoparticles 2 (500 ⁇ g), or nanoparticles 3 (500 ⁇ g) loaded with cancer cell whole cell components were mixed with DCs (5 million cells) and B cells (5 million cells) in 20 mL of high-glucose DMEM.
  • the culture medium was incubated for a total of 72 hours (37°C, 5% CO 2 ); the incubation system contained granulocyte-macrophage colony-stimulating factor (GM-CSF, 2000U/mL), IL-2 (500U/mL), IL- 7 (200U/mL), IL-12 (200U/mL) and CD86 antibody (10ng/mL).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL-2 500U/mL
  • IL- 7 200U/mL
  • IL-12 200U/mL
  • CD86 antibody 10ng/mL
  • B16F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse on days 8, 10, 12, 14, 16, 18, and 20
  • the mice were injected subcutaneously with 100 ⁇ L of ⁇ PD-1 antibody (10 mg/kg) on each day.
  • the mice were sacrificed on the 24th day, and the tumor tissues of the mice in each group were collected to prepare a single cell suspension of the tumor tissue, and then the viable cells in the single cell suspension of the tumor tissue were sorted using a magnetic bead sorting method (using viable Dead cell dye marks dead cells to remove dead cells) CD3 + T cells.
  • the sorted T cells (500,000), allogeneic B cells (2.5 million), and the nanoparticles (100 ⁇ g) prepared in step (5) were incubated in 10 mL RPMI1640 complete medium for 48 hours. (37°C, 5% CO 2 ), and then use flow cytometry to sort the incubated CD3 + CD8 + CD69 + T cells and CD3 + CD4 + CD69 + T cells, which are cancer cells activated by whole cell antigens of cancer cells.
  • Cell-specific T cells The cancer cell-specific T cells sorted above were incubated with IL-2 (2000U/mL), ⁇ CD-3 antibody (20ng/mL) and ⁇ CD-28 antibody (20ng/mL) in RPMI1640 complete medium for 11 days. (Change the medium every two days) to amplify and sort the cancer cell-specific T cells.
  • mice were prepared by selecting 6-8 week old female C57BL/6 as model mice. One day before transplantation of mouse cancer cell-specific T 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 800,000 expanded cancer cell-specific CD8 + T cells and 200,000 expanded cancer cell-specific CD4 + 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.
  • nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with cancer cell whole cell antigens assisted in the isolation and expansion of cancer cell-specific T cells.
  • the tumor growth rate of the transplanted mice was significantly slowed down, and most of the tumors in the mice disappeared after the cancer cells were inoculated.
  • nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with two types of B CpG and Poly(I:C) as mixed adjuvants are more effective in assisting in the isolation and expansion of T cells than those loaded with two types of A CpG and Poly(I:C).
  • Nanoparticles prepared from nanoparticles containing Poly(I:C) as a mixed adjuvant or antigen-presenting cells activated by nanoparticles loaded with only Poly(I:C) as an adjuvant assist in the isolation of expanded T cells.
  • This example uses MC38 mouse colon cancer as a cancer model to illustrate how to use nanoparticles prepared by nanoparticle-activated antigen-presenting cells to assist in the isolation of broad-spectrum cancer cell-specific T cells for the treatment of colon cancer.
  • Colon cancer tumor tissue and lung cancer cells are first lysed to prepare water-soluble antigens, and the antigens are first degraded into peptides using protease in vitro.
  • other enzymes or other methods can also be used to first degrade the proteins in the whole cell components into polypeptides.
  • Water-insoluble antigens are converted to soluble in 8M urea aqueous solution. Add trypsin (0.5 mg/mL) and chymotrypsin (Chymotrypsin, 0.5 mg/mL) to the water-soluble antigen (80 mg/mL) and incubate for 1 hour, then heat at 95°C for 10 minutes to inactivate the proteases for later use.
  • Water-soluble antigens from colon cancer tumor tissue and lung cancer cancer cells were mixed at a mass ratio of 1:1; water-insoluble antigens dissolved in 8M urea were also mixed at a mass ratio of 1:1. Then, the water-soluble antigen mixture and the water-insoluble antigen mixture are mixed at a mass ratio of 1:1, and this mixture is the source of raw materials for preparing nanoparticles.
  • Collect BCG use 8M urea aqueous solution to cleave BCG, and then dissolve the cleavage components for later use.
  • nanoparticle 1 is prepared by solvent evaporation method.
  • the molecular weight of PLA, the material for preparing nanoparticle 1, is 20KDa.
  • the nanoparticles are loaded with tumor tissue and cancer cell lysate, bacterial lysate and immune adjuvant inside, and the tumor tissue and cancer cell lysate components are loaded on the surface.
  • the immune adjuvants used were CpGM362, CPG1018 and poly ICLC, and the adjuvants were loaded inside the nanoparticles.
  • the mass ratio of tumor tissue and cancer cell lysate to bacterial lysate used in preparing the nanoparticles was 1:1. The preparation method is as mentioned above.
  • the lysate mixture, bacterial lysate components and adjuvants are first loaded inside the nanoparticles using the double emulsion method. Then 100 mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and 10 mL containing 4% is used. Trehalose was resuspended in ultrapure water and freeze-dried for 48 h. Before use, resuspend 20 mg of nanoparticles in 0.9 mL of PBS and mix and incubate at room temperature for 5 minutes with 0.1 mL of samples containing equal amounts of cancer cell and tumor tissue lysate mixture and bacterial lysate components (80 mg/mL). use.
  • the average particle size of nanoparticles 1 is about 290nm.
  • Each 1 mg of PLGA nanoparticles 1 is loaded with approximately 140 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA nanoparticles contains 0.04 mg each of CpGM362, CPG1018 and Poly ICLC immune adjuvant.
  • PBMC Peripheral blood mononuclear cells
  • BMDC and BMDM were used simultaneously as antigen-presenting cells.
  • the preparation method of BMDC is the same as that in Example 2.
  • the preparation method of BMDM is as follows: C57 mice are anesthetized and killed by dislocation. The mice are disinfected with 75% ethanol. Then use scissors to cut a small opening on the back of the mouse. Tear the skin directly to the calf joint of the mouse and remove it. Mouse foot joints and skin.
  • M-CSF Macrophage colony-stimulating factor
  • Nanoparticle 1 (1000 ⁇ g) was incubated with peripheral blood-derived DC (20 million) and BMDC (20 million) in RPMI1640 complete medium for 72 hours (37°C, 5% CO 2 ); the incubation system contained GM- CSF (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL), IL-12 (500U/mL) and CD80 antibody (10ng/mL).
  • GM- CSF 500U/mL
  • IL-2 500U/mL
  • IL-7 500U/mL
  • IL-12 500U/mL
  • CD80 antibody 10ng/mL
  • incubate nanoparticle 1 1000 ⁇ g
  • peripheral blood-derived DCs 10 million
  • BMDC 10 million
  • BMDM 10 million
  • B cells 10 million
  • the incubation system contains GM-CSF (500U/mL), IL-2 (500U/mL), IL-7 (500U/mL), and IL-12 (500U/mL) and CD80 antibody (10ng/mL).
  • peripheral blood-derived DCs (20 million) and BMDC (20 million) were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors.
  • PBS 4°C phosphate buffer solution
  • the cells were resuspended in PBS water and treated in a high-pressure homogenizer (5000 bar) for 5 minutes.
  • the sample was then centrifuged at 2000g for 15 minutes and the supernatant was collected.
  • the supernatant was centrifuged at 8000g for 15 minutes and the supernatant was collected.
  • the supernatant was incubated with the nanoparticle 1 prepared in step (3) at 4°C for 16 hours, and then use a 0.45 ⁇ m filter to repeatedly co-extrude. Centrifuge the extrudate at 13000g for 20 minutes, discard the supernatant and collect the precipitate. Resuspend the precipitate in PBS to obtain nanoparticle 2, with a particle
  • each C57BL/6 mouse was subcutaneously inoculated with 1.5 ⁇ 10 5 MC38 cells on the back.
  • the mice were injected subcutaneously with 100 ⁇ L of 1 mg PLGA nanoparticles 1.
  • the mice were sacrificed on day 24, and the draining lymph nodes and spleens of the mice were collected. Single-cell suspensions of draining lymph nodes and spleen cells were prepared and T cells were isolated from them using the magnetic bead method.
  • T cells (4 million), 200 ⁇ g of nanoparticles (nanoparticle 1, or nanoparticle 2, or nanoparticle 3), IL-2 (500 U/mL), IL-7 (500 U/mL) and IL- 15 (5000U/mL) was incubated in 5mL DMEM complete medium for a total of 96 hours, and then flow cytometry was used to sort the incubated CD3 + CD8 + CD69 + T cells, which are cancer cell-specific cells activated by cancer cell whole cell antigens. T cells.
  • the cancer cell-specific T cells obtained by the above sorting were mixed with IL-2 (1000U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and ⁇ CD-3 antibody (10ng/mL).
  • the cells were incubated in DMEM complete medium for a total of 8 days (the medium was changed every two days) to amplify the sorted cancer cell-specific CD8 + T cells.
  • the cancer cell-specific T cells using nanoparticles 1 to assist in sorting and amplification are T cell vaccine 1;
  • the cancer cell-specific T cells using nanoparticles 2 to assist in sorting and amplification are T cell vaccines 2;
  • Particle 3 assists in sorting and amplifying cancer cell-specific T cells as T cell vaccine 3.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer tumor-bearing mice. On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 MC38 cells, and on days 4, 7, 10, 15, and 20, mice were injected with 100 ⁇ L containing 2 million cancer cells. Specific CD8 + T cells. The methods for monitoring mouse tumor volume and survival were the same as above.
  • T cell vaccine of the present invention has good therapeutic effect on colon cancer.
  • Example 7 Cancer cell-specific T cells for the prevention of breast cancer
  • This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how micron particles loaded with cancer cell whole cell antigens activate antigen-presenting cells and then are prepared into micron particles to assist in the isolation of specific T cells from peripheral blood cancer cells and used to prevent breast cancer. cancer.
  • the cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water, and then repeatedly frozen and thawed 5 times with ultrasound to lyse cancer cells.
  • the double emulsion method is used to prepare micron particles.
  • the molecular weight of the micron particle 1 framework material PLGA is 38KDa-54KDa, and the immune adjuvants used are CpG2395 (Category C), CpGM362 (Category C) and Poly (I:C).
  • the double emulsion method is used to prepare micron particles loaded with lysate components and adjuvants internally, and then 100 mg of micron particles are centrifuged at 9000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and dried for 48 hours before use.
  • the average particle size of this microparticle system is about 2.5 ⁇ m, and the surface potential is about -6mV; each 1 mg of PLGA micron particles is loaded with approximately 110 ⁇ g of protein or polypeptide components, and each of CpG2395, CpGM362 and Poly(I:C) is loaded with 0.02 mg.
  • the preparation materials and preparation method of control micron particles 2 are the same as above, the particle size is about 2.5 ⁇ m, and the surface potential is about -6mV; every 1 mg of PLGA micron particles is loaded with approximately 110 ⁇ g of protein or peptide components, and every 1 mg of PLGA is loaded with CpG1585 (Class A), CpG2336 ( Class A) and Poly(I:C) 0.02mg each.
  • mice peripheral splenocytes were used. After the mice were sacrificed, the spleens were removed, and then a single cell suspension of mouse spleen cells was prepared, and the CD19 + B cells in the single cell suspension were sorted using magnetic bead sorting.
  • micron particles (800 ⁇ g) loaded with cancer cell whole cell antigen components were incubated with the B cells (10 million) prepared in step (3) in 15 mL high-sugar DMEM complete medium for 48 hours (37°C, 5% CO 2 ) , the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL) and CD86 antibody (10ng/mL).
  • GM-CSF 2000U/mL
  • IL-2 500U/mL
  • IL-7 200U/mL
  • IL-12 200U/mL
  • CD86 antibody 10ng/mL
  • the incubated B cells (10 million) were collected by centrifugation at 400g for 5 minutes, then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors, and resuspended in PBS water at 4°C. Ultrasonicate at low power (20W) for 1 minute and then use a homogenizer at 1000rpm for 3 minutes. The sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000g for 15 minutes and the supernatant was collected. The supernatant was mixed with the microparticles (60mg) prepared in step (2) and DSPE-PEG.
  • PBS 4°C phosphate buffer solution
  • each BALB/c mouse was subcutaneously inoculated with 1 ⁇ 10 6 4T1 cells on the back.
  • the mice were injected subcutaneously with 100 ⁇ L of 1 mg PLGA micron particles.
  • the mice were sacrificed on day 30, their peripheral blood was collected, peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood, and then CD3 + T cells were isolated from the PBMC using flow cytometry.
  • T cells (1 million cells), DC2.4 (2 million cells) and micron particles (50 ⁇ g) were incubated in 2 mL DMEM complete medium for 72 hours (37°C, 5% CO 2 ), and then flow cytometry was performed.
  • CD3 + CD69 + T cells are sorted, which are cancer cell-specific T cells activated by cancer cell whole cell antigens.
  • the cancer cell-specific T cells sorted above were incubated with IL-2 (4000U/mL), IL-7 (2000U/mL) and ⁇ CD-3 antibody (20ng/mL) in DMEM complete medium for 12 days. (Change the medium every two days) Expand the sorted cancer cell-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. Mice were injected subcutaneously on day 0 with 100 ⁇ L of 1.5 million expanded cancer cell-specific T cells. At the same time, 1 ⁇ 10 6 4T1 cells were subcutaneously injected into each mouse on day 0, and the tumor volume of the mice was recorded every 3 days starting from day 3. The method for monitoring tumor growth and survival in mice is the same as above.
  • mice treated with cancer cell-specific T cells prepared by micron particles-assisted isolation of antigen-presenting cells activated by micron particles was significantly slower and the survival period was significantly longer.
  • the effect of microparticles prepared from antigen-presenting cells activated by microparticles using two types of CpG and Poly(I:C) as mixed adjuvants was better than that of two types of type A CpG and Poly(I:C) as mixed adjuvants.
  • the micron particles are prepared by activating the antigen-presenting cells with the micron particles.
  • Mannose is used as an active target in the micron vaccine of this embodiment. In practical applications, any target with the ability to target target cells, such as CD32 antibodies, mannans, CD205 antibodies, and CD19 antibodies, can also be used.
  • This example uses mannose as the target to illustrate how to use active targeting nanoparticles to activate the nanoparticles prepared by antigen-presenting cells to assist in the isolation of cancer cell-specific T cells and to prevent cancer.
  • the specific dosage form, adjuvant, administration time, administration frequency, and dosage regimen can be adjusted according to the situation.
  • Actively targeting nanoparticles can be taken up into dendritic cells via mannose receptors on their surface.
  • the cultured Pan02 pancreatic cancer cells were collected and 10% octylglucoside was used to lyse the cancer cells and dissolve the whole cell antigens derived from the cancer cells.
  • the nanoparticle system was prepared using the double emulsion method.
  • the nanoparticle preparation materials are PLGA and mannose-modified PLGA, both of which have molecular weights of 7KDa-17KDa. When the two are used together to prepare nanoparticles with a target, the mass ratio is 4:1.
  • the immune adjuvants used were Poly(I:C) and CpG SL03. 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.
  • Nanoparticles 1 with the target is about 270nm.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein and peptide components, including 0.04 mg each of Poly(I:C) and CpGSL03.
  • Nanoparticles 2 that are not loaded with adjuvants but have a mannose target are also about 270nm in size. They are prepared using the same amount of cell lysis components but do not contain any immune adjuvants.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein and peptides. components.
  • This example uses BMDC and BMDM as antigen-presenting cells.
  • the preparation methods of BMDC and BMDM are the same as above.
  • Nanoparticle 1 (1000 ⁇ g) or nanoparticle 2 (1000 ⁇ g) were incubated with BMDC (10 million pcs), BMDM (10 million pcs) and IL-7 (500 U/mL) respectively in 15 mL high-glucose DMEM complete medium for 48 hours. (37°C, 5% CO 2 ). Or incubate BMDC (10 million pcs), BMDM (10 million pcs) and IL-7 (500U/mL) in 15mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO2). Both of the above incubation systems contain IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN- ⁇ (500U/mL) and CD80 antibody (10ng/mL ).
  • the one prepared using mixed antigen-presenting cells that have not been activated by nanoparticles is nanoparticle 3, with a particle size of 110 nanometers;
  • the one prepared using mixed antigen-presenting cells activated with nanoparticles 2 is nanoparticle 4, with a particle size of 110 nanometers.
  • Nanoparticle 5 was prepared using mixed antigen-presenting cells activated by nanoparticle 1, with a particle size of 110 nanometers.
  • each C57BL/6 mouse was subcutaneously inoculated with 1 ⁇ 10 6 Pan02 pancreatic cancer cells on the back.
  • day 10 day 15, day 20, and day 27, mice were injected subcutaneously with 100 ⁇ L of 1 mg PLGA. Nanoparticles.
  • day 15 day 15, day 20, and day 27, mice were sacrificed and the tumor tissues and lymph nodes of the mice were removed.
  • Mouse tumor tissues and lymph nodes were prepared into single cell suspensions. Then flow cytometry was used to separate CD45 + CD3 + T cells from the tumor tissue single cell suspension and the lymph node single cell suspension respectively, and the T cells from the tumor tissue and lymph node were mixed.
  • T cells (5 million) were incubated with 100 ⁇ g of nanoparticles (nanoparticle 1, or nanoparticle 3, or nanoparticle 4, or nanoparticle 5) in DMEM high-glucose medium for 72 hours (37°C, 5% CO 2 ), the incubation system contains IL-2 (500U/mL), IL-7 (500U/mL) and IFN- ⁇ (500U/mL). Then flow cytometry is used to sort out CD3 + CD69 + T cells from the incubated cells, which are cancer cell-specific T cells.
  • IL-2 500U/mL
  • IL-7 500U/mL
  • IFN- ⁇ 500U/mL
  • the T cells obtained above were cultured with IL-2 (2000U/mL), IL-7 (2000U/mL), IL-15 (1000U/mL) and ⁇ CD-3 antibody (50ng/mL) in DMEM high glucose.
  • the resulting cancer cell-specific T cells were amplified by incubation for a total of 12 days (the medium was changed every two days).
  • the cancer cell-specific T cells using nanoparticles 1 to assist in sorting and amplification are T cell vaccine 1; the cancer cell-specific T cells using nanoparticles 3 to assist in sorting and amplification are T cell vaccines 2;
  • the cancer cell-specific T cells assisted by particles 4 in sorting and amplifying are T cell vaccines 3;
  • the cancer cell-specific T cells assisted by nanoparticles 5 in sorting and amplifying are T cell vaccines 4.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected as model mice to prepare pancreatic cancer tumor-bearing mice.
  • the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate the recipient mice. immune cells in mice.
  • step 5 million cancer cell-specific T cells (T cell vaccine 1, or T cell vaccine 2, or T cell vaccine 3, or T cell vaccine 4) or 100 ⁇ L PBS were intravenously injected into the recipient mice.
  • each recipient mouse was inoculated subcutaneously with 1 ⁇ 10 6 Pan02 pancreatic cancer cells in the lower right side of the back. Monitor mouse tumor growth rate and mouse survival time. Tumor growth and survival monitoring methods are the same as above.
  • T cell vaccine 1 and T cell vaccine 2 treatment groups grew rapidly, and the mice died quickly.
  • the tumor growth rate of mice treated with T cell vaccine 3 and T cell vaccine 4 was significantly slower.
  • T cell vaccine 4 is more effective than T cell vaccine 3.
  • the prepared nanoparticles can effectively assist in the isolation of cancer cell-specific T cells from tumor-infiltrating lymphocytes, but with adjuvant Nanoparticles prepared by activating antigen-presenting cells with nanoparticles of the agent are more effective. This shows that the cancer cell-specific T cells of the present invention can effectively prevent cancer.
  • This example illustrates that calcified nanoparticles assist in the isolation of cancer cell-specific T cells from tumor-infiltrating lymphocytes.
  • other biomineralization technologies cross-linking, gelation and other modified particles can also be used.
  • mouse lung cancer tumor tissue was lysed with 8M urea (containing 200mM sodium chloride), dissolved and loaded into a nanoparticle system. After using the particles to activate antigen-presenting cells, the antigen-presenting cells were prepared into nanoparticles. , assists in the isolation of tumor tissue infiltrating lymphocytes and cancer cell-specific T cells in peripheral blood and amplifies them for the prevention of lung cancer.
  • mice Inoculate 1 ⁇ 10 LLC mouse lung cancer cells on the back of female C57BL/6 mice aged 6 to 8 weeks. When the tumor volume reaches 1000 mm3 , the mice will be sacrificed. The mouse tumor tissue will be removed, cut into pieces and ground. Then filter it through a cell mesh to prepare a single cell suspension, use ultraviolet irradiation for 5 minutes and then heat at 80°C for 10 minutes, and then use 8M urea (containing 200mM sodium chloride) to lyse and dissolve the tumor tissue single cell suspension to obtain Cancer cell whole cell antigen.
  • 8M urea containing 200mM sodium chloride
  • the nanoparticles are loaded with cancer cell whole cell antigens inside and on the surface, and then the nanoparticles are biocalcified.
  • the nanoparticles are prepared by a solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA is 7KDa-17KDa.
  • the immune adjuvants CpG2006 and Poly(I:C) are loaded inside the nanoparticles.
  • the preparation method is as follows.
  • the double emulsion method is first used to load the antigen inside the nanoparticles, then 100mg PLGA nanoparticles are centrifuged at 13000g for 20 minutes and resuspended in 18mL PBS, and then 2mL of tumor tissue dissolved in 8M urea and Cancer cell lysate (60 mg/mL) was incubated at room temperature for 10 minutes, centrifuged at 12,000 g for 20 minutes, and the precipitate was collected. 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 peptide components, including 0.03 mg of CpG2006 and Poly(I:C).
  • This example uses BMDC and B as antigen-presenting cells.
  • the preparation method of BMDC is the same as in Example 1.
  • B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.
  • Nanoparticles (1000 ⁇ g) loaded with cancer cell whole cell components were incubated with BMDC (5 million) and B cells (5 million) in 15 mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO 2 ) ;
  • the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN- ⁇ (500U/mL) and CD80 antibody (10ng/mL) and CD40 antibody (20mg/mL) or as a control did not contain any cytokines and antibodies in the incubation system.
