[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2022007665A1 - Procédé non viral pour la préparation de cellules nk stables avec une expression de récepteur chimérique élevée - Google Patents

Procédé non viral pour la préparation de cellules nk stables avec une expression de récepteur chimérique élevée Download PDF

Info

Publication number
WO2022007665A1
WO2022007665A1 PCT/CN2021/103235 CN2021103235W WO2022007665A1 WO 2022007665 A1 WO2022007665 A1 WO 2022007665A1 CN 2021103235 W CN2021103235 W CN 2021103235W WO 2022007665 A1 WO2022007665 A1 WO 2022007665A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
natural killer
car
chimeric receptor
Prior art date
Application number
PCT/CN2021/103235
Other languages
English (en)
Chinese (zh)
Inventor
肖�琳
裘新红
彭群武
张亮
朱苏闽
徐芳芳
Original Assignee
杭州优凯瑞医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 杭州优凯瑞医药科技有限公司 filed Critical 杭州优凯瑞医药科技有限公司
Publication of WO2022007665A1 publication Critical patent/WO2022007665A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/50—Colon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • A61K39/4613—
    • A61K39/4631—
    • A61K39/464417—
    • A61K39/464429—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

Definitions

  • the present application belongs to the technical field of medical bioengineering, and relates to a method for preparing NK cells modified by stable and high expression of chimeric receptors by using a non-viral method and its application.
  • NK cells Natural killer cells are an important part of the innate immune system. The proportion of NK cells in peripheral blood lymphocytes is about 10%, and they are the third largest lymphocyte population after B cells and T cells. NK cells have multiple biological functions and are the body's first line of defense against infections and tumors. NK cell function is regulated by a sophisticated and complex network of activating and inhibitory receptors. Most circulating NK cells are in a quiescent phase and can be activated by cytokines and infiltrate pathogen-infected or malignant cells-containing tissues. When the receptors of NK cells are combined with the corresponding ligands, NK cells can also secrete some cytokines, such as interferon (IFN)- ⁇ , to play an immunoregulatory role.
  • IFN interferon
  • NK cells are involved in controlling the occurrence and development of various diseases.
  • Several in vitro studies on mammalian cells, including human cells, and in vivo studies in mice and rats have shown that NK cells can recognize tumor cells as targets and control tumor cell growth, metastasis, and spread in vivo. Decreased peripheral blood NK cell activity increases the risk of cancer in adults, an 11-year follow-up epidemiological survey showed.
  • NK cells As host immunity has also been studied in various cases of flavivirus infection such as Japanese encephalitis virus, yellow fever virus, dengue virus, tick-borne encephalitis virus and West Nile virus (WNV) ; their role in viral hepatitis, influenza virus and HIV-1 infection has also been well documented in several studies; likewise, their role in preventing respiratory tract infections by viruses such as bacteria, respiratory syncytial virus (RSV) and influenza
  • RSV respiratory syncytial virus
  • the role of Aspects has also been described in detail in mouse studies. (For details, please refer to: Mandal A, Viswanathan C. et al., Natural killer cells: In health and diseas[J]. Hematology/oncology & Stem Cell Therapy, 2015, 8(2): 47-55.).
  • NK cells do not require HLA matching, so they can be used as heterologous off-the-shelf cell drugs for immunotherapy of patients, which has important clinical application prospects.
  • Adoptive therapy based on autologous or allogeneic NK cells has been clinically applied to anti-tumor and anti-viral infection, especially allogeneic NK cells have shown good therapeutic effects in the treatment of hematological cancers (Reference: Lupo K B, Matosevic S. et al., Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer Immunotherapy [J]. Cancers, 2019, 11(6).
  • NK cells have powerful immune effector functions
  • many cancer cells and viruses suppress the function of NK cells through mechanisms such as antigen escape and immunosuppression.
  • immunosuppression such as by expressing endogenous cytokines to enhance NK after adoptive reinfusion Cell proliferation ability, inhibiting immunosuppressive signals or enhancing the killing function of NK cells, etc.
  • CARs chimeric antigen receptors
  • a chimeric antigen receptor is a fusion of an antibody's scFv with an intracellular signal transduction structure, and the scFv is used to mediate the recognition and binding of tumor cell surface antigens.
  • primary NK cells are more resistant to gene transfection, which results in a low rate of uptake by NK cells to various vector systems, low transgene expression in NK cells, and only viral vectors achieve high levels in primary NK cells gene transfer efficiency.
  • the modification of chimeric receptors in primary NK cells is generally achieved by electrotransformation of mRNA encoding genes of chimeric receptors or by lentivirus/retrovirus infection. mRNA electrotransformation can only make the gene encoding the chimeric receptor transiently expressed, and the cost of synthesizing mRNA in vitro is relatively high, requiring multiple injections in clinical practice, and the treatment cost is high.
  • Lentiviral/retroviral transfection can produce NK cells with stable expression of chimeric receptor genes.
  • primary NK cells are extremely difficult to infect. So far, only a few scientific research teams have successfully applied NK cells.
  • Virus/retrovirus prepares chimeric antigen receptor-modified primary NK cells. After repeated infection, the expression ratio of its transgene in the final product is between 20% and 80%. Among them, retrovirus transfection The efficiency is higher than that of lentivirus (see literature: Sandro et al., Viral and Nonviral Engineering of Natural Killer Cells as Emerging Adoptive Cancer Immunotherapies [J]. Journal of Immunology Research, 2018.).
  • retroviruses and/or lentiviruses are themselves pathogenic and have the potential for insertional mutagenesis, they pose a significant regulatory hurdle to the implementation of human clinical trials.
  • the large-scale production of viral vectors in cell lines has the problems of high cost and low efficiency, which constitutes an obstacle to the clinical transformation of chimeric receptor-modified NK cells and significantly increases the production cost.
  • the non-viral vector system is an artificial synthesis system, which does not depend on any viral components or mammalian cells for production, and is low in cost, and at the same time, it is beneficial to avoid the immune problems caused by the use of viral vectors.
  • DNA transposon is a typical non-viral vector system with the advantages of sustainable gene expression, low immunogenicity, low cost and high efficiency.
  • DNA transposons are discrete DNA segments containing transposase genes flanked by inverted terminal repeats (ITRs) containing transposase binding sites.
  • ITRs inverted terminal repeats
