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WO2017188790A1 - Procédé de mise en prolifération et de culture de cellules immunitaires dans des conditions hypoxiques - Google Patents

Procédé de mise en prolifération et de culture de cellules immunitaires dans des conditions hypoxiques Download PDF

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WO2017188790A1
WO2017188790A1 PCT/KR2017/004595 KR2017004595W WO2017188790A1 WO 2017188790 A1 WO2017188790 A1 WO 2017188790A1 KR 2017004595 W KR2017004595 W KR 2017004595W WO 2017188790 A1 WO2017188790 A1 WO 2017188790A1
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cells
conditions
peripheral blood
cancer
expression
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English (en)
Korean (ko)
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이경미
박현성
문윤원
임선아
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Korea University Research and Business Foundation
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Korea University Research and Business Foundation
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1114—T cells

Definitions

  • the present invention relates to a method for proliferating and culturing immune cells using hypoxic conditions that allow peripheral blood mononuclear cell-derived NK cells to be cultured under normal oxygen conditions and further cultured under hypoxic conditions to improve cancer cell killing ability and to survive in the body for a long time. .
  • NK cells are one of the innate immune cells, which are capable of killing various types of cancer cells and recognize cancer cells regardless of the presence or absence of antigens.
  • the immune cells injected due to the immune evasion mechanism that occurs in the large tumor tissues cause deactivation, and the cancer cell attack ability is lost.
  • the major cause of this immune evasion mechanism is that the interior of the tumor is a hypoxic environment and this environment is a problem in the treatment of solid cancer patients by providing a condition that NK cells can not effectively kill cancer cells.
  • hypoxic oxygen partial pressures of 3% (peripheral tissue) to 14% (lung) depending on tissue, and lower oxygen levels are referred to as hypoxic, often 0.5% to 3%. It has been reported that tumor cells maintain and promote growth under hypoxic conditions, whereas normal cells and immune cells do not grow well under hypoxia. In addition, in the case of immune cells, hypoxic conditions are known to inhibit the function of immune cells. In NK cells, NKp30, NKp44, NKp46, and NKG2D, which are the major activation receptors, are downregulated when cultured at 1% O 2 , and cancer cell killing ability is lowered compared to NK cells grown in normal cell conditions.
  • the present inventors have previously exposed NK cells to various concentrations of hypoxic conditions in vitro expansion to adapt them in a hypoxic environment, and when the NK cells are injected into the patient's body, they can maintain anticancer activity even in hypoxic conditions in cancer tissues.
  • the present invention was completed by establishing culture conditions.
  • Another object of the present invention is to provide a prophylactic or therapeutic use of cancer of the NK cells with improved cancer cell killing ability.
  • the present invention is to culture the peripheral blood monocytes and feeder cells under cytokine for 3 to 13 days under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, and then the oxygen partial pressure is increased. It provides a method for inducing and proliferating NK cells comprising the step of inducing and proliferating natural killer cells (NK cells) by further culture for 5 to 30 days in low oxygen conditions of 1% to 5%.
  • NK cells natural killer cells
  • the present invention also includes NK cells with improved cancer cell killing ability compared to peripheral blood mononuclear cells-derived NK cells cultured under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, wherein the NK cells with improved cancer cell killing ability are in normal oxygen conditions.
  • the invention also provides the use of said NK cells for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer.
  • the present invention also provides a method for treating cancer comprising administering to a cancer patient a pharmaceutically effective amount of the pharmaceutical composition for preventing or treating the cancer.
  • NK cells induced and expanded under normal oxygen conditions in the case of NK cells induced and expanded by incubating for a certain period of time under normal oxygen conditions and further cultured under hypoxic conditions.
  • cell death is reduced, aging is inhibited, cancer cell killing ability can be promoted.
  • the NK cells have a long survival time and excellent cancer cell killing ability in vivo, and thus can be used as an adoptive immune cell therapy for the prevention or treatment of cancer.
  • NK cells 1 is a result of confirming the effect of oxygen conditions in the induction and proliferation of NK cells from peripheral blood monocytes, peripheral blood mononuclear cells cultured for 3 weeks in normal oxygen conditions (20%) or hypoxic conditions (0.5%, 1.5%) or After culturing for 9 days in normal oxygen conditions, and further cultured in low oxygen conditions (1.5%) shows the measured cell number.
  • Figure 2a is a graph comparing the proliferation of NK cells induced in normal oxygen conditions, and NK cells according to the present invention induced by further culture in hypoxic conditions after culture in normal oxygen conditions
  • Figure 2b is induced in normal oxygen conditions
  • Figure 2c is a graph comparing the cancer cell killing ability of NK cells induced in normal oxygen conditions and NK cells according to the present invention.
  • FIG. 3 is a graph comparing the killing ability of NK cells induced in normal oxygen conditions and A375 cells, a melanoma cell line under various oxygen conditions of the NK cells according to the present invention.
