WO2022145832A1 - Cellule tueuse naturelle dérivée d'une cellule souche pluripotente induite et son utilisation - Google Patents

Cellule tueuse naturelle dérivée d'une cellule souche pluripotente induite et son utilisation Download PDF

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WO2022145832A1
WO2022145832A1 PCT/KR2021/019255 KR2021019255W WO2022145832A1 WO 2022145832 A1 WO2022145832 A1 WO 2022145832A1 KR 2021019255 W KR2021019255 W KR 2021019255W WO 2022145832 A1 WO2022145832 A1 WO 2022145832A1
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cells
added
medium
culturing
cultured
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Korean (ko)
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이미연
나득채
김경운
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주식회사 온코인사이트
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • 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]
    • C12N2501/2313—Interleukin-13 (IL-13)
    • 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]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • 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
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • step (f) Stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine kinase 3 ligand (FLT-3L) in the cultured cells of step (e) Culturing by replacing with the added medium.
  • SCF Stem cell factor
  • IL-7 interleukin-7
  • IL-15 interleukin-15
  • FLT-3L FMS-like tyrosine kinase 3 ligand
  • BMP4 is added at a concentration of 30 to 70 ng/mL, such as 50 ng/mL,
  • VEGF and bFGF are each added at a concentration of 30 to 70 ng/mL, such as 50 ng/mL, or
  • IL-3, SCF, IL-7, IL-15 and FLT-3L are each added at a concentration of 30 to 70 ng/mL, such as 50 ng/mL, or
  • step (f) SCF, IL-7, IL-15 and FLT-3L may each be added at a concentration of 30 to 70 ng/mL, such as 50 ng/mL.
  • the manufacturing method of the present invention comprises:
  • the present invention also provides NK cells prepared according to the above production method, and a pharmaceutical composition for treating or preventing cancer comprising the NK cells.
  • iPSCs are cultured in a medium comprising BMP4, followed by culture in a medium comprising VEGF and bFGF, and then cultured in a medium comprising SB431542, VEGF and bFGF to another cell, such as hematopoietic stem cells or hematopoietic cells. It can differentiate into progenitor cells.
  • the method for producing NK cells of the present invention may include one or more of the following steps (b) to (d), for example, all of the steps (b) to (d).
  • the iPSC-cultured cells may be differentiated into other cells, such as hematopoietic stem cells or hematopoietic progenitor cells.
  • IL-3, SCF, IL-7, IL-15 and FLT-3L are 30 to 70 ng/mL, preferably 40 to 60 ng/mL, more preferably 50 added at a concentration of ng/mL, or
  • the NK cells produced according to the method of the present invention are CD2(+)/CD56(+) NK cells, CD16(+)/CD56(+) NK cells, CD94(+)/CD56(+) NK cells. cells, or KIR2D, CD158(+)/CD56(+) NK cells.
  • a method for preventing or treating cancer comprising administering to a subject an anticancer cell therapy composition comprising NK cells prepared according to the production method of the present invention.
  • the subject means a mammal, preferably a human.
  • mTeSR-1 medium was added to a 24-well plate coated with Matrigel, and iPSCs were seeded at a density of 5 x 10 4 cells per well.
  • the seeded cells were differentiated while replacing the medium by time as shown in FIG. 1 .
  • the medium was replaced with STEMdiff APEL 2 (Stem cell Technologies), and BMP4 (50 ng/mL, Peprotech) was added to the medium.
  • VEGF 50 ng/mL, Peprotech
  • bFGF 50 ng/mL, Peprotech
  • SB431542 (20 ⁇ M, Reagents Direct, Encinitas, CA, USA), VEGF (50 ng/mL) and bFGF (50 ng/mL) were added, and the medium was replaced with fresh STEMdiff APEL.
  • IL-3 50 ng/mL, Peprotech
  • SCF 50 ng/mL, Peprotech
  • IL-7 50 ng/mL, Peprotech
  • IL-15 50 ng/mL, Peprotech
  • FLT-3L 50 ng/mL, Peprotech
  • the cells were transferred to a 15 mL conical tube as many as the number of cells shown in Table 4, centrifuged at 516 x g for 5 minutes, the supernatant was suctioned, and the medium as much as the volume in Table 4 was added and resuspended.
  • NK cell preparation iPSC-derived NK cells: Effector cell