  • the process of transposition is that the transposase binds to the ITRs, "cuts” the transposition from one location, and "pastes” it to another new location.
  • Transposon-based vector systems such as Sleeping Beauty (SB) system, PiggyBac (PB) system, and Tol transposon system, use the method of transposition to introduce transgenes into the host genome.
  • the present application provides a non-viral preparation of chimeric receptors
  • the receptor-modified NK cell method uses a transposon system to introduce stable and highly expressed chimeric receptors into NK cells to achieve efficient genetic modification of NK cells.
  • the present invention provides a method for preparing natural killer cells modified by chimeric receptors by utilizing a non-viral transposon system and artificial antigen-presenting cells, comprising:
  • the NK cells are primary human NK cells, which can be derived from human peripheral blood, umbilical cord blood, placental tissue or induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • the chimeric receptors include chimeric antigen receptors, chimeric switching receptors, or other chimeric receptors synthesized by recombinant DNA technology.
  • the transposon system of step (1) includes the PiggyBac transposon system, the Sleeping Beauty transposon system, the Tol2 transposon system or other transposon systems using transposase.
  • the transposon system in step (1) includes a transposon element and a transposase element; the transposon element includes a transposon 5' inverted terminal repeat (5' ITR) and a 3' inverse terminal repeat.
  • the transposon can Is a plasmid or a linearized nucleic acid fragment; the transposase element can be encoded by a plasmid or a linearized nucleic acid fragment, or can be encoded by mRNA, or can be a protein.
  • the nucleic acid sequence of the transposase and the nucleic acid sequence of the transposon may be on one vector or on two different vectors.
  • genes encoding cytokines that promote the survival of NK cells can be added to the transposon element.
  • the cytokine can be secreted, membrane-bound, or a combination of cytokine-linked receptors; the cytokine encoding gene can be in the same transposon expression vector with the chimeric receptor encoding gene , also in different transposon expression vectors.
  • the IL-15 encoding gene that promotes the survival of NK cells is added to the transposon element, and is in the same transposon expression vector as the encoding gene of the chimeric receptor.
  • the method for using the transposon system to transfer the encoding gene of the chimeric receptor into NK cells includes electroporation, chemical transfection, and other non-viral transfection methods.
  • the relevant vectors of the transposon system are transferred into primary NK cells by electroporation.
  • the NK cells may or may not be activated in advance.
  • the NK cells are activated in advance.
  • the NK cells are activated in advance, it can be 0 to 14 days after the activation of the NK cells, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.
  • the gene encoding the chimeric receptor was transferred into NK cells using the transposon system.
  • the gene encoding the chimeric receptor is transferred into the NK cells using a transposon system.
  • the gene encoding the chimeric receptor is transferred into the NK cells using a transposon system.
  • NK cells are activated in advance, artificial antigen-presenting cells, feeder cells, cytokines, antibodies or compounds can be used for activation.
  • the artificial antigen-presenting cells include cell-based, artificially synthesized or exosome-based artificial antigen-presenting cells.
  • the cell-based artificial antigen presenting cells are selected from human myeloid leukemia K562 cells, human Burkitt lymphoma Daudi cells, EBV transformed B lymphoblastoid cells (EBV-LCL) cells or mouse embryonic fibroblasts Cell line NIH/3T3 cells.
  • the cell-based artificial antigen presenting cells are selected from K562 cells.
  • the cell-based artificial antigen presenting cell is an engineered cell that expresses an antigen recognized by a chimeric receptor.
  • the engineered cells express ligand molecules, cytokines, etc. required for NK cell activation, wherein the cytokines may be secreted or membrane-bound.
  • the ligand molecules required for NK cell activation include CD137L and/or OX40L
  • the cytokines required for NK activation include any one of human IL-12, human IL-15, human IL-18 or human IL-21 or a combination of at least two.
  • the cell-based artificial antigen presenting cells and feeder cells are treated with gamma radiation (100 Gy) or mitomycin C (20 ⁇ g/mL).
  • both NK cell activation and CAR-NK cell expansion can be performed using artificial antigen presenting cells.
  • step (1) 0 to 14 days after the gene encoding the chimeric receptor is transferred into NK cells using the transposon system, for example, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, or 14 days, using artificial antigen-presenting cells to stimulate the proliferation of chimeric receptor-modified NK cells.
  • the transposon system for example, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, or 14 days, using artificial antigen-presenting cells to stimulate the proliferation of chimeric receptor-modified NK cells.
  • the chimeric receptor-modified NK cells in step (2) are expanded multiple times, and each expansion cycle is 3 to 14 days, such as 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13 or 14 days.
  • magnetic sorting can be used to enrich NK cells or CD3+ cells can be removed before step (1); or 0 to 14 days after step (1), magnetic sorting can be used to enrich NK cells cells or remove CD3+ cells; magnetic sorting can also be used to enrich NK cells or remove CD3+ cells after step (2).
  • the method further comprises the step of sorting CAR+ cells.
  • the method further includes the step of sorting CAR+ cells.
  • step (2) that is, after one round of co-culture with artificial antigen-presenting cells, sorting of CAR+ cells is performed.
  • a cytokine composition such as a combination of hrIL-12 and hrIL-18 or a combination of hrIL-12, hrIL-15 and hrIL-18, can be added during the preparation of chimeric receptor-modified NK cells using the transposon system The combination.
  • the cytokine composition can be added on any day during the preparation of the chimeric receptor-modified NK cells.
  • the cytokine composition can be removed by centrifugation one day after being added during the production of chimeric receptor-modified NK cells.
  • the final concentration of hrIL-12 is 1-100 ng/mL
  • the final concentration of hrIL-15 is 1-100 ng/mL
  • the final concentration of hrIL-18 is 1-100 ng/mL
  • the final concentration of hrIL-12 is 1-100 ng/mL.
  • the final concentration of hrIL-15 or hrIL-18 is the same or different, for example, it can be 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100ng/mL.
  • the chimeric receptor-modified NK cell culture is carried out using a liquid medium
  • the liquid medium can be a cell culture medium commonly used in the art, such as AIM V TM (Gibco), X-VIVO TM 15 ( Lonza), SCGM TM (CellGenix), RPMI, NK MACS TM (Miltenyi), OpTmizer TM (Gibco), ImmunoCult TM -XF T Cell Expansion Medium (STEMCELL) or StemSpan TM (STEMCELL).
