WO2023217182A1 - 一种过表达CD16a的NK细胞的制备方法及其应用 - Google Patents

一种过表达CD16a的NK细胞的制备方法及其应用 Download PDF

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WO2023217182A1
WO2023217182A1 PCT/CN2023/093245 CN2023093245W WO2023217182A1 WO 2023217182 A1 WO2023217182 A1 WO 2023217182A1 CN 2023093245 W CN2023093245 W CN 2023093245W WO 2023217182 A1 WO2023217182 A1 WO 2023217182A1
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plasmid
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曾皓宇
沈振波
蒋碧愉
卫佳琦
刘康志
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广东普罗凯融生物医药科技有限公司
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present application belongs to the field of cellular immunotherapy, and specifically relates to a preparation method and application of NK cells overexpressing CD16a.
  • ADCC Antibody-dependent cell mediatedcytotoxicity
  • Immune cells and their receptors that mediate ADCC include mononuclear leukocytes (such as natural killer cells (NK cells), macrophages, and ⁇ T cells) and polymorphonuclear cells (neutrophils). cells, basophils and eosinophils).
  • NK cells are the main cells that mediate ADCC, and they are stronger than ADCC mediated by cells such as monocytes, neutrophils, and macrophages.
  • CD16a is a member of the immunoglobulin superfamily and is a low-affinity IgG receptor expressed on the surface of immune effector cells such as natural killer cells (NK cells), macrophages, mast cells, and neutrophils.
  • the transmembrane protein is a relatively important binding site for the body's cellular immune function.
  • CD16a binds to the Fc segment of human IgG1 and IgG3, inducing the cytotoxicity of immune effector cells (mainly NK cells) - that is, antibody-dependent cell-mediated cytotoxicity (ADCC) killing target cells, and can also directly mediate NK cells.
  • NK cells natural killer cells
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the killing effect of cells on tumor target cells can also promote the secretion of cytokines such as IFN- ⁇ , TNF and matrix metalloproteinases, affecting the function of cellular immunity.
  • cytokines such as IFN- ⁇ , TNF and matrix metalloproteinases
  • Monoclonal antibody-mediated molecular targeted therapy based on ADCC is increasingly used in malignant tumors, and CD16a as a binding site has attracted increasing attention.
  • CD16a could be overexpressed on the surface of NK cells through gene transfer technology to increase the cytotoxicity of NK cells - that is, antibody-dependent cell-mediated cytotoxicity (ADCC) or enhance the killing effect of NK cells on tumor cells.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Electroporation instantaneously applies a certain high electric field pulse to cells or tissues to instantly create permeability on the cell membrane surface, thereby causing charged molecules to enter the cells.
  • electroporation-based transgenic operations exogenous DNA is introduced into the cell through reversible electroporation.
  • the exogenous gene is expressed in its new host cell and is inherited as the cell divides.
  • the method of electroporation is combined with a non-viral gene modification system that can induce stable expression of transgenes.
  • Electroporation has the advantages of simple operation, low immunogenicity and genotoxicity, and low safety risks, but its problems are also relatively obvious: it is easy to cause excessive cell death under instantaneous high voltage, and the transfection efficiency is low, specifically with cells. Depends on the type and electroconversion conditions.
  • Viral methods mainly include using retroviral vectors or lentiviral vectors to express genes, introducing genes into immune effector cells through packaged virus particles, and integrating them into the cell genome through the integration system of the retrovirus or lentivirus itself.
  • the transfection efficiency of viral vector systems is also related to the cell type and specific transfection process. Therefore, there is still a need for a method that can improve the survival rate, stability and functionality (eg, killing effect) of immune effector cells, especially NK cells, after viral transfection.
  • the purpose of this application is to provide a method for preparing NK cells that overexpress CD16a, so as to effectively improve the survival rate of NK cells, the overexpression efficiency of CD16a, enhance the killing effect of NK cells on tumor cells, or enhance the cytotoxicity of NK cells. Effect-i.e., antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • a method for preparing NK cells overexpressing CD16a comprising the following steps:
  • HEK293T cells are human embryonic kidney cells and can be purchased commercially
  • step (4) Add the mixed solution obtained in step (4) dropwise to the culture flask supernatant containing HEK 293T cells, Mix thoroughly and place in an incubator for 6 to 9 hours. Aspirate and discard the supernatant. Add cell culture medium containing 0.1-5vol% fetal bovine serum and continue culturing;
  • the preparation method of this application uses a variety of carefully configured culture media, the first culture medium, the second culture medium, and the third culture medium, combined with the polyethylenimine transfection reagent and polybrene co-infection agent used , and specific operating methods, effectively improve the survival rate of NK cells and the overexpression efficiency of CD16a, and enable NK cells to stably overexpress CD16a after multiple passages, enhancing the killing effect of NK cells on tumor cells.
  • the first packaging plasmid is psPAX2 plasmid
  • the second packaging plasmid is pMD2.G plasmid
  • the target plasmid with CD16a is (1 to 3): (1 to 5 ):(2 ⁇ 8).
  • psPAX2 and pMD2.G encode HIV-1 gag-pol and vesicular stomatitis virus glycoprotein (VSV-G) respectively.
  • the pMD2.G plasmid is an envelope plasmid and can infect a wider range of cell types.
  • the psPAX2 plasmid is a packaging plasmid and reduces recombination.
  • the self-replication ability of the virus improves the safety of use.
  • the mass ratio of the three is within the range of (1 ⁇ 3):(1 ⁇ 5):(2 ⁇ 8), which can effectively improve the packaging efficiency of the entire lentivirus transfection system. , to make preliminary preparations for achieving a higher NK cell transfection positivity rate in the future.
  • the packaging plasmid includes a first packaging plasmid, a second packaging plasmid and a third packaging plasmid.
  • the first packaging plasmid is pLP1 plasmid
  • the second packaging plasmid is pLP2 plasmid
  • the third packaging plasmid is pLP/VSVG plasmid and has CD16a.
  • the target plasmid is an expression vector inserted into pLenti; the weight ratio of pLP1 plasmid, pLP2 plasmid, pLP/VSVG plasmid, and the target plasmid with CD16a is 1.5 ⁇ 7.5:1.2 ⁇ 5.8:3.2 ⁇ 9.3:6.8 ⁇ 10 .
  • the expression vector inserted into pLenti is used to insert the CD16a gene, as above It includes the ⁇ packaging signal and the truncated HIV 3' and 5'LTR to facilitate virus packaging; the pLP1 plasmid expresses the gag gene necessary to form a lentiviral structure and the pol gene necessary for viral replication and integration; the pLP2 plasmid is used to express Rev protein , it can work with the response element on pLP1 to induce gag and pol expression and guide the nuclear transport of viral RNA; the pLP/VSVG plasmid expresses VSV-G, making the host range wider. These four plasmids must work together to produce a virus with the ability to infect.
  • the total ratio of polyethylenimine to the three plasmids in step (3) is 1 to 10:1.
  • This application uses polyethylenimine as the transfection reagent and controls the ratio of it to the plasmid mixture, which can effectively improve the virus titer and packaging efficiency of the entire lentiviral transfection system.