  • Figure 4 is a result confirming the change in the NK cells induced in normal oxygen conditions and the activating receptors and inhibitory receptors of NK cells according to the present invention.
  • 5 is a result confirming the expression of p16, an aging marker in NK cells induced in normal oxygen conditions and NK cells according to the present invention.
  • Figure 6 is a result confirming the cancer cell killing ability of NK cells induced in normal oxygen conditions and NK cells according to the present invention in xenograft hematological cancer model.
  • the present invention is cultured peripheral blood monocytes and feeder cells under cytokines for 3 days to 13 days under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, hypoxia with an oxygen partial pressure of 1% to 5%
  • the present invention relates to a method of inducing and proliferating NK cells, which further comprises inducing and proliferating natural killer cells (NK cells) by further culturing for 5 to 30 days under conditions.
  • NK cells natural killer cells
  • the normal oxygen condition means a cell culture condition having an oxygen partial pressure of 20% to 22%
  • a low oxygen condition means a cell culture condition having an oxygen partial pressure of 1% to 5%, or 1.5% to 3%.
  • Induction and proliferation culture method of NK cells of the present invention is characterized in that the peripheral blood monocytes are cultured for a certain period of time under normal oxygen conditions, and further cultured under low oxygen conditions to induce and proliferate NK cells.
  • peripheral blood monocytes under cytokines were cultured for 3 days to 13 days under vegetative cells and normal oxygen conditions with an oxygen partial pressure of 20% to 22%, and 5 days under hypoxic conditions with an oxygen partial pressure of 1% to 5%. It may be further incubated for 30 days.
  • peripheral blood monocytes may be derived from peripheral blood of normal or cancer patients.
  • the feeder cells may be irradiated Jurkat cells and irradiated EBV-LCL cells. More specifically, the feeder cells may be a mixture of irradiated Jurkat cells and irradiated EBV-LCL cells in a ratio of about 1: 1 cell number, but is not particularly limited thereto.
  • peripheral blood monocytes and feeder cells may be co-cultured by mixing at a ratio of about 1: 1 cells, but are not particularly limited thereto.
  • the cytokine may comprise interleukin-2 (IL-2).
  • IL-2 interleukin-2
  • the NK cells derived above may be cells having a CD3 - CD56 + phenotype.
  • the medium used for the induction and proliferation culture of the NK cells can be used without limitation, known cell culture medium added with serum.
  • the serum can be used without limitation the kind of known serum used for cell culture.
  • NK cells according to the induction and proliferation culture method of the NK cells of the present invention has a larger number of cells than NK cells derived from peripheral blood monocytes cultured under normal oxygen conditions.
  • NK cells when transferred to hypoxic conditions (1.5% or 0.5%) when less than 3 days of culture
  • the growth rate of NK cells was significantly higher than that of the control group when the cells were transferred to hypoxic conditions after 9 days of culture under normal oxygen conditions.
  • the proliferation rate was increased by about 60%
  • the cell death confirmation experiment was reduced by about 50%
  • the cancer cell killing ability was increased by 20% to 40% by the investigation of cancer cell killing ability.
  • the cancer cell killing ability of the NK cells in the hypoxic condition was tested, compared with the NK cells cultured continuously in the normal oxygen condition, the cancer cell killing ability for solid cancer was superior, the NK cells have a high expression of CD107a (killing capacity measure) In addition, the secretion of IFN- ⁇ is increased.
  • NKp44 an activation receptor and molecules related to cancer cell killing ability, perforin and granzyme B are increased.
  • the expression of the aging marker p16 is reduced, it can be seen that less aging compared to NK cells continuously cultured under normal oxygen conditions.
  • NK cells induced by culturing under normal oxygen conditions and further cultured under hypoxic conditions showed significantly better cancer cell killing ability than NK cells induced under normal conditions.
  • NK cells of the present invention can be used as an active ingredient in the composition for the prevention or treatment of cancer, the present invention compared to NK cells derived from peripheral blood monocytes cultured in normal oxygen conditions with an oxygen partial pressure of 20% to 22% NK cells with improved cancer cell killing ability, wherein the NK cells with improved cancer cell killing ability compared to peripheral blood monocyte-derived NK cells cultured under normal oxygen conditions, CD107a, NKp44, perforin, and granzyme B It provides a pharmaceutical composition for the prevention or treatment of cancer, wherein the expression of is increased, the secretion of IFN-gamma is increased, and the expression of p16 is reduced.
  • the present invention provides the use of said NK cells for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer.
  • the present invention provides a method for treating cancer, comprising administering to a cancer patient a pharmaceutically effective amount of the pharmaceutical composition for preventing or treating the cancer.
  • the pharmaceutical composition may comprise an active ingredient or an active or inactive pharmaceutically acceptable carrier which constitutes a composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the cancer includes solid cancer, blood cancer, and the like, but is not particularly limited thereto.