  • the present application provides a method for preparing chimeric receptor-modified NK cells using the PiggyBac transposon system, the method comprising:
  • PBMCs peripheral blood mononuclear cells
  • the helper plasmid encoding the PiggyBac (PB) transposon system and the donor plasmid encoding the chimeric acceptor were electroporated into NK cells;
  • chimeric receptor-modified NK cells were co-cultured with ⁇ -ray-treated engineered K562 cells, and added at the beginning of the culture.
  • the cell culture medium is AIMV medium supplemented with 1-5% human AB serum/human autologous plasma, and hrIL-2 is present during the culture.
  • the method for preparing chimeric receptor-modified NK cells using the PiggyBac transposon system according to the present invention is:
  • the helper plasmid encoding the PiggyBac (PB) transposon system and the donor plasmid encoding the chimeric acceptor were electroporated into NK cells;
  • the cell culture medium is AIMV medium supplemented with 1-5% human AB serum/human autologous plasma, and hrIL-2 is present during the culture.
  • lentiviral vectors or retroviral vectors also brings certain safety hazards to operators. Not only that, when using lentiviral vectors or retroviral vectors to prepare chimeric receptor-modified NK cells, There is also a potential risk of contamination of NK cell products by replicating retrovirus (RCR) or replicating lentivirus (RCL). Although the viral vector design and production system are constantly improving, this risk has been greatly reduced, but it cannot be completely ruled out. , the transposon system used in this application is a non-viral method, which is very safe and will not have these hidden dangers;
  • the integration efficiency of the gene encoding the chimeric receptor in NK cells is between 40% and 80%, which has reached the same level as that of using retrovirus to modify primary NK cells in the prior art.
  • FIG. 1A is the map of plasmid 2
  • FIG. 1B is the map of plasmid 4;
  • Figure 2A is the expression of membrane-bound human interleukin 15 on the surface of K562-NK1 cells
  • Figure 2B is the expression of membrane-bound human interleukin 21 on the surface of K562-NK1 cells
  • the abscissa is the membrane-bound human interleukin IL-15 or IL-21
  • the expression intensity of , the ordinate is the cell count;
  • Fig. 3 is the flow chart that utilizes PiggyBac transposon system to prepare CAR-NK;
  • Figure 4A shows the proportion of NK cells in the cell population on the 10th, 17th, and 24th days of culture for different CAR-NK preparation methods
  • Figure 4B shows the proportion of CAR+ cells in the cell population for different CAR-NK preparation methods on the 10th, 17th, and 24th days of culture. the proportion of NK cells;
  • Figure 5 shows the cumulative expansion fold of the total cell number on the 10th, 17th, and 24th days of culture for different CAR-NK preparation methods
  • Figure 6A shows the proportion of NK cells in the cell population of mononuclear cells (MNCs) from different sources at the end of the 1st, 2nd, 3rd, and 4th rounds of co-culture on day 0, and Figure 6B shows the ratios of MNCs from different sources in the first At the end of 2, 3, and 4 rounds of co-culture, the proportion of CAR+ cells in NK cells;
  • MNCs mononuclear cells
  • Figure 7 is the cumulative expansion fold of the total cell number of MNCs from different sources at the end of the first, second, third, and fourth rounds of co-culture;
  • Figure 8A shows the real-time killing map of NK and NKG2D CAR-NK cells on the human colorectal cancer cell line HCT116 on the 21st day after the third round of co-culture
  • Figure 8B shows the NK and NKG2D CAR on the 21st day after the third round of co-culture -
  • Figure 10A shows the expression of GFP in NK cells 1 day after the pmax-GFP plasmid was electroporated.
  • the NK cells used were NK cells on the 17th day of expansion.
  • the abscissa is the expression intensity of GFP
  • the ordinate is the cell count
  • Figure 10B is pmax -Number of NK cells before and after 1 day of electroporation of GFP plasmid
  • the NK cells used are NK cells on the 17th day of expansion
  • the abscissa is before and 1 day after electroporation, respectively
  • the ordinate is the number of cells;
  • Figure 11A shows the proportion of NK cells in the cell population on the 10th, 17th, 24th and 31st days of culture by different CAR-NK preparation methods
  • Figure 11B shows the ratio of NK cells in the cell population by different CAR-NK preparation methods on the 10th, 17th, 24th and 31st days of culture , the proportion of CAR+ cells in NK cells;
  • Figure 12 shows the cumulative expansion fold of the total cell number on the 10th, 17th, 24th, and 31st days of culture for different CAR-NK preparation methods
  • Figure 13A is the real-time killing map of human ovarian cancer cell line SKOV3 by NK, CAR-NK and eCAR-NK cells on the 17th day.
  • the coordinate is the cell index
  • Figure 13B shows the data analysis after adding effector cells for 24 hours to calculate the tumor growth inhibition rate, the abscissa is the different experimental groups, and the ordinate is the tumor growth inhibition rate;
  • Figure 14A is the detection of the survival of NK, NKG2D CAR-NK and NKG2D eCAR-15NK on the 22nd day of culture in the presence or absence of IL-2 for one week in vitro;
  • Figure 14B is the detection of the NK on the 21st day of culture , NKG2D eCAR-15NK and NKG2D eCAR-15 NK(sorted) were cultured in vitro for one week in the presence or absence of IL-2;
  • Figure 14C is the detection of NK, NKG2D eCAR-NK ( sorted) and NKG2D eCAR-mbIL15 NK(sorted) in vitro cultured without IL-2 for one week; ) Survival of one week in vitro culture with or without IL-2; among them, "-IL2" in the abscissa is the culture group without IL-2, and "+IL2" is the culture group with IL-2 added , the ordinate is the percentage change relative to the number of cells before one week
  • Figure 15A shows the killing results of NK, NKG2D CAR-mbIL15 NK, and NKG2D eCAR-mbIL15 NK (sorted) on the 24th day after the third round of co-culture on human acute myeloid leukemia cell line KG1, the abscissa is the effect-target ratio (E : T), the killing percentage on the ordinate;
  • Figure 15B is the killing result of NK and NKG2D eCAR-mbIL15 NK (sorted) on the human colorectal cancer cell line HCT116 on the 24th day after the third round of co-culture, the abscissa is the tumor cell plating
  • the time after (that is, after the start of the experiment), in hours, the ordinate is the cell index;
  • Figure 16 is the expression of BCMA on the surface of K562-NK2 cells
  • Figure 17A shows the proportion of NK cells in the cell population of peripheral blood mononuclear cells (PBMC) derived from a healthy donor at the end of the 1st, 2nd and 3rd rounds of co-culture on day 0;
  • Figure 17B shows a healthy donor The proportion of CAR+ cells in NK cells at the end of the 1st, 2nd and 3rd rounds of co-culture of the derived PBMC;
  • PBMC peripheral blood mononuclear cells
  • Figure 18 is the expansion fold of the total cell number in each round of PBMC derived from a healthy donor at the end of the 1st, 2nd, and 3rd rounds of co-culture;