  • the NK cells in step (8) are NK92MI cells, and the final concentration of polybrene in step (8) is 8-100 ⁇ g/mL. Compared with other NK cells, NK92MI cells are easier to culture and have high viability after transfection. Polybrene is a polycationic polymer that further enhances transfection efficiency.
  • the specific composition of the first cell culture medium in step (8) is as follows: add inositol 0.005-0.6mM, folic acid 0.00005-0.6mM, mercaptoethanol 0.0005-0.6mM, and fetal bovine serum 0. ⁇ 2vol%, horse serum 0 ⁇ 2vol%, double antibody 0 ⁇ 2vol%. Use this first culture medium to improve subsequent transfection efficiency.
  • the specific operation of centrifugation in step (9) is: adjust the constant temperature centrifuge to 600-1000g, 30-36°C, centrifuge for 10 minutes to preheat to 30-36°C, and transfer the preheated culture plate to the centrifuge. Medium, centrifuge at 600 ⁇ 1000g for 30min ⁇ 3h, 30 ⁇ 36°C. Using this centrifugation method, the virus transfection system can be used to transfect cells.
  • the specific composition of the second cell culture medium in step (10) is as follows: add inositol 0.5-50mM, folic acid 0.05-5mM, mercaptoethanol 0.05-5mM, and fetal bovine serum 8-50vol% in the MEM basal medium. , horse serum 8 ⁇ 50vol%, double antibody 0.5 ⁇ 5vol%. Using this second culture medium, the survival rate of NK cells can be improved.
  • the final concentration of polybrene in step (10) is 8-100 ⁇ g/mL.
  • the specific composition of the third cell culture medium in step (10) is as follows: add inositol 0.05-5mM, folic acid 0.005-0.5mM, mercaptoethanol 0.005-0.5mM, and fetal bovine serum 5-5mM in the MEM basal medium. 20vol%, horse serum 5 ⁇ 20vol%, double antibody 0.5 ⁇ 5vol%.
  • this application uses different carefully configured culture media to culture cells more precisely at different transfection and culture stages, instead of using the same culture medium at different stages, which can efficiently improve cell culture. High post-transfection NK cell survival rate and transfection stability.
  • the specific operation of filtering and concentrating the virus liquid in step (7) is as follows: filter the supernatant after virus transfection with a 0.45um filter membrane; add 5 to 10 ml of PEG-6000NaCl stock solution for every 10 to 80 ml of the filtered initial virus solution. , place at 4°C, mix and shake every 20 to 30 minutes, 3 to 5 times in total; after leaving at 4°C overnight; centrifuge at 4°C, 4000 ⁇ 6000g for 10 to 40 minutes; discard the supernatant, let stand for 1 to 2 minutes, and aspirate Remove the remaining liquid, add an appropriate amount of lentivirus dissolving solution to dissolve the lentivirus precipitate, mix well, and aliquot. Using this filtration and concentration method, the viral titer of lentivirus can be increased.
  • the present application also provides a CD16a-overexpressing NK cell prepared by the above method.
  • the preparation method of this application uses a variety of carefully configured culture media, combined with the polyethylenimine transfection reagent, polybrene transfection enhancement reagent, and specific operating methods, to effectively improve the survival of NK cells. rate and CD16a overexpression efficiency, and enables NK cells to stably overexpress CD16a after multiple passages, enhancing the killing effect of NK cells on tumor cells.
  • Figure 1 is a flow cytometry diagram of the NK92MI cell clone 1 overexpressing CD16a constructed in Example 1;
  • Figure 2 is a flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 1 constructed in Example 1 after culture for 2 months;
  • Figure 3 is a flow cytometry diagram of the NK92MI cell clone 2 overexpressing CD16a constructed in Example 2;
  • Figure 4 is a flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 2 constructed in Example 2 after culture for 2 months;
  • Figure 5 is a flow cytometry diagram of the NK92MI cell clone 3 overexpressing CD16a constructed in Comparative Example 1;
  • Figure 6 is a flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 3 constructed in Comparative Example 1 after culture for 2 months;
  • Figure 7 is a flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 4 constructed in Comparative Example 2;
  • Figure 8 is a flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 4 constructed in Comparative Example 2 after culture for 2 months;
  • Figure 9 is a flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 5 constructed in Comparative Example 3;
  • Figure 10 is a flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 5 constructed in Comparative Example 3 after culture for 2 months;
  • Figure 11 is a flow cytometry diagram of the NK92MI cell clone 6 overexpressing CD16a constructed in Comparative Example 4;
  • Figure 12 is a flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 6 constructed in Comparative Example 4 after culture for 2 months;
  • Figure 13 is a statistical graph showing the killing effect of multiple clones of NK92MI cells overexpressing CD16a on different tumor cells.
  • compositions and methods include the recited elements but do not exclude other elements.
  • definition used in compositions and methods, “consisting essentially of” shall be understood to exclude other elements than having any essential significance for the combination. For example, a composition consisting essentially of each element defined in the application will not exclude other elements that do not substantially affect the basic new features of the application. "Consisting of” is to be understood as excluding more than trace amounts of other listed components and substantial process steps. Examples of these transitional term definitions fall within the scope of this application.
  • overexpression means that the content (such as expression level) of CD16a in cells significantly exceeds the level of initial cells (cells that have not been transferred with the exogenous gene); for example, compared with initial cells, its The content is 20% higher, preferably 50% higher; more preferably more than 100% higher, such as 200%, 300%...500% or higher.
  • a situation of "overexpression” is that the gene encoding an exogenous transcription factor is transferred into the cell and expressed.
  • the NK cells may be isolated from the body, including autologous and allogeneic NK cells; the NK cells may be cultured in vitro, and may be primary cultured or subcultured cells.
  • NK92MI natural killer cells
  • ATCC CRL-2408 ATCC CRL-2408
  • other NK cell lines established in this field include: NK92, NKL, YT, HANK-1, NK-YS and SNK-6, etc. It should be understood that they can all be used in this application.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from lentiviruses, or portions thereof.
  • lentiviral vector or “lentiviral expression vector” may be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles. It should be understood that nucleic acid sequence elements (such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.) exist in the form of RNA in the lentiviral particles of the present application and in the form of DNA in the DNA plasmids of the present application.
  • Example 1 Preparation of NK916 cells (NK92MI cells overexpressing CD16)
  • the three-plasmid system is the target plasmid for overexpressing CD16, the psPAX2 plasmid and the pMD2.G plasmid.
  • step (6) Add the mixed solution obtained in step (5) drop by drop to the supernatant of the culture bottle containing the tool cells.
  • the pipette tip is suspended in the air and the movement is gentle. Be careful not to blow up the cells.
  • step (6) Gently shake the culture bottle to fully mix the virus packaging ingredients and culture medium, and place it in the incubator for 6 to 9 hours. Aspirate and discard the supernatant.
  • PEG-6000NaCl mother solution For every 10 to 80 ml of filtered initial virus solution, add 7.5 ml of PEG-6000NaCl stock solution. Place at 4°C, mix once every 20 to 30 minutes, shake in the figure 8, 3 to 5 times in total; after leaving at 4°C overnight, centrifuge at 4000 to 6000g for 10 to 40 minutes.