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  • Animal Behavior & Ethology (AREA)
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  • Wood Science & Technology (AREA)
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  • Developmental Biology & Embryology (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une cellule tueuse naturelle (cellule NK) dérivée d'une cellule souche pluripotente induite (iPSC), son utilisation et son procédé de préparation.
PCT/KR2021/019255 2020-12-29 2021-12-17 Cellule tueuse naturelle dérivée d'une cellule souche pluripotente induite et son utilisation WO2022145832A1 (fr)

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KR1020200186564A KR102292843B1 (ko) 2020-12-29 2020-12-29 역분화줄기세포(iPSC) 유래 자연 살해 세포 및 이의 용도
KR10-2020-0186564 2020-12-29

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115873795A (zh) * 2023-02-02 2023-03-31 苏州血霁生物科技有限公司 一种分化造血干/祖细胞的方法、培养基及应用
CN115896019A (zh) * 2023-02-23 2023-04-04 山东兴瑞生物科技有限公司 一种诱导性多能干细胞诱导分化为nk细胞的方法
CN115975925A (zh) * 2023-03-21 2023-04-18 天九再生医学(天津)科技有限公司 一种变速悬浮诱导人多能干细胞分化为自然杀伤细胞的方法
WO2023233339A1 (fr) * 2022-06-01 2023-12-07 Crispr Therapeutics Ag Compositions et procédés de différenciation de cellules souches en cellules nk

Families Citing this family (5)

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KR102292843B1 (ko) * 2020-12-29 2021-08-25 주식회사 온코인사이트 역분화줄기세포(iPSC) 유래 자연 살해 세포 및 이의 용도
CN113388579A (zh) * 2021-07-07 2021-09-14 杭州原生生物科技有限公司 一种含有细胞因子的可将造血干细胞分化为自然杀伤细胞的培养基及其分化方法
CN113801846B (zh) * 2021-09-22 2024-06-04 南京艾尔普再生医学科技有限公司 一种从人诱导多能干细胞分化为自然杀伤细胞的方法
WO2023191597A1 (fr) * 2022-04-01 2023-10-05 (주) 테라베스트 Cellules tueuses naturelles produites à partir de cellules souches pluripotentes induites, leur procédé de production et leur utilisation
KR20240076715A (ko) * 2022-11-17 2024-05-30 주식회사 씨티셀즈 미분화 줄기세포로부터 자연 살해 세포로의 분화 방법 및 이의 용도

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023233339A1 (fr) * 2022-06-01 2023-12-07 Crispr Therapeutics Ag Compositions et procédés de différenciation de cellules souches en cellules nk
CN115873795A (zh) * 2023-02-02 2023-03-31 苏州血霁生物科技有限公司 一种分化造血干/祖细胞的方法、培养基及应用
CN115873795B (zh) * 2023-02-02 2023-09-01 苏州血霁生物科技有限公司 一种分化造血干/祖细胞的方法、培养基及应用
CN115896019A (zh) * 2023-02-23 2023-04-04 山东兴瑞生物科技有限公司 一种诱导性多能干细胞诱导分化为nk细胞的方法
CN115975925A (zh) * 2023-03-21 2023-04-18 天九再生医学(天津)科技有限公司 一种变速悬浮诱导人多能干细胞分化为自然杀伤细胞的方法
CN115975925B (zh) * 2023-03-21 2023-09-08 天九再生医学(天津)科技有限公司 一种变速悬浮诱导人多能干细胞分化为自然杀伤细胞的方法

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