  • Figure 19 shows the killing results of human multiple myeloma cell line U266 by NK and BCMA eCAR-NK on the 36th day at the end of the fifth round of co-culture, wherein the abscissa is the effect-target ratio (E:T), and the ordinate is the killing percentage.
  • PBMC peripheral blood of solid tumor patients or healthy donors
  • MNC derived from umbilical cord blood was purchased from Maishun Biotechnology Co., Ltd.
  • human chronic myeloid leukemia cell line wild-type K562 cells were purchased from ATCC
  • human multiple myeloma cell line U266 was purchased from Wuhan Proceeds Life Technology Co., Ltd.
  • the other reagents or consumables not mentioned were all from conventional reagent manufacturers in the field or prepared by conventional means in the field;
  • the medium of human ovarian cancer cell line SKOV3 and human colorectal cancer cell line HCT116 is McCoy 5A (Gibco) + 10% FBS (Gibco)
  • the medium of human gastric cancer cell lines MGC803 and U266 is RPMI (Gibco) + 10% FBS (Gibco)
  • the medium of K562 and human AML cell line KG1 is IMDM (Gibco) + 10% FBS (Gibco);
  • the K562-NK1 cells used in the examples are ⁇ -ray-treated (100 Gy) genetically engineered K562 encoding membrane-bound human IL-15 (mbIL15), membrane-bound human IL-21 (mbIL21), eGFP and Puromycin cell. Since K562 cells naturally express NKG2D ligands, the antigens recognized by NKG2D CAR are not additionally loaded into K562-NK1 cells here.
  • Membrane-bound human IL-15 is composed of the GM-CSF Ra signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-15 (amino acids 30-162 of UniprotKB P40933) and the hinge transmembrane region of human CD8 (UniprotKB P01732 Amino acids 128-213) are fused together; membrane-bound human IL-21 is composed of GM-CSF R ⁇ signal peptide (amino acids 1-22 of UniprotKB P15509), human IL-21 (amino acids 25-162 of UniprotKB Q9HBE4) ), the hinge constant region of human IGHG4 (the 99th to 327th amino acids of UniprotKB P01861), the transmembrane region of human CD4 (the 397th to 418th amino acids of UniprotKB P01730) are fused together, and the amino acid sequence of eGFP is as shown in SEQ ID No:3 Shown, the amino acid sequence of Puro
  • the K562-NK2 cells used in the examples were genetically engineered to encode membrane-bound human IL-15 (mbIL15), membrane-bound human IL-21 (mbIL21), truncated BCMA (tBCMA), eGFP and Puromycin K562 cells.
  • mbIL15 membrane-bound human IL-15
  • mbIL21 membrane-bound human IL-21
  • tBCMA truncated BCMA
  • eGFP Puromycin K562 cells.
  • the sequence of tBCMA is the amino acid sequence of positions 1-77 of UniProt KB-Q02223.
  • Human recombinant IL-2 (hrIL2) was purchased from Beijing Shuanglu Company, and human recombinant IL-12 (hrIL12), human recombinant IL-15 (hrIL15), and human recombinant IL-18 (hrIL18) were purchased from Nearshore Protein Company;
  • NK cell culture medium AIM medium (Gibco company) + 5% human AB serum (Gemini company);
  • P3 Primary Cell 4D-Nucleofector X Kit was purchased from Lonza Company;
  • PE-conjugated anti-human CD3 antibody, APC-conjugated anti-human CD56 antibody were purchased from BD
  • FITC-conjugated anti-Strep-tag II antibody was purchased from GenScript
  • Biotin-conjugated anti-human IL-15 antibody and APC Conjugated Streptavidin was purchased from Biolegend Company
  • Biotin-conjugated anti-human IL-21 antibody was purchased from eBioscience Company
  • anti-Biotin microbeads and Streptavidin micobeads were purchased from Miltenyi Company
  • Flow cytometer was purchased from BD Company, model C6 Sampler; real-time killing detector was purchased from ACEA Bio Company, model xCELLigence RTCA DP; human IFN ⁇ ELISA detection kit was purchased from Biolegend Company; Calcein-AM was purchased from Yisheng Biotechnology; pmax-GFP was purchased from Lonza Company;
  • the amino acid sequence of PiggyBac transposase is from GenBank: AAA87375.2; the NKG2D CAR is: NKG2D-CD28-4-1BB-CD3 ⁇ , the antigen-binding domain is from amino acids 83-216 of NKG2D (UniProtKB-P26718), and the hinge region is from Amino acids 99-110 of IgG4 (UniProtKB-P01861), transmembrane region from amino acids 153-179 of CD28 (UniProtKB-P10747), intracellular domain from 209-255 of 4-1BB (UniProtKB-Q07011) BCMA CAR is: anti-BCMA ScFv-CD28-4-1BB-DAP12 ⁇ , the antigen binding domain is from the scFv, hinge of Anti-BCMA antibody C11D5.3 The region is derived from amino acids 99-110 of IGHG4 (UniProtKB-P01861), the transmembrane region is derived from amino acids 153
  • DNA fragment 1 CMV promoter nucleotide sequence, puromycin coding nucleotide sequence, T2A, EGFP coding nucleotide sequence were synthesized by conventional biotechnology service companies in the field;
  • DNA fragment 2 CMV promoter nucleotide sequence, mbIL-15 coding nucleotide sequence, P2A coding nucleotide sequence, mbIL-21 coding nucleotide sequence are synthesized by conventional biotechnology service companies in the field;
  • DNA Fragment 1 and DNA Fragment 2 were cloned into pFastBac1 plasmid (purchased from Thermofisher) and designated as plasmid 1 .
  • the 5' inverted terminal repeat of the PiggyBac transposon (SEQ ID NO: 1), the chicken ⁇ -globin chromatin insulator cHS4 (GenBank: AY040835.1), the EF1 ⁇ promoter, the EcoRI and SalI restriction site sequences , SV40 polyA sequence, reverse complementary cHS4 sequence and 3' reverse terminal repeat sequence (SEQ ID NO: 2) were fused, and synthesized by an outsourced service company, cloned into pmaxCloning vector (purchased from Lonza company) through BsaI, named For pZTS4, the expression of the fusion gene is controlled by the EF1 ⁇ promoter;
  • SEQ ID NO: 1 5'-ccctagaaagataatcatattgtgacgtacgttaagataatcatgtgtaaaattgacgcatg-3';
  • SEQ ID NO: 2 5'-catgcgtcaattttacgcagactatctttctaggg-3';
  • the extracellular antigen-binding domain (ED) of NKG2D was fused with the IGHG4 hinge region, CD28 transmembrane region, 4-1BB and CD3 ⁇ signaling domain to generate the second-generation NKG2D CAR vector; CAR expression was detected by cytometry, and 3 repeats of Strept-tag II (ST2) were added to the sequence of the CAR; the CAR fragment was synthesized by a conventional biotechnology service company in the field, and the 5' and 3' ends of the sequence included restriction Sexual restriction sites EcoRI and SalI; after the synthesized DNA fragment was digested by EcoRI and SalI, it was cloned into pFastBac1 plasmid (purchased from Thermofisher) and named as plasmid 3.
  • pFastBac1 plasmid purchased from Thermofisher
  • the CAR sequence in plasmid 2 was digested with EcoRI and SalI and cloned into pZTS4, named plasmid 4. As shown in Figure 1B, the expression of the CAR gene is controlled by the EF1 ⁇ promoter. control.
  • the sequence of IRES and human IL-15 (UniprotKB: P40933) was fused, and the 5' and 3' ends of the sequence included the restriction site SalI .
  • the synthesized DNA fragment was digested with SalI and cloned into plasmid 4, which was named plasmid 5.
  • the NKG2D CAR transposon donor vector containing the membrane-bound human IL-15 (mbIL-15) gene the IRES and the sequence of membrane-bound human IL-15 were fused, including restriction at the 5' and 3' ends of the sequence The cleavage site SalI.
  • the synthesized DNA fragment was digested with SalI and cloned into plasmid 4, which was named plasmid 6.