  • the preparation method of PEG-6000NaCl mother solution is: NaCl 20 ⁇ 40g; PEG6000100 ⁇ 300g; 500ml ultrapure water; after dissolution, place the liquid in 121°C high temperature sterilization for 30 minutes; filter the sterilized mother solution with a 0.45um filter membrane and store it at 4°C;
  • the specific composition of the first cell culture medium is as follows: add 0.5mM myo-inositol, 0.5mM folic acid, 0.05mM mercaptoethanol, 2vol% fetal bovine serum, 2vol% horse serum, and 2vol% double antibodies to the MEM basic medium.
  • the specific composition of the second cell culture medium is as follows: add 1mM myo-inositol, 0.5mM folic acid, 0.1mM mercaptoethanol, 20vol% fetal bovine serum, 30vol% horse serum, and 1vol% double antibody to the MEM basic medium.
  • the specific composition of the third cell culture medium is as follows: add 2mM myo-inositol, 0.1mM folic acid, 0.005mM mercaptoethanol, 20vol% fetal bovine serum, 5vol% horse serum, and 0.5vol% double antibody to the MEM basal medium.
  • FIG. 1 The flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 1 constructed in Example 1 is shown in Figure 1, and the flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 1 constructed in Example 1 after culture for 2 months is as shown in Figure 1 2 shown.
  • Example 2 The operation mode of Example 2 is basically the same as that of Example 1. The main difference is that the first, second and third culture media in Example 1 are replaced as follows; and the mass ratio of pMD2.G, psPAX2 and the target expression plasmid. The total ratio of polyethylenimine to the three plasmids was 1:1, and the final concentration of polybrene was 8 ⁇ g/mL.
  • the specific composition of the first cell culture medium is as follows: add 0.1mM myo-inositol to the MEM basic medium, Acid 0.2mM, mercaptoethanol 0.1mM, fetal calf serum 2vol%, horse serum 2vol%, double antibody 2vol%.
  • the specific composition of the second cell culture medium is as follows: add 1mM myo-inositol, 0.05mM folic acid, 0.1mM mercaptoethanol, 25vol% fetal bovine serum, 25ol% horse serum, and 1vol% double antibody to the MEM basic medium.
  • the specific composition of the third cell culture medium is as follows: add 0.05mM myo-inositol, 0.05mM folic acid, 0.1mM mercaptoethanol, 10vol% fetal bovine serum, 10vol% horse serum, and 5vol% double antibodies to the MEM basal medium.
  • FIG. 3 The flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 2 constructed in Example 2 is shown in Figure 3, and the flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 2 constructed in Example 2 after culture for 2 months is shown in Figure 3 4 shown.
  • Comparative Example 1 The operation mode of Comparative Example 1 is basically the same as that of Example 1. The main difference is that the first, second and third culture media in Example 1 are replaced as follows, and the final concentration of polybrene is 15 ⁇ g/mL.
  • the specific composition of the first cell culture medium is as follows: add 0.05mM folic acid, 0.05mM mercaptoethanol, 1vol% fetal bovine serum, 2vol% horse serum, and 1vol% double antibody to the MEM basic medium.
  • the specific composition of the second cell culture medium is as follows: add 2mM folic acid, 0.5mM mercaptoethanol, 15vol% fetal bovine serum, 30vol% horse serum, and 15vol% double antibodies to the MEM basic medium.
  • the specific composition of the third cell culture medium is as follows: add 0.5mM folic acid, 0.5mM mercaptoethanol, 10vol% fetal bovine serum, 5vol% horse serum, and 15vol% double antibodies to the MEM basal medium.
  • the flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 3 constructed in Comparative Example 1 is shown in Figure 5, and the flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 3 constructed in Comparative Example 1 was cultured for 2 months. As shown in Figure 6.
  • Comparative Example 2 The operation mode of Comparative Example 2 is basically the same as that of Example 1. The main difference is that the first, second and third culture media in Example 1 are replaced as follows; and the quality of pMD2.G, psPAX2 and the target expression plasmid The ratio was 3:5:2, the total ratio of polyethylenimine to the three plasmids was 10:1, and the final concentration of polybrene was 20 ⁇ g/mL.
  • the specific composition of the first cell culture medium is as follows: add 0.6mM myo-inositol, 0.6mM mercaptoethanol, 2vol% fetal bovine serum, 1vol% horse serum, and 1vol% double antibody to the MEM basic medium.
  • the specific composition of the second cell culture medium is as follows: add 0.5mM myo-inositol, 1mM mercaptoethanol, 40vol% fetal bovine serum, 10vol% horse serum, and 1vol% double antibody to the MEM basic medium.
  • the specific composition of the third cell culture medium is as follows: add 0.1mM myo-inositol, 0.5mM mercaptoethanol, 5vol% fetal bovine serum, 5vol% horse serum, and 1vol% double antibody to the MEM basic medium.
  • Comparative Example 3 The operation mode of Comparative Example 3 is basically the same as that of Example 1. The main difference is that the first, second and third culture media in Example 1 are replaced as follows; and the quality of pMD2.G, psPAX2 and the target expression plasmid The ratio was 3:5:8, the total ratio of polyethylenimine to the three plasmids was 5:1, and the final concentration of polybrene was 50 ⁇ g/mL.
  • the specific composition of the first cell culture medium is as follows: add 0.01mM myo-inositol, 0.05mM folic acid, 2vol% fetal bovine serum, 1vol% horse serum, and 1vol% double antibody to the MEM basic medium.
  • the specific composition of the second cell culture medium is as follows: add 0.5mM myo-inositol, 0.05mM folic acid, 30vol% fetal bovine serum, 30vol% horse serum, and 2vol% double antibodies to the MEM basic medium.
  • the specific composition of the third cell culture medium is as follows: add 0.1mM myo-inositol, 0.05mM folic acid, 4vol% fetal bovine serum, 6vol% horse serum, and 2vol% double antibodies to the MEM basic medium.
  • the flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 5 constructed in Comparative Example 3 is shown in Figure 9, and the flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 5 constructed in Comparative Example 3 was cultured for 2 months. As shown in Figure 10.
  • Comparative Example 4 is basically the same as that of Example 1. The main difference is that the first, second and third culture media in Example 1 are replaced as follows; and the quality of pMD2.G, psPAX2 and the target expression plasmid The ratio is 1:1:8, the total ratio of polyethylenimine to the three plasmids is 1:1, and the final concentration of polybrene is 8 ⁇ g/mL.
  • the specific composition of the first cell culture medium is as follows: add 0.005mM mercaptoethanol, 2vol% fetal bovine serum, 2vol% horse serum, and 1vol% double antibody to the MEM basic medium.
  • the specific composition of the second cell culture medium is as follows: add 0.051mM mercaptoethanol, 50vol% fetal bovine serum, 8vol% horse serum, and 2vol% double antibodies to the MEM basic medium.