  • the incubated DC and B cells were collected by centrifugation at 400g for 5 minutes, and then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors.
  • PBS 4°C phosphate buffer solution
  • the cells were resuspended in PBS water and incubated at 4°C at low power (PBS). 20W) Ultrasonic for 2 minutes.
  • the sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected.
  • the supernatant was centrifuged at 5000g for 10 minutes and the supernatant was collected.
  • the supernatant was filtered through a 0.45 ⁇ m membrane and ultrafiltrated using an ultrafiltration membrane (molecular weight cutoff 50KDa). Filter, centrifuge, filter and concentrate.
  • step (2) Mix the filtered and concentrated sample with the nanoparticles prepared in step (2) and treat it with a high-pressure homogenizer (10000bar) for 3 minutes. Then centrifuge at 13000g for 30 minutes and then discard the supernatant to collect the precipitate. , resuspend the precipitate in PBS to obtain nanoparticles with a particle size of 300 nm.
  • a high-pressure homogenizer 10000bar
  • each C57BL/6 mouse was subcutaneously inoculated with 1 ⁇ 10 6 LLC lung cancer cells on the back.
  • day 10 day 15, day 20, and day 27, the mice were subcutaneously injected with 100 ⁇ L of 1 mg PLGA nanoparticles. particle.
  • the mice were sacrificed and the tumor tissues and peripheral blood of the mice were removed.
  • Mouse tumor tissue was prepared into a single cell suspension and CD45 + CD3 + T cells were isolated from the tumor tissue single cell suspension using flow cytometry; PBMC were isolated from peripheral blood and PBMC were isolated using flow cytometry.
  • CD45 + CD3 + T cells T cells from tumor tissue and peripheral blood are mixed.
  • T cells (5 million) and nanoparticles (100 ⁇ g) prepared by antigen-presenting cells were incubated in DMEM high-glucose medium for 72 hours (37°C, 5% CO 2 ).
  • the system contained IL- 2 (500U/mL) and IL-7 (500U/mL); or T cells (5 million) and nanoparticles (100 ⁇ g) prepared from antigen-presenting cells were incubated in DMEM high-glucose medium for 72 hours (37°C, 5% CO2), and the incubation system does not contain any cytokines or antibodies.
  • flow cytometry is used to sort out CD3 + CD69 + T cells from the incubated cells, which are cancer cell-specific T cells.
  • T cells obtained above were cultured with IL-2 (2000U/mL), IL-7 (2000U/mL), IL-15 (1000U/mL) and ⁇ CD-3 antibody (50ng/mL) in DMEM high glucose.
  • the base was incubated for a total of 12 days (the medium was changed every two days) to expand T cells.
  • mice Select 6-8 weeks old female C57BL/6 as model mice to prepare lung cancer tumor-bearing mice.
  • the recipient mice were intraperitoneally injected with cyclophosphamide at a dose of 100 mg/kg to eliminate the recipients. Immune cells in mice. Then, 5 million cancer cell-specific T cells were injected intravenously into the recipient mice. The next day, 1 ⁇ 10 6 LLC lung cancer cells were subcutaneously inoculated into the lower right side of the back of each recipient mouse. Monitor mouse tumor growth rate and mouse survival time. Tumor growth and survival monitoring methods are the same as above.
  • nanoparticles prepared from antigen-presenting cells activated by calcified nanoparticles assisted in the isolation and expansion of cancer cell-specific T cells, which can prolong the survival of mice and effectively prevent cancer.
  • the system containing cytokines and/or antibodies is better than the system without cytokines and/or antibodies; moreover, the nanoparticles prepared by antigen-presenting cells are better than those without cytokines and/or antibodies.
  • T cells are co-incubated, the system containing cytokines and/or antibodies is better than the system without cytokines and/or antibodies.
  • Example 10 Cancer cell-specific T cells for the treatment of melanoma
  • the supernatant part is the water-soluble antigen; the precipitate part uses 10% sodium deoxycholate aqueous solution to dissolve the non-water-soluble antigen.
  • the water-soluble antigen and the non-water-soluble antigen dissolved in sodium deoxycholate are miscible at a mass ratio of 1:1, which is the source of the antigen raw material for preparing the nanoparticle system.
  • the nanoparticles are prepared by the double emulsion method and have the ability to target dendritic cells.
  • the materials used to prepare nanoparticle 1 are PLGA and mannan-modified PLGA, both of which have molecular weights of 24KDa-38KDa. When used, the mass ratio of unmodified PLGA to mannan-modified PLGA is 9:1.
  • the immune adjuvants used are poly(I:C), CpG1018 and CpG2216.
  • the substance that increases lysosomal immune escape is KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA), and the adjuvants and KALA polypeptide 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 KALA peptides inside the nanoparticle 1, and then 100 mg of the nanoparticle is centrifuged at 12,000g for 25 minutes, and 10 mL containing 4% Trehalose was resuspended in ultrapure water and freeze-dried for 48 h.
  • the average particle diameter of the nanoparticles 1 is about 250nm, and the surface potential is about -5mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA nanoparticles is loaded with poly(I:C), CpG1018 and CpG2216.
  • the immune adjuvant is 0.02mg each, and the loaded KALA polypeptide is 0.05mg.
  • the preparation materials and methods of nanoparticle 2 are the same as those of nanoparticle 1. Its particle size is about 250nm and its surface potential is about -5mV. Nanoparticle 2 does not load KALA polypeptide, but it loads equal amounts of adjuvants and cell lysis components.
  • the preparation materials and preparation methods of nanoparticle 3 are the same as those of nanoparticle 1, which is about 250nm, and the surface potential is about -5mV.
  • Each 1mg PLGA nanoparticle is loaded with approximately 100 ⁇ g of protein and polypeptide components.
  • the poly( I:C)0.02mg, loaded CpG1018 is 0.04mg
  • loaded KALA polypeptide is 0.05mg.
  • This example uses BMDC and BMDM as mixed antigen-presenting cells.
  • the preparation methods of BMDC and BMDM are the same as above.
  • Nanoparticles (1000 ⁇ g) loaded with cancer cell whole cell components were incubated with BMDC (10 million) and BMDM (10 million) in 15 mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO 2 );
  • the incubation system contains GM-CSF (2000U/mL), M-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN- ⁇ (500U/mL) and CD80 antibody (10ng/mL).
  • the sample was then filtered through membranes with pore diameters of 30 ⁇ m, 10 ⁇ m, 5 ⁇ m, 2 ⁇ m, and 0.45 ⁇ m.
  • the filtrate was centrifuged at 12,000g for 45 minutes and the supernatant was discarded to collect the precipitate.
  • the precipitate was placed in physiological saline containing 4% mannitol. Resuspend and then freeze-dry to obtain nanoparticles.
  • the nanoparticles prepared by using antigen-presenting cells activated by nanoparticle 1 are nanoparticles 4, with a particle size of 260 nanometers
  • the nanoparticles prepared by using antigen-presenting cells activated by nanoparticles 2 are nanoparticles 5, with a particle size of 260 nanometers.
  • the nanoparticles prepared using antigen-presenting cells activated by nanoparticle 3 are nanoparticles 6, with a particle size of 260 nanometers.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected and injected subcutaneously with 0.5 mg of PLGA nanoparticles (lysate-loaded group) on days 0, 7, 14, 21 and 28. points, Poly(I:C) and two CpG adjuvants and KALA polypeptide). The mice were sacrificed on day 32, and their peripheral blood and lymph nodes were collected. Gradient centrifugation was used to isolate PBMCs from the peripheral blood of mice. The lymph nodes were cut into small pieces and ground through a cell mesh to prepare a single cell suspension. The PBMCs were then mixed with the single cell suspension of lymph node cells. CD45 + CD3 + T cells were then sorted using magnetic bead sorting.
  • the sorted CD3 + T cells (5 million cells) were co-incubated with 40 ⁇ g of nanoparticles (nanoparticle 4, or nanoparticle 5, or nanoparticle 6) and IL-7 (10 ng/mL) in 2 mL of RPMI1640 complete medium. 96 hours. Then flow cytometry is used to sort the CD3 + OX40 + T cells in the incubated T cells, which are cancer cell-specific T cells that can recognize cancer cell whole cell antigens.
  • the CD3 + OX40 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-15 (1000U/mL), IL-21 (1000U/mL) and ⁇ CD-3 antibody (20ng/mL).
  • the cancer cell-specific T cells assisted in sorting and amplification by nanoparticles 4 are T cell vaccine 1; the cancer cell-specific T cells assisted in sorting and amplification by nanoparticles 5 are T cell vaccines 2; the cancer cell-specific T cells assisted by nanoparticles 5 are sorted and amplified.
  • the cancer cell-specific T cells that assist sorting and amplification are T cell vaccines 3.
  • Expanded cancer cell-specific T cells are used to treat cancer
  • 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. 1.5 million expanded cancer cell-specific T cells were injected intravenously on days 4, 7, 10, 15 and 20 after melanoma inoculation. In the experiment, the mouse tumor volume and survival period were monitored as above.
  • the tumors in the PBS control group grew quickly.
  • the tumor growth rate of mice treated with nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with whole cell components assisted in the isolation and expansion of cancer cell-specific T cells was significantly slower and the survival period was significantly prolonged.
  • the nanoparticles 4 prepared by activated antigen-presenting cells by adding nanoparticles 1 that increase lysosomal escape substances to assist in the isolation and expansion of cancer cell-specific T cell vaccines 1 are better than nanoparticles 2 that do not add lysosomal escape substances.
  • Nanoparticles 5 prepared from activated antigen-presenting cells assist in the isolation and expansion of cancer cell-specific T cell vaccines 2; activated antigen presentation using nanoparticles 1 using two CpGs and Poly(I:C) as mixed adjuvants
  • the therapeutic effect of cell-prepared nanoparticles 4 assisted in the isolation and expansion of cancer cell-specific T cell vaccines 1 is better than that of nanoparticles 3 activated with only one CpG and Poly(I:C) mixed adjuvant.
  • the prepared nanoparticles 6 assist in sorting and amplifying the T cell vaccine 3.
  • the T cells of the present invention have good therapeutic effects on cancer.
  • Example 11 Cancer cell-specific T cells for prevention of breast cancer
  • This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use nanoparticles prepared from antigen-presenting cells activated by micron particles loaded with cancer cell whole-cell antigens to assist in the sorting of cancer cell-specific T cells for prevention.
  • 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 cleave the non-water-soluble antigens in the cancer cells.
  • PLGA was used as the micron particle skeleton material
  • CpG2007, CpG1018, and Poly ICLC were used as immune adjuvants
  • polyarginine and polylysine were used as substances that enhance lysosomal escape to prepare whole cell antigens loaded with cancer cells.
  • Micron particles are used to prepare nanoparticles after activating antigen-presenting cells. Nanoparticles are used to assist in the isolation and expansion of cancer cell-specific T cells and are used for cancer prevention.
  • 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 at 60°C for 5 minutes, and then ultrapure water was added and repeatedly frozen and thawed 5 times, supplemented by ultrasound to lyse the cancer cells.
  • the cell lysate was centrifuged at 5000g for 10 minutes, and the supernatant The liquid is the water-soluble antigen.
  • the precipitate is dissolved with 10% octyl glucoside to become the original dissolved non-water-soluble antigen.
  • the double emulsion method is used to prepare micron particles.
  • the molecular weight of the micron particle 1 skeleton material PLGA is 38KDa-54KDa.
  • the immune adjuvants used are CpG2007, CpG1018 and Poly ICLC.
  • the lysosomal escape-increasing substance used is polyacrylate. amino acids and polylysine.
  • During preparation first use the double emulsion method to prepare micron particles internally loaded with lysate components, adjuvants and KALA polypeptides, then centrifuge 100 mg of micron particles at 9000g for 20 minutes, resuspend in 10 mL of ultrapure water containing 4% trehalose and dry Reserve after 48 hours.
  • the average particle size of the micron particles 1 is about 1.5 ⁇ 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 each of CpG2007, CpG1018, and Poly ICLC, and polyacrylamide. 0.02mg each of amino acid and polylysine.
  • This example uses BMDC and mouse splenocyte-derived B cells as antigen-presenting cells.
  • the preparation method of BMDC and B cells is the same as above.
  • Micron particles (1000 ⁇ g) loaded with cancer cell whole cell components were incubated with 20 million mixed antigen-presenting cells (including 10 million BMDC + 10 million DC2.4 cells) in 15 mL high-glucose DMEM complete medium for 48 hours ( 37°C, 5% CO 2 ); the incubation system contains GM-CSF (500U/mL), M-CSF (500U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL -12 (200U/mL), IFN- ⁇ (500U/mL) and CD80 antibody (10ng/mL).
  • GM-CSF 500U/mL
  • M-CSF 500U/mL
  • IL-2 500U/mL
  • IL-7 200U/mL
  • IL -12 200U/mL
  • IFN- ⁇ 500U/mL
  • CD80 antibody 10ng/mL
  • step (2) Incubate the filtered and concentrated sample with 40 mg of the nanoparticles prepared in step (2) for 10 minutes and then repeatedly extrude it using a 2 ⁇ m filter membrane. Then, centrifuge the extruded liquid at 10,000g for 20 minutes and collect it. The supernatant was removed to collect the precipitate, and the precipitate was resuspended in PBS to obtain micron particles 2 with a particle size of 1.6 ⁇ m.
  • the sorted CD3 + T cells (1 million), 100 ⁇ g micron particles 2 and IL-7 (500 U/mL) were incubated in 5 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ); or The sorted CD3 + T cells (1 million) and 100 ⁇ g micron particles 2 were incubated in 5 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ); or the sorted CD3 + T cells (100 10,000), 1 million BMDC prepared in step (3), 100 ⁇ g micron particles 1 and IL-7 (500 U/mL) were incubated in 5 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ).
  • CD8 + CD69 + T cells and CD4 + CD69 + T cells among the incubated CD3 + T cells, which are cancer cell-specific T cells that can recognize cancer cell whole cell antigens.
  • the CD8 + CD69 + T cells or CD4 + CD69 + T cells obtained above were combined with IL-2 (1000U/mL), IL-6 (1000U/mL), IL-12 (1000U/mL) and ⁇ CD28 antibody respectively. (10ng/mL) in RPMI1640 complete medium for 14 days to expand cancer cell-specific T cells.
  • the cancer cell-specific T cells obtained by co-incubating and sorting using CD3 + T cells, micron particles 2 and IL-7 are T cell vaccine 1; the cancer cells obtained by co-incubating and sorting using CD3 + T cells and micron particles 2 Specific T cells are T cell vaccine 2; cancer cell specific T cells obtained by co-incubation and sorting with CD3 + T cells, DCs, micron particles 1 and IL-7 are T cell vaccine 3.
  • 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 T cell vaccine (containing 1 million expanded CD8 + T cells and 200,000 expanded CD4 + ) or 100 ⁇ L of PBS. At the same time, 1 ⁇ 10 6 4T1 cells were injected subcutaneously into each mouse on day 0. The method for monitoring tumor growth and survival in mice is the same as above.
  • the tumor growth rate of the cancer cell-specific T cell vaccine treatment group obtained by micron particle-assisted sorting prepared by micron particle-activated antigen-presenting cells was significantly slower and the survival time of mice was significantly slower.
  • the cancer cell-specific T cell vaccine 1 obtained by adding IL-7 to assist sorting during the co-incubation of nanoparticles prepared from antigen-presenting cells and immune cells containing T cells is more effective than the co-incubation process without adding IL.
  • T cell vaccine 1 is more effective than T cell vaccine 3.
  • cancer cell-specific T cells This shows that adding the cytokine IL-7 during co-incubation is beneficial to assist in the isolation of cancer cell-specific T cells, and that micron particles prepared from activated mixed antigen-presenting cells can assist in the isolation without antigen-presenting cells.
  • the effect of cancer cell-specific T cells is better than that of cancer cell-specific T cells obtained by co-incubation and sorting of nanoparticles loaded with whole cell components and DC+T cells. It can be seen that the cancer cell-specific T cell vaccine of the present invention has a preventive effect on breast cancer.
  • Example 12 Cancer cell-specific T cells for prevention of breast cancer
  • the cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water.
  • the obtained cancer cells 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.
  • Centrifuge Lactobacillus acidophilus at 5000g for 30 minutes, then discard the precipitate and collect the supernatant. Filter the supernatant with a 1 ⁇ m filter membrane, and then centrifuge at 16000g for 90 minutes. The precipitate is treated with 8M urea aqueous solution (containing 500mM chlorine). Sodium chloride) cleaves and solubilizes bacterial outer vesicle components.
  • the double emulsion method is used to prepare micron particles.
  • the skeleton material of micron particle 1 is unmodified PLA and mannose-modified PLA, both with a molecular weight of 40KDa.
  • the ratio of unmodified PLA to mannose-modified PLA is 4:1.
  • the immune adjuvants used were CpG2006, CpG2216 and Poly ICLC, and the lysosomal escape-increasing substances used were arginine and histidine.
  • the mass ratio of cancer cell lysate components and bacterial external vesicle components used in the preparation of micron particles is 1:1.
  • the double emulsion method is first used to prepare micron particles that are internally loaded with cancer cell lysate components, bacterial external vesicle components, adjuvants, arginine and histidine. Then, 100mg of micron particles are centrifuged at 9000g for 20 minutes. Use 10 mL of ultrapure water containing 4% trehalose to resuspend and dry for 48 hours to obtain micron particles 1. The average particle size is about 1.5 ⁇ m. Each 1 mg of PLGA micron particles 1 is approximately loaded with 100 ⁇ g of protein or peptide components, including CpG2006 and CpG2216. and Poly ICLC 0.02mg each, containing 0.05mg each of arginine and histidine.
  • control microparticle 2 The preparation materials and methods of control microparticle 2 are the same as those of microparticle 1.
  • the particle size is about 1.5 ⁇ m. It is loaded with equal amounts of arginine, histidine and equal amounts of cancer cell lysate components and bacterial outer vesicle components. However, Does not contain any adjuvants.
  • This example uses BMDC, B cells and BMDM as antigen-presenting cells.
  • the preparation methods of BMDC and BMDM are the same as above.
  • B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.
  • microparticles 1 or 2 were incubated with 40 million mixed antigen-presenting cells (containing 20 million BMDCs, 10 million B cells and 10 million BMDMs) in 15 mL of high-glucose DMEM complete medium for 48 hours. (37°C, 5% CO 2 ); the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN- ⁇ (500U/mL) and CD80 antibody (10ng/mL) and CD40 antibody (20mg/mL).
  • mixed antigen-presenting cells containing 20 million BMDCs, 10 million B cells and 10 million BMDMs
  • Nanoparticles Use a high-pressure homogenizer (10,000 bar) to process the filtered and concentrated sample for 3 minutes. Then, centrifuge at 13,000g for 30 minutes. Discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS. Nanoparticles. Among them, the mixed antigen-presenting cells activated using micron particles 1 were prepared as nanoparticles 1, with a particle size of 250 nanometers; the mixed antigen-presenting cells activated using micron particles 2 were prepared as nanoparticles 2, with a particle size of 250 nanometers.
  • step (2) Use a high-pressure homogenizer (10,000 bar) to treat the filtered and concentrated sample for 3 minutes, and then react it with 60 mg of micron particles 1 or 2 prepared in step (2) for 10 minutes before use.
  • the 2 ⁇ m filter membrane was repeatedly co-extruded, and then the extruded liquid was centrifuged at 10,000 g for 20 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS to obtain micron particles.
  • the mixed antigen-presenting cell membrane component activated by micron particle 1 and the micron particle 1 are combined to prepare micron particle 3, with a particle size of 1.6 ⁇ m; the mixed antigen-presenting cell membrane component activated by micron particle 2 and micron particle 2 are prepared by the combined action of micron particles 4, with a particle size of 1.6 ⁇ m.
  • mice Select 6-8 week old female BALB/c mice and subcutaneously inject 100 ⁇ L of micron particles containing 0.2 mg of PLGA prepared in step (2) on days 0, 7, 14, 21, and 28. 1.
  • the mice were sacrificed on day 32, peripheral blood and spleen were collected, and then a single cell suspension of PBMC and spleen cells was prepared and mixed, and then CD3 + T cells were sorted out using magnetic bead sorting method.
  • nanoparticles 1, or nanoparticles 2, or microparticles 3 or microparticles 4 are used to assist in sorting and amplifying cancer cell-specific T cells respectively.
  • the sorted CD3 + T cells (2 million), nanoparticles or microparticles (100 ⁇ g), and DC2.4 cells (1 million) were incubated in 10 mL RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ), the incubation system contains IL-2 (200U/mL), IL-7 (200U/mL), IL-15 (200U/mL) and CD80 antibody (10ng/mL). Then flow cytometry is used to sort the CD3 + CD8 + CD69 + T cells among the incubated CD3 + T cells and the CD4 + CD69 + T cells among the CD4 + T cells, which are cancer cells that can recognize the whole cell antigen of cancer cells. specific T cells.
  • the CD8 + CD69 + T cells or CD4 + CD69 + T cells obtained above are mixed according to the quantity ratio of 2:1 and then mixed with IL-2 (1000U/mL), IL-7 (1000U/mL) and ⁇ CD-3 respectively.
  • Antibodies (10ng/mL) were incubated in RPMI1640 complete medium for a total of 14 days to expand cancer cell-specific T cells, during which the medium was changed every two days.
  • the cancer cell-specific T cells assisted by nanoparticle 1 to assist in isolation and expansion are T cell vaccine 1;
  • the cancer cell-specific T cells assisted by nanoparticles 2 in isolation and expansion are T cell vaccine 2; micron particles 3 assist in the isolation and expansion of cancer cell-specific T cells.
  • the amplified cancer cell-specific T cells are T cell vaccine 3; the cancer cell-specific T cells assisted by micron particles 4 in isolating and amplifying are T cell vaccine 4.
  • anti-mouse CD3 antibodies, anti-mouse CD3 antibodies, and anti-mouse CD69 antibodies were used to label the incubated T cells, and then flow cytometry was used to analyze the co-incubation of different nanoparticles with T cells and antigen-presenting cells. The proportion of CD69 + T cells among post-T cells.
  • the T cells in the splenocytes without nanoparticle-assisted sorting were incubated with different nanoparticles prepared in step (6) in DMEM high-glucose complete medium for 48 hours, and then the incubated cells were collected and used with The IFN- ⁇ antibody of the fluorescent probe labels the incubated T cells, and then flow cytometry is used to analyze the proportion of IFN- ⁇ + T cells in the T cells.