  • the anti-BCMA scFv (C11D5.3) was fused with the IGHG44 hinge region, CD28 transmembrane region, 4-1BB and CD3 ⁇ signaling domains to generate a second-generation anti-BCMA CAR vector; in order to To facilitate the detection of CAR expression by flow cytometry, 3 repeats of Strept-tag II (ST2) are added to the CAR sequence; the CAR fragment is synthesized by an outsourced service company, and the 5' and 3' ends of the sequence include restriction Sex restriction sites EcoRI and SalI; the synthesized DNA fragment was digested by EcoRI and SalI, and then cloned into pFastBac1 plasmid (purchased from Thermofisher), named plasmid 7.
  • ST2 Strept-tag II
  • the CAR sequence in plasmid 7 was digested with EcoRI and SalI and cloned into pZTS4, which was named plasmid 8.
  • the expression of the CAR gene was controlled by the EF1 ⁇ promoter.
  • plasmid 9 the DNA sequence of tBCMA was synthesized by an outsourcing company, and cloned into pZTS4 by EcoRI and SalI, named plasmid 9.
  • the wild-type K562 cells were resuspended in 10 mL Opti-MEM, centrifuged at 200 ⁇ g for 5 min, the cell pellet was resuspended in 100 ⁇ L P3 buffer, 10 ⁇ g plasmid 1 was added, and after mixing, it was transferred to the Lonza electric shock cup;
  • puromycin screening was performed at a concentration of 200 ⁇ g/mL for 1 month, and the medium was changed every 2 days;
  • K562-NK1 cells expressed high levels of human IL-15 and human-IL21 on the surface, with positive rates of 100.0% and 78.9%, respectively.
  • PBMCs and 5 ⁇ 10 6 K562-NK1 were resuspended in 10 mL of NK cell culture medium, seeded into T25 cell culture flasks, and hrIL-2 was added to make the final concentration is 100IU/mL;
  • the suspended cells were taken out and counted, and centrifuged at 300 ⁇ g for 10 min; resuspended the cell pellet with 10 mL Opti-MEM and centrifuged at 300 ⁇ g for 10 min; resuspended the cell pellet in 100 ⁇ L P3 buffer, and added 5 ⁇ g plasmid 2 and 10 ⁇ g Plasmid 4, mix well and transfer to the electroporation Lonza electroporation cup; place the electroporation cup in the Lonza 4D-NucleofectorTM X Unit (in a single electroporation cup module), and perform electroporation.
  • NK cell suspension After the electroporation, slowly transfer the NK cell suspension in one electroporation cup Transfer to a new T25 cell culture flask, add 10 mL of NK cell culture medium containing a final concentration of 100IU/mL hrIL-2, mix gently, and place the T25 cell culture flask in a 37°C cell incubator for cultivation;
  • This example compares the ratio of NK cells in CAR-NK, the ratio of CAR expression in NK cells, and the total cells obtained by electroporation before NK cell activation or on the 2nd or 4th day after NK cell activation. the amplification fold.
  • Example 3 Electrically At day 2 activated NK cells transfected is the procedure of Example 3 is referred to as Day2 EP; method of electroporation prior to activation of NK cells is different from the embodiment 3 in that the 5 ⁇ 10 6 PBMC were as described in embodiment The method of Example 3 was electroporated. Immediately after electroporation, it was co-cultured with 5 ⁇ 10 6 K562-NK1 (100Gy ⁇ -ray irradiation), which was recorded as Day0 EP; electroporation was performed on the 4th day after the activation of NK cells, which was different from that in Example 3.
  • 5 ⁇ 10 6 K562-NK1 100Gy ⁇ -ray irradiation
  • the above three methods were used to prepare CAR-NK cells from a normal donor PBMC, and the obtained NK cell purity (CD3-CD56+) was compared.
  • the results are shown in Figure 4A.
  • the purity of NK cells was 68%, 77%, and 85% on the 10th, 17th, and 24th days of culture, respectively, while the purity of NK cells in the CAR-NK cells prepared by electroporation before NK cell activation was on the 10th, 17th day of culture. were 11% and 13%, respectively, and the purity of NK cells in CAR-NK cells prepared by electroporation 4 days after NK cell activation were 46%, 81%, and 88% on culture days 10, 17, and 24, respectively. It shows that the purity of NK cells is higher in the CAR-NK cells prepared by electroporation after NK cell activation than that prepared by electroporation before NK activation.
  • the expression ratio of CAR in the obtained CAR-NK cells was compared, and the results were shown in Figure 4B. were 14%, 30%, and 45%, while the proportion of CAR+ NK cells in CAR-NK cells prepared by electroporation before NK cell activation was 14% and 27% on the 10th and 17th days of culture, respectively.
  • the proportion of CAR+ NK cells in the CAR-NK cells prepared by electroporation on day 4 was 2%, 15%, and 19% on the 10th, 17th, and 24th days of culture, respectively. It shows that the earlier electroporation is performed, the higher the proportion of CAR+ NK cells.
  • This example also compares the amplification multiples of the total number of cells obtained.
  • the results are shown in Figure 5.
  • the amplification multiples of the total number of cells obtained by the method of Example 3 for 24 days is 3022 times; After a total of 17 days of expansion, the total number of cells was expanded by 133 times; 4 days after the activation of NK cells, electroporated for 24 days, the total number of cells was expanded by 736 times. This indicated that the cells obtained by electroporation on the second day after the activation of NK cells had the highest expansion fold.
  • Example 3 Based on the comparison of these three parameters, the method of Example 3, that is, electroporation was performed 2 days after the activation of NK cells, the prepared CAR-NK cells, the purity of NK cells, the proportion of CAR+ NK cells, and the amplification fold of the total number of cells. Relatively best.
  • Example 5 Mononuclear cells from multiple sources can use the PiggyBac transposon system to prepare NKG2D CAR-NK
  • NKG2D CAR-NK 7 mononuclear cells (MNCs) from different sources were used to prepare NKG2D CAR-NK according to the method described in Example 3, wherein HD001, HD002, HD003, HD004, HD005, and HD006 were extracted from peripheral blood of healthy donors.
  • PBMC, CB001 was derived from MNC extracted from umbilical cord blood.
  • Example 6 Direct electroporation of NK cells, transgene expression efficiency and NK cell survival rate
  • the green fluorescent protein GFP gene was used as the reporter gene, and the pmax-GFP plasmid was directly used to electroporate the NK cells amplified for 17 days.
  • the NK cell expansion procedure is as follows: On day 0, 2 ⁇ 10 6 PBMCs and 2 ⁇ 10 6 K562-NK1 (100Gy ⁇ -ray irradiated) were resuspended in 10 mL of NK cell culture medium, and seeded in T75 cell culture flasks (vertical).
  • hrIL-2 was added to make the final concentration 100IU/mL; from the 2nd day to the 10th day, according to the cell growth, an appropriate amount of fresh NK cell culture medium containing hrIL-2 was added; on the 10th day, all cells were collected Counting, taking 2 ⁇ 10 6 cells for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD56 antibody); 2 ⁇ 10 6 cells and 2 ⁇ 10 6 K562-NK1 (100Gy ⁇ -ray irradiation) ) was resuspended in 10 mL of medium, and hrIL-2 was added to make its final concentration 100 IU/mL; from the 10th day to the 17th day, according to the cell growth, an appropriate amount of fresh NK cell culture medium containing hrIL-2 was added; the 17th day On the next day, all cells were collected and counted, and 2 ⁇ 10 6 cells were taken for flow cytometric phenotype analysis (anti-human CD3 antibody, anti-human CD