  • the specific composition of the third cell culture medium is as follows: add 0.5mM mercaptoethanol, 20vol% fetal bovine serum, 5vol% horse serum, and 2vol% double antibodies to the MEM basic medium.
  • the flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 6 constructed in Comparative Example 4 is shown in Figure 11, and the flow cytometry diagram of the CD16a-overexpressing NK92MI cell clone 6 constructed in Comparative Example 4 was cultured for 2 months. As shown in Figure 12.
  • the test subjects of this test example are the CD16a-overexpressing NK cells prepared in Examples 1 to 2 and Comparative Examples 1 to 4 (the transfection system contains lentivirus and co-infectious agent), and the viruses are removed and samples are taken.
  • Use flow cytometry for detection Since the lentivirus is labeled with GFP (green fluorescent protein), GFP-positive cells are successfully transfected cells during flow cytometry.
  • GFP-positive cells are successfully transfected cells during flow cytometry.
  • NK916 cells The CD16a-overexpressing NK cells (referred to as NK916 cells) prepared in Examples 1 to 2 and Comparative Examples 1 to 4 were After 2 months of subculture (cells), their killing properties were tested as follows:
  • wash the plate Aspirate the liquid in the plate, wash each well twice with high-pressure sterilized water; soak in NaOH solution for 1-4 hours, wash twice with high-pressure sterilized water; finally rinse once with DPBS.
  • transfected NK cells of Examples 1 and 2 have significantly enhanced killing properties against these four tumor cells compared with the negative control.
  • the killing performance of the transfected NK cells in Comparative Examples 1-4 against these four tumor cells was also enhanced to a certain extent, but the degree of enhancement was obviously lower than that in Examples 1 and 2.

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Abstract

本申请提供一种制备过表达CD16a的NK细胞的方法，包括以下步骤：(1)构建具有CD16a的目标质粒的慢病毒转染体系；(2)培养HEK 293T细胞；(3)将转染试剂和质粒混均；(5)进行慢病毒包装；(6)收集、过滤和浓缩病毒液；(7)转染第二细胞培养基重悬的NK细胞，加入聚凝胺，预热；(8)离心；(9)加入含有聚凝胺的第二细胞培养基，混匀，采用第三细胞培养基继续培养；(11)取细胞做流式检测阳性率，做流式分选，筛选出阳性细胞，接种相应体系继续扩增培养并建库。采用本申请方法制备的过表达CD16a的NK细胞阳性率高和细胞存活率高，且使得NK细胞多次传代后依然能够稳定过表达CD16a，对于癌细胞具有良好的杀伤性能。

Description

一种过表达CD16a的NK细胞的制备方法及其应用 技术领域
本申请属于细胞免疫治疗领域，具体地，涉及一种过表达CD16a的NK细胞的制备方法及其应用。
背景技术
抗体依赖的细胞毒性(antibody-dependent cell mediatedcytotoxicity，ADCC)作用是指具有杀伤活力的免疫细胞，通过细胞表面的FC受体和单克隆抗体结合，识别被单克隆抗体结合的靶抗原(如细菌或者肿瘤细胞)，直接杀伤靶细胞。研究发现，ADCC作用是单克隆抗体治疗肿瘤或其他疾病的重要机制和手段之一。
介导ADCC作用的免疫细胞及其受体:机体发挥ADCC作用的免疫细胞主要包括单核白细胞(如自然杀伤细胞(NK细胞)、巨噬细胞和γδT细胞)与多形核细胞(中性粒细胞、嗜碱性粒细胞和嗜酸性粒细胞)。其中，NK细胞是介导ADCC作用的主要细胞，比单核细胞、中性粒细胞和巨噬细胞等细胞介导的ADCC作用要强。
FcγRⅢa(CD16a)属于免疫球蛋白的超家族成员，为低亲和力的IgG受体，是一种表达于自然杀伤细胞(NK细胞)、巨噬细胞、肥大细胞和中性粒细胞等免疫效应细胞表面的跨膜蛋白，是机体细胞免疫功能发挥作用的一个比较重要的结合位点。CD16a与人IgG1和IgG3的Fc段结合，诱导免疫效应细胞(主要是NK细胞)的细胞毒作用-即抗体依赖的细胞介导的细胞毒作用(ADCC)杀伤靶细胞，也可直接介导NK细胞对肿瘤靶细胞的杀伤作用；还可促进IFN-γ、TNF和基质金属蛋白酶等细胞因子的分泌，影响细胞免疫功能发挥。以ADCC为作用基础的单克隆抗体介导分子靶向治疗在恶性肿瘤中应用越来越广泛，作为结合位点的CD16a越来越受到关注。
曾有人设想通过基因转移技术在NK细胞的表面过表达CD16a，以增加NK细胞的细胞毒作用-即抗体依赖的细胞介导的细胞毒性作用(ADCC)或增强NK细胞对肿瘤细胞的杀伤效果。