  • the cancer cell whole cell antigens loaded by the nanoparticles can be degraded into antigen epitopes after being phagocytosed by the antigen-presenting cells and presented to the surface of the antigen-presenting cell membrane.
  • the nanoparticles prepared by the antigen-presenting cells are loaded with the above-mentioned degraded and presented products.
  • the antigenic epitope can be recognized by cancer cell-specific T cells and activate cancer cell-specific T cells. After activation, they secrete killer cytokines.
  • IFN- ⁇ is the most important cytokine secreted by antigen-specific T cells that are activated after recognizing antigens.
  • CD3 + IFN- ⁇ + T cells analyzed using flow cytometry are cancer cell-specific T cells that can recognize and kill cancer 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 T cell vaccine (containing 600,000 expanded CD8 + T cells and 300,000 expanded CD4 + T cells) or 100 ⁇ L PBS. At the same time, 1 ⁇ 10 6 4T1 cells were subcutaneously injected into each mouse on day 0. The mouse tumor volume and survival period were monitored as above.
  • T cell vaccine containing 600,000 expanded CD8 + T cells and 300,000 expanded CD4 + T cells
  • T cell vaccine 1 is better than T cell vaccine 2; T cell vaccine 3 is better than T cell vaccine 4.
  • T cell vaccine 4 This shows that the effect of particles prepared from antigen-presenting cells activated by micron particles containing substances that increase lysosomal escape function and mixed adjuvants is better than that of substances that only contain lysosomal escape function.
  • the particles prepared from antigen-presenting cells activated by micron particles without mixed adjuvants assist in the isolation of cancer cell-specific T cells.
  • T cell vaccine 3 is better than T cell vaccine 1
  • T cell vaccine 4 is better than T cell vaccine 2.
  • the isolated cancer cell-specific T cells are better than the cancer cell-specific T cells isolated only with the help of vesicle particles loaded with activated antigen-presenting cell components. It can be seen that the cancer cell-specific T cells of the present invention have the ability to kill breast cancer and can be used to prevent or treat cancer. Moreover, the use of mixed adjuvants and the internal loading of whole cell components of cancer cells are helpful in assisting in the isolation of cancer cell-specific T cells.
  • the proportion of cancer cell-specific T cells that can be activated when using different particles to assist sorting is related to the efficacy in figures a and b, which illustrates that using the particles of the present invention to assist sorting
  • the selected T cells are cancer cell-specific T cells that can specifically recognize and kill cancer cells.
  • This example uses mouse colon cancer as a cancer model to illustrate how to use nanoparticles loaded with colon cancer whole-cell antigens to activate antigen-presenting cells prepared by nanoparticles to assist in sorting cancer cell-specific T cells and use them to treat 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), CpG2336 and CpG2006 are used as adjuvants, and NH 4 HCO 3 is used as the adjuvant. Add lysosomal escape substances and prepare nanoparticles.
  • nanoparticles to activate antigen-presenting cells and prepare the antigen-presenting cells into nanoparticles. Then use nanoparticles to assist in sorting cancer cell-specific T cells. After two-step sorting The resulting cancer cell-specific T cells are expanded and used for cancer treatment.
  • the nanoparticles were prepared using the double emulsion method.
  • the preparation material of nanoparticles is PLGA with a molecular weight of 7KDa-17KDa, Poly(I:C) and CpG as adjuvants, NH 4 HCO 3 as a substance that increases lysosomal escape, and the adjuvants and NH 4 HCO 3 are loaded on the nanoparticles Within; the preparation method is as described above.
  • the lysis solution components and adjuvants are first loaded inside the nanoparticles, and then 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL of ultrapure water containing 4% trehalose is used to rehydrate the nanoparticles.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein and polypeptide components, and each 1 mg of PLGA nanoparticles is loaded with poly(I :C), CpG2336 and CpG2006 immune adjuvant 0.02mg each, loaded with NH 4 HCO 3 0.01mg.
  • the preparation materials and methods of nanoparticle 2 are the same as nanoparticle 1.
  • the particle size is about 260nm and the surface potential is about -7mV.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 90 ⁇ g of protein and peptide components.
  • Each 1 mg PLGA nanoparticle is loaded with NH 4 HCO. 3 0.01mg, loaded with 0.03mg each of CpG2336 and CpG2006.
  • This example uses BMDC and B as antigen-presenting cells.
  • the preparation method of BMDC is the same as in Example 1.
  • B cells were derived from mouse peripheral blood PBMC, and the preparation method was the same as above.
  • Nanoparticles (1000 ⁇ g) loaded with cancer cell whole cell components were incubated with BMDC (5 million) and B cells (5 million) in 15 mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO 2 ) ;
  • the incubation system contains GM-CSF (2000U/mL), IL-2 (500U/mL), IL-7 (200U/mL), IL-12 (200U/mL), IFN- ⁇ (500U/mL) and CD80 antibody (10ng/mL) and CD40 antibody (20mg/mL).
  • the incubated DC and B cells were collected by centrifugation at 400g for 5 minutes, and then washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors.
  • PBS 4°C phosphate buffer solution
  • the cells were resuspended in PBS water and incubated at 4°C at low power (PBS). 20W) Ultrasonic for 2 minutes.
  • the sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected.
  • the supernatant was centrifuged at 5000g for 10 minutes and the supernatant was collected.
  • the supernatant was filtered through a 0.45 ⁇ m membrane and ultrafiltrated using an ultrafiltration membrane (molecular weight cutoff 50KDa). Filter, centrifuge, filter and concentrate.
  • step (2) Mix the filtered and concentrated sample with the nanoparticles prepared in step (2) and treat it with a high-pressure homogenizer (10000bar) for 3 minutes. Then centrifuge at 13000g for 30 minutes and then discard the supernatant to collect the precipitate. , resuspend the precipitate in PBS to obtain nanoparticles, with a particle size of 300 nm.
  • a high-pressure homogenizer 10000bar
  • mice Female C57BL/6 mice aged 6-8 weeks were selected, and 2 ⁇ 10 6 MC38 colon cancer cells were subcutaneously inoculated into the back on day 0. On days 14 and 28, 100 ⁇ L of nanoparticles containing 0.4 mg PLGA were injected subcutaneously. (Loading lysate components, mixed adjuvants and substances that increase lysosomal escape). The mice were sacrificed on the 32nd day, and the tumor tissues and peripheral blood of the mice were removed. The mouse tumor tissue is prepared into a tumor tissue single cell suspension; PBMC are isolated from the peripheral blood of the mouse, and then the tumor tissue single cell suspension and PBMC are mixed, and then flow cytometry is used to sort out CD3 + from the above mixed cells.
  • CD8 + T cells and sorted CD3 + CD4 + T cells The sorted CD8 + T cells (200,000 cells), CD4 + T cells (100,000 cells), nanoparticles prepared from antigen-presenting cells (50 ⁇ g), B cells (1 million cells), and IL-7 (10 ng /mL) were incubated in 2 mL RPMI1640 complete medium for a total of 48 hours (37°C, 5% CO 2 ), and then flow cytometry was used to sort the CD8 + CD69 + T cells and CD4 + T cells in the incubated CD8 + T cells.
  • CD4 + CD69 + T cells in the cells are cancer cell-specific T cells that can recognize cancer cell whole cell antigens.
  • CD8 + CD69 + T cells or CD4 + CD69 + T cells obtained above were mixed with IL-2 (1000U/mL), IL-12 (1000U/mL), IL-15 (1000U/mL) and ⁇ CD- 3 antibodies (10ng/mL) were incubated in RPMI1640 complete medium for a total of 14 days (the medium was changed every two days) to amplify cancer cell-specific T cells.
  • Cancer cell-specific T cells are used to treat cancer
  • the tumor growth rate of mice treated with nanoparticle-assisted isolation and expansion of cancer cell-specific T cells prepared by nanoparticle-activated antigen-presenting cells was significantly slower and the mice Survival period was significantly prolonged.
  • CD8 + T cells and CD4 + T cells obtained by simultaneously using nanoparticles prepared from antigen-presenting cells to assist in the isolation and expansion are better than CD8 + T cells obtained by using only nanoparticles to assist in the isolation and expansion.
  • nanoparticles prepared from antigen-presenting cells activated by nanoparticles loaded with mixed adjuvants, lysate components and lysosomal escape substances are more effective in assisting in the isolation of cancer cell-specific T cells than those loaded with lysate components and both.
  • Nanoparticles of CpG adjuvants and lysosomal escape substances It can be seen that the cancer cell-specific T cells of the present invention have excellent therapeutic effects on cancer.

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Abstract

涉及一种癌细胞特异性T细胞疫苗、以及激活癌细胞特异性T细胞的方法，将外周血、外周免疫器官或肿瘤浸润淋巴细胞中的T细胞与被激活的抗原提呈细胞制备的粒子共孵育以激活癌细胞特异性T细胞。克服了临床上目前无法有效筛选肿瘤浸润淋巴细胞中广谱和多克隆的癌细胞特异性T细胞的难题，能够从外周血、外周免疫器官或者肿瘤浸润淋巴细胞中分离广谱的具有特异性肿瘤杀伤功能的效应性癌细胞特异性T细胞，且具有易分离获取和特异性高的特点，可用于癌症的预防和治疗。

Description

一种癌细胞特异性T细胞疫苗、以及激活癌细胞特异性T细胞的方法 技术领域
本发明涉及免疫治疗领域，尤其涉及一种癌细胞特异性T细胞疫苗、以及激活癌细胞特异性T细胞的方法。
背景技术
T细胞尤其是癌细胞特异性T细胞发挥着抗癌主力军的作用。T细胞是机体特异性识别和杀灭癌细胞的主要细胞，每一种癌细胞特异性T细胞的克隆可以特异性识别一种抗原表位。癌症患者体内尤其是经过免疫治疗或者放疗的患者体内都含有一定数量的癌细胞特异性T细胞。但肿瘤患者体内癌细胞特异性T细胞普遍数量不足，因此限制了癌细胞特异性T细胞杀伤癌细胞的能力，如果能够将体内的癌细胞特异性T细胞分离出来之后扩增重新回输给患者，那么就能调控免疫微环境，较好的控制癌症进展。
但是，如何从上百万种甚至上千万种不同克隆的T细胞中分选出广谱的癌细胞特异性T细胞中的具有癌细胞识别和杀伤功能的效应性癌细胞特异性T细胞就显得非常关键。目前没有特别高效的手段从众多T细胞中较全面的分离出该部分具有特异性肿瘤杀伤功能的效应性癌细胞特异性T细胞，因此提出本发明。
发明内容
为解决上述技术问题，本发明提供了一种使用被负载癌细胞全细胞抗原的纳米粒子(NP)或微米粒子(MP)激活过的抗原提呈细胞制备的纳米粒子和/或微米粒子辅助分离和扩增杀伤性(效应性)癌细胞特异性T细胞(T eff)的方法，该方法使用被激活的抗原提呈细胞制备的纳米粒子或微米粒子先激活广谱多克隆的癌细胞特异性T细胞，然后将利用被激活的杀伤性癌细胞特异性T细胞(T eff)特异性表达的标志物分离提取上述癌细胞特异性T细胞，回输给患者预防或者治疗癌症，有效地解决了如何从外周血、外周免疫器官或肿瘤浸润淋巴细胞中特异性分离提取具有识别和杀伤癌细胞能力的广谱和多克隆的癌细胞特异性T细胞的问题。而且，由于辅助分离和扩增癌细胞特异性T细胞的纳米粒子或微米粒子负载抗原提呈细胞膜组分，所以在使用本发明所述纳米粒子或微米粒子与T细胞共孵育时体系中可以没有抗原提呈细胞的辅助。
本发明的第一个目的是提供一种由粒子激活的抗原提呈细胞制备的癌症T细胞疫苗的制备方法，包括以下步骤：
S1、将抗原提呈细胞与第一粒子共孵育，得到激活后的抗原提呈细胞；其中，第一粒子负载肿瘤组织和/或癌细胞全细胞组分；
S2、将激活后的抗原提呈细胞的细胞膜组分制备成纳米囊泡；或将激活后的抗原提呈 细胞的细胞膜组分与第二粒子共作用，使细胞膜组分负载于第二粒子上，得到负载细胞膜组分的粒子；其中，第二粒子负载肿瘤组织和/或癌细胞全细胞组分；
S3、将S2的纳米囊泡和/或负载细胞膜组分的粒子与含有T细胞的免疫细胞共孵育，将其中可以识别癌细胞抗原的癌细胞特异性T细胞激活，再使用特定手段分选出该部分被激活的癌细胞特异性T细胞，即可得到癌症T细胞疫苗。
进一步地，在步骤S3中，分选出被激活的癌细胞特异性T细胞之后，还包括对癌细胞特异性T细胞进行扩增的步骤。
进一步地，上述分选为利用被癌细胞全细胞组分激活的癌细胞特异性T细胞的特异性表面标志物进行筛选。特异性表面标志物包括但不限于CD69、PD-1、TIM-3、LAG-3、CD25、OX40(CD134)、TCF-1、CD137、CD44、CD39、CD103、CD56、CD279、CD278、CD244、CD27、CD154、CD28等。利用表面标志物分离癌细胞特异性T细胞的技术包括但不限于流式细胞术和磁珠分选法。
进一步地，含有T细胞的免疫细胞可来源于外周血、外周免疫器官或肿瘤浸润淋巴细胞。当其与S2的产物共孵育之前，可对以上免疫细胞进行分选，分选出其中的T细胞，具体地，使用流式细胞术或者磁珠分选法从外周血、外周免疫组织、肿瘤浸润淋巴细胞中分选CD3 +的细胞、分选出CD45 +CD3 +的细胞、分选出CD3 +CD8 +的细胞、分选出CD45 +CD3 +CD8 +的细胞、分选出CD3 +CD4 +的细胞或分选出CD45 +CD3 +CD4 +的细胞。