  • the NK cells on the 17th day of expansion were taken, and the electroporation method in Example 3 was used.
  • the number of electroporated NK cells was 2 ⁇ 10 6 , and the plasmid was replaced by the pmax-GFP plasmid.
  • cell counting and flow cytometry were performed to detect the proportion of GFP in NK cells.
  • Figure 10A and Figure 10B on the second day of electroporation, although the expression ratio of GFP was 17.99%, the survival rate of NK cells was only 3.2%.
  • PBMCs and 5 ⁇ 10 6 K562-NK1 were resuspended in 10 mL of NK cell culture medium, seeded into T25 cell culture flasks, and hrIL-2 was added to make the final concentration is 100IU/mL;
  • the suspended cells were taken out for counting, and centrifuged at 300 ⁇ g for 10 min; resuspended the cell pellet with 10 mL Opti-MEM and centrifuged at 300 ⁇ g for 10 min; resuspended the cell pellet in 100 ⁇ L P3 buffer, and added 5 ⁇ g plasmid 2 and 10 ⁇ g Plasmid 4, mix well and transfer to the Lonza electroporation cup; place the electroporation cup in the Lonza 4D-NucleofectorTM X Unit (in a single electroporation cup module) and perform electroporation.
  • NK cell culture medium containing hrIL-2 After the electroporation, slowly transfer the NK cell suspension in one electroporation cup to a new T25 cell culture flask, add 10 mL of NK cell culture medium containing hrIL-2 with a final concentration of 100IU/mL, mix gently, and place the T25 cell culture flask in a 37°C cell incubator for cultivation;
  • Example 8 Effects of different cytokine compositions on the expansion fold and cell phenotype of CAR-NK cells
  • This example compares the effect of adding cytokine compositions (IL-12, IL-15, IL-18) on the growth of CAR-NK cells during the second co-culture with K562-NK1 on the 10th day of CAR-NK preparation. Impact.
  • cytokine compositions IL-12, IL-15, and IL-18 were added (Example 7).
  • NK cell purity (CD3-CD56+) was compared.
  • the purity of NK cells in NK cells was 79%, 81%, 90%, and 93% on the 10th, 17th, 24th, and 31st days of culture, respectively, while the purity of NK cells in the eCAR-NK cells prepared in Example 4 was on the 1st day of culture. At 10, 17, 24, and 31 days, they were 79%, 87%, 93%, and 93%, respectively.
  • cytokines IL-12, IL-15 and IL-18 can slightly increase the proportion of NK cells during the second co-culture with K562-NK1 on the 10th day of CAR-NK preparation.
  • the ratio of NK cells of CAR+ in the obtained cells was compared.
  • the results are shown in Figure 11B.
  • the ratio of NK cells of CAR+ in the CAR-NK cells prepared in Example 3 was on the 10th, 17th, 24th, and 31st days of culture, respectively. were 34%, 47%, 49%, and 74%, while the proportion of CAR+ NK cells in the eCAR-NK cells prepared in Example 7 was 34%, 57%, and 58% on the 10th, 17th, 24th, and 31st days of culture, respectively. , 85%.
  • the total cell expansion folds obtained in the preparation process of CAR-NK and eCAR-NK cells are compared.
  • the results are shown in Figure 12.
  • the total expansion fold of the CAR-NK cells prepared in Example 3 is in 0-10 days, 10-17 days, 17-24 days and 24-31 days were 27, 21, 9, and 8 times respectively; while the eCAR-NK cells prepared in Example 7 had a total expansion fold at the 0- 10 days, 10-17 days, 17-24 days and 24-31 days were 27, 27, 9, and 7 times, respectively.
  • Example 7 Comprehensively comparing the data of the above three aspects, the method described in Example 7, that is, adding the cytokine compositions IL-12, IL-15, IL- 18. Contribute to the expansion of CAR+ NK cells.
  • Example 9 The effect of adding cytokine composition on the 10th day of CAR-NK preparation on the killing of human ovarian cancer cell line SKOV3 in vitro (RTCA)
  • This example compares the effect of adding cytokine compositions (IL-12, IL-15, IL-18) on the in vitro effects of CAR-NK on the 10th day of NKG2D CAR-NK preparation, when it was co-cultured with K562-NK1 for the second time. impact on lethality.
  • cytokine compositions IL-12, IL-15, IL-18
  • SKOV3 cells were plated in a 16-well electrode plate (ACEA Bio Company), 5000 cells/well; about 24 hours later, the NKG2D CAR-NK cells, NKG2D eCAR-NK cells and NKG2D eCAR-NK cells were cultured on the 17th day.
  • NK cells effector cells
  • SKOV3:NK 1:1” in the figure or CAR-NK cells (shown as “SKOV3:CAR-NK 1:1” in the figure)
  • the growth of tumor cells can be significantly inhibited, in which the killing of CAR-NK cells
  • the tumor effect was significantly better than that of NK cells, which indicated the effect of CAR in NKG2D CAR-NK cells; while adding eCAR-NK cells (shown as "SKOV3:eCAR-NK 1:1” in the figure) or CAR-NK cells (Fig. shown as "SKOV3:CAR-NK 1:2"), the tumor cells were almost completely killed.
  • the specific steps include: on the 0th day, 2 ⁇ 10 7 PBMC cells were taken, and CD3+ cells were removed with CD3 magnetic beads (Miltenyi) according to the manufacturer’s recommended procedure; 5 ⁇ 10 6 sorted PBMCs and 5 ⁇ 10 6 cells were removed K562-NK1 (100Gy ⁇ -ray irradiation) was resuspended in 10mL of NK cell culture medium, inoculated into a T25 cell culture flask, and hrIL-2 was added to make the final concentration 50IU/mL;
  • the suspended cells were taken out for counting, and centrifuged at 300 ⁇ g for 10 min; resuspended the cell pellet with 10 mL Opti-MEM and centrifuged at 300 ⁇ g for 10 min; resuspended the cell pellet in 100 ⁇ L P3 buffer, and added 5 ⁇ g plasmid 2 and 10 ⁇ g Plasmid 4, mix well and transfer to the Lonza electroporation cup; place the electroporation cup in the Lonza 4D-NucleofectorTM X Unit (in a single electroporation cup module) and perform electroporation.
  • NK cell culture medium containing hrIL-2 After the electroporation, slowly transfer the NK cell suspension in one electroporation cup to a new T25 cell culture flask, add 10 mL of NK cell culture medium containing hrIL-2 with a final concentration of 50IU/mL, mix gently, and place the T25 cell culture flask in a 37°C cell incubator for cultivation;
  • CAR-NK cells were resuspended in 80 ⁇ L of DPBS+1% AB serum buffer, added with 20 ⁇ L of anti-Strep tag II antibody, mixed and incubated at 2-8°C for 20 min; after incubation, 5 mL of buffer was added and centrifuged at 300g 10min, discard the supernatant and resuspend in 80 ⁇ L DPBS+1% AB serum buffer, add 20 ⁇ L anti-Biotin magnetic beads, mix well, incubate at 2-8°C for 15min; add 5mL buffer after incubation, centrifuge at 300g for 10min , discard the supernatant and resuspend in 500 ⁇ L of buffer, and then follow the steps recommended by the Mil
  • Example 11 CAR+ cell sorting can significantly increase the proportion of CAR+ NK cells
  • the NKG2D CAR-NK cells on the 10th day after the first round of co-culture were taken and divided into three groups: in the first group, 2 ⁇ 10 6 cells were co-cultured with 2 ⁇ 10 6 K562-NK1 (100Gy ⁇ -ray irradiation) cells1 Week, named as the unsorted group; in the second group, 1 ⁇ 10 7 cells were sorted according to the method described in Example 10.