基因转移技术主要包括病毒方法与电转方法。电转是通过瞬时向细胞或组织施加一定的高电场脉冲，在细胞膜表面瞬时形成通透，进而导致带电荷的分子进入细胞。基于电穿孔的转基因操作中，外源DNA通过可逆的电穿孔被引入胞内，外源基因在其新的宿主细胞内表达，并随着细胞分裂而被遗传。用电转的方法结合能够诱导稳定表达转基因的非病毒基因修饰系统。电转具有操作简单、免疫源性和基因毒性低、安全风险低的优势，但其存在的问题也比较明显：在于瞬时高电压下容易造成细胞的过量死亡，且转染效率低，具体地与细胞类型及电转条件有关。
病毒方法主要包括使用逆转录病毒载体或慢病毒载体来表达基因，通过包装的病毒颗粒将基因导入免疫效应细胞内，并通过逆转录病毒或慢病毒自身的整合系统整合至细胞基因组上。病毒载体系统的转染效率高低也与细胞类型和具体的转染过程有关。因此目前仍需要一种能够提高免疫效应细胞，尤其是NK细胞，在病毒转染后存活率、稳定性和功能性(例如，杀伤作用)的方法。
发明内容
本申请的目的在于提供一种制备过表达CD16a的NK细胞的方法，以有效提高NK细胞的存活率，CD16a的过表达效率，增强NK细胞对肿瘤细胞的杀伤效果，或者增强NK细胞的细胞毒作用-即抗体依赖的细胞介导的细胞毒性作用(ADCC)。
根据本申请的一个方面，提供一种制备过表达CD16a的NK细胞的方法，包括以下步骤：
(1)构建具有CD16a的目标质粒和包装质粒的慢病毒转染体系；
(2)培养用于慢病毒的HEK 293T细胞，对所述HEK 293T细胞进行饥饿处理(HEK293T细胞为人胚肾细胞，可以商业化购买)；
(3)将减血清培养基或者无血清培养基平均分成两份，一份加入具有CD16a的目标质粒、第一包装质粒和第二包装质粒的慢病毒转染体系，混合均匀，得到质粒混合物；另一份加入聚乙烯亚胺，混合均匀，然后室温放置1～10min，得到转染试剂；
(4)将转染试剂逐滴加入质粒混合物中，混合均匀后，室温放置5～40min，得到含有聚乙烯亚胺和质粒的混合溶液；
(5)将步骤(4)得到的混合溶液逐滴加入到含有HEK 293T细胞的培养瓶上清中， 充分混匀，并放入培养箱中培养6～9h，吸弃上清，加入含有0.1-5vol％胎牛血清的细胞培养基，继续培养；
(6)每隔24h收集一次病毒转染后的上清，共收取1～3次；
(7)过滤和浓缩病毒液；
(8)取第一细胞培养基重悬培养好的NK细胞，按Moi＝5～100加入浓缩后慢病毒液，再加入聚凝胺，预热；
(9)离心；
(10)加入含有聚凝胺的第二细胞培养基，混匀，继续培养，培养期间根据培养基颜色和细胞数量判断是否半换液或传代，培养期间采用第三细胞培养基。
(11)当细胞传代至起始数10倍以上时，取细胞做流式检测阳性率；当细胞生长至5-10×106万个细胞左右时，做流式分选，筛选出阳性细胞，接种相应体系继续扩增培养并建库。
本申请的制备方法采用了精细化配置的多种培养基，第一培养基、第二培养基、第三培养基，结合其所采用的聚乙烯亚胺转染试剂、聚凝胺助感染剂，以及特定的操作方式，有效提高了NK细胞的存活率和CD16a的过表达效率，且使得NK细胞多次传代后依然能够稳定过表达CD16a，增强NK细胞对肿瘤细胞的杀伤效果。
优选的，其中第一包装质粒为psPAX2质粒，第二包装质粒为pMD2.G质粒，且pMD2.G、psPAX2、具有CD16a的目标质粒三者的质量比为(1～3):(1～5):(2～8)。psPAX2和pMD2.G分别编码HIV-1gag-pol和水疱性口炎病毒糖蛋白(VSV-G，pMD2.G质粒是包膜质粒，能感染更广泛的细胞类型，psPAX2质粒是包装质粒，减少重组病毒自我复制能力，提升使用的安全性。使得三者的质量比在(1～3):(1～5):(2～8)范围内，可以有效提高整个慢病毒转染体系的包装效率，为之后实现较高的NK细胞转染阳性率做好前期准备。
优选的，包装质粒包括第一包装质粒、第二包装质粒和第三包装质粒，第一包装质粒为pLP1质粒，第二包装质粒为pLP2质粒，第三包装质粒为pLP/VSVG质粒，且具有CD16a的目标质粒为插入pLenti的表达载体；其中pLP1质粒、、pLP2质粒，、pLP/VSVG质粒、具有CD16a的目标质粒四者的重量比为1.5～7.5:1.2～5.8:3.2～9.3:6.8～10。插入pLenti的表达载体，用于插入CD16a基因，上 面包括ψ包装信号以及截短的HIV 3'及5'LTR，便于病毒包装；pLP1质粒表达形成慢病毒结构所必需的gag基因以及病毒复制和整合必需的pol基因；pLP2质粒用以表达Rev蛋白，它能与pLP1上的反应元件共同作用诱导gag和pol表达，并指导病毒RNA的核运输；pLP/VSVG质粒表达VSV-G，使宿主范围更广。必须这4个质粒共同作用，才能产生有感染能力的病毒。
优选的，其中步骤(3)中聚乙烯亚胺与三种质粒的总量比为1～10:1。本申请采用聚乙烯亚胺作为转染试剂，且控制其和质粒混合物的比例，可以有效提高整个慢病毒转染体系的病毒滴度和包装效率。
优选的，其中步骤(8)中NK细胞是NK92MI细胞，且步骤(8)中聚凝胺终浓度为8～100μg/mL。NK92MI细胞相对于其他NK细胞而言，更易培养，且转染后的存活性高。聚凝胺是一种多聚阳离子聚合物，可以进一步增强转染效率。
优选的，其中步骤(8)中第一细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.005～0.6mM，叶酸0.00005～0.6mM，巯基乙醇0.0005～0.6mM，胎牛血清0～2vol％，马血清0～2vol％，双抗0～2vol％。采用该第一培养基，提高后续的转染效率。
优选的，其中步骤(9)离心的具体操作为：将恒温离心机调成600～1000g，30～36℃，10min离心预热至30～36℃，将预热后的培养板转移至离心机中，600～1000g离心30min～3h，30～36℃。采用该离心方式，可以促进病毒转染体系转染细胞。
优选的，其中步骤(10)中第二细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.5～50mM，叶酸0.05～5mM，巯基乙醇0.05～5mM，胎牛血清8～50vol％，马血清8～50vol％，双抗0.5～5vol％。采用该第二培养基，可以提高NK细胞的存活率。
优选的，其中步骤(10)中聚凝胺终浓度为8～100μg/mL。
优选的，其中步骤(10)中第三细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.05～5mM，叶酸0.005～0.5mM，巯基乙醇0.005～0.5mM，胎牛血清5～20vol％，马血清5～20vol％，双抗0.5～5vol％。
优选的，本申请在不同的转染和培养阶段，采用精细化配置的不同培养基对细胞进行更精细化的培养，而不是在不同的阶段均采用同一种培养基，可以高效地提 高转染后NK细胞的存活率和转染稳定性。
优选的，其中步骤(7)过滤和浓缩病毒液的具体操作为：病毒转染后的上清用0.45um滤膜过滤；每10～80ml过滤后的病毒初始液加入PEG-6000NaCl母液5～10ml，4℃放置，每20～30min混合摇匀，共进行3～5次；4℃放置过夜后；4℃，4000～6000g，离心10～40min；吸弃上清，静置1～2min，吸走残余液体，加入适量的慢病毒溶解液溶解慢病毒沉淀，混匀，分装。采用该过滤和浓缩方法，可以提高慢病毒的病毒滴度。
另一方面，本申请还提供了一种采用上述方法制备得到的过表达CD16a的NK细胞。