进一步地，上述扩增为体外扩增，即将癌细胞特异性T细胞与细胞因子和/或抗体进行共孵育。
进一步地，步骤S1的抗原提呈细胞与第一粒子的共孵育体系中、步骤S3的纳米囊泡和负载细胞膜组分的粒子与免疫细胞共孵育体系中，均可含有细胞因子和/或抗体。
优选地，孵育体系中含有IL-2和IL-7。
进一步地，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14(IL-14)、白介素4(IL-4)、白介素15(IL-15)、白介素21(IL-21)、粒细胞－巨噬细胞集落刺激因子(GM-CSF)、白介素17(IL-17)、IL-12、白介素12(IL-12)、白介素6(IL-6)、巨噬细胞集落刺激因子(M-CSF)、白介素33(IL-33)、γ干扰素(IFN-γ)、TNF-α。
进一步地，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。
进一步地，在步骤S3中，共孵育体系中还可含有未经激活的抗原提呈细胞。
进一步地，在上述制备方法中，第一粒子或第二粒子上还可负载细菌裂解组分和/或细菌外囊泡裂解组分，该抗细菌裂解组分和/或细菌外囊泡裂解组分经含有裂解剂的裂解液裂解细菌或细菌外囊泡得到，裂解剂为尿素、盐酸胍、脱氧胆酸盐、十二烷基硫酸盐(如SDS)、甘油、蛋白质降解酶、白蛋白、卵磷脂、Triton、吐温、氨基酸、糖苷、胆碱等的水溶液，细菌包括但不限于卡介苗、大肠杆菌、长双歧杆菌、短双歧杆菌、乳双歧杆菌、嗜酸乳杆 菌、格式乳杆菌、罗伊氏乳杆菌、鼠李糖乳杆菌等。
进一步地，第一粒子或第二粒子上还负载有免疫增强佐剂，免疫增强佐剂包括但不限于模式识别受体激动剂、卡介苗、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、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、植物油、内毒素、脂质体佐剂、MF59、双链RNA、双链DNA、CAF01、人参、黄芪的有效成分等。
优选地，免疫增强佐剂两种或两种以上Toll样受体激动剂，如包括(1)Poly(I:C)或Poly(ICLC)；(2)CpG-ODN，其中，CpG-ODN为A类CpG-ODN、B类CpG-ODN和C类CpG-ODN中的至少两种，且至少其中一种为B类CpG-ODN或C类CpG-ODN。其中，A类CpG-ODN选自CpG-ODN 2216、CpG-ODN 1585或CpG-ODN 2336，B类CpG-ODN选自CpG-ODN 1018、CpG-ODN 2006、CpG-ODN 1826、CpG-ODN 1668、CpG-ODN 2007、CpG-ODN BW006或CpG-ODN SL01，C类CpG-ODN选自CpG-ODN 2395、CpG-ODN SL03或CpG-ODN M362。
进一步地，第一粒子或第二粒子上还负载有带正电荷的多肽(如KALA多肽、RALA多肽、蜂毒肽等)、精氨酸、聚精氨酸、赖氨酸、聚赖氨酸、组氨酸、聚组氨酸、NH 4HCO 3、鱼精蛋白或组蛋白等。
进一步地，第一粒子或第二粒子上还负载有主动靶向抗原提呈细胞的靶头，靶头可为甘露糖、甘露聚糖、CD19抗体、CD20抗体、BCMA抗体、CD32抗体、CD11c抗体、CD103抗体、CD44抗体等。
进一步地，第一粒子或第二粒子可由以下材料制备得到：有机合成高分子材料包括但不限于PLGA、PLA、PGA、PEG、PCL、Poloxamer、PVA、PVP、PEI、PTMC、聚酸酐、PDON、PPDO、PMMA、聚氨基酸、合成多肽等；天然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、细胞膜成分、淀粉、糖类、多肽等；无机材料包括但不限于三氧化二铁、四氧化三铁、碳酸盐、磷酸盐等。
进一步地，第一粒子或第二粒子的粒径大小为纳米级或微米级，这样能保证粒子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。纳米粒子的粒径大小为1nm-1000nm，更优选地，粒径大小为30nm-1000nm，最优选地，粒径大小为50nm-600nm；微米粒子的粒径大小为1μm-1000μm，更优选地，粒径大小为1μm-100μm，更优选地，粒径大小为1μm-10μm，最优选地，粒径大小为1μm-5μm。
进一步地，在步骤S2中，将被激活的抗原提呈细胞经过机械破坏、膜过滤或梯度离心制备得到纳米囊泡，或将被激活的抗原提呈细胞经过机械破坏、膜过滤或梯度离心，将产物与第二粒子共作用，得到包裹细胞膜组分的粒子。
进一步地，机械破坏方式选自超声、均质化、匀浆、高速搅拌、高压破坏、高剪切力破坏、溶胀、化学物质、皱缩中的一种或多种。共作用方式选自共孵育、共挤出、超声、搅拌、透析、超滤、均质化和匀浆中的一种或多种，与纳米粒子或微米粒子共作用后抗原提呈细胞组分覆盖于原有纳米粒子或微米粒子表层形成新的纳米粒子或微米粒子。
进一步地，肿瘤组织和/或癌细胞全细胞组分由以下步骤制备得到：将癌细胞和/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，得到的上清液为水溶性组分，沉淀中经含有溶解剂的溶液溶解后转为可溶的部分为非水溶性组分，水溶性组分和非水溶性组分合并后得到肿瘤组织和/或癌细胞全细胞组分；或将癌细胞和/或肿瘤组织经含有溶解剂的溶解液裂解并溶解后得到可溶组分，该可溶组分为肿瘤组织和/或癌细胞全细胞组分。溶解剂选自尿素、盐酸胍、脱氧胆酸盐、十二烷基硫酸盐(如SDS)、甘油、蛋白质降解酶、白蛋白、卵磷脂、无机盐(0.1-2000mg/mL)、Triton、吐温、氨基酸、糖苷、胆碱中的至少一种。
进一步地，抗原提呈细胞包括B细胞、树突状细胞(DC)和巨噬细胞中的至少一种，优选为两种及以上，更优选为三种细胞的组合。
进一步地，得到的癌细胞特异性T细胞包括CD4 +T细胞和/或CD8 +T细胞，优选为同时包括CD4 +T细胞和CD8 +T细胞。
本发明中，使用负载癌细胞全细胞抗原的纳米粒子和/或微米粒子先特异性激活抗原提呈细胞，在将抗原提呈细胞制备成纳米粒子或微米粒子，所制备成的纳米粒子或微米粒子就负载癌细胞全细胞抗原表位，然后使用由抗原提呈细胞制备而成的纳米粒子或微米粒子激活外周血、外周免疫组织或者肿瘤浸润淋巴细胞中预存的已在淋巴结中被激活过的癌细胞特异性T细胞，再利用被激活的癌细胞特异性T细胞分泌特定细胞因子或者高表达某些表面分子的特性，利用流式细胞术等手段分离得到癌细胞特异性T细胞，经体外扩增后回输给患者使用，能够分离和扩增到最多样和广谱的具有识别和杀伤癌细胞功能的癌细胞特异性T细胞。
本发明的第二个目的是提供上述癌症T细胞疫苗在制备治疗或预防癌症的药物中的应用。
进一步地，外周血、外周免疫组织或肿瘤浸润淋巴细胞中的T细胞来源于自体或同种异体。
进一步地，抗原提呈细胞可以与癌细胞特异性T细胞来源于同体、同种异体，细胞系或由干细胞转化而来。
进一步地，第一粒子或第二粒子中，用于制备抗原的癌细胞或肿瘤组织中至少有一种与上述药物治疗的目标疾病类型相同。本发明制备的疫苗具有特异性。
本发明的第三个目的是提供一种体外激活癌细胞特异性T细胞的方法，该方法包括以下步骤：
S1、将抗原提呈细胞与第一粒子共孵育，得到激活后的抗原提呈细胞；其中，第一粒 子负载肿瘤组织和/或癌细胞全细胞组分；
S2、将激活后的抗原提呈细胞的细胞膜组分制备成纳米囊泡；或将激活后的抗原提呈细胞的细胞膜组分与第二粒子共作用，使细胞膜组分负载于第二粒子上，得到负载细胞膜组分的粒子；其中，第二粒子负载肿瘤组织和/或癌细胞全细胞组分；
S3、将S2的纳米囊泡和/或负载细胞膜组分的粒子与含有T细胞的细胞共孵育，其中的可以识别癌细胞抗原的癌细胞特异性T细胞被激活，使用特定方法分选出被激活的癌细胞特异性T细胞后经体外扩增即得。
本发明突破现有激活方式的限制，使粒子上负载全部抗原和经激活的抗原提呈细胞细胞膜，能够辅助分离更广谱和多样的癌细胞特异性T细胞，且高度特异，在免疫治疗中效果更佳，从而为细胞治疗提供更有力的备选药物。
借由上述方案，本发明至少具有以下优点：
本发明提供了一种使用纳米级或微米级粒子递送系统体外辅助激活后分离免疫细胞中癌细胞特异性T细胞的技术，所分离得到的癌细胞特异性T细胞广谱而且高度特异，包含所有克隆的可以特异性识别和杀伤癌细胞效应性(杀伤性)癌细胞特异性T细胞(T eff)，将癌细胞特异性T细胞扩增后，所得细胞可以用于预防和治疗癌症。并在此基础上对抗原提呈细胞激活过程、与T细胞孵育过程、第一粒子和第二粒子负载物质进行优化，得到一种治疗和预防效果极佳的癌症疫苗。
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。
附图说明
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。
图1为本发明细胞系统的制备过程及应用示意图；其中，a为水溶性抗原和非水溶性抗原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解液溶解癌细胞全细胞抗原和制备纳米粒子或微米粒子的示意图；c为使用a或b中制备的纳米粒子和/或微米粒子激活抗原提呈细胞，并将被激活的抗原提呈细胞制备的粒子激活癌细胞特异性T细胞后，利用T细胞被激活后的特征分离提取癌细胞特异性T细胞，尔后扩增该类T细胞，并用该类细胞预防或治疗癌症的示意图；
图2-14分别为实施例1-13中用分离扩增的癌细胞特异性T细胞预防或治疗癌症时小鼠肿瘤生长速度和生存期实验结果；图2-14中，a为预防或治疗癌症时的肿瘤生长速度实验结果(n≥8)；b为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)；图3、4和13中，c和d为使用流式细胞术分析被癌细胞全细胞抗原激活的癌细胞特异性T细胞占相应T细胞比例的结果；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；*表示与PBS空白对照组相比p＜0.05，有显著性差异；εε代表与使用特定细胞因子组分加入纳米粒/微米粒激活抗原提呈细胞共孵育体系，所制备的纳米粒子/微米粒子辅助分离的癌细胞特异性T细胞组相比p＜0.01，有显著性差异；###表示单独孵育而无任何纳米粒激活的抗原提呈细胞制备的纳米粒子辅助分离的T细胞对照组相比p＜0.005，有显著性差异；
Figure PCTCN2022108966-appb-000001
表示与纳米粒/微米粒激活的抗原提呈细胞制备的空心纳米粒子(内部不负载癌细胞全细胞组分)辅助分离的T细胞对照组相比p＜0.005，有显著性差异；&&&表示与空白纳米粒/微米粒+游离裂解液激活的抗原提呈细胞制备的纳米粒子和/或微米粒子辅助分离的T细胞对照组相比p＜0.005，有显著性差异；δδδ代表与多肽纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子/微米粒子辅助分离的T细胞组相比p＜0.005，有显著性差异；ω代表与纳米粒/微米粒激活的DC细胞或者B细胞制备的纳米粒子辅助分离的T细胞组相比p＜0.05，有显著性差异；χ代表与负载癌细胞全细胞组分和吐温80裂解和溶解的细菌组分的纳米粒/微米粒激活的抗原提呈细胞制备的粒子辅助分离的T细胞组相比p＜0.05，有显著性差异；θ代表与使用两种A类CpG和Poly ICLC/Poly(I:C)作为混合佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子/微米粒子辅助分离的T细胞组相比p＜0.05，有显著性差异；ττ代表与使用A类CpG和B类CpG作为混合佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子辅助分离的T细胞组相比p＜0.01，有显著性差异；μμ代表与只使用Poly(I:C)作为佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子辅助分离的T细胞组相比p＜0.01，有显著性差异；ΟΟΟ代表与使用内部负载癌细胞全细胞组分但是表面不负载任何抗原提呈细胞膜组分的纳米粒/微米粒激活的辅助分离的T细胞组相比p＜0.005，有显著性差异；φ代表与纳米粒/微米粒激活的DC制备的纳米粒子辅助分离的T细胞组相比p＜0.05，有显著性差异；
Figure PCTCN2022108966-appb-000002
代表与负载抗原不负载佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子/微米粒子辅助分离的T细胞组相比p＜0.005，有显著性差异；
Figure PCTCN2022108966-appb-000003
代表与负载抗原不负载佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子/微米粒子辅助分离的T细胞组相比p＜0.01，有显著性差异；Δ代表将负载癌细胞全细胞组分的纳米粒/微米粒、DC细胞及T细胞共孵育后辅助分离的T细胞组相比p＜0.05，有显著性差异；ρ代表纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子/微米粒子辅助分离T细胞时不加入细胞因子的组相比p＜0.05，有显著性差异；η代表纳米粒/微米粒激活抗原提呈细胞时不加入细胞因子所制备的基于抗原提呈细胞的纳米粒子/微米粒子辅助分离的T细胞组相比p＜0.05，有显著性差异；β代表与不负载溶酶体逃逸物质的纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子/微米粒子辅助分离的T细胞组相比p＜0.05，有显著性差异；ξ代表与只负载一种CpG+Poly(I:C)混合佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子/微米粒子辅助分离的T细胞组相比p＜0.05，有显著性差异；υυυ代表与表面负载膜细胞组分但是内部不负载癌细胞全细胞组分的纳米粒子或微米粒子辅助分离的T细胞组相比p＜0.005，有显著性差异；ΣΣ代表与内部不负载佐剂但负载癌细胞全细胞组分，表面负载使用不负载佐剂的纳米粒子激活的抗原提呈细胞的膜 组分的纳米粒子/微米粒子辅助分离的T细胞组相比p＜0.01，有显著性差异；λλ代表与只负载两类CpG作为佐剂的纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子/微米粒子辅助分离的T细胞组相比p＜0.01，有显著性差异；π代表与只使用被纳米粒/微米粒激活的抗原提呈细胞制备的纳米粒子/微米粒子辅助分离的癌细胞特异性CD8 +T细胞组相比p＜0.05，有显著性差异。
具体实施方式
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。
本发明所述的用于预防或治疗癌症的T细胞系统，其包含来自于外周血、外周免疫组织或者肿瘤浸润淋巴细胞中的经过特异性分离和扩增的癌细胞特异性T细胞，该类癌细胞特异性T细胞在分离时先被激活过的抗原提呈细胞所制备的纳米粒子和/或微米粒子激活，然后利用被激活后高表达的特异性分子被分离。分离后扩增的癌细胞特异性T细胞可以来自同种同体或者同种异体。其中，用于制备纳米粒子或微米粒子的抗原提呈细胞先被负载肿瘤组织和/或癌细胞全细胞抗原或其混合物的纳米粒子和/或微米粒子激活。制备预防或治疗癌症的T细胞系统，其制备过程及应用领域如图1所示。
在制备激活抗原提呈细胞的纳米粒子或微米粒子时，可裂解细胞或组织后先分别收集水溶性抗原和水不溶性抗原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解液直接裂解细胞或组织并溶解癌细胞全细胞抗原并制备纳米或微米粒子系统。本发明所述癌细胞全细胞抗原在裂解前或(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、蛋白酶内切或降解、核酸酶处理等处理后再制备纳米粒子或微米粒子；也可细胞裂解前或(和)裂解后不经过任何灭活或(和)变性、固化、生物矿化、离子化、化学修饰、蛋白酶内切或降解、核酸酶处理直接制备纳米粒子或微米粒子。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、核酸酶处理、蛋白酶内切或降解、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。
在将被激活的抗原提呈细胞制备成纳米粒子或微米粒子时，先对抗原提呈细胞进行机械破坏，然后使用离心和/或一定孔径的滤膜过滤，可选地，与纳米粒子或微米粒子共同作用。
被激活的抗原提呈细胞在经过机械破坏后含有一定的细胞膜结构。
被激活的抗原提呈细胞在经过机械破坏后，与纳米粒子或微米粒子共作用后所形成的新的纳米粒子或微米粒子中抗原提呈细胞的组分位于粒子外层。
在使用抗原提呈细胞制备的纳米粒子和/或微米粒子体外激活癌细胞特异性T细胞时，体系中也可以同时加入抗原提呈细胞的辅助。制备成纳米粒子或微米粒子的抗原提呈细胞以及用来加入与T细胞共孵育的抗原提呈细胞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。抗原提呈细胞可以是DC细胞、B细胞、巨噬细胞或者上述三者的任意混合物，也可以是其他具有抗原提呈功能的细胞。
在使用负载肿瘤组织和/或癌细胞全细胞组分激活抗原提呈细胞时，体系中可含有细胞因子和/或抗体以提高激活效率。
在使用被激活的抗原提呈细胞制备成的纳米粒子和/或微米粒子激活癌细胞特异性T细胞时，体系中可含有细胞因子和/或抗体以提高激活效率。
在一些实施方案中，采用负载癌细胞全细胞抗原的纳米粒子或微米粒子先激活抗原提呈细胞，再将抗原提呈细胞制备成纳米粒子或微米粒子，使用抗原提呈细胞制备的纳米粒子或微米粒子辅助分离扩增来自外周血、外周免疫组织或肿瘤浸润淋巴细胞的癌细胞特异性T细胞的具体制备方法如下：
步骤1，将第一预定体积的含有第一预定浓度的水相溶液加入第二预定体积的含有第二预定浓度制备粒子原材料的有机相中。
在一些实施例中，水相溶液可含有癌细胞/肿瘤组织裂解物中的各组分以及免疫增强佐剂；裂解物中的各组分在制备时分别为水溶性抗原或是溶于含有尿素或盐酸胍等溶解剂的溶解液中的原非水溶性抗原。水相溶液所含有的水溶性抗原的浓度或原非水溶性抗原的浓度，也即第一预定浓度要求蛋白质多肽浓度含量大于1ng/mL，能负载足够癌细胞全细胞抗原以激活相关细胞。免疫增强佐剂在初始水相中的浓度为大于0.01ng/mL。
在一些实施例中，有机溶剂选用二氯甲烷。另外，在一些实施例中，制备粒子原材料的第二预定浓度的范围为0.5mg/mL-5000mg/mL，优选为100mg/mL。
实际中，有机相的第二预定体积根据其和水相的第一预定体积的比例进行设定，在本发明中，水相的第一预定体积和有机相的第二预定体积之比的范围为1:1.1-1:5000，优选地为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整以调整制备的纳米粒或微米粒的尺寸大小。
优选地，水相溶液为裂解物组分溶液时，其中蛋白质和多肽的浓度大于1ng/mL，优选1mg/mL～100mg/mL；水相溶液为裂解物组分/免疫佐剂溶液时，其中蛋白质和多肽的浓度大于1ng/mL，优选1mg/mL～100mg/mL，免疫佐剂的浓度大于0.01ng/mL，优选0.01mg/mL～20mg/mL。有机相溶液中，溶剂为DMSO、乙腈、乙醇、氯仿、甲醇、DMF、异丙醇、二氯甲烷、丙醇、乙酸乙酯等，优选二氯甲烷；有机相的浓度为0.5mg/mL～5000mg/mL，优选为100mg/mL。
步骤2，将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌或均质处理或微流控处理。优选地，搅拌为机械搅拌或者磁力搅拌时，搅拌速度大于50rpm，搅拌时间大于1分钟，比如搅拌速度为50rpm～1500rpm，搅拌时间为0.1小时～24小时； 超声处理时，超声功率大于5W，时间大于0.1秒，比如2～200秒；均质处理时使用高压/超高压均质机或高剪切均质机，使用高压/超高压均质机时压力大于5psi，比如20psi～100psi，使用高剪切均质机时转速大于100rpm，比如1000rpm～5000rpm；使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化和/或微米化，超声时间长短或搅拌速度或均质处理压力及时间能控制制备的微纳粒子大小，过大或过小都会带来粒径大小的变化。
步骤3，将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于2秒的超声处理或大于1分钟的搅拌或进行均质处理或微流控处理。该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌纳米化或微米化。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。优选地，搅拌为机械搅拌或者磁力搅拌时，搅拌速度大于50rpm，搅拌时间大于1分钟，比如搅拌速度为50rpm～1500rpm，搅拌时间为0.5小时～5小时；超声处理时，超声功率为50W～500W，时间大于0.1秒，比如2～200秒；均质处理时使用高压/超高压均质机或高剪切均质机，使用高压/超高压均质机时压力大于20psi，比如20psi～100psi，使用高剪切均质机时转速大于1000rpm，比如1000rpm～5000rpm；使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化或微米化，超声时间长短或搅拌速度或均质处理压力及时间能控制制备的纳米或微米粒子大小，过大或过小都会带来粒径大小的变化。
在一些实施例中，乳化剂水溶液为聚乙烯醇(PVA)水溶液，第三预定体积为5mL，第三预定浓度为20mg/mL。第三预定体积根据其与第二预定体积的比例进行调整。在本发明中，第二预定体积与第三预定体积之的范围为1:1.1-1:1000进行设定，优选地可以为2:5。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸，可以对第二预定体积和第三预定体积之比进行调整。同样地，本步骤的超声时间或搅拌时间、乳化剂水溶液的体积以及浓度的取值根据，均为了得到尺寸大小合适的纳米粒或微米粒。
步骤4，将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件。
本步骤中，乳化剂水溶液为PVA溶液或其他溶液。
第四预定浓度为5mg/mL，第四预定浓度的选择，以得到尺寸大小合适的纳米粒或微米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本发明中，第三预定体积与第三预定体积之比为范围为1:1.5-1:2000，优选地为1:10。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。
在本发明中，本步骤的预定搅拌条件为直至有机溶剂挥发完成，也即步骤1中的二氯甲烷挥发完成。
步骤5，将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于 1分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有冻干保护剂的水溶液中或者第六预定体积的PBS(或生理盐水)中。
步骤6，将步骤5得到的含有冻干保护剂的混悬液进行冷冻干燥处理后，将冻干物质备用。
步骤7，将第六预定体积的步骤5中得到的重悬于PBS(或生理盐水)中的含纳米粒的混悬液或者采用第六预定体积的PBS(或生理盐水)重悬步骤6得到的冷冻干燥后的含有纳米粒或微米粒和冻干保护剂的冻干物质直接使用；或者上述样品与第七预定体积的水溶性抗原或者溶解的原非水溶性抗原混合后使用。
在本发明中，第六预定体积与第七预定体积的体积比为1:10000到10000:1，优先体积比为1:100到100:1，最优体积比为1:30到30:1。
步骤8，将抗原提呈细胞与上述制备的纳米粒子和/或微米粒子共孵育一定时间。制备纳米粒子和/微米粒子的肿瘤组织和/或癌细胞与抗原提呈细胞可以来自于自体或者同种异体。
步骤9，收集共孵育后的细胞，进行超声、均质化、机械搅拌等机械破坏。
步骤10，将超声完的样品进行离心和/或使用一定孔径的滤膜过滤和/或与负载癌细胞全细胞组分的纳米粒子和/或微米粒子共同作用，制备得到基于抗原提呈细胞的纳米粒子或微米粒子。
步骤11，取得外周血、外周免疫组织或者肿瘤组织，收集上述组织中T细胞或含有T细胞的免疫细胞。以上外周血、外周免疫组织或者肿瘤组织可以来自于自体或者同种异体。
步骤12，将步骤10制备的纳米和/或微米粒子与步骤11得到的含有T细胞的免疫细胞混合后共孵育一定时间。
步骤13，采用流式细胞术、磁珠分选法等分离被抗原激活的T细胞。
步骤14，将分离得到的被癌细胞全细胞抗原激活的T细胞进行体外扩增。
步骤15，将扩增后的癌细胞特异性T细胞，回输到患者体内预防或治疗癌症。
实施例1癌细胞特异性T细胞分离扩增后用于黑色素瘤的预防
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用纳米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离扩增肿瘤浸润淋巴细胞中的癌细胞特异性T细胞后用于预防黑色素瘤。本实施例中，裂解B16F10黑色素瘤肿瘤组织以制备肿瘤组织的水溶性抗原和非水溶性抗原，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抗原和非水溶性抗原的纳米粒子系统，然后使用纳米粒子激活抗原提呈细胞，并将抗原提呈细胞机械破坏后离心制备纳米粒子，使用该纳米粒子辅助分离肿瘤浸润淋巴细胞中的癌细胞特异性T细胞，并经扩增后注射到体内预防黑色素瘤。