  • treatment means any action that inhibits, alleviates or beneficially alters the clinical situation associated with a disease. Treatment can also mean increased survival compared to the expected survival if untreated. Treatment includes simultaneously prophylactic measures in addition to therapeutic means.
  • an "individual” may be a vertebrate, preferably a mammal, such as a dog, cat, mouse, human, or the like.
  • composition of the present invention may be formulated further comprising a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not significantly irritate an organism and does not inhibit the biological activity and properties of the administered component.
  • the pharmaceutically acceptable carrier in the present invention may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, If desired, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added to formulate them in the form of injectables suitable for infusion into tissues or organs. It may also be formulated as an isotonic sterile solution, or as a dry preparation (particularly a lyophilized preparation), which may optionally be an injectable solution with the addition of sterile water or physiological saline.
  • composition of the present invention may further include a filler, an excipient, a disintegrant, a binder and a lubricant.
  • the compositions of the present invention may also be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the term "administration" refers to introducing a composition of the present invention to a patient in any suitable manner, wherein the route of administration of the composition of the present invention is via oral or parenteral various routes as long as the target tissue can be reached. May be administered. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.
  • an effective amount means an amount necessary to delay or entirely stop the onset or progression of the particular disease to be treated.
  • the composition may be administered in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment.
  • the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved and whether other agents are used in some cases, the age, weight, general state of health, sex of the patient. And various factors and similar factors well known in the medicinal art, including diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or concurrent with the specific composition.
  • peripheral blood monocytes were isolated from the buff coat. Then, 10% FBS and 1% penicillin / streptomycin were added to RPMI1640 medium at a ratio of 1: 0.5: 0.5 in the presence of IL-2 500 U / ml using 100 Gy irradiated Jurkat cell line and irradiated EBV-LCL cell line The culture was co-cultured in hRPMI medium and exchanged with hRPMI medium added with 500 U / ml of IL-2 once every 2-3 days.
  • Ficoll Ficoll-paqueTM PLUS, GE healthcare
  • the culture was carried out for 9 days at normal oxygen conditions and then moved to 1.5% O 2 conditions for about 3 weeks. At this time, it was incubated while exchanging IL-2 with hRPMI medium added at 500 U / ml.
  • peripheral blood monocytes were cultured for 3 weeks in normal oxygen conditions (20%) and hypoxic conditions (0.5% and 1.5%), or normal oxygen as described in Example 1. After culturing for 9 days under conditions (20%), the cells were moved to hypoxic conditions (1.5%), and cultured for 3 weeks, and the cell number was measured by a hematocytometer. At this time, it was incubated while exchanging IL-2 with hRPMI medium added at 500 U / ml.
  • the number of cells increased under normal oxygen conditions, but the cells did not proliferate or exhibited insufficient growth effects under hypoxic conditions.
  • NK cells normal oxygen conditions (20% O 2 ) and NK cells cultured after 5 days of culture under normal oxygen conditions (1.5% O 2 ) (Hypoxia) Cell proliferation was confirmed. To this end, 3 ⁇ 10 5 cells were cultured from each of the cultured cells, and then stained with Percp-labeled CD3 mAb and APC-labeled CD56mAb to confirm cell proliferation of cells that are CD3 - CD56 + .
  • the cell death was confirmed by staining Annexin V and 7AAD in the experimental and control groups, respectively.
  • the cells were labeled with Chromium for 1 hour, co-cultured with NK cells at a ratio of 1: 1, and after 4 hours, the supernatant was taken to a gamma counter. Isotope values were identified.
  • the growth rate of NK cells was increased by about 60% when cultured in hypoxic conditions, and cell death was reduced by about 50% when cultured in hypoxic conditions.
  • cancer cell killing ability was increased by 20-40% compared to cells cultured under normal oxygen conditions when cultured in hypoxic conditions.
  • NK cells and target cells were mixed at a 1: 1 ratio, incubated at 37 ° C. for 1 hour, 2.5 ⁇ l of FITC labeled anti CD107a mAb, Golgi stop (BD pharmingen), and 37 for 5 hours. Incubated at °C. After incubation, Percp-labeled CD3 mAb and APC-labeled CD56mAb were added and reacted at 4 ° C. for 20 minutes.
  • NK cells cultured in a hypoxic condition had high expression of CD107a (death killing scale) and increased secretion of IFN- ⁇ .
  • NKp44 As shown in FIG. 4, when cultured in hypoxic conditions, NKp44, an NK cell activation receptor, and perforin and granzyme B, molecules related to cancer cell killing ability, were increased.
  • NK cells cultured by moving to hypoxic conditions are less aging than NK cells cultured under normal oxygen conditions.
  • PKH-labeled human T lymphoblastoid cell line CEM in 8-week-old NSG mice was injected 5 ⁇ 10 6 into the mouse abdominal cavity and 1 ⁇ 10 7 NK cells were injected in 48 hours after the cancer cells remained in the abdominal cavity. Collected and analyzed by flow cytometry.
  • NK cells cultured by moving to hypoxic conditions were superior to cancer cell killing ability against blood cancer compared to NK cells cultured under normal oxygen conditions.
  • the present invention can be used in the field of adoptive immune cell therapy.