本申请的制备方法采用了精细化配置的多种培养基，结合其所采用的聚乙烯亚胺转染试剂、聚凝胺转染增强试剂，以及特定的操作方式，有效提高了NK细胞的存活率和CD16a的过表达效率，且使得NK细胞多次传代后依然能够稳定过表达CD16a，增强NK细胞对肿瘤细胞的杀伤效果。
附图说明
图1为实施例1构建的过表达CD16a的NK92MI细胞克隆1的流式细胞图；
图2为实施例1构建的过表达CD16a的NK92MI细胞克隆1培养2个月后的流式细胞图；
图3为实施例2构建的过表达CD16a的NK92MI细胞克隆2的流式细胞图；
图4为实施例2构建的过表达CD16a的NK92MI细胞克隆2培养2个月后的流式细胞图；
图5为对比实施例1构建的过表达CD16a的NK92MI细胞克隆3的流式细胞图；
图6为对比实施例1构建的过表达CD16a的NK92MI细胞克隆3培养2个月后的流式细胞图；
图7为对比实施例2构建的过表达CD16a的NK92MI细胞克隆4的流式细胞图；
图8为对比实施例2构建的过表达CD16a的NK92MI细胞克隆4培养2个月后的流式细胞图；
图9为对比实施例3构建的过表达CD16a的NK92MI细胞克隆5的流式细胞图；
图10为对比实施例3构建的过表达CD16a的NK92MI细胞克隆5培养2个月后的流式细胞图；
图11为对比实施例4构建的过表达CD16a的NK92MI细胞克隆6的流式细胞图；
图12为对比实施例4构建的过表达CD16a的NK92MI细胞克隆6培养2个月后的流式细胞图；
图13为过表达CD16a的NK92MI细胞多个克隆对于不同肿瘤细胞的杀伤性统计图。
具体实施方式
为了使本技术领域的人员更好地理解本申请方案，下面将对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分的实施例，而不是全部的实施例。
本申请公开和描述之前，应该了解的是，下面描述的各个方面并不限于具体的组合物、制备所述组合物的方法或其用途，因为这些方面当然可能发生变化。还应该了解的是，本申请使用的术语仅仅是为了描述特定方面，而并非用于限制。
除非另有规定，本申请使用的所有技术和科学术语的意义与本申请所属领域的普通技术人员通常理解的相同。
本申请使用的术语仅仅是为了描述特定实施例，并不是用于限制本申请。如在本申请中使用的，除非上下文中清楚表明，否则，单数形式的“一”、“一个”和“所述”也包括复数形式。所有数值表示，例如，pH、温度、时间、浓度、剂量和分子量，包括范围，若合适的话，是在(+)或(-)10％、1％或0.1％变化的近似值。应该了解的是，虽然并未总是明确指出，但是，所有数值前面都可以加上术语“大约”。还应该了解的是，虽然并不总是明确指出，但是，本申请描述的试剂只是示例性的，并且所述试剂的等同试剂是本领域已知的。
“任选的”或“任选地”指的是随后描述的事件或环境可以出现或不出现，所述描述包括其中事件或环境出现的情况及不出现的情况。
术语“包括”指的是组合物和方法包括引用的要素，但不排除其它要素。定义 组合物和方法时使用的“基本上由……组成”应理解为排除对组合具有任何基本意义以外的其它要素。例如，基本上由本申请定义各要素组成的组合物将不排除实质上不影响所述申请基本新特点的其它要素。“由……组成”应理解为不包括列举的超过痕量的其它组分和实质性方法步骤。这些过渡术语定义的实施例属于本申请的范围。
如本文所用，所述的“过表达”是指细胞内CD16a的含量(如表达量)显著地超过初始细胞(未转入该外源基因的细胞)的水平；如与初始细胞相比，其含量高20％，较佳地高50％；更佳地高100％以上，如高200％，300％...500％或更高。一种“过表达”的情形是将外源的转录因子的编码基因转入细胞中且发生表达。
本申请中，所述的NK细胞可以是分离自机体的，包括自体和异体来源的NK细胞；所述的NK细胞可以是体外培养的，可以是原代培养或传代培养的细胞。现在，也已经有一些商品化的NK细胞，可以供本领域技术人员方便地获得，例如，人恶性非霍奇金淋巴瘤患者的自然杀伤细胞NK92MI，其可获自ATCC(ATCC CRL-2408)；此外，其它本领域已建立的NK细胞系还包括有：NK92，NKL，YT，HANK-1，NK-YS和SNK-6等，应理解，它们均可被应用于本申请中。
术语“慢病毒载体”指的是包含主要来源于慢病毒的结构和功能基因元件或其一部分的病毒载体或质粒。
术语“慢病毒载体”或“慢病毒表达载体”可用于指慢病毒转移质粒和/或感染慢病毒颗粒。应该了解的是，核酸序列元件(如克隆位点、启动子、调节元件、异源核酸等)在本申请的慢病毒颗粒中以RNA形式存在，在本申请的DNA质粒中以DNA形式存在。
实施例1：NK916细胞(过表达CD16的NK92MI细胞)的制备
(1)准备三质粒系统，三质粒系统分别是过表达CD16的目标质粒、psPAX2质粒和pMD2.G质粒。
(2)配制基本培养基：5-20vol％胎牛血清、1vol％青霉素-链霉素的DMEM完全培养基。
(3)将约(1～10)×106个293T细胞接种到T75培养瓶中，加入基本培养基，培 养过夜，当细胞密度达到50～90％时，吸弃培养瓶中的上清，加入10mL只含0.1-2vol％胎牛血清的DMEM培养基对细胞进行饥饿处理1-3h，得到工具细胞；
(4)按照每份病毒1～10mL的量吸取Opti-MEM培养基，平均分成两等分。一份加入慢病毒包装质粒psPAX2、pMD2.G以及目标表达质粒，混合均匀，得到质粒混合物；另一份加入聚乙烯亚胺，混合均匀，然后室温放置1～10min，得到转染试剂；其中，pMD2.G、psPAX2、目标表达质粒三者的质量比为1:1:8，聚乙烯亚胺与三种质粒的总量比为10:1；
(5)将转染试剂逐滴加入质粒混合物中，混合均匀后，室温放置5～40min，得到含有聚乙烯亚胺和质粒的混合溶液；
(6)将步骤(5)得到的混合溶液逐滴加入到含有工具细胞的培养瓶上清中，滴加时枪头悬空，动作轻柔，注意不能将细胞吹起，加完后，按照十字法轻轻晃动培养瓶，使病毒包装成分和培养基充分混匀，并放入培养箱中培养6～9h，吸弃上清，每个培养瓶中加入5～20mL新鲜提前预热的含有0.1-5vol％胎牛血清的DMEM培养基，继续培养72h；
(7)自加入新鲜的含有0.1-5vol％胎牛血清的DMEM培养基开始算，每隔24h收集一次病毒转染后的上清并用0.45um滤膜过滤，并更换新鲜的含有0.1-5vol％胎牛血清的DMEM培养基，共收取3次，暂存于4℃；
(8)每10～80ml过滤后的病毒初始液，加入PEG-6000NaCl母液7.5ml。4℃放置，每20～30min混合一次，8字摇匀，共进行3～5次；4℃放置过夜后；4℃，4000～6000g，离心10～40min。其中PEG-6000NaCl母液的配置方法为：NaCl 20～40g；PEG6000100～300g；500ml超纯水；溶解后将液体置于121℃高温灭菌30min；将灭菌后母液用0.45um滤膜过滤后保存在4℃；
(9)吸弃上清，静置管子1～2min，吸走残余液体，加入适量的慢病毒溶解液(DMEM基础培养基)溶解慢病毒沉淀。将混匀后溶解病毒悬液分装，得到NK916慢病毒，储存在-80℃，按需取用。
(10)取适量生长状态较好的NK92MI细胞悬液，300g离心2-5min，去上清，收集NK92MI细胞，用第一培养基重悬细胞，向12孔板一个孔中加入(5～500)×104万个细胞(不超过1mL)，按MOI＝5～100向12孔板中加入NK916慢病毒，再加入 聚凝胺(聚凝胺终浓度为100μg/mL)，并加入第一培养基至总体积为3mL，将孔板置于37℃培养箱预热10min，