(1)肿瘤组织的裂解及各组分的收集
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超纯水并反复冻融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抗原；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抗原转化为在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。
(2)负载全细胞组分的纳米粒子的制备
本实施例中纳米粒子1采用溶剂挥发法中的复乳法制备。在制备时负载癌细胞全细胞抗原中水溶性抗原的纳米粒子和负载癌细胞全细胞抗原中非水溶性抗原的纳米粒子分别制备，然后使用时一起使用。所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的免疫佐剂为poly(I:C)且poly(I:C)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细胞组分和佐剂，然后将100mg纳米粒子在10000g离心20分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1平均粒径为280nm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。
(3)骨髓来源的树突状细胞(BMDC)的制备
本实施例以从小鼠骨髓细胞制备树突状细胞为例来说明如何制备BMDC。首先，取1只6-8周龄C57小鼠颈椎脱臼处死，手术取出后腿的胫骨和股骨放入PBS中，用剪刀和镊子将骨周围的肌肉组织剔除干净。用剪刀剪去骨头两端，再用注射器抽取PBS溶液，针头分别从骨头两端插入骨髓腔，反复冲洗骨髓到培养皿中。收集骨髓溶液，400g离心3min后加入1mL红细胞裂解液裂红。加入3mL RPMI 1640(10％FBS)培养基终止裂解，400g离心3min，弃上清。将细胞放置10mm培养皿中培养，使用RPMI 1640(10％FBS)培养基，同时加入重组小鼠GM-CSF(20ng/mL)，37度，5％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。
(4)抗原提呈细胞的激活
将负载来源于肿瘤组织的癌细胞全细胞组分的纳米粒子(250μg负载水溶性组分的纳米粒子+250μg负载非水溶性组分的纳米粒子)与BMDC(1000万个)在15mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)；孵育体系中含有细胞因子组合1：粒细胞－巨噬细胞集落刺激因子(GM-CSF，500U/mL)、IL-2(500U/mL)、IL-7(500U/mL)、IL-12(500U/mL)或者含有细胞因子组分2：GM-CSF(500U/mL)、IL-4(500U/mL)、肿瘤坏死因子α(TNF-α，500U/mL)、IL10(500U/mL)。
(5)DC来源的纳米粒子的制备
通过在400g离心5分钟收集添加细胞因子组分1的孵育后的DC(1000万个)，然后使用生理盐水洗涤细胞两遍，将细胞重悬在生理盐水中后在在4℃和7.5W下超声20分钟以破坏细 胞并制备含有细胞膜组分的样品。然后将样品在2000g离心20分钟并收集上清液，将上清液在7000g离心20分钟后收集上清液，然后在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米粒子2，纳米粒子2粒径为120纳米。
或者收集步骤(3)制备的未经任何纳米粒子或微米粒子激活的DC(1000万个)，然后使用生理盐水洗涤细胞两遍，将细胞重悬在生理盐水中后在4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。然后将样品在2000g离心20分钟并收集上清液，将上清液在7000g离心20分钟后收集上清液，将上清液与40mg步骤(2)制备的负载癌细胞全细胞组分的纳米粒子1(负载水溶性组分的纳米粒子20mg+负载非水溶性组分的纳米粒子20mg)共孵育10分钟，然后使用0.45μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米粒子3，粒径为300nm。
或者通过在400g离心5分钟收集添加细胞因子组分2的孵育后的DC(1000万个)，然后使用生理盐水洗涤细胞两遍，将细胞重悬在生理盐水中后在在4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。然后将样品在2000g离心20分钟并收集上清液，将上清液在7000g离心20分钟后收集上清液，将上清液与40mg步骤(2)制备的负载癌细胞全细胞组分的纳米粒子1(负载水溶性组分的纳米粒子20mg+负载非水溶性组分的纳米粒子20mg)共孵育10分钟，然后使用0.45μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米粒子。其中，使用纳米粒子1与膜组分共孵育得到的为纳米粒子4，粒径为300nm。
或者通过在400g离心5分钟收集添加细胞因子组分1的孵育后的DC(1000万个)，然后使用生理盐水洗涤细胞两遍，将细胞重悬在生理盐水中后在在4℃和7.5W下超声20分钟以破坏细胞并制备含有细胞膜组分的样品。然后将样品在2000g离心20分钟并收集上清液，将上清液在7000g离心20分钟后收集上清液，将上清液与40mg步骤(2)制备的负载癌细胞全细胞组分的纳米粒子1(负载水溶性组分的纳米粒子20mg+负载非水溶性组分的纳米粒子20mg)共孵育10分钟，然后使用0.45μm的滤膜反复共挤出，将挤出液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米粒子5，粒径为300nm。
(6)癌细胞特异性T细胞的分离和扩增
在每只C57BL/6小鼠背部皮下接种0.5×10 5个B16F10细胞，在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取小鼠肿瘤组织和脾细胞。将小鼠肿瘤组织切成小块后使用胶原酶消化15分钟，然后通过细胞筛网制备单细胞悬液，离心并用PBS洗涤后使用流式细胞术从肿瘤组织单细胞悬液中分离活细胞中(使用活死细胞染料标记死细胞以去除死细胞)的CD3 +T细胞。将纳米粒子2(100μg)或者纳米粒子3(100μg)或者纳米粒子4(100μg)或者纳米粒子5(100μg)与来自肿瘤浸润淋巴细胞的T细胞(50万个)在3mL RPMI 1649完全培养基中共孵育96小时(37℃，5％CO 2)。然后采用流式细胞术分选孵育后的细胞中的CD3 +CD8 +CD69 +T细胞，即为癌细胞特异性CD8 +T细胞。将上述分选得到的癌细胞特异性T细胞与IL-2(2000U/mL)、IL-7(2000U/mL)、IL-12(200U/mL)、IL-15(200U/mL) 和αCD-3抗体(10ng/mL)共孵育10天(每两天换液一次)以扩增分选得到癌细胞特异性T细胞，即为T细胞疫苗。其中，由纳米粒子2辅助分选和扩增得到的癌细胞特异性T细胞疫苗为T细胞疫苗1；由纳米粒子3辅助分选和扩增得到的癌细胞特异性T细胞疫苗为T细胞疫苗2；由纳米粒子4辅助分选和扩增得到的癌细胞特异性T细胞疫苗为T细胞疫苗3；由纳米粒子5辅助分选和扩增得到的癌细胞特异性T细胞疫苗为T细胞疫苗4。
(7)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠，在小鼠过继转移细胞前1天，给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。然后，将步骤(6)制备得到的400万个癌细胞特异性T细胞(T细胞疫苗1，或者T细胞疫苗2，或者T细胞疫苗3，或者T细胞疫苗4)或者100μL PBS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。
(8)实验结果
如图2所示，PBS对照组的小鼠其肿瘤生长速度很快，生存期很短。接受几种T细胞疫苗处理的小鼠肿瘤生长速度都明显变慢，小鼠生存期变长。其中，T细胞疫苗4效果最好，T细胞疫苗4的效果优于T细胞疫苗1、T细胞疫苗2和T细胞疫苗3。这说明：在激活抗原提呈细胞过程中加入细胞因子组合1效果优于细胞因子组分2；辅助分离T细胞的纳米粒子表面负载抗原提呈细胞膜组分，内部为负载细胞组分的实心纳米粒子效果优于只是表面负载膜组分的纳米囊泡结构；被负载癌细胞全细胞组分的纳米粒子激活的抗原提呈细胞所制备的纳米粒子效果远好于未被激活的抗原提呈细胞制备的纳米粒子。综上所述，本发明所述的癌细胞特异性T细胞对黑色素瘤具有良好的预防效果。被负载癌细胞全细胞组分的纳米粒子激活的抗原提呈细胞会降解和提呈所吞噬纳米粒子负载的癌细胞全细胞组分中的全细胞抗原，被抗原提呈细胞提呈到细胞膜表面的癌细胞抗原表位已经与主要组织相容性复合物(MHC)分子结合。将上述抗原提呈细胞经过机械破坏后，抗原提呈细胞的细胞膜组分中含有与MHC结合的抗原表位。通过离心和/或使用一定孔径的滤膜过滤和/或与纳米粒子或微米粒子共作用，上述抗原提呈细胞中的细胞膜组分会形成纳米粒子或微米粒子，并负载了MHC分子和被降解提呈的癌细胞抗原表位，因而可以不经过抗原提呈细胞的辅助而直接激活癌细胞特异性T细胞。
实施例2癌细胞特异性T细胞分离扩增后用于黑色素瘤的预防
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用纳米粒子激活的抗原提呈细胞制备纳米粒子辅助分离扩增癌细胞特异性T细胞后用于预防黑色素瘤。本实施例中，裂解B16F10黑色素瘤肿瘤组织以制备肿瘤组织的水溶性抗原和非水溶性抗原，然后，以PLGA为纳米粒骨架材料，以poly(I:C)和CpG1018为免疫佐剂采用溶剂挥发法制备负载有肿瘤组 织的水溶性抗原和非水溶性抗原的纳米粒子系统，然后使用纳米粒子激活抗原提呈细胞，并将被激活的抗原提呈细胞制备成纳米粒子辅助分离肿瘤浸润淋巴细胞中的癌细胞特异性T细胞，并经扩增后注射到体内预防黑色素瘤。
(1)肿瘤组织的裂解及各组分的收集
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抗原；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抗原转化为在8M尿素水溶液中可溶。将水溶性组分和非水溶性组分按质量比1:1混合后即为制备纳米粒子系统的抗原原料来源。
(2)纳米粒子的制备
本实施例中纳米粒子及作为对照的空白纳米粒子和多肽纳米粒子采用溶剂挥发法制备。负载全细胞组分的纳米粒子1制备材料PLGA分子量为7Da-17KDa，所采用的免疫佐剂为poly(I:C)和CpG1018且佐剂包载于纳米粒子内部。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抗原和佐剂，然后将100mg纳米粒子在10000g离心20分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子平均粒径为280nm左右；每1mg PLGA纳米粒子约负载100μg蛋白质和多肽组分，每1mgPLGA纳米粒所使用的poly(I:C)和CpG1018免疫佐剂各0.02mg。本实施例中，采用等质量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)的多肽纳米粒子2作为对照纳米粒子使用，其制备材料和制备方法同纳米粒子1，对照纳米粒2的粒径为280nm左右，负载100μg多肽组分，负载等量佐剂。空白纳米粒3的制备材料和制备方法同纳米粒子1，粒径为280nm左右，只负载等量的免疫佐剂却不负载任何抗原组分。
(3)抗原提呈细胞的制备
采用骨髓来源的树突状细胞(BMDC)和B细胞作为抗原提呈细胞。BMDC的制备同实施例1。B细胞提取流程如下：处死小鼠后摘取小鼠脾脏，然后制备小鼠脾细胞单细胞悬液，然后使用磁珠分选法从脾细胞单细胞悬液中分选出CD19 +B细胞。将BMDC和B细胞按数量比1:1混合后作为混合抗原提呈细胞使用。
(4)抗原提呈细胞的激活
将纳米粒子1(500μg)或多肽纳米粒子2(500μg)或空白纳米粒3(500μg)+游离裂解液与2000万个混合抗原提呈细胞(1000万个BMDC+1000万个B细胞)在15mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)；孵育体系中含有细胞因子组合：IL-15(500U/mL)、IL-2(500U/mL)、IL-7(500U/mL)、IL-12(1000U/mL)。
或者纳米粒子1与2000万个BMDC在15mL RPMI1640完全培养基中共孵育48小时 (37℃，5％CO 2)；孵育体系中含有细胞因子组合：IL-15(500U/mL)、IL-2(500U/mL)、IL-7(500U/mL)、IL-12(1000U/mL)。
(5)基于抗原提呈细胞的纳米粒子的制备
通过在400g离心5分钟收集孵育后的2000万个混合抗原提呈细胞(1000万个BMDC+1000万个B细胞)，然后使用生理盐水洗涤细胞两遍，将细胞重悬在生理盐水中后在4℃下使用低功率7.5W超声10分钟以破坏细胞并制备含有细胞膜组分的样品。然后将样品一次通过滤膜孔径为50μm、10μm、5μm、1μm、0.45μm、0.22μm的滤膜过滤，收集所得滤液后与相对应的步骤(2)制备的负载癌细胞全细胞组分的纳米粒子1(50mg)或者多肽纳米粒子2(50mg)或者空白纳米粒子3(50mg)共孵育10分钟，然后使用0.45μm的滤膜反复共挤出，将挤出液并在15000g离心60分钟，弃去上清后使用生理盐水重悬所得沉淀即为纳米粒子。其中，使用空白纳米粒子3激活的混合抗原提呈细胞膜组分与空白纳米粒子3共作用后所制得的纳米粒子为纳米粒子4，粒径为300nm；使用多肽纳米粒子2激活的混合抗原提呈细胞膜组分与多肽纳米粒子2共作用后所制得的纳米粒子为纳米粒子5，粒径为300nm；使用纳米粒子1激活的混合抗原提呈细胞膜组分与纳米粒子1共作用后所制得的纳米粒子为纳米粒子6，粒径为300nm。
或者通过在400g离心5分钟收集与纳米粒子1孵育后的2000万个BMDC，然后使用生理盐水洗涤BMDC两遍，将细胞重悬在生理盐水中后在4℃下使用低功率7.5W超声10分钟以破坏细胞并制备含有细胞膜组分的样品。然后将样品一次通过滤膜孔径为50μm、10μm、5μm、1μm、0.45μm、0.22μm的滤膜过滤，收集所得滤液后与步骤(2)制备的纳米粒子1(50mg)共孵育10分钟，然后使用0.45μm的滤膜反复共挤出，将挤出液并在15000g离心60分钟，弃去上清后使用生理盐水重悬所得沉淀即为纳米粒子7，粒径为300nm。
(6)癌细胞特异性T细胞的分离和扩增
第0天，在每只C57BL/6小鼠背部皮下接种5×10 5个B16F10细胞，在第7天，第14天，第21天和第28天分别给小鼠皮下注射100μL负载癌细胞全细胞组分的1mgPLGA纳米粒子。在第32天处死小鼠，收集小鼠的脾脏和肿瘤组织。将小鼠肿瘤组织切成小块后通过细胞筛网制备单细胞悬液，离心并用PBS洗涤后使用流式细胞术从肿瘤组织单细胞悬液中分离活细胞中(使用活死细胞染料标记死细胞以去除死细胞)的CD3 +T细胞。与此同时，将小鼠脾脏通过细胞筛网和裂解红细胞后制备脾细胞单细胞悬液，使用流式细胞术从脾细胞单细胞悬液中分选活细胞中(使用活死细胞染料标记死细胞以去除死细胞)的CD19 +B细胞。将步骤(5)制备的基于抗原提呈细胞的100μg纳米粒子(纳米粒子4，或者纳米粒子5，或者纳米粒子6，或者纳米粒子7)与B细胞(500万个)和来自肿瘤浸润淋巴细胞的T细胞(40万个)在5mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)，然后采用流式细胞术分选孵育后的CD3 +CD134 +T细胞，即为被癌细胞全细胞抗原激活的癌细胞特异性T细胞；其中使用纳米粒子4辅助分选得到的癌细胞特异性T细胞为T细胞疫苗1；其中使用纳米粒子5辅助分选得到的癌细胞特异性T细胞为T细胞疫苗2；其中使用纳米粒子6辅助分选得 到的癌细胞特异性T细胞为T细胞疫苗3；其中使用纳米粒子7辅助分选得到的癌细胞特异性T细胞为T细胞疫苗4。或者将步骤(5)制备的基于抗原提呈细胞的100μg纳米粒子6与来自肿瘤浸润淋巴细胞的T细胞(40万个)在5mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)，然后采用流式细胞术分选孵育后的CD3 +CD134 +T细胞，即为被癌细胞全细胞抗原激活的癌细胞特异性T细胞，为T细胞疫苗5。
与此同时，分别使用抗小鼠CD3抗体、抗小鼠CD4抗体、抗小鼠CD8抗体和抗小鼠CD134抗体标记孵育后的T细胞，然后使用流式细胞术分析不同纳米粒子与T细胞和抗原提呈细胞共孵育后CD8 +T细胞亚型和CD4 +T细胞亚型中CD134 +的T细胞所占的比例。纳米粒子所负载的癌细胞全细胞抗原在被抗原提呈细胞吞噬后可被降解成抗原表位被提呈到抗原提呈细胞表面，可以识别癌细胞全细胞抗原的特异性T细胞即可以识别癌细胞全细胞抗原表位后被激活并表达特异性表面标志物，通过流式细胞术分析高表达特异性表面标志物的T细胞的比例，即可以知道被激活和可以分选出来的可以识别和具有杀伤效能的癌细胞特异性T细胞的数量。
将上述分选得到的T细胞疫苗分别与IL-2(2000U/mL)和αCD-3抗体(20ng/mL)共孵育14天(每两天换液一次)以扩增分选得到的癌细胞特异性T细胞疫苗。
(7)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠，在小鼠过继转移细胞前1天，给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。然后，将步骤(6)制备得到的100万个癌细胞特异性T细胞疫苗(T细胞疫苗1，或者T细胞疫苗2，或者T细胞疫苗3，或者T细胞疫苗4，或者T细胞疫苗5)或者100μL PBS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。
(8)实验结果
如图3所示，PBS对照组和T细胞疫苗1组小鼠肿瘤生长速度都很快，小鼠生存期很短。与上述两组对照组相比，其他T细胞疫苗组小鼠的肿瘤生长速度都明显变慢，而且部分小鼠肿瘤消失痊愈。T细胞疫苗3和T细胞疫苗5效果最好，T细胞疫苗3和T细胞疫苗5都明显好于T细胞疫苗2和T细胞疫苗4；而且T细胞疫苗3和T细胞疫苗5效果相当。这说明，使用负载癌细胞全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米粒子有利于分离得到更好的癌细胞特异性T细胞；而且DC和B细胞的混合抗原提呈细胞效果好于单一DC。T细胞疫苗5优于T细胞疫苗4说明本发明所述的辅助分离广谱癌细胞特异性T细胞的方法可以不依赖于抗原提呈细胞。而且，在辅助分离得到癌细胞特异性T细胞的过程中，不需要添加抗原提呈细胞即可得到与添加抗原提呈细胞辅助分离癌细胞特异 性T细胞一样的效果，这也是本发明所述的由被激活的抗原提呈细胞制备的纳米粒子或微米粒子辅助分选和扩增癌细胞特异性T细胞的一个优势。
负载癌细胞全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离扩增的癌细胞特异性T细胞对癌症的预防效果优于负载四种抗原多肽的纳米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离和扩增的癌细胞特异性T细胞。这说明负载四种新生抗原多肽纳米粒子激活的抗原提呈细胞所制备的纳米粒子辅助分离的癌细胞特异性T细胞种类有限，因而扩增后的T细胞系统所含有的T细胞克隆数很少，所能识别和杀灭的癌细胞也就较少。而负载癌细胞全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米粒子能辅助分离更广谱的癌细胞特异性T细胞，因而扩增后所能得到的T细胞克隆数也就更广谱，所能识别和杀灭的癌细胞也就越多，治疗或预防癌症的效果也越好。
如图3中c和d所示为5个T细胞疫苗体外激活癌细胞特异性T细胞的情况，负载全细胞组分的纳米粒子激活的抗原提呈细胞制备的纳米粒子所能激活的CD8 +CD134 +T细胞和CD4 +CD134 +T细胞占CD8 +T细胞和CD4 +T细胞的比例明显高于对照组。由此可见，本发明所述的负载全细胞组分的纳米粒子激活的抗原提呈细胞制备的纳米粒子可以更好的辅助分离具有识别癌细胞和杀伤癌细胞能力的癌细胞特异性T细胞。
实施例3分选扩增的癌细胞特异性T细胞后用于黑色素瘤的治疗
本实施例以小鼠黑色素瘤为癌症模型来说明如何使用纳米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离扩增小鼠外周免疫细胞中的癌细胞特异性T细胞后用于治疗黑色素瘤。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抗原混合物(质量比1:1)和非水溶性抗原混合物(质量比1:1)，并将水溶性抗原混合物和非水溶性抗原混合物按质量比1:1混合。然后，以PLGA为纳米粒骨架材料，以Poly(I:C)和CpG2006为佐剂制备负载裂解物组分的纳米粒子，然后将纳米粒子与抗原提呈细胞共孵育一段时间后激活抗原提呈细胞，并将抗原提呈细胞制备成纳米粒子辅助分离激活癌细胞特异性T细胞，并扩增后用于治疗黑色素瘤。
(1)肿瘤组织和癌细胞的裂解及各组分的收集
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抗原；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抗原转化为在8M尿素水溶液中可溶。将肿瘤组织的水溶性抗原和癌细胞的水溶性抗原按质量比1:1混合；肿瘤组织的非水溶性抗原和癌细胞的非水溶性抗原按质量比1:1混合。将水溶性抗原混合物和非水溶性抗原混合物按质量比1:1混合，即为制备纳米粒子的抗原原料来源。
(2)细菌细胞外囊泡(OMV)和癌细胞外囊泡的制备
将长双歧杆菌在5000g离心30分钟，然后弃去沉淀后收集上清液，将上清液使用1μm的滤膜过滤，在4℃下使用20W超声处理5分钟，然后在16000g离心90分钟，将沉淀在PBS中重悬后即为收集到的细菌外囊泡膜组分，然后使用8M尿素水溶液裂解和溶解细菌外囊泡膜组分。
或者将长双歧杆菌在5000g离心30分钟，然后弃去沉淀后收集上清液，将上清液使用1μm的滤膜过滤，在4℃下使用20W超声处理5分钟，然后在16000g离心90分钟，将沉淀在PBS中重悬后即为收集到的细菌外囊泡膜组分，然后使用吐温80水溶液裂解和溶解细菌膜组分。
(3)纳米粒子的制备
本实施例中纳米粒子1采用复乳法制备，制备材料PLGA分子量为7KDa-17KDa，所采用的免疫佐剂为poly(I:C)和CpG2006且佐剂包载于纳米粒子内。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，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备用。纳米粒子1平均粒径为250nm左右，每1mg PLGA纳米粒子1约负载130μg蛋白质或多肽组分，每1mg PLGA纳米粒子1所负载的poly(I:C)和CpG2006免疫佐剂各0.02mg。
本实施例中纳米粒子2制备材料和制备方法同纳米粒子1。纳米粒子2内部同时负载步骤(1)所制备的抗原组分和步骤(2)所制备的8M尿素溶解的细菌外囊泡膜组分，且二者质量比为1:1。所采用的免疫佐剂为poly(I:C)和CpG2006且佐剂包载于纳米粒子内。在制备过程中首先采用复乳法在纳米粒子内部负肿瘤组织载裂解液组分、细菌外囊泡组分和佐剂，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备用。纳米粒子2平均粒径为250nm左右，每1mg PLGA纳米粒子2约负载130μg蛋白质或多肽组分，每1mg PLGA纳米粒2所负载的poly(I:C)和CpG2006免疫佐剂各0.02mg。
本实施例中纳米粒子3制备材料和制备方法同纳米粒1。纳米粒子3内部同时负载步骤(1)所制备的抗原组分和步骤(2)所制备的吐温80溶解的细菌外囊泡膜组分，且二者质量比为1:1。所采用的免疫佐剂为poly(I:C)和CpG2006且佐剂包载于纳米粒子内。在制备过程中首先采用复乳法在纳米粒子内部负肿瘤组织载裂解液组分、细菌外囊泡组分和佐剂，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备用。纳米粒子3平均粒径为250nm左右，每1mg PLGA纳米粒子3约负载130μg蛋白质或多肽组分，每1mg PLGA纳米粒3所负载的poly(I:C)和CpG2006免疫佐剂各0.02mg。
空白纳米粒子4的制备材料和制备方法同纳米粒子1，但是空白纳米粒子4只负载等量的佐剂而不负载任何的肿瘤组织裂解物组分。纳米粒子4的粒径为250nm左右。
(4)B细胞的分离
处死C57BL/6小鼠后摘取小鼠脾脏，制备小鼠脾细胞单细胞悬液，使用磁珠分选法分离脾细胞中的CD19 +B细胞。