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Abstract

L'invention concerne un procédé de mise en prolifération et de culture de cellules immunitaires dans des conditions hypoxiques. Lorsque les cellules NK, qui sont induites par la co-culture de cellules mononucléaires du sang périphérique et de cellules nourricières en présence de cytokines dans des conditions normoxiques, sont transférées dans des conditions hypoxiques de manière à les faire proliférer et à les cultiver, la prolifération cellulaire augmente, l'apoptose s'atténue, la capacité de destruction des cellules cancéreuses augmente, et la sénescence se réduit par comparaison avec les cellules NK cultivées en continu uniquement dans des conditions normoxiques. Ainsi, les cellules NK mises en prolifération et cultivées dans des conditions hypoxiques sont utilisables comme d'excellents agents thérapeutiques de cellules immunitaires adoptives pour la prévention ou le traitement du cancer.
PCT/KR2017/004595 2016-04-29 2017-04-28 Procédé de mise en prolifération et de culture de cellules immunitaires dans des conditions hypoxiques Ceased WO2017188790A1 (fr)

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WO2018165265A1 (fr) * 2017-03-08 2018-09-13 Nantcell, Inc. Cellules nk hypoxiques et procédés associés
CN111757745A (zh) * 2018-02-01 2020-10-09 Nkmax有限公司 产生天然杀伤细胞的方法和用于治疗癌症的组合物
EP3822341A4 (fr) * 2018-05-31 2022-03-09 Korea University Research and Business Foundation Croissance et culture de cellules tueuses naturelles dérivées de l'être humain à l'aide d'igfbp2

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KR20250124466A (ko) 2024-02-13 2025-08-20 인하대학교 산학협력단 스트레스로 유도된 배양보조세포를 통한 자연살해세포의 효과적인 활성화 방법 및 이의 용도

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WO2018165265A1 (fr) * 2017-03-08 2018-09-13 Nantcell, Inc. Cellules nk hypoxiques et procédés associés
US11221328B2 (en) 2017-03-08 2022-01-11 Nantcell, Inc. Hypoxic NK cells and methods therefor
CN111757745A (zh) * 2018-02-01 2020-10-09 Nkmax有限公司 产生天然杀伤细胞的方法和用于治疗癌症的组合物
EP3746095A4 (fr) * 2018-02-01 2021-04-21 Nkmax Co., Ltd. Procédé de production de cellules tueuses naturelles et composition pour le traitement du cancer
US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
CN111757745B (zh) * 2018-02-01 2022-11-04 Nkmax有限公司 产生天然杀伤细胞的方法和用于治疗癌症的组合物
IL276365B1 (en) * 2018-02-01 2023-06-01 Nkmax Co Ltd A method for the production of natural killer cells and a preparation for cancer treatment
IL276365B2 (en) * 2018-02-01 2023-10-01 Nkmax Co Ltd A method for the production of natural killer cells and a preparation for cancer treatment
US12098388B2 (en) 2018-02-01 2024-09-24 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
EP3822341A4 (fr) * 2018-05-31 2022-03-09 Korea University Research and Business Foundation Croissance et culture de cellules tueuses naturelles dérivées de l'être humain à l'aide d'igfbp2

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