(11)同时将恒温离心机调成600～1000g，30～36℃，10min离心预热至30～36℃。将预热后的12孔板转移至离心机中，600～1000g离心30min～3h，30～36℃；
(12)离心结束后，将12孔板取出，按1mL/孔加入聚凝胺(聚凝胺终浓度100μg/mL)的第二培养基，移液枪于孔正中间轻轻吹打混匀3-5次，将孔板转移回培养箱中培养，培养期间根据培养基颜色和细胞数量判断是否半换液或传代，培养基为第三培养基。
(13)当细胞传代至起始数10倍以上时，取20万个细胞做流式检测阳性率。当细胞生长至(5～10)×106万个细胞左右时，准备细胞做流式分选，分选出阳性细胞接种相应体系继续扩增培养并建库。
其中，第一细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.5mM，叶酸0.5mM，巯基乙醇0.05mM，胎牛血清2vol％，马血清2vol％，双抗2vol％。
第二细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇1mM，叶酸0.5mM，巯基乙醇0.1mM，胎牛血清20vol％，马血清30vol％，双抗1vol％。
第三细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇2mM，叶酸0.1mM，巯基乙醇0.005mM，胎牛血清20vol％，马血清5vol％，双抗0.5vol％。
实施例1构建的过表达CD16a的NK92MI细胞克隆1的流式细胞图如图1所示，且实施例1构建的过表达CD16a的NK92MI细胞克隆1培养2个月后的流式细胞图如图2所示。
实施例2
实施例2的操作方式与实施例1基本相同，主要区别在于将实施例1中的第一、第二和第三培养基替换如下；且pMD2.G、psPAX2、目标表达质粒三者的质量比为3:5:2，聚乙烯亚胺与三种质粒的总量比为1:1，聚凝胺终浓度为8μg/mL。
第一细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.1mM，叶 酸0.2mM，巯基乙醇0.1mM，胎牛血清2vol％，马血清2vol％，双抗2vol％。
第二细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇1mM，叶酸0.05mM，巯基乙醇0.1mM，胎牛血清25vol％，马血清25ol％，双抗1vol％。
第三细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.05mM，叶酸0.05mM，巯基乙醇0.1mM，胎牛血清10vol％，马血清10vol％，双抗5vol％。
实施例2构建的过表达CD16a的NK92MI细胞克隆2的流式细胞图如图3所示，且实施例2构建的过表达CD16a的NK92MI细胞克隆2培养2个月后的流式细胞图如图4所示。
对比实施例1
对比实施例1的操作方式与实施例1基本相同，主要区别在于将实施例1中的第一、第二和第三培养基替换如下，且其中聚凝胺终浓度为15μg/mL。
第一细胞培养基的具体组成如下：在MEM基础培养基中加入叶酸0.05mM，巯基乙醇0.05mM，胎牛血清1vol％，马血清2vol％，双抗1vol％。
第二细胞培养基的具体组成如下：在MEM基础培养基中加入叶酸2mM，巯基乙醇0.5mM，胎牛血清15vol％，马血清30vol％，双抗15vol％。
第三细胞培养基的具体组成如下：在MEM基础培养基中加入叶酸0.5mM，巯基乙醇0.5mM，胎牛血清10vol％，马血清5vol％，双抗15vol％。
对比实施例1构建的过表达CD16a的NK92MI细胞克隆3的流式细胞图如图5所示，且对比实施例1构建的过表达CD16a的NK92MI细胞克隆3培养2个月后的流式细胞图如图6所示。
对比实施例2
对比实施例2的操作方式与实施例1基本相同，主要区别在于将实施例1中的第一、第二和第三培养基替换如下；且pMD2.G、psPAX2、目标表达质粒三者的质量比为3:5:2，聚乙烯亚胺与三种质粒的总量比为10:1，聚凝胺终浓度为20μg/mL。
第一细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.6mM，巯基乙醇0.6mM，胎牛血清2vol％，马血清1vol％，双抗1vol％。
第二细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.5mM，巯基乙醇1mM，胎牛血清40vol％，马血清10vol％，双抗1vol％。
第三细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.1mM，巯基乙醇0.5mM，胎牛血清5vol％，马血清5vol％，双抗1vol％。
对比实施例2构建的过表达CD16a的NK92MI细胞克隆4的流式细胞图如图7所示，且对比实施例2构建的过表达CD16a的NK92MI细胞克隆4培养2个月后的流式细胞图如图8所示。
对比实施例3
对比实施例3的操作方式与实施例1基本相同，主要区别在于将实施例1中的第一、第二和第三培养基替换如下；且pMD2.G、psPAX2、目标表达质粒三者的质量比为3:5:8，聚乙烯亚胺与三种质粒的总量比为5:1，聚凝胺终浓度为50μg/mL。
第一细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.01mM，叶酸0.05mM，胎牛血清2vol％，马血清1vol％，双抗1vol％。
第二细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.5mM，叶酸0.05mM，胎牛血清30vol％，马血清30vol％，双抗2vol％。
第三细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.1mM，叶酸0.05mM，胎牛血清4vol％，马血清6vol％，双抗2vol％。
对比实施例3构建的过表达CD16a的NK92MI细胞克隆5的流式细胞图如图9所示，且对比实施例3构建的过表达CD16a的NK92MI细胞克隆5培养2个月后的流式细胞图如图10所示。
对比实施例4
对比实施例4的操作方式与实施例1基本相同，主要区别在于将实施例1中的第一、第二和第三培养基替换如下；且pMD2.G、psPAX2、目标表达质粒三者的质量比为1:1:8，聚乙烯亚胺与三种质粒的总量比为1:1，聚凝胺终浓度为8μg/mL。
第一细胞培养基的具体组成如下：在MEM基础培养基中加入巯基乙醇0.005mM，胎牛血清2vol％，马血清2vol％，双抗1vol％。
第二细胞培养基的具体组成如下：在MEM基础培养基中加入巯基乙醇0.051mM，胎牛血清50vol％，马血清8vol％，双抗2vol％。
第三细胞培养基的具体组成如下：在MEM基础培养基中加入巯基乙醇0.5mM，胎牛血清20vol％，马血清5vol％，双抗2vol％。
对比实施例4构建的过表达CD16a的NK92MI细胞克隆6的流式细胞图如图11所示，且对比实施例4构建的过表达CD16a的NK92MI细胞克隆6培养2个月后的流式细胞图如图12所示。
测试：阳性率检测