(5)抗原提呈细胞的激活
将500μg的纳米粒子1，或者500μg的纳米粒子2，或者500μg的纳米粒子3，或者500μg的纳米粒子4分别与B细胞(1000万个)在15mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)，孵育体系中含GM-CSF(2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)、白蛋白(50ng/mL)和CD80抗体(10ng/mL)。
(6)抗原提呈细胞来源的纳米粒子的制备
通过在400g离心5分钟收集孵育后的B细胞，然后使用PBS洗涤细胞三遍，将细胞重悬在PBS水中后在低功率(10W)超声15分钟。然后将样品在500g离心5分钟并收集上清液，将上清液依次过孔径为30um、10um、5um、0.45um、0.22um的膜过滤后，将所得滤液样品在18000g离心60分钟后弃去上清液并将沉淀使用PBS重悬后即得纳米粒子。其中，使用纳米粒子1激活的抗原提呈细胞制备的纳米粒子为纳米粒子5，粒径为110纳米；使用纳米粒子2激活的抗原提呈细胞制备的纳米粒子为纳米粒子6，粒径为110纳米；使用纳米粒子3激活的抗原提呈细胞制备的纳米粒子为纳米粒子7，粒径为110纳米；使用纳米粒子4激活的抗原提呈细胞制备的纳米粒子为纳米粒子8，粒径为110纳米。
(7)癌细胞特异性T细胞的分离和扩增
在第0天给每只C57BL/6小鼠背部皮下接种5×10 5个B16F10细胞，在第10天，第17天和第24天分别给小鼠皮下注射0.5mg步骤(3)制备的PLGA纳米粒子1。第31天处死小鼠，摘取小鼠脾脏并制备小鼠脾脏单细胞悬液，使用磁珠分选法分选脾脏细胞中活细胞中(使用活死细胞染料标记死细胞以去除死细胞)的CD45 +CD3 +T细胞。将分离得到的T细胞(400万个)与步骤(6)制备的100μg的纳米粒子(纳米粒子5，或者纳米粒子6，或者纳米粒子7，或者纳米粒子8)在40mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO 2)，然后采用流式细胞术分选孵育后的CD3 +CD134 +T细胞，即为被癌细胞全细胞抗原特异性激活的癌细胞特异性T细胞。其中，使用纳米粒子5辅助分选得到的癌细胞特异性T细胞为T细胞疫苗1；使用纳米粒子6辅助分选得到的癌细胞特异性T细胞为T细胞疫苗2；使用纳米粒子7辅助分选得到的癌细胞特异性T细胞为T细胞疫苗3；使用纳米粒子8辅助分选得到的癌细胞特异性T细胞为T细胞疫苗4。
与此同时，在流式细胞术分选T细胞的同时分析脾脏T细胞与不同的纳米粒子在DMEM高糖完全培养基中共孵育48小时后T细胞中CD3 +CD134 +T细胞的比例。
与此同时，将未经纳米粒子辅助分选的脾细胞中的T细胞与步骤(6)制备的不同纳米粒子在DMEM高糖完全培养基中共孵育48小时，然后收集孵育后的细胞并用带有荧光探针的IFN-γ抗体标记孵育后的T细胞，尔后使用流式细胞术分析T细胞中IFN-γ +T细胞所占比例。纳米粒子所负载的癌细胞全细胞抗原在被抗原提呈细胞吞噬后可被降解成抗原表位被提呈到抗原提呈细胞膜表面，抗原提呈细胞制备的纳米粒子负载有上述降解提呈后的抗原表位，可以被癌细胞特异性T细胞识别并激活癌细胞特异性T细胞，被激活后分泌杀伤性细胞因子。IFN-γ是抗原特异性T细胞识别抗原后被激活所分泌的最主要的细胞因子。使用流式 细胞术分析的CD3 +IFN-γ +T细胞即为可以识别和杀伤癌细胞的癌细胞特异性T细胞。
将上述分选得到的癌细胞特异性T细胞疫苗分别与IL-2(2000U/mL)和IL-7(2000U/mL)在DMEM高糖完全培养基中共孵育7天(37℃，5％CO 2，每两天换液一次)以扩增分选得到的癌细胞特异性T细胞疫苗。
(8)癌细胞特异性T细胞用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1.5×10 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、第20天和第25天分别静脉注射200万个癌细胞特异性T细胞疫苗(T细胞疫苗1，或者T细胞疫苗2，或者T细胞疫苗3，或者T细胞疫苗4)或者100μL PBS。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。
(9)实验结果
如图4中a和b所示，PBS对照组和空白纳米粒激活的抗原提呈细胞制备的纳米粒子辅助分离扩增的T细胞疫苗4对照组小鼠的肿瘤生长速度很快，生存期很短。而T细胞疫苗1、T细胞疫苗2和T细胞疫苗3处理的小鼠其肿瘤生长速度明显变慢，而且部分小鼠肿瘤消失痊愈。而且，T细胞疫苗2的效果优于T细胞疫苗1和T细胞疫苗3，这说明使用适当方法裂解和溶解的细菌外囊泡组分负载到纳米粒子后所激活的抗原提呈细胞有利于辅助分离癌细胞特异性T细胞。综上所述，本发明所述的细胞系统对癌症具有优异的治疗效果。
如图4中c和d所示为T细胞疫苗体外激活癌细胞特异性T细胞的情况，使用不同粒子辅助分选时所能激活的癌细胞特异性T细胞的比例高低与a和b图中的疗效相关。而且，使用CD134作为激活的表面标志物分选得到的被激活的癌细胞特异性T细胞比例与遇到癌细胞抗原后可以释放杀伤性细胞因子IFN-γ的T细胞的比例相一致，这说明，使用CD134作为激活的表面标志物分选得到的T细胞就是可以特异性识别和杀伤癌细胞的癌细胞特异性T细胞。由此可见，本发明所述的分离方法可以有效的分选肿瘤组织中具有识别癌细胞和杀伤癌细胞能力的癌细胞特异性T细胞。
实施例4纳米粒子辅助分离扩增的癌细胞特异性T细胞用于预防癌症
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抗原。然后，以PLGA为微米粒骨架材料，以CpG BW006(B类)、CPG2216(A类)和Poly ICLC为免疫佐剂制备负载有癌细胞全细胞抗原的微米粒子系统。使用微米粒子激活抗原提呈细胞后，将抗原提呈细胞制备成纳米粒子辅助分离癌细胞特异性T细胞，并扩增后用于预防癌症。
(1)癌细胞的裂解
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，癌细胞全细胞抗原裂解并溶于6M盐酸 胍后即为制备微米粒子系统的抗原原料来源。
(2)微米粒子的制备
本实施例中微米粒子采用复乳法制备。所采用的微米粒子1制备材料PLGA分子量为38KDa-54KDa，所采用的免疫佐剂为CpG BW006、CPG2216和Poly ICLC。Poly ICLC是toll样受体3激动剂，而各类CpG是Toll样受体9激动剂，而Toll样受体3和Toll样受体9均位于细胞内的内吞体膜结构中。首先将裂解物组分和免疫佐剂共负载于微米粒子内，然后在10000g离心15分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h；在粒子使用前将其用7mL PBS重悬然后加入3mL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微米粒子1。该微米粒子平均粒径为2.50μm左右，表面电位为-2mV左右；每1mg PLGA微米粒子1约负载140μg蛋白质或多肽组分，负载的CpG BW006(B类)、CPG2216(A类)和Poly ICLC各0.02mg。
对照微米粒子2制备材料和制备方法相同，负载的免疫佐剂为CpG2336(A类)、CPG2216(A类)和Poly ICLC。对照微米粒子2粒径为2.50μm左右，表面电位为-2mV左右，每1mg PLGA微米粒子约负载140μg蛋白质或多肽组分，每1mgPLGA微米粒所负载的CpG2336(A类)、CPG2216(A类)和Poly ICLC免疫佐剂各为0.02mg。
对照微米粒子3制备材料和制备方法相同，负载的免疫佐剂为CpG BW006(B类)和CPG2216(A类)。对照微米粒子3每1mgPLGA微米粒所使用的佐剂为0.02mg，粒径为2.50μm左右，表面电位为-2mV左右，每1mg PLGA微米粒子约负载140μg蛋白质或多肽组分，每1mgPLGA微米粒所负载的CpG BW006(B类)和CPG2216(A类)各0.03mg。
(3)抗原提呈细胞的制备
处死小鼠后收集小鼠淋巴结和脾脏，将小鼠淋巴结或者脾脏切碎研磨分别通过细胞筛网过滤制备单细胞悬液，将淋巴结单细胞悬液和脾脏单细胞悬液混合后，使用流式细胞术从中分选出CD19 +B细胞和CD11c +的DC。
(4)抗原提呈细胞的激活
将负载癌细胞全细胞组分的微米粒子(500μg)与制备的DC(1000万个)和B细胞(1000万个)在20mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO2)，孵育体系中含有粒细胞－巨噬细胞集落刺激因子(GM-CSF，2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)和CD86抗体(10ng/mL)。
(5)抗原提呈细胞来源的纳米粒子的制备
通过在400g离心5分钟收集孵育后的DC和B细胞，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(22.5W)超声1分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液，然后在16000g离心90分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米粒子，纳米粒子粒径为110纳米。
(6)癌细胞特异性T细胞的分离和扩增
第0天，在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞。在第10天，第15天，第20天使用射线照射肿瘤部位对小鼠进行射线照射治疗。在第25天处死小鼠，收集各组小鼠的肿瘤组织，将小鼠肿瘤组织切成小块后过细胞筛网，制备单细胞悬液，然后使用磁珠分选法分选肿瘤组织单细胞悬液中的活细胞中(使用活死细胞染料标记死细胞以去除死细胞)的CD3 +T细胞。将分选所得T细胞(500万个)、步骤(5)所制备的纳米粒子(100μg)在2mL RPMI1640完全培养基中共孵育24小时(37℃,5％CO 2)，尔后采用磁珠分选法分选T细胞中的CD69 +T细胞，即为被癌细胞全细胞抗原激活的癌细胞特异性T细胞。将上述分选得到的癌细胞特异性T细胞与IL-2(2000U/mL)、αCD-3抗体(20ng/mL)及αCD-28抗体(20ng/mL)在RPMI1640完全培养基中共孵育7天(两天换液一次)以扩增分选得到的癌细胞特异性T细胞。
(7)癌细胞特异性T细胞扩增后用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在小鼠过继转移细胞前1天，给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠静脉注射100μL含300万个癌细胞特异性T细胞。同时在第0天给每只小鼠皮下注射接种1.5×10 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。
(8)实验结果
如图5所示，对照组小鼠的肿瘤都长大，而使用癌细胞特异性T细胞处理小鼠肿瘤生长速度都明显变慢且生存期明显延长。而且，负载CpG佐剂和Poly ICLC混合佐剂的微米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离扩增的T细胞对黑色素瘤的预防效果优于负载两种CpG混合佐剂的微米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离扩增的T细胞。而且，负载一种B类CpG、一种A类CpG和Poly ICLC混合佐剂的微米粒子激活的抗原提呈细胞制备的纳米粒子效果好于使用负载两种A类CpG和PolyICLC混合佐剂的微米粒子激活的抗原提呈细胞制备的纳米粒子。这说明负载两种不同toll样受体的混合佐剂的微米粒子激活的抗原提呈细胞能制备的纳米粒子效果更好，而且，含有B类CpG与Toll样受体3激动剂作为混合佐剂的微米粒子激活的抗原提呈细胞制备的纳米粒子效果更好。
实施例5癌细胞特异性T细胞用于癌症的预防
本实施例中，首先使用8M尿素裂解B16F10黑色素瘤肿瘤组织，并溶解肿瘤组织裂解物组分。然后，以PLGA为纳米粒骨架材料，以Poly(I:C)、CpG2006(B类)和CpGSL01(B类)为免疫佐剂制备负载有癌细胞全细胞抗原的纳米粒子，使用纳米粒子激活抗原提呈细胞后制备纳米粒子，然后辅助分离肿瘤浸润淋巴细胞中的癌细胞特异性T细胞后，将上述细胞扩增后用于预防癌症。
(1)肿瘤组织的收集及裂解
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8M尿素裂解细胞，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。
(2)纳米粒子的制备
本实施例中纳米粒子采用溶剂挥发法制备。纳米粒子1所采用制备材料PLGA分子量为7KDa-17KDa，所采用的免疫佐剂为Poly(I:C)、CpG2006和CpGSL01，且裂解物组分和佐剂包载于纳米粒子内部。制备方法如前所述，在纳米粒子内部负载裂解物组分和佐剂后，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-3mV左右；每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分，每1mg PLGA纳米粒所使用的Poly(I:C)、CpG2006和CpGSL01各为0.02mg。
对照纳米粒2制备材料和制备方法同上，粒径为270nm左右，负载等量的裂解物组分，负载免疫佐剂为Poly(I:C)，每1mg PLGA负载Poly(I:C)0.06mg。
对照纳米粒子3粒径为270nm左右，负载等量的裂解物组分，负载免疫佐剂为Poly(I:C)、CpG1585(A类)和CpG2216(A类)，每1mg PLGA负载Poly(I:C)、CpG1585(A类)和CpG2216(A类)各0.02mg。
(3)DC和B细胞的制备
处死C57BL/6后摘取小鼠淋巴结，制备小鼠淋巴结单细胞悬液，然后使用流式细胞术从淋巴结细胞单细胞悬液中分选出CD11c +DC和CD19 +B细胞。
(4)抗原提呈细胞的激活
将负载癌细胞全细胞组分的纳米粒子1(500μg)、纳米粒子2(500μg)或纳米粒子3(500μg)与DC(500万个)和B细胞(500万个)在20mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO 2)；孵育体系中含有粒细胞－巨噬细胞集落刺激因子(GM-CSF，2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)和CD86抗体(10ng/mL)。
(5)基于抗原提呈细胞的纳米粒子的制备
通过在400g离心5分钟收集孵育后的DC和B细胞，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃使用匀浆机在2000rpm搅拌破坏处理25分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液，然后在15000g离心30分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米例子，纳米粒子粒径为150纳米。
(6)癌细胞特异性T细胞的分离和扩增
第0天，在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10细胞，在第8天，第10天，第12天，第14天，第16天，第18天，第20天分别给小鼠皮下注射100μL的αPD-1抗体(10mg/kg)。在第24天处死小鼠，分别收集各组小鼠的肿瘤组织，制备肿瘤组织单细胞悬液，然后使用 磁珠分选法分选得到肿瘤组织单细胞悬液中的活细胞中(使用活死细胞染料标记死细胞以去除死细胞)的CD3 +T细胞。然后，将分选得到的T细胞(50万个)与来源于同种异体的B细胞(250万个)、步骤(5)制备的纳米粒子(100μg)在10mL RPMI1640完全培养基中共孵育48小时(37℃,5％CO 2)，然后采用流式细胞术分选孵育后的CD3 +CD8 +CD69 +T细胞和CD3 +CD4 +CD69 +T细胞，即为被癌细胞全细胞抗原激活的癌细胞特异性T细胞。将上述分选得到的癌细胞特异性T细胞与IL-2(2000U/mL)、αCD-3抗体(20ng/mL)以及αCD-28抗体(20ng/mL)在RPMI1640完全培养基中共孵育11天(每两天换液一次)以扩增分选得到的癌细胞特异性T细胞。
(7)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在小鼠癌细胞特异性T细胞移植前1天，给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠皮下注射100μL的80万个扩增得到的癌细胞特异性CD8 +T细胞和20万个扩增得到的癌细胞特异性CD4 +T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。
(8)实验结果
如图6所示，对照组小鼠的肿瘤都长大，而经负载癌细胞全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离扩增得到的癌细胞特异性T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。而且，负载两种B类CpG与Poly(I:C)作为混合佐剂的纳米粒子激活的抗原提呈细胞所制备的纳米粒子辅助分离扩增的T细胞效果好于负载两种A类CpG与Poly(I:C)作为混合佐剂的纳米粒子或者只负载Poly(I:C)作为佐剂的纳米粒子激活的抗原提呈细胞所制备的纳米粒子辅助分离扩增的T细胞。
实施例6癌细胞特异性T细胞用于治疗结肠癌
本实施例以MC38小鼠结肠癌为癌症模型来说明如何使用纳米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离广谱的癌细胞特异性T细胞用于治疗结肠癌。首先裂解结肠癌肿瘤组织和肺癌癌细胞以制备水溶性抗原，并将抗原使用蛋白酶在体外先降解为多肽。在实际应用中也可以使用其它酶或者其它方法先将全细胞组分中的蛋白质降解为多肽。然后再制备水溶性抗原混合物(质量比1:1)和非水溶性抗原(质量比1:1)混合物，并将水溶性抗原混合物和非水溶性抗原混合物按质量比1:1混合。然后，以PLA为纳米粒骨架材料，以CpGM362、CPG1018和和Poly ICLC为免疫佐剂制备纳米粒子，并用该纳米粒子体外激活癌细胞特异性T细胞，然后分离提取扩增癌细胞特异性T细胞用于治疗结肠癌。
(1)肿瘤组织和癌细胞的裂解及各组分的收集
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1000 mm 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抗原；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抗原转化为在8M尿素水溶液中可溶。在水溶性抗原(80mg/mL)中加入胰蛋白酶(Trypsin，0.5mg/mL)和糜蛋白酶(Chymotrypsin，0.5mg/mL)共孵育1小时，然后在95℃加热10分钟灭活蛋白酶备用。
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抗原；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抗原转化为在8M尿素水溶液中可溶。在水溶性抗原(80mg/mL)中加入胰蛋白酶(Trypsin，0.5mg/mL)和糜蛋白酶(Chymotrypsin，0.5mg/mL)共孵育1小时，然后在95℃加热10分钟灭活蛋白酶备用。
将来自结肠癌肿瘤组织的和肺癌癌细胞的水溶性抗原按质量比1:1混合；溶解于8M尿素中的非水溶性抗原也按质量比1:1混合。然后将水溶性抗原混合物和非水溶性抗原混合物按照质量比1:1混合，该混合物为制备纳米粒子的原料来源。
(2)卡介苗(BCG)的裂解和溶解
收集BCG，使用8M尿素水溶液裂解BCG后溶解裂解组分备用。
(3)纳米粒子的制备
本实施例中纳米粒子1采用溶剂挥发法制备。纳米粒子1制备材料PLA分子量为20KDa，纳米粒子内部负载肿瘤组织和癌细胞裂解物、细菌裂解物以及免疫佐剂，表面负载肿瘤组织和癌细胞裂解物组分。所采用的免疫佐剂为CpGM362、CPG1018和poly ICLC，且佐剂负载于纳米粒子内部，制备纳米粒子时使用的肿瘤组织和癌细胞裂解物与细菌裂解物的质量比为1:1。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、细菌裂解物组分和佐剂，然后将100mg纳米粒子在10000g离心20分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h。使用前将20mg纳米粒重悬于0.9mL PBS中，并于0.1mL含有等量癌细胞和肿瘤组织裂解物混合物和细菌裂解物组分(80mg/mL)的样品室温混合孵育5分钟后即可使用。纳米粒子1平均粒径为290nm左右，每1mg PLGA纳米粒子1约负载140μg蛋白质或多肽组分，每1mgPLGA纳米粒含有CpGM362、CPG1018和Poly ICLC免疫佐剂各0.04mg。
(4)抗原提呈细胞的制备
处死C57BL/6后收集小鼠外周血，从外周血中分离外周血单核细胞(PBMC)，然后使用流式细胞术从PBMC中分选出CD11c +DC和CD19 +B细胞。本实施例中同时使用BMDC和BMDM作为抗原提呈细胞。BMDC制备方法同实施例2。BMDM制备方法如下：将C57小鼠麻醉后脱臼处死，将小鼠使用75％乙醇的消毒，然后用剪刀在小鼠背部剪开一小口，用手直接撕开皮肤至小鼠小腿关节处，去除小鼠足关节以及皮肤。用剪刀沿着小鼠大腿根 部大转子将后肢拆下来，去掉肌肉组织后放置在含有75％乙醇的培养皿内浸泡5min，更换新的75％乙醇的培养皿移入超净台中。将乙醇浸泡的腿骨移入冷的PBS浸泡，洗去胫骨、股骨表面的乙醇，此过程可重复3次。将清洗好的股骨、胫骨分开，并用剪刀分别将股骨、胫骨两端剪断，使用1mL注射器吸取冷的诱导培养基将骨髓从股骨、胫骨中吹出，反复吹洗3次，直至腿骨内看不到明显的红色为止。用5mL移液枪将含有骨髓细胞的培养基反复吹打，使细胞团块分散，然后使用70μm细胞滤器将细胞过筛，转移至15mL离心管内，1500rpm/min离心5min，弃上清，加入红细胞裂解液重悬静置5min后1500rpm/min离心5min，弃上清用冷的配置好的骨髓巨噬细胞诱导培养基(含有15％L929培养基的DMEM高糖培养基)重悬，铺板。将细胞培养过夜，以去除贴壁较快的其他杂细胞如纤维细胞等等。收集未贴壁细胞按实验设计安排种入皿或细胞培养板内。巨噬细胞集落刺激因子(M-CSF)以40ng/mL浓度刺激使骨髓细胞向单核巨噬细胞分化。培养8天，光镜下观察巨噬细胞形态变化。8天后消化收集细胞，用抗小鼠F4/80抗体和抗小鼠CD11b抗体，4℃避光孵育30min后，使用流式细胞术鉴定所诱导成功的巨噬细胞的比例即可。
(5)抗原提呈细胞的激活
将纳米粒子1(1000μg)与外周血来源的DC(2000万个)、BMDC(2000万个)在RPMI1640完全培养基中共孵育72小时(37℃，5％CO 2)；孵育体系中含有GM-CSF(500U/mL)、IL-2(500U/mL)、IL-7(500U/mL)、IL-12(500U/mL)和CD80抗体(10ng/mL)。
或者将纳米粒子1(1000μg)与外周血来源的DC(1000万个)、BMDC(1000万个)、BMDM(1000万个)、B细胞(1000万个)在20mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)，孵育体系中含有GM-CSF(500U/mL)、IL-2(500U/mL)、IL-7(500U/mL)、IL-12(500U/mL)和CD80抗体(10ng/mL)。
(6)基于抗原提呈细胞的纳米粒子的制备
通过在400g离心5分钟收集孵育后的外周血来源的DC(2000万个)和BMDC(2000万个)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在高压均质机中(5000bar)中处理5分钟。然后将样品在2000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液，将上清液与步骤(3)所制备的纳米粒子1在4℃共孵育16小时，然后使用0.45μm的滤膜反复共挤出，将挤出液在13000g离心20分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米粒子2，粒径为310纳米。
或者通过在400g离心5分钟收集孵育后的外周血来源的DC(1000万个)、BMDC(1000万个)、B细胞(1000万个)及BMDM(1000万个)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在高压均质机中(5000bar)中处理5分钟。然后将样品在2000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液，将上清液与步骤(3)所制备的纳米粒子1(50mg)在4℃共孵育16小时，然后使用0.45μm的滤膜反复共挤出，将挤出液在13000g离心20分钟后弃去上清液收集沉淀， 将沉淀在PBS中重悬后即得纳米粒子3，粒径为310纳米。