  • NK cells:artificial antigen-presenting cells 1:2 and K562 -NK1 (100Gy ⁇ -ray irradiation) was co-cultured for 1 week, named Biotin Anti-Strep tag II Ab+anti-Biotin microbeads group; 1 ⁇ 10 7 cells were taken from the third group and resuspended in 90 ⁇ L DPBS+1% AB serum Add 10 ⁇ L of Streptavidin microbeads to the buffer, mix well and incubate at 2-8 °C for 20 min; after incubation, add 5 mL of buffer, centrifuge at 300 ⁇ g for 10 min, discard the supernatant and resuspend in 500 ⁇ L of buffer, and then proceed with magnetic beads The labeled positive cells were sorted, and the remaining cells were taken after sorting and co-cultured with K562-NK1 (100Gy ⁇ -ray irradiation) for 1 week according to NK cells:art
  • the sorting yield of Biotin Anti-Strep tag II Ab+anti-Biotin microbeads is higher, and the proportion of CAR+ cells in NK cells after one round of expansion can be increased to more than 90%.
  • Example 12 Adding secreted or membrane-bound human IL-15 to the CAR carrier can promote the survival of CAR-NK cells in vitro
  • NK NKG2D CAR-NK
  • eCAR-IL15 NKG2D eCAR-IL15 NK
  • NKG2D CAR-NK refers to Example 3; the preparation of NKG2D eCAR-IL15 NK refers to Example 7, and plasmid 4 is replaced with plasmid 5. Since the CAR vector contains IL-15, the cytokine composition on the 17th day IL-15 was removed. Take 2 ⁇ 10 6 cells in each group on the 22nd day after the third round of co-culture, and count them after 1 week of in vitro culture with and without addition of hrIL-2 (50 IU/mL), and calculate the percentage change of cells ( 100% ⁇ the number of cells after 1 week of culture/2 ⁇ 10 6 ).
  • NK, NKG2D eCAR-IL15 NK and NKG2D eCAR-IL15 NK were prepared from umbilical cord blood, respectively.
  • NKG2D eCAR-IL15 NK refer to Example 7, and replace plasmid 4 with plasmid 5. Since the CAR vector contains IL-15, IL-15 was removed from the cytokine composition on day 17; NKG2D eCAR-IL15 NK (sorted) Preparation Refer to Example 10, replace plasmid 4 with plasmid 5, since IL-15 is contained in the CAR vector, IL-15 is removed from the cytokine composition on day 17.
  • the percentage of cell change in NK, NKG2D eCAR-NK(sorted), NKG2D eCAR-mbIL15 NK(sorted) were 6%, 13%, and 140%, respectively, and the membrane Binding of IL-15 (mbIL-15) can also promote the survival of CAR-NK in vitro.
  • NK, NKG2D CAR-mbIL15 NK and NKG2D eCAR-mbIL15 NK were prepared from PBMCs, respectively.
  • the preparation of NKG2D CAR-mbIL15 NK refers to Example 3, and plasmid 4 is replaced with plasmid 6. Since the CAR vector contains mbIL-15, IL-15 is removed from the cytokine composition on day 17; NKG2D eCAR-mbIL15 NK (sorted ) Preparation of ) Refer to Example 10, replace plasmid 4 with plasmid 6, since mbIL-15 is contained in the CAR vector, IL-15 is removed from the cytokine composition on day 17.
  • NK, NKG2D eCAR-mIL15 NK and NKG2D eCAR-mbIL15 NK were prepared respectively with PBMC as the source, wherein the preparation of NKG2D eCAR-mbIL15 NK refers to Example 7, and plasmid 4 was replaced with plasmid 6, because CAR The vector contains mbIL-15, and IL-15 is removed from the cytokine composition on day 17; the preparation of NKG2D eCAR-mbIL15 NK (sorted) refers to Example 10, and plasmid 4 is replaced with plasmid 6, because the CAR vector contains mbIL -15, Depletion of IL-15 in the cytokine composition on day 17.
  • the cells on the 16th day after the second round of co-culture were taken for in vitro killing test.
  • the human AML cell line KG1 cells were taken and resuspended in DPBS at 1 ⁇ 10 6 cells/ml, and Calcein-AM (final concentration 0.2 ⁇ M) was added for staining at 37° C. for 15 min. After staining, FBS was added in an equal volume to stop the staining, and washed three times with DPBS.
  • the stained KG1 was resuspended in the medium, the cell density was adjusted to 2 ⁇ 10 5 cells/ml, and KG1 was plated in 100 ⁇ L per well in a U-shaped 96-well plate.
  • the tumor growth inhibition rate was calculated according to the following formula:
  • the K562-NK1 cells were resuspended in 10 mL Opti-MEM, centrifuged at 300 ⁇ g for 10 min, the cell pellet was resuspended in 100 ⁇ L P3 buffer, 5 ⁇ g plasmid 2 and 10 ⁇ g plasmid 9 were added, and the cells were transferred to the Lonza electric shock cup; The cups were placed in a Lonza 4D-NucleofectorTM X Unit (in a single shock cup module) for electroporation.
  • K562 cell suspension in an electroporation cup was slowly transferred to a well of a 6-well plate, and K562 medium (IMDM+10% FBS) was pre-added to the well; on the 7th day after electroporation, the cells were used
  • the sorting instrument BD Fusion sorts single cells into 96-well plates, and analyzes the amplified single cell clones by flow cytometry to detect the expression of BCMA.
  • the detection antibody is APC-conjugated anti-human BMCA.
  • Antibody, the screened single cell clone was named K562-NK2.
  • K562-NK2 cells expressed high levels of BCMA on the surface, with a positive rate of 99.4%.
  • the suspended cells were taken out for counting, and centrifuged at 300 ⁇ g for 10 min; resuspended the cell pellet with 10 mL Opti-MEM and centrifuged at 300 ⁇ g for 10 min; resuspended the cell pellet in 100 ⁇ L P3 buffer, and added 5 ⁇ g plasmid 2 and 10 ⁇ g Plasmid 8, after mixing, transfer to the electroporation Lonza electroporation cup; place the electroporation cup in the Lonza 4D-NucleofectorTM X Unit (in a single electroporation cup module), and perform electroporation.
  • NK cell suspension After the electroporation, slowly transfer the NK cell suspension in one electroporation cup Transfer to a new T25 cell culture flask, add 10 mL of NK cell culture medium containing hrIL-2 with a final concentration of 50IU/mL, mix gently, and place the T25 cell culture flask in a 37°C cell incubator for cultivation;
  • Figure 18 shows the expansion fold of the total number of cells in each round at the end of the first, second, and third rounds of co-culture during the preparation of BCMA eCAR-NK from a healthy donor-derived PBMC, and the expansion of the first round of co-culture.
  • the multiplication factor was 13 times
  • the second round of co-culture was 60 times
  • the third round of co-culture was 46 times.
  • U266 cells were taken and resuspended in DPBS at 1 ⁇ 10 6 cells/mL, and Calcein-AM (final concentration 0.2 ⁇ M) was added for staining at 37° C. for 15 min. After staining, FBS was added in an equal volume to stop the staining, and washed three times with DPBS. The stained U266 cells were resuspended in the medium, the cell density was adjusted to 2 ⁇ 10 5 cells/mL, and 100 ⁇ L per well of U266 was plated in a U-shaped 96-well plate.
  • the present application adopts a non-viral method and uses a transposon system to introduce a stable and highly expressed transgene into NK cells to achieve efficient genetic modification of NK cells.
  • the production cycle is short and the production cost is low.
  • the prepared transgenic modification NK cells have high purity and good safety, and have significant killing and/or inhibitory effects on tumor cells.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Hematology (AREA)
  • Hospice & Palliative Care (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Pregnancy & Childbirth (AREA)