本测试例的参试对象为实施例1～2和对比例1～4所制得的过表达CD16a的NK细胞(转染体系中含有慢病毒和助感染剂)，对其去病毒并取样，利用流式细胞仪进行检测，由于慢病毒经过GFP(绿色荧光蛋白)标记，因此进行流式检测时GFP阳性的细胞为转染成功的细胞，对转染成功的细胞和阴性对照(未经处理)的细胞进行计数，通过计算过表达CD16aDE细胞与未经处理的细胞的比值，得到感染阳性率，即NK细胞的慢病毒转染效率。计算公式如下：NK细胞的慢病毒转染效率＝过表达CD16a的NK细胞/总NK细胞。
流式细胞仪的检测结果如图1，图3，图5，图7，图9和图11所示。可以看出，初始转染的NK细胞的阳性率均很高，均可达到90％以上。
将这些转染后的细胞继续传代培养2个月后，再次利用流式细胞仪检测阳性率。流式细胞仪的检测结果如图2，图4，图6，图8，图10和图12所示。可以看出，除了实施例1和实施例2的细胞克隆能够稳定的高表达CD16a外，其他实施例的细胞克隆均发生了不同程度的脱靶。尤其是对比实施例3和4，经2个月培养后，阳性率跌落到了5％以下；但实施例1和实例2的NK细胞在培养2个月后，依然能够高效稳定的过表达CD16a。
测试：杀伤性能检测
将实施例1～2和对比例1～4所制得的过表达CD16a的NK细胞(称为NK916细 胞)传代培养2个月后，按照以下方式检测其杀伤性能：
1.洗板：吸掉板内液体，每个孔用高压灭菌水洗涤2次；再用NaOH溶液浸泡1-4h，用高压灭菌水洗涤2次；最后用DPBS润洗1次。
2.铺板：将培养好的目标肿瘤细胞系(BxPC-3、OVCAR-3、AGS、HO8910这四种肿瘤细胞)消化、离心、重悬、台盼蓝染色计数，按1-10万活细胞/孔接种电流板，补加适量培养基，上机放入培养箱中监测。细胞生长曲线为平滑上升，且孔间差异不大，则视为细胞生长正常。
3.加入实施例1～2和对比例1～4所制得的过表达CD16a的NK细胞：以肿瘤细胞系生长曲线平缓期开端点为加NK细胞时间点。将待测NK细胞取出离心、重悬、台盼蓝染色计数，按肿瘤细胞接种量：NK细胞＝3：1的比例加入电流板中，上机(实时细胞分析仪)放入培养箱中持续监测杀伤情况，且采用未经转染的NK92MI细胞作为阴性对照。
由图13可知，实施例1和2的转染后的NK细胞对于这四种肿瘤细胞相对于阴性对照，均有明显增强的杀伤性能。而对比例1-4的转染后的NK细胞对于这四种肿瘤细胞的杀伤性能也有一定的增强，但是增强程度明显较实施例1和2更低一点。
以上实施例仅用以说明本申请的技术方案而非对本申请保护范围的限制，尽管参照较佳实施例对本申请作了详细说明，本领域的普通技术人员应当理解，可以对本申请的技术方案进行修改或者等同替换，而不脱离本申请技术方案的实质和范围。

Claims (10)

  1. 一种制备过表达CD16a的NK细胞的方法，包括以下步骤：
    (1)构建具有CD16a的目标质粒和包装质粒的慢病毒转染体系；
    (2)培养用于慢病毒的HEK 293T细胞，对所述HEK 293T细胞进行饥饿处理；
    (3)将减血清培养基或者无血清培养基平均分成两份，一份加入具有CD16a的目标质粒、第一包装质粒和第二包装质粒的慢病毒转染体系，混合均匀，得到质粒混合物；另一份加入聚乙烯亚胺，混合均匀，然后室温放置1～10min，得到转染试剂；
    (4)将转染试剂逐滴加入质粒混合物中，混合均匀后，室温放置5～40min，得到含有聚乙烯亚胺和质粒的混合溶液；
    (5)将步骤(4)得到的混合溶液逐滴加入到含有HEK 293T细胞的培养瓶上清中，充分混匀，并放入培养箱中培养6～9h，吸弃上清，加入含有0.1-5vol％胎牛血清的细胞培养基，继续培养；
    (6)每隔24h收集一次病毒转染后的上清，共收取1～3次；
    (7)过滤和浓缩病毒液；
    (8)取第一细胞培养基重悬培养好的NK细胞，按Moi＝5～100加入浓缩后慢病毒液，再加入聚凝胺，预热；
    (9)离心；
    (10)加入含有聚凝胺的第二细胞培养基，混匀，继续培养，培养期间根据培养基颜色和细胞数量判断是否半换液或传代，培养期间采用第三细胞培养基。
    (11)当细胞传代至起始数10倍以上时，取细胞做流式检测阳性率；当细胞生长至5-10×106万个细胞左右时，做流式分选，筛选出阳性细胞，接种相应体系继续扩增培养并建库。
  2. 如权利要求1所述的制备过表达CD16a的NK细胞的方法，其中包装质粒包括第一包装质粒和第二包装质粒，其中第一包装质粒为psPAX2质粒，第二包装质粒为pMD2.G质粒，且pMD2.G、psPAX2、具有CD16a的目标质粒三者的质量比为1～3:1～5:2～8；
    或者，其中包装质粒包括第一包装质粒、第二包装质粒和第三包装质粒，第一 包装质粒为pLP1质粒，第二包装质粒为pLP2质粒，第三包装质粒为pLP/VSVG质粒，且具有CD16a的目标质粒为插入pLenti的表达载体；其中pLP1质粒、、pLP2质粒，、pLP/VSVG质粒、具有CD16a的目标质粒四者的重量比为1.5～7.5:1.2～5.8:3.2～9.3:6.8～10。
  3. 如权利要求2所述的制备过表达CD16a的NK细胞的方法，其中步骤(3)中聚乙烯亚胺与三种质粒的总量比为1～10:1。
  4. 如权利要求1所述的制备过表达CD16a的NK细胞的方法，其中步骤(8)中NK细胞是NK92MI细胞，且步骤(8)中聚凝胺终浓度为8～100μg/mL。
  5. 如权利要求1所述的制备过表达CD16a的NK细胞的方法，其中步骤(8)中第一细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.005～0.6mM，叶酸0.00005～0.6mM，巯基乙醇0.0005～0.6mM，胎牛血清0～2vol％，马血清0～2vol％，双抗0～2vol％。
  6. 如权利要求1所述的制备过表达CD16a的NK细胞的方法，其中步骤(9)离心的具体操作为：将恒温离心机调成600～1000g，30～36℃，10min离心预热至30～36℃，将预热后的培养板转移至离心机中，600～1000g离心30min～3h，30～36℃。
  7. 如权利要求1所述的制备过表达CD16a的NK细胞的方法，其中步骤(10)中第二细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.5～50mM，叶酸0.05～5mM，巯基乙醇0.05～5mM，胎牛血清8～50vol％，马血清8～50vol％，双抗0.5～5vol％。
  8. 如权利要求1所述的制备过表达CD16a的NK细胞的方法，其中步骤(10)中聚凝胺终浓度为8～100μg/mL。
  9. 如权利要求1所述的制备过表达CD16a的NK细胞的方法，其中步骤(10)中第三细胞培养基的具体组成如下：在MEM基础培养基中加入肌醇0.05～5mM，叶酸0.005～0.5mM，巯基乙醇0.005～0.5mM，胎牛血清5～20vol％，马血清5～20vol％，双抗0.5～5vol％。
  10. 一种采用权利要求1～9任一项所述的制备方法制备的过表达CD16a的NK细胞。
PCT/CN2023/093245 2022-05-12 2023-05-10 一种过表达CD16a的NK细胞的制备方法及其应用 WO2023217182A1 (zh)

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