(7)癌细胞特异性T细胞的分离和扩增
第0天每只C57BL/6小鼠背部皮下接种1.5×10 5个MC38细胞，在第10天，第15天和第21天分别给小鼠皮下注射100μL的1mg PLGA纳米粒子1。在第24天处死小鼠，收集小鼠的引流淋巴结和脾脏，制备引流淋巴结和脾细胞的单细胞悬液并使用磁珠法从中分选出T细胞。将所得T细胞(400万个)、200μg的纳米粒子(纳米粒子1，或者纳米粒子2，或者纳米粒子3)、IL-2(500U/mL)、IL-7(500U/mL)及IL-15(5000U/mL)在5mLDMEM完全培养基中共孵育96小时，然后采用流式细胞术分选孵育后的CD3 +CD8 +CD69 +T细胞，即为被癌细胞全细胞抗原激活的癌细胞特异性T细胞。将上述分选得到的癌细胞特异性T细胞与IL-2(1000U/mL)、IL-7(200U/mL)、IL-15(200U/mL)和αCD-3抗体(10ng/mL)在DMEM完全培养基中共孵育8天(每两天换液一次)以扩增分选得到的癌细胞特异性CD8 +T细胞。其中，使用纳米粒子1辅助分选和扩增的癌细胞特异性T细胞为T细胞疫苗1；使用纳米粒子2辅助分选和扩增的癌细胞特异性T细胞为T细胞疫苗2；使用纳米粒子3辅助分选和扩增的癌细胞特异性T细胞为T细胞疫苗3。
(8)癌细胞特异性CD8 +T细胞用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备结肠癌荷瘤小鼠。在第0天给每只小鼠皮下接种2×10 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200万个癌细胞特异性CD8 +T细胞。小鼠肿瘤体积及生存期监测方法同上。
(9)实验结果
如图7所示，PBS对照组和T细胞疫苗1组的小鼠的肿瘤都生长很快小鼠生存期很短。与上述两组相比，T细胞疫苗2和T细胞疫苗3处理的小鼠其肿瘤生长速度都明显变慢，而且部分小鼠肿瘤消失痊愈。而且，T细胞疫苗3效果好于T细胞疫苗2。这说明粒子表面负载被激活的抗原提呈细胞膜组分以及使用混合抗原提呈细胞膜组分均可以提高纳米粒子或微米粒子辅助分离和扩增癌细胞特异性T细胞的效果。综上所述，本发明所述T细胞疫苗对结肠癌具有良好的治疗效果。
实施例7癌细胞特异性T细胞用于乳腺癌的预防
本实施例以4T1小鼠三阴性乳腺癌为癌症模型来说明如何负载癌细胞全细胞抗原的微米粒子激活抗原提呈细胞后制备成微米粒子辅助分离来自外周血癌细胞特异性T细胞并用于预防乳腺癌。
(1)癌细胞的裂解
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，然后反复冻融5遍并伴有超声以裂解癌细胞。在所裂解系细胞中加入1mg/mL的核酸酶降解裂解物中的核酸，然后在95℃加热10分钟灭活核酸酶，然后在5000g离心5分钟收集上清液即为水溶性抗原组分，沉淀使用10％脱氧胆酸钠(含10M精氨酸)溶解即为非水溶性抗原组分，将水溶性抗原组分与非水溶性抗原组分按质量比3：1混合即为制备粒子系统的原料来源。
(2)微米粒子系统的制备
本实施例中制备微米粒子采用复乳法。微米粒子1骨架材料PLGA分子量为38KDa-54KDa，所采用的免疫佐剂为CpG2395(C类)、CpGM362(C类)和Poly(I:C)。制备时采用复乳法制备内部负载裂解物组分和佐剂的微米粒子,然后将100mg微米粒子在9000g离心20分钟，使用10mL含4％海藻糖的超纯水重悬后干燥48h后备用。该微米粒子系统平均粒径为2.5μm左右，表面电位为-6mV左右；每1mg PLGA微米粒子约负载110μg蛋白质或多肽组分，负载CpG2395、CpGM362和Poly(I:C)各0.02mg。对照微米粒子2制备材料和制备方法同上，粒径为2.5μm左右，表面电位为-6mV左右；每1mg PLGA微米粒子约负载110μg蛋白质或多肽组分，每1mgPLGA负载CpG1585(A类)、CpG2336(A类)和Poly(I:C)各0.02mg。
(3)B细胞的制备
使用来自外周脾细胞中的B细胞。处死小鼠后摘取脾脏，然后制备小鼠脾细胞单细胞悬液，使用磁珠分选法分选单细胞悬液中的CD19 +B细胞。
(4)抗原提呈细胞的激活
将负载癌细胞全细胞抗原组分的微米粒子(800μg)与步骤(3)制备的B细胞(1000万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO 2)，孵育体系中含有GM-CSF(2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)和CD86抗体(10ng/mL)。
(5)基于抗原提呈细胞的微米粒子的制备
通过在400g离心5分钟收集孵育后的B细胞(1000万个)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声1分钟后使用匀浆机在1000rpm处理3分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液，将上清液与步骤(2)所制备的微米粒子(60mg)以及DSPE-PEG-甘露糖(1mg)在100W超声处理2分钟，然后在8000g离心20分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得微米粒子，粒径为2.6μm。
(6)癌细胞特异性T细胞的分离和扩增
第0天每只BALB/c小鼠背部皮下接种1×10 6个4T1细胞，在第10天，第17天和第24天分别给小鼠皮下注射100μL的1mg PLGA微米粒子。在第30天处死小鼠，收集小鼠的外周血，从外周血中分离外周血单个核细胞(PBMC)，然后使用流式细胞术从PBMC中分离CD3 +T细胞。将T细胞(100万个)、DC2.4(200万个)以及微米粒子(50μg)在2mL DMEM完全培养基中共孵育72小时(37℃，5％CO 2)，然后采用流式细胞术从中分选出CD3 +CD69 +T细胞，即为被癌细胞全细胞抗原激活的癌细胞特异性T细胞。将上述分选得到的癌细胞特异性T细胞分别与IL-2(4000U/mL)、IL-7(2000U/mL)和αCD-3抗体(20ng/mL)在DMEM完全培养基中共孵育12天(每两天换液一次)扩增分选得到的癌细胞特异性T细胞。
(4)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。在小鼠过继转移细胞前1天，给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠皮下注射100μL的150万个扩增的癌细胞特异性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。小鼠肿瘤生长和生存期监测方法同上。
(5)实验结果
如图8所示，与对照组相比，微米粒子激活的抗原提呈细胞所制备的微米粒子辅助分离得到的癌细胞特异性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，使用两种C类CpG与Poly(I:C)作为混合佐剂的微米粒子激活的抗原提呈细胞制备的微米粒子效果好于两种A类CpG与Poly(I:C)作为混合佐剂的微米粒子激活的抗原提呈细胞制备的微米粒子。本实施例微米疫苗中使用甘露糖作为主动靶向的靶头，在实际应用中也可以使用CD32抗体、甘露聚糖、CD205抗体、CD19抗体等任何具有靶向靶细胞能力的靶头。
实施例8癌细胞特异性T细胞用于癌症的预防
本实施例以甘露糖为靶头说明如何使用主动靶向纳米粒激活抗原提呈细胞制备的纳米粒子辅助分离癌细胞特异性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。主动靶向纳米粒子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。
(1)癌细胞的裂解
收集培养的Pan02胰腺癌癌细胞后采用10％辛基葡萄糖苷裂解癌细胞和溶解来源于癌细胞的癌细胞全细胞抗原。
(2)纳米粒子的制备
本实施例中纳米粒子系统使用复乳法制备。纳米粒子制备材料为PLGA和甘露糖修饰的PLGA，二者分子量都为7KDa-17KDa。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。所采用的免疫佐剂为Poly(I:C)和CpG SL03。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。带有靶头的纳米粒子1的平均粒径均为270nm左右，每1mg PLGA纳米粒子约负载80μg蛋白质和多肽组分，含Poly(I:C)和CpGSL03各0.04mg。不负载佐剂但带有甘露糖靶头的纳米粒子2粒径也为270nm左右，制备时采用等量细胞裂解组分但是不含任何免疫佐剂，每1mg PLGA纳米粒子约负载80μg蛋白质和多肽组分。
(3)抗原提呈细胞的制备
本实施例使用BMDC和BMDM作为抗原提呈细胞。BMDC和BMDM制备方法同上。
(4)抗原提呈细胞的激活
将纳米粒子1(1000μg)或者纳米粒子2(1000μg)分别与BMDC(1000万个)、BMDM (1000万个)及IL-7(500U/mL)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO 2)。或者将BMDC(1000万个)、BMDM(1000万个)及IL-7(500U/mL)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)。上述两种孵育系统中都含有IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)、IFN-γ(500U/mL)和CD80抗体(10ng/mL)。
(5)抗原提呈细胞来源的纳米粒子的制备
通过在400g离心5分钟收集孵育后的DC和巨噬细胞，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(10W)超声20分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液依次过孔径为30μm、10μm、5μm、2μm、1μm、0.45μm、0.22μm的膜过滤后，收集滤液，然后将滤液在18000g离心50分钟后收集弃去上清液收集沉淀，将沉淀在4％海藻糖水溶液中重悬，尔后冷冻干燥48小时后即得纳米粒子。其中，使用未被纳米粒子激活的混合抗原提呈细胞制备的为纳米粒子3，粒径为110纳米；使用纳米粒子2激活的混合抗原提呈细胞制备的为纳米粒子4，粒径为110纳米；使用纳米粒子1激活的混合抗原提呈细胞制备的为纳米粒子5，粒径为110纳米。
(6)癌细胞特异性T细胞的分离和扩增
在第0天给每只C57BL/6小鼠背部皮下接种1×10 6个Pan02胰腺癌细胞，第10天，第15天、第20天和第27天分别给小鼠皮下注射100μL的1mg PLGA纳米粒子。第24天处死小鼠并摘取小鼠肿瘤组织和淋巴结。将小鼠肿瘤组织和淋巴结分别制备成单细胞悬液。然后从肿瘤组织单细胞悬液中和淋巴结单细胞悬液中分别使用流式细胞术分离CD45 +CD3 +T细胞，将来自肿瘤组织的和淋巴结的T细胞混合。然后将T细胞(500万个)与100μg纳米粒子(纳米粒子1，或者纳米粒子3，或者纳米粒子4，或者纳米粒子5)在DMEM高糖培养基中共孵育72小时(37℃，5％CO 2)，孵育体系中含有IL-2(500U/mL)、IL-7(500U/mL)和IFN-γ(500U/mL)。然后采用流式细胞术从孵育后细胞中分选出CD3 +CD69 +T细胞，即为癌细胞特异性T细胞。将上述分选得到的T细胞与IL-2(2000U/mL)、IL-7(2000U/mL)、IL-15(1000U/mL)和αCD-3抗体(50ng/mL)在DMEM高糖培养基中共孵育12天(每两天换液一次)扩增所得癌细胞特异性T细胞。其中，使用纳米粒子1辅助分选和扩增的癌细胞特异性T细胞为T细胞疫苗1；使用纳米粒子3辅助分选和扩增的癌细胞特异性T细胞为T细胞疫苗2；使用纳米粒子4辅助分选和扩增的癌细胞特异性T细胞为T细胞疫苗3；使用纳米粒子5辅助分选和扩增的癌细胞特异性T细胞为T细胞疫苗4。
(7)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备胰腺癌荷瘤小鼠，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。然后，将步骤500万个癌细胞特异性T细胞(T细胞疫苗1，或者T细胞疫苗2，或者T细胞疫苗3，或者T细胞疫苗4)或100μL PBS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1×10 6个Pan02胰腺癌细胞。监测小鼠肿瘤生长速度和小鼠生存期。肿瘤生长和生存 期监测方法同上。
(8)实验结果
如图9所示，PBS组、T细胞疫苗1及T细胞疫苗2处理组小鼠肿瘤生长很快，小鼠很快死亡。与上述几组相比，T细胞疫苗3和T细胞疫苗4处理的小鼠其肿瘤生长速度明显变慢。而且，T细胞疫苗4效果好于T细胞疫苗3。综上所述，不管是否被带有佐剂的纳米粒子激活的抗原提呈细胞，其所制备的纳米粒子都可以有效辅助分离肿瘤浸润淋巴细胞中的癌细胞特异性T细胞，但是带有佐剂的纳米粒子激活的抗原提呈细胞制备的纳米粒子效果更佳。这说明本发明所述的癌细胞特异性T细胞可以有效预防癌症。
实施例9纳米粒子辅助分离癌细胞特异性T细胞用于预防肺癌
本实施例说明钙化的纳米粒子辅助分离肿瘤浸润淋巴细胞中的癌细胞特异性T细胞，在实际使用时也可以使用其他生物矿化技术、交联、凝胶化等修饰粒子。本实施例中，将小鼠肺癌肿瘤组织以8M尿素(含200mM氯化钠)裂解后溶解并负载于纳米粒子系统，使用该粒子激活抗原提呈细胞后，将抗原提呈细胞制备成纳米粒子，辅助分离肿瘤组织浸润淋巴细胞和外周血中的癌细胞特异性T细胞并扩增后用于肺癌的预防。
(1)肿瘤组织和癌细胞的裂解
在6-8周的雌性C57BL/6小鼠后背接种1×10 6个LLC小鼠肺癌细胞，待肿瘤体积长到1000mm 3时处死小鼠摘取小鼠肿瘤组织，将肿瘤组织切块研磨后通过细胞筛网过滤制备成单细胞悬液，使用紫外照射5分钟后在80℃高温加热10分钟，然后采用8M尿素(含200mM氯化钠)裂解和溶解肿瘤组织单细胞悬液，即得癌细胞全细胞抗原。
(2)纳米粒子的制备
本实施例在纳米粒子内部和表面负载癌细胞全细胞抗原后生物钙化纳米粒子。本实施例中纳米粒子采用溶剂挥发法制备，所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa，所采用免疫佐剂CpG2006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，然后将100mg PLGA纳米粒子在13000g离心20min后使用18mL PBS重悬，然后加入2mL溶解于8M尿素的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20mL DMEM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，CpG2006和Poly(I:C)各0.03mg。
(3)抗原提呈细胞的制备
本实施例使用BMDC和B作为抗原提呈细胞。BMDC制备方法同实施例1。B细胞来自小鼠外周血PBMC，制备方法同上。
(4)抗原提呈细胞的激活
将负载癌细胞全细胞组分的纳米粒子(1000μg)与BMDC(500万个)及B细胞(500 万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO 2)；孵育体系中含有GM-CSF(2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)、IFN-γ(500U/mL)和CD80抗体(10ng/mL)和CD40抗体(20mg/mL)或者作为对照在孵育体系中不含有任何细胞因子和抗体。
(5)基于抗原提呈细胞的纳米粒子的制备
通过在400g离心5分钟收集孵育后的DC和B细胞，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在5000g离心10分钟后收集上清液，将上清液通过0.45μm的膜过滤后使用超滤膜(截留分子量50KDa)超滤离心过滤和浓缩，将过滤和浓缩后的样品与步骤(2)制备的纳米粒子混合后使用高压均质机(10000bar)处理3分钟，然后在13000g离心30分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米粒子，粒径为300纳米。
(6)癌细胞特异性T细胞的分离和扩增
在第0天给每只C57BL/6小鼠背部皮下接种1×10 6个LLC肺癌细胞，第10天，第15天、第20天和第27天分别给小鼠皮下注射100μL的1mg PLGA纳米粒子。第24天处死小鼠并摘取小鼠肿瘤组织和收集小鼠外周血。将小鼠肿瘤组织制备成单细胞悬液并从肿瘤组织单细胞悬液中使用流式细胞术分离CD45 +CD3 +T细胞；从外周血中分离PBMC，并使用流式细胞术从PBMC中分离CD45 +CD3 +T细胞；将来自肿瘤组织和外周血的T细胞混合。然后将T细胞(500万个)与抗原提呈细胞制备的纳米粒子(100μg)在DMEM高糖培养基中共孵育72小时(37℃，5％CO 2)，在孵育过程中系统中含有IL-2(500U/mL)和IL-7(500U/mL)；或者T细胞(500万个)与抗原提呈细胞制备的纳米粒子(100μg)在DMEM高糖培养基中共孵育72小时(37℃，5％CO2)，且孵育体系中不含任何细胞因子或抗体。然后采用流式细胞术从孵育后细胞中分选出CD3 +CD69 +T细胞，即为癌细胞特异性T细胞。将上述分选得到的T细胞与IL-2(2000U/mL)、IL-7(2000U/mL)、IL-15(1000U/mL)和αCD-3抗体(50ng/mL)在DMEM高糖培养基中共孵育12天(每两天换液一次)扩增T细胞。
(7)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备肺癌荷瘤小鼠，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。然后，将步骤500万个癌细胞特异性T细胞静脉注射给受体小鼠。隔天，给每只受体小鼠背部右下方皮下接种1×10 6个LLC肺癌细胞。监测小鼠肿瘤生长速度和小鼠生存期。肿瘤生长和生存期监测方法同上。
(8)实验结果
如图10所示，与对照组相比，钙化纳米粒激活的抗原提呈细胞制备的纳米粒子辅助分离扩增得到的癌细胞特异性T细胞可以延长小鼠生存期有效预防癌症。而且，负载癌细胞全细胞抗原纳米粒子激活抗原提呈细胞时体系中含有细胞因子和/或抗体优于体系中不 含细胞因子和/或抗体的；而且，抗原提呈细胞制备的纳米粒子与T细胞共孵育时体系中含有细胞因子和/或抗体的优于体系中不含有细胞因子和/或抗体的。
实施例10癌细胞特异性T细胞后用于黑色素瘤的治疗
(1)肿瘤组织和癌细胞的裂解及各组分的收集
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，加入超纯水后反复冻融并伴有超声裂解上述细胞，然后加入核酸酶(0.5mg/mL)、胰蛋白酶(Trypsin，0.5mg/mL)和糜蛋白酶(Chymotrypsin，0.5mg/mL)作用15分钟，再在95℃作用10分钟灭活核酸酶。尔后在8000g离心3分钟，上清液部分即为水溶性抗原；沉淀部分使用10％脱氧胆酸钠水溶液溶解非水溶性抗原。将水溶性抗原和脱氧胆酸钠溶解后的非水溶性抗原按质量比1:1混溶即为制备纳米粒子系统的抗原原料来源。
(2)纳米粒子系统的制备
本实施例中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1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9:1。所采用的免疫佐剂为poly(I:C)、CpG1018和CpG2216，增加溶酶体免疫逃逸的物质为KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)，且佐剂、KALA多肽包载于纳米粒子内。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1内部负载裂解液组分、佐剂、KALA多肽，然后将100mg纳米粒子在12000g离心25分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h。该纳米粒子1平均粒径为250nm左右，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)、CpG1018和CpG2216免疫佐剂各0.02mg,负载KALA多肽0.05mg。纳米粒子2的制备材料和方法与纳米粒子1相同，其粒径为250nm左右，表面电位为-5mV左右，纳米粒子2不负载KALA多肽，但是负载等量佐剂和细胞裂解组分。纳米粒子3的制备材料和制备方法与纳米粒子1相同，为250nm左右，表面电位为-5mV左右，每1mg PLGA纳米粒子约负载100μg蛋白质和多肽组分，每1mg PLGA纳米粒所负载的poly(I:C)0.02mg,负载CpG1018为0.04mg,负载KALA多肽0.05mg。
(3)抗原提呈细胞的制备
本实施例使用BMDC和BMDM作为混合抗原提呈细胞。BMDC和BMDM制备方法同上。
(4)抗原提呈细胞的激活
将负载癌细胞全细胞组分的纳米粒子(1000μg)与BMDC(1000万个)和BMDM(1000万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO 2)；孵育体系中含有GM-CSF(2000U/mL)、M-CSF(2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)、,IFN-γ(500U/mL)和CD80抗体(10ng/mL)。
(5)基于抗原提呈细胞的纳米粒子的制备
通过在400g离心5分钟收集孵育后的BMDC和BMDM，然后使用含有0.0759M蔗糖和0.225M甘露醇的30mM pH 7.0Tris-HCl缓冲液中1200rpm 3min离心清洗三次，然后在磷酸酶抑制剂和蛋白酶抑制剂的存在下超声3分钟(25W)机械破坏抗原提呈细胞。经过离心后所获细胞膜用10mM pH 7.5的Tris-HCl和1mM EDTA的溶液清洗。然后将样品依次过孔径为30μm、10μm、5μm、2μm、0.45μm的膜过滤后，将滤液在12000g离心45分钟后弃去上清液收集沉淀，将沉淀在含有的4％甘露醇的生理盐水中重悬后冷冻干燥，即得纳米粒子。其中使用纳米粒子1激活的抗原提呈细胞制备的纳米粒子为纳米粒子4，粒径为260纳米；使用纳米粒子2激活的抗原提呈细胞制备的纳米粒子为纳米粒子5，粒径为260纳米；使用纳米粒子3激活的抗原提呈细胞制备的纳米粒子为纳米粒子6，粒径为260纳米。
(6)癌细胞特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠，在第0天、第7天、第14天、第21天和第28天分别给小鼠皮下注射0.5mg的PLGA纳米粒子(负载裂解物组分、Poly(I:C)和两种CpG佐剂及KALA多肽)。在第32天处死小鼠,收集小鼠的外周血和淋巴结。使用梯度离心法分离小鼠外周血中的PBMC，将淋巴结切成小块后研磨通过细胞筛网制备单细胞悬液，尔后把PBMC和淋巴结细胞单细胞悬液混合。然后使用磁珠分选法分选出CD45 +CD3 +的T细胞。将分选得到的CD3 +T细胞(500万个)和40μg纳米粒子(纳米粒子4、或者纳米粒子5、或者纳米粒子6)以及IL-7(10ng/mL)在2mL RPMI1640完全培养基中共孵育96小时。然后采用流式细胞术分选孵育后的T细胞中的CD3 +OX40 +T细胞，即为可识别癌细胞全细胞抗原的癌细胞特异性T细胞。将上述分选得到的CD3 +OX40 +T细胞与IL-2(1000U/mL)、IL-15(1000U/mL)、IL-21(1000U/mL)以及αCD-3抗体(20ng/mL)在RPMI1640完全培养基中共孵育14天(每两天换液一次)以扩增癌细胞特异性T细胞。其中，由纳米粒子4辅助分选扩增的癌细胞特异性T细胞为T细胞疫苗1；由纳米粒子5辅助分选扩增的癌细胞特异性T细胞为T细胞疫苗2；由纳米粒子6辅助分选扩增的癌细胞特异性T细胞为T细胞疫苗3。
(7)扩增后的癌细胞特异性T细胞用于治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1.5×10 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150万个扩增后的癌细胞特异性T细胞。在实验中，小鼠肿瘤体积和生存期监测方法同上。
(8)实验结果