Abstract

Nous proposons un procédé pour préparer des cellules tueuses naturelles (NK) stables avec une expression élevée de récepteurs chimériques au moyen d'un procédé non viral et leur utilisation, comprenant spécifiquement les étapes suivantes : (1) transfert du gène codant du récepteur chimérique dans une cellule tueuse naturelle en utilisant un système de transposon pour obtenir une cellule tueuse naturelle initiale modifiée par un récepteur chimérique ; et (2) amplification de la cellule tueuse naturelle initiale modifiée par un récepteur chimérique en utilisant une cellule présentatrice d'antigène artificielle. La cellule NK modifiée par un récepteur chimérique préparée au moyen du procédé présente une capacité de destruction de tumeur relativement forte, et présente un effet anti-tumoral relativement bon in vitro.
PCT/CN2021/103235 2020-07-09 2021-06-29 Procédé non viral pour la préparation de cellules nk stables avec une expression de récepteur chimérique élevée WO2022007665A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010657827.5 2020-07-09
CN202010657827.5A CN113913458A (zh) 2020-07-09 2020-07-09 非病毒方法制备稳定高表达嵌合受体的nk细胞

Publications (1)

Publication Number Publication Date
WO2022007665A1 true WO2022007665A1 (fr) 2022-01-13

Family

ID=79231926

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/103235 WO2022007665A1 (fr) 2020-07-09 2021-06-29 Procédé non viral pour la préparation de cellules nk stables avec une expression de récepteur chimérique élevée

Country Status (2)

Country Link
CN (1) CN113913458A (fr)
WO (1) WO2022007665A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024108614A1 (fr) * 2022-11-22 2024-05-30 浙江康佰裕生物科技有限公司 Gène de fusion contenant le gène codant d'un récepteur antigénique chimérique et gène codant pour un récepteur de commutation chimérique et son utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0814838A1 (fr) * 1995-03-08 1998-01-07 The Scripps Research Institute Systeme et procedes de presentation des antigenes pour l'activation des lymphocytes t
CN106414748A (zh) * 2014-02-14 2017-02-15 得克萨斯州大学系统董事会 嵌合抗原受体及制备方法
CN107523545A (zh) * 2016-06-20 2017-12-29 上海细胞治疗研究院 一种高效稳定表达抗体的杀伤性细胞及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0814838A1 (fr) * 1995-03-08 1998-01-07 The Scripps Research Institute Systeme et procedes de presentation des antigenes pour l'activation des lymphocytes t
CN106414748A (zh) * 2014-02-14 2017-02-15 得克萨斯州大学系统董事会 嵌合抗原受体及制备方法
CN107523545A (zh) * 2016-06-20 2017-12-29 上海细胞治疗研究院 一种高效稳定表达抗体的杀伤性细胞及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RAN YUE, ZIYANG LIU, YA ZHENG, XIAODAN LU, SHANSHAN HU, BINGYONG ZHANG, XIULING LI, JINGGUO LI, SHUANGYIN HAN: "Nanocarrier-mediated PiggyBac transposon system for preparation of CAR-NK cells", CHINESE JOURNAL OF CANCER BIOTHERAPY, vol. 27, no. 2, 25 February 2020 (2020-02-25), Publishing House of Chinese Journal of Cancer Biotherapy, CN, pages 109 - 114, XP055886315, ISSN: 1007-385X, DOI: 10.3872/j.issn.1007-385x.2020.02.002 *
YOU FENGTAO: "CD7-CAR-NK-92MI Cell Targeted Therapy for T Cell Tumor and A Bispecific CAR-T Cell Targeted Therapy for B Cell Tumor", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE-SOOCHOW UNIVERSITY, 1 June 2019 (2019-06-01), XP055886320, [retrieved on 20220202] *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024108614A1 (fr) * 2022-11-22 2024-05-30 浙江康佰裕生物科技有限公司 Gène de fusion contenant le gène codant d'un récepteur antigénique chimérique et gène codant pour un récepteur de commutation chimérique et son utilisation

Also Published As

Publication number Publication date
CN113913458A (zh) 2022-01-11

Similar Documents

Publication Publication Date Title
AU2016206868B2 (en) Modified hepatitis post-transcriptional regulatory elements
CN111566221B (zh) 用于nk细胞转导的方法
US20210154231A1 (en) Method for producing t cells modified by chimeric antigen receptor
WO2022007665A1 (fr) Procédé non viral pour la préparation de cellules nk stables avec une expression de récepteur chimérique élevée
CN112210539A (zh) 一种第四代car-t细胞及其应用
US11359012B1 (en) Specific chimeric antigen receptor cells targeting human CLDN18A2, preparation method and application thereof
US11912987B2 (en) Methods for screening for cancer targets
CN111378046A (zh) 一种免疫效应细胞转换受体
CN116410336B (zh) 一种嵌合抗原受体的编码核苷酸、car-nk细胞及其构建方法和应用
CN112063653A (zh) 一种基于电转重编程质粒制备nk样细胞的方法
CN112592898B (zh) 一种重编程nk饲养细胞及其制备方法和应用
EP4349969A1 (fr) Composition contenant une cellule nourricière pour la prolifération de cellules tueuses naturelles
EP3986428B1 (fr) Vecteur viral tout-en-un pour molécules car et effectrices thérapeutiques
CN112626028B (zh) 一种激活nk样细胞的工程化细胞及其制备方法和应用
EP2267118A1 (fr) Procédé de production d'une cellule transfectée
CN114269929A (zh) 生产含有表达car的免疫细胞的细胞群体的方法
CN117467619B (zh) 一种可诱发自崩解型人工抗原呈递细胞及其制备方法和应用
WO2023125860A1 (fr) Technique de préparation d'une cellule car-t universelle, et application d'une cellule car-t universelle
CN115820697B (zh) 一种免疫细胞及其制备方法和应用
US20220228164A1 (en) Engineered antigen presenting cells
WO2023082640A1 (fr) Procédé d'amélioration de la durabilité d'une cellule immunitaire
CN113913384A (zh) 一种制备靶点特异性nk细胞的方法及其应用
CN113913459A (zh) 一种应用非病毒方法制备高纯度嵌合抗原受体修饰的t细胞的方法及其应用
CN112608901A (zh) 一种人工抗原呈递细胞及其制备方法和应用
WO2023133398A2 (fr) Polypeptides cd40 chimériques et méthodes d'utilisation en immunothérapie

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21837744

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21837744

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 21837744

Country of ref document: EP

Kind code of ref document: A1