如图11所示，PBS对照组的肿瘤很快都长大。与对照组相比，被负载全细胞组分的纳米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离扩增的癌细胞特异性T细胞处理的小鼠肿瘤生长速度明显变慢生存期明显延长。而且，加入增加溶酶体逃逸物质的纳米粒子1激活的抗原提呈细胞制备的纳米粒子4辅助分离和扩增的癌细胞特异性T细胞疫苗1好于未加入溶酶体逃逸的纳米粒子2激活的抗原提呈细胞制备的纳米粒子5辅助分离和扩增的癌细胞特异性T细胞疫苗2；使用两种CpG和Poly(I:C)作为混合佐剂的纳米粒子1激活的抗原提 呈细胞制备的纳米粒子4辅助分离和扩增的癌细胞特异性T细胞疫苗1的治疗效果好于只使用一种CpG和Poly(I:C)混合佐剂的纳米粒子3激活的抗原提呈细胞制备的纳米粒子6辅助分选扩增的T细胞疫苗3。综上所述，本发明所述的T细胞对癌症具有良好的治疗效果。
实施例11癌细胞特异性T细胞用于乳腺癌的预防
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癌细胞全细胞抗原的微米粒子激活的抗原提呈细胞制备的纳米粒子辅助分选癌细胞特异性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抗原。然后，以PLGA为微米粒子骨架材料，以CpG2007、CpG1018、和Poly ICLC为免疫佐剂，以聚精氨酸和聚赖氨酸为增强溶酶体逃逸的物质，制备负载有癌细胞全细胞抗原的微米粒子，并使用该粒子激活抗原提呈细胞后制备纳米粒子，使用纳米粒子辅助分离和扩增癌细胞特异性T细胞并用于癌症预防。
(1)癌细胞的裂解
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60℃高温加热进行灭活和变性处理5分钟，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，上清液即为水溶性抗原，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抗原，将水溶性抗原和非水溶性抗原按质量比2:1混合，即为制备微米粒子所需的裂解物组分。
(2)微米粒子系统的制备
本实施例中制备微米粒子采用复乳法，微米粒子1骨架材料PLGA分子量为38KDa-54KDa，所采用的免疫佐剂为CpG2007、CpG1018和Poly ICLC，所采用的溶酶体逃逸增加物质为聚精氨酸和聚赖氨酸。制备时先采用复乳法制备内部负载裂解物组分、佐剂和KALA多肽的微米粒子,然后将100mg微米粒子在9000g离心20分钟，使用10mL含4％海藻糖的超纯水重悬后干燥48h后备用。该微米粒子1平均粒径为1.5μm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CpG2007、CpG1018和Poly ICLC各0.01mg，含聚精氨酸和聚赖氨酸各0.02mg。
(3)抗原提呈细胞的制备
本实施例使用BMDC和小鼠脾细胞来源的B细胞作为抗原提呈细胞。BMDC及B细胞制备方法同上。将BMDC和B细胞按数量比1:1混合后即为混合抗原提呈细胞。
(4)混合抗原提呈细胞的激活
将负载癌细胞全细胞组分的微米粒子(1000μg)与2000万个混合抗原提呈细胞(含BMDC1000万个+DC2.4细胞1000万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO 2)；孵育体系中含有GM-CSF(500U/mL)、M-CSF(500U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)、IFN-γ(500U/mL)和CD80抗体(10ng/mL)。
(5)抗原提呈细胞来源的微米粒子的制备
通过在400g离心5分钟收集孵育后的混合抗原提呈细胞2000万个，然后使用含有蛋白 酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在5000g离心10分钟后收集上清液，将上清液通过0.45μm的膜过滤后使用超滤膜(截留分子量50KDa)超滤离心过滤和浓缩，将过滤和浓缩后的样品与40mg步骤(2)制备的纳米粒子共孵育10分钟后使用2μm的滤膜反复挤出，然后将挤出液在10000g离心20分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得微米粒子2，粒径为1.6μm。
(6)癌细胞特异性T细胞的制备
选取6-8周的雌性BALB/c小鼠，在第0天小鼠后背皮下接种2×10 6个4T1乳腺癌细胞；在第7天、第14天、第21天和第28天分别皮下注射0.3mg PLGA的微米粒子(负载裂解物组分、佐剂及增加溶酶体逃逸的物质)。在第32天处死小鼠,收集小鼠肿瘤组织，将肿瘤组织切成小块后通过细胞筛网制备单细胞悬液。使用流式细胞术从肿瘤组织单细胞悬液中分选CD3 +的肿瘤浸润T细胞。将分选得到的CD3 +T细胞(100万个)、100μg微米粒子2以及IL-7(500U/mL)在5mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)；或者将分选得到的CD3 +T细胞(100万个)、100μg微米粒子2在5mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)；或者将分选得到的CD3 +T细胞(100万个)、100万个步骤(3)制备的BMDC、100μg微米粒子1以及IL-7(500U/mL)在5mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)。然后采用流式细胞术分选孵育后的CD3 +T细胞中的CD8 +CD69 +T细胞以及CD4 +CD69 +T细胞，即为可识别癌细胞全细胞抗原的癌细胞特异性T细胞。将上述分选得到的CD8 +CD69 +T细胞或者CD4 +CD69 +T细胞分别与IL-2(1000U/mL)、IL-6(1000U/mL)、IL-12(1000U/mL)以及αCD28抗体(10ng/mL)在RPMI1640完全培养基中共孵育14天以扩增癌细胞特异性T细胞。其中，使用CD3 +T细胞、微米粒子2及IL-7共孵育分选得到的癌细胞特异性T细胞为T细胞疫苗1；使用CD3 +T细胞及微米粒子2共孵育分选得到的癌细胞特异性T细胞为T细胞疫苗2；使用CD3 +T细胞、DC、微米粒子1以及IL-7共孵育分选得到的癌细胞特异性T细胞为T细胞疫苗3。
(7)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。在小鼠过继转移细胞前1天,给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠皮下注射100μL T细胞疫苗(内含100万个扩增得到的CD8 +T细胞以及20万个扩增得到的CD4 +)或者100μL PBS。同时在第0天给每只小鼠皮下注射接种1×10 6个4T1细胞。小鼠肿瘤生长和生存期监测方法同上。
(8)实验结果
如图12所示，与PBS对照组相比，微米粒子激活的抗原提呈细胞制备的微米粒子辅助分选得到的癌细胞特异性T细胞疫苗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，在抗原提呈细胞制备成的纳米粒子与含有T细胞的免疫细胞共孵育过程中加入IL-7辅助分选得到的癌细胞特异性T细胞疫苗1效果好于共孵育过程中不加IL-7辅助分选得到的 癌细胞特异性T细胞疫苗2。而且，T细胞疫苗1的效果好于T细胞疫苗3。这说明在共孵育中加入细胞因子IL-7有利于辅助分离癌细胞特异性T细胞，而且，被激活的混合抗原提呈细胞制备成的微米粒子在无抗原提呈细胞的情况下辅助分离得到的癌细胞特异性T细胞效果好于使用负载全细胞组分的纳米粒子与DC+T细胞共孵育分选得到的癌细胞特异性T细胞。由此可见，本发明所述的癌细胞特异性T细胞疫苗对乳腺癌具有预防效果。
实施例12癌细胞特异性T细胞用于乳腺癌的预防
(1)癌细胞及细菌外囊泡的裂解
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。
将长嗜酸乳杆菌在5000g离心30分钟，然后弃去沉淀后收集上清液，将上清液使用1μm的滤膜过滤，然后在16000g离心90分钟，将沉淀使用8M尿素水溶液(含500mM氯化钠)裂解和溶解细菌外囊泡组分。
(2)微米粒子的制备
本实施例中制备微米粒子采用复乳法。微米粒子1骨架材料为未修饰的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4:1。所采用的免疫佐剂为CpG2006、CpG2216和Poly ICLC，所采用的溶酶体逃逸增加物质为精氨酸和组氨酸。微米粒子制备时所使用的癌细胞裂解物组分和细菌外囊泡组分质量比为1:1。制备时先采用复乳法制备内部负载癌细胞裂解物组分、细菌外囊泡组分、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后即得微米粒子1，平均粒径为1.5μm左右，每1mg PLGA微米粒子1约负载100μg蛋白质或多肽组分，含CpG2006，CpG2216和Poly ICLC各0.02mg，含精氨酸和组氨酸各0.05mg。对照微米粒子2制备材料和制备方法同微米粒子1，粒径为1.5μm左右，负载等量精氨酸、组氨酸和等量的癌细胞裂解物组分和细菌外囊泡组分，但是不负载任何佐剂。
(3)抗原提呈细胞的制备
本实施例使用BMDC、B细胞以及BMDM作为抗原提呈细胞。BMDC及BMDM制备制备方法同上。B细胞来自小鼠外周血PBMC，制备方法同上。将BMDC、B细胞和BMDM按数量比2:1:1混和后即为混合抗原提呈细胞。
(4)抗原提呈细胞的激活
将1000μg的微米粒子1或微米粒子2分别与4000万个混合抗原提呈细胞(含2000万个BMDC，1000万个B细胞及1000万个BMDM)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO 2)；孵育体系中含有GM-CSF(2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)、IFN-γ(500U/mL)和CD80抗体(10ng/mL)和CD40抗体(20mg/mL)。
(5)抗原提呈细胞来源的粒子的制备
通过在400g离心5分钟收集孵育后的4000万个混合抗原提呈细胞，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在5000g离心10分钟后收集上清液，将上清液通过0.45μm的膜过滤后使用超滤膜(截留分子量50KDa)超滤离心过滤和浓缩，将过滤和浓缩后的样品使用高压均质机(10000bar)处理3分钟，然后在13000g离心30分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米粒子。其中，使用微米粒子1激活的混合抗原提呈细胞制备的为纳米粒子1，粒径为250纳米；使用微米粒子2激活的混合抗原提呈细胞制备的为纳米粒子2，粒径为250纳米。
通过在400g离心5分钟收集与微米粒子1或者微米粒子2孵育后的4000万个混合抗原提呈细胞，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在5000g离心10分钟后收集上清液，将上清液通过0.45μm的膜过滤后使用超滤膜(截留分子量50KDa)超滤离心过滤和浓缩，将过滤和浓缩后的样品使用高压均质机(10000bar)处理3分钟，然后与60mg相对应的步骤(2)制备的微米粒子1或微米粒子2共作用10分钟后使用2μm滤膜反复共挤出，然后将挤出液在10000g离心20分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得微米粒子。其中，使用微米粒子1激活的混合抗原提呈细胞膜组分与微米粒子1共作用制备的为微米粒子3，粒径为1.6μm；使用微米粒子2激活的混合抗原提呈细胞膜组分与微米粒子2共作用制备的为微米粒子4，粒径为1.6μm。
(6)癌细胞特异性T细胞的制备及分析
选取6-8周的雌性BALB/c小鼠，在第0天、第7天、第14天、第21天和第28天分别皮下注射100μL含0.2mg步骤(2)制备的PLGA的微米粒子1。在第32天处死小鼠,收集外周血和脾脏，然后制备PBMC和脾细胞单细胞悬液并将二者混合，然后使用磁珠分选法从中分选出CD3 +T细胞。然后使用纳米粒子1、或者纳米粒子2、或者微米粒子3或者微米粒子4分别辅助分选和扩增癌细胞特异性T细胞。将分选得到的CD3 +T细胞(200万个)、纳米粒子或微米粒子(100μg)、DC2.4细胞(100万个)在10mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)，孵育体系中含有IL-2(200U/mL)、IL-7(200U/mL)、IL-15(200U/mL)以及CD80抗体(10ng/mL)。然后采用流式细胞术分选孵育后的CD3 +T细胞中的CD3 +CD8 +CD69 +T细胞以及CD4 +T细胞中CD4 +CD69 +T细胞，即为可识别癌细胞全细胞抗原的癌细胞特异性T细胞。将上述分选得到的CD8 +CD69 +T细胞或者CD4 +CD69 +T细胞按照数量比2:1混合后分别与IL-2(1000U/mL)、IL-7(1000U/mL)以及αCD-3抗体(10ng/mL)在RPMI1640完全培养基中共孵育14天以扩增癌细胞特异性T细胞，期间每两天换液一次。其中使用纳米粒子1辅助分离和扩增的癌细胞特异性T细胞为T细胞疫苗1；纳米粒子2辅助分离和扩增的癌细胞特异性T细胞为T细胞疫苗2；微米粒子3辅助分离和扩增的癌细胞特异性T细胞为T细胞疫苗3；微米粒子4辅助分离和扩增的癌细胞特异性T细胞为T细胞疫苗4。
与此同时，分别使用抗小鼠CD3抗体、抗小鼠CD3抗体和抗小鼠CD69抗体标记孵育后的T细胞，然后使用流式细胞术分析不同纳米粒子与T细胞和抗原提呈细胞共孵育后T细胞中CD69 +的T细胞所占的比例。
与此同时，将未经纳米粒子辅助分选的脾细胞中的T细胞与步骤(6)制备的不同纳米粒子在DMEM高糖完全培养基中共孵育48小时，然后收集孵育后的细胞并用带有荧光探针的IFN-γ抗体标记孵育后的T细胞，尔后使用流式细胞术分析T细胞中IFN-γ +T细胞所占比例。纳米粒子所负载的癌细胞全细胞抗原在被抗原提呈细胞吞噬后可被降解成抗原表位被提呈到抗原提呈细胞膜表面，抗原提呈细胞制备的纳米粒子负载有上述降解提呈后的抗原表位，可以被癌细胞特异性T细胞识别并激活癌细胞特异性T细胞，被激活后分泌杀伤性细胞因子。IFN-γ是抗原特异性T细胞识别抗原后被激活所分泌的最主要的细胞因子。使用流式细胞术分析的CD3 +IFN-γ +T细胞即为可以识别和杀伤癌细胞的癌细胞特异性T细胞。
(7)癌细胞特异性T细胞用于癌症的预防
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。在小鼠过继转移细胞前1天，给受体小鼠腹腔注射100mg/kg剂量的环磷酰胺以清除受体小鼠体内的免疫细胞。在第0天给小鼠皮下注射T细胞疫苗(内含60万个扩增后的CD8 +T细胞和30万个扩增后的CD4 +T细胞)或者100μL PBS。同时在第0天给每只小鼠皮下注射接种1×10 6个4T1细胞，小鼠肿瘤体积以及生存期监测方法同上。
(8)实验结果
如图13中a和b所示，与对照组相比，使用T细胞疫苗处理的小鼠，其肿瘤生长速度明显变慢且小鼠生存期明显延长。而且，T细胞疫苗1好于T细胞疫苗2；T细胞疫苗3好于T细胞疫苗4。这说明含有增加溶酶体逃逸功能的物质和混合佐剂的微米粒子激活的抗原提呈细胞制备的粒子所辅助分离得到的癌细胞特异性T细胞效果好于只含有溶酶体逃逸功能的物质而不含有混合佐剂的微米粒子激活的抗原提呈细胞制备的粒子所辅助分离得到的癌细胞特异性T细胞。而且，T细胞疫苗3好于T细胞疫苗1，T细胞疫苗4好于T细胞疫苗2，这说明内部负载癌细胞裂解组分而表面负载被激活的抗原提呈细胞组分的实心粒子所辅助分离得到的癌细胞特异性T细胞好于只是负载被激活的抗原提呈细胞组分的囊泡粒子所辅助分离的癌细胞特异性T细胞。由此可见，本发明所述的癌细胞特异性T细胞对乳腺癌具有杀伤能力，可以用于预防或者治疗癌症。而且混合佐剂的使用以及内部负载癌细胞全细胞组分均有助于辅助分离癌细胞特异性T细胞。
如图13中c和d所示,使用不同粒子辅助分选时所能激活的癌细胞特异性T细胞的比例高低与a和b图中的疗效相关，这说明使用本发明所述粒子辅助分选得到的T细胞就是可以特异性识别和杀伤癌细胞的癌细胞特异性T细胞。
实施例13癌细胞特异性T细胞用于结肠癌的治疗
本实施例以小鼠结肠癌为癌症模型来说明如何使用负载结肠癌全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米粒子辅助分选癌细胞特异性T细胞并用于治疗结肠癌。 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)、CpG2336和CpG2006为佐剂，以NH 4HCO 3为增加溶酶体逃逸物质，制备纳米粒子，使用该纳米粒子激活抗原提呈细胞后将抗原提呈细胞制备成纳米粒子，然后使用纳米粒子辅助分选癌细胞特异性T细胞，经过两步分选得到的癌细胞特异性T细胞经过扩增后用于癌症治疗。
(1)肿瘤组织的裂解及各组分的收集
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。
(2)纳米粒子系统的制备
本实施例中纳米粒子采用复乳法制备。纳米粒子的制备材料PLGA分子量为7KDa-17KDa，以Poly(I:C)和CpG为佐剂，以NH 4HCO 3为增加溶酶体逃逸物质，且佐剂和NH 4HCO 3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，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)、CpG2336和CpG2006免疫佐剂各0.02mg，负载NH 4HCO 30.01mg。纳米粒子2的制备材料和制备方法同纳米粒子1,粒径为260nm左右，表面电位为-7mV左右，每1mg PLGA纳米粒子约负载90μg蛋白质和多肽组分，每1mg PLGA纳米粒负载NH 4HCO 30.01mg,负载CpG2336和CpG2006各0.03mg。
(3)抗原提呈细胞的制备
本实施例使用BMDC和B作为抗原提呈细胞。BMDC制备方法同实施例1。B细胞来自小鼠外周血PBMC，制备方法同上。
(4)抗原提呈细胞的激活
将负载癌细胞全细胞组分的纳米粒子(1000μg)与BMDC(500万个)及B细胞(500万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO 2)；孵育体系中含有GM-CSF(2000U/mL)、IL-2(500U/mL)、IL-7(200U/mL)、IL-12(200U/mL)、IFN-γ(500U/mL)和CD80抗体(10ng/mL)和CD40抗体(20mg/mL)。
(5)基于抗原提呈细胞的纳米粒子的制备
通过在400g离心5分钟收集孵育后的DC和B细胞，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在5000g离心10分钟后收集上清液，将上清液通过0.45μm的膜过滤后使用超滤膜(截留分子量50KDa)超滤离心过滤和浓缩，将过滤和浓缩后的样品与步骤(2)制备的纳米粒子混合后使用高压均质机 (10000bar)处理3分钟，然后在13000g离心30分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米粒子，纳米粒子粒径为300纳米。
(6)癌细胞特异性T细胞的制备
选取6-8周的雌性C57BL/6小鼠，第0天在后背部皮下接种2×10 6个MC38结肠癌细胞，在第14天和第28天分别皮下注射100μL含0.4mg PLGA的纳米粒子(负载裂解物组分、混合佐剂及增加溶酶体逃逸的物质)。在第32天处死小鼠,摘取小鼠肿瘤组织和收集小鼠外周血。将小鼠肿瘤组织制备成肿瘤组织单细胞悬液；从小鼠外周血中分离PBMC，然后将肿瘤组织单细胞悬液和PBMC混合，然后使用流式细胞术从上述混合细胞中分选出CD3 +CD8 +T细胞和分选出CD3 +CD4 +T细胞。将分选得到的CD8 +T细胞(20万个)、CD4 +T细胞(10万个)、抗原提呈细胞制备的纳米粒子(50μg)、B细胞(100万个)以及IL-7(10ng/mL)在2mL RPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)，然后采用流式细胞术分选孵育后的CD8 +T细胞中的CD8 +CD69 +T细胞以及CD4 +T细胞中CD4 +CD69 +T细胞，即为可识别癌细胞全细胞抗原的癌细胞特异性T细胞。将上述分选得到的CD8 +CD69 +T细胞或者CD4 +CD69 +T细胞分别与IL-2(1000U/mL)、IL-12(1000U/mL)、IL-15(1000U/mL)以及αCD-3抗体(10ng/mL)在RPMI1640完全培养基中共孵育14天(每两天换液一次)以扩增癌细胞特异性T细胞。
(7)癌细胞特异性T细胞用于治疗癌症
选取6-8周的雌性C57BL/6为模型小鼠制备结肠癌小鼠。在第0天给每只小鼠背部右下方皮下接种2×10 6个MC38细胞。在接种结肠癌细胞后第6天、第9天、第12天、第15天、第20天和第25天分别静脉注射80万个CD8 +癌细胞特异性T细胞和40万个CD4 +癌细胞特异性T细胞；或者在上述天数注射120万个CD8 +癌细胞特异性T细胞。小鼠肿瘤生长和生存期监测方法同上。
(8)实验结果
如图14所示，与对照组相比，纳米粒子激活的抗原提呈细胞制备的纳米粒子辅助分离扩增得到的癌细胞特异性T细胞处理小鼠后其肿瘤生长速度明显变慢且小鼠生存期明显延长。而且，同时使用抗原提呈细胞制备成的纳米粒子辅助分离和扩增得到的CD8 +T细胞和CD4 +T细胞好于只使用纳米粒子辅助分离和扩增得到的CD8 +T细胞。而且，负载混合佐剂、裂解物组分和溶酶体逃逸物质的纳米粒子激活的抗原提呈细胞制备成的纳米粒子辅助分离的癌细胞特异性T细胞效果好于负载裂解物组分、两种CpG佐剂和溶酶体逃逸物质的纳米粒子。由此可见，本发明所述的癌细胞特异性T细胞对癌症具有优异治疗效果。
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他不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种由粒子激活的抗原提呈细胞制备的癌症T细胞疫苗的制备方法，其特征在于，包括以下步骤：
    S1、将抗原提呈细胞与第一粒子共孵育，得到激活后的抗原提呈细胞；其中，第一粒子负载肿瘤组织和/或癌细胞全细胞组分；
    S2、将激活后的抗原提呈细胞的细胞膜组分制备成纳米囊泡；或将激活后的抗原提呈细胞的细胞膜组分与第二粒子共作用，使所述细胞膜组分负载于第二粒子上，得到负载细胞膜组分的粒子；其中，第二粒子负载肿瘤组织和/或癌细胞全细胞组分；
    S3、将S2所述的纳米囊泡和/或负载细胞膜组分的粒子与含有T细胞的免疫细胞共孵育，将其中可以识别癌细胞抗原的癌细胞特异性T细胞激活，再分选出该部分被激活的癌细胞特异性T细胞，得到所述癌症T细胞疫苗。
  2. 根据权利要求1所述的制备方法，其特征在于：在步骤S3中，共孵育体系中含有抗原提呈细胞、细胞因子和抗体中的一种或多种。
  3. 根据权利要求1所述的制备方法，其特征在于：在步骤S3中，分选出被激活的癌细胞特异性T细胞之后，还包括对所述癌细胞特异性T细胞进行体外扩增的步骤；所述的分选为利用T细胞被激活后细胞表面高表达的标志物对所述癌细胞特异性T细胞进行筛选，所述的体外扩增为将所述癌细胞特异性T细胞与细胞因子和/或抗体共孵育。
  4. 根据权利要求1所述的制备方法，其特征在于：在步骤S1中，共孵育体系中含有细胞因子和/或抗体。
  5. 根据权利要求1所述的制备方法，其特征在于：所述抗原提呈细胞选自树突状细胞、B细胞和巨噬细胞中的一种或多种。
  6. 根据权利要求1所述的制备方法，其特征在于：所述第一粒子或第二粒子还负载有细菌裂解组分和/或细菌外囊泡裂解组分，所述细菌裂解组分和/或细菌外囊泡裂解组分经含有裂解剂的裂解液裂解细菌和/或细菌外囊泡得到；所述裂解剂选自尿素、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、Triton、吐温、氨基酸、糖苷和胆碱的水溶液中的一种或多种。
  7. 根据权利要求1所述的制备方法，其特征在于：所述第一粒子或第二粒子还负载有免疫增强佐剂。
  8. 权利要求1-7任一项所述的制备方法制备得到的癌症T细胞疫苗。
  9. 权利要求8所述的癌症T细胞疫苗在制备用于治疗或预防癌症药物中的应用。
  10. 一种体外激活癌细胞特异性T细胞的方法，其特征在于，包括以下步骤：
    S1、将抗原提呈细胞与第一粒子共孵育，得到激活后的抗原提呈细胞；其中，第一粒子负载肿瘤组织和/或癌细胞全细胞组分；
    S2、将激活后的抗原提呈细胞的细胞膜组分制备成纳米囊泡；或将激活后的抗原提呈 细胞的细胞膜组分与第二粒子共作用，使所述细胞膜组分负载于第二粒子上，得到负载细胞膜组分的粒子；其中，第二粒子负载肿瘤组织和/或癌细胞全细胞组分；
    S3、将S2所述的纳米囊泡和/或负载细胞膜组分的粒子与含有T细胞的细胞共孵育，分选出被激活的癌细胞特异性T细胞。
PCT/CN2022/108966 2022-07-01 2022-07-29 一种癌细胞特异性t细胞疫苗、以及激活癌细胞特异性t细胞的方法 WO2024000725A1 (zh)

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