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1. CN106399242 - Method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells

Office Chine
Numéro de la demande 201610821709.7
Date de la demande 13.09.2016
Numéro de publication 106399242
Date de publication 15.02.2017
Numéro de délivrance 106399242
Date de délivrance 22.01.2019
Type de publication B
CIB
C12N 5/0783
CCHIMIE; MÉTALLURGIE
12BIOCHIMIE; BIÈRE; SPIRITUEUX; VIN; VINAIGRE; MICROBIOLOGIE; ENZYMOLOGIE; TECHNIQUES DE MUTATION OU DE GÉNÉTIQUE
NMICRO-ORGANISMES OU ENZYMES; COMPOSITIONS LES CONTENANT; CULTURE OU CONSERVATION DE MICRO-ORGANISMES; TECHNIQUES DE MUTATION OU DE GÉNÉTIQUE; MILIEUX DE CULTURE
5Cellules non différenciées humaines, animales ou végétales, p.ex. lignées cellulaires; Tissus; Leur culture ou conservation; Milieux de culture à cet effet
07Cellules animales ou tissus animaux
071Cellules ou tissus de vertébrés, p.ex. cellules humaines ou tissus humains
078Cellules du sang ou du système immunitaire
0783Cellules T; Cellules NK; Progéniteurs de cellules T ou NK
C12N 15/867
CCHIMIE; MÉTALLURGIE
12BIOCHIMIE; BIÈRE; SPIRITUEUX; VIN; VINAIGRE; MICROBIOLOGIE; ENZYMOLOGIE; TECHNIQUES DE MUTATION OU DE GÉNÉTIQUE
NMICRO-ORGANISMES OU ENZYMES; COMPOSITIONS LES CONTENANT; CULTURE OU CONSERVATION DE MICRO-ORGANISMES; TECHNIQUES DE MUTATION OU DE GÉNÉTIQUE; MILIEUX DE CULTURE
15Techniques de mutation ou génie génétique; ADN ou ARN concernant le génie génétique, vecteurs, p.ex. plasmides, ou leur isolement, leur préparation ou leur purification; Utilisation d'hôtes pour ceux-ci
09Technologie d'ADN recombinant
63Introduction de matériel génétique étranger utilisant des vecteurs; Vecteurs; Utilisation d'hôtes pour ceux-ci; Régulation de l'expression
79Vecteurs ou systèmes d'expression spécialement adaptés aux hôtes eucaryotes
85pour cellules animales
86Vecteurs viraux
867Vecteurs rétroviraux
C12N 5/10
CCHIMIE; MÉTALLURGIE
12BIOCHIMIE; BIÈRE; SPIRITUEUX; VIN; VINAIGRE; MICROBIOLOGIE; ENZYMOLOGIE; TECHNIQUES DE MUTATION OU DE GÉNÉTIQUE
NMICRO-ORGANISMES OU ENZYMES; COMPOSITIONS LES CONTENANT; CULTURE OU CONSERVATION DE MICRO-ORGANISMES; TECHNIQUES DE MUTATION OU DE GÉNÉTIQUE; MILIEUX DE CULTURE
5Cellules non différenciées humaines, animales ou végétales, p.ex. lignées cellulaires; Tissus; Leur culture ou conservation; Milieux de culture à cet effet
10Cellules modifiées par l'introduction de matériel génétique étranger, p.ex. cellules transformées par des virus
CPC
C12N 5/0636
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor;
06Animal cells or tissues; Human cells or tissues
0602Vertebrate cells
0634Cells from the blood or the immune system
0636T lymphocytes
C12N 5/0646
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor;
06Animal cells or tissues; Human cells or tissues
0602Vertebrate cells
0634Cells from the blood or the immune system
0646Natural killers cells [NK], NKT cells
C12N 15/86
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
79Vectors or expression systems specially adapted for eukaryotic hosts
85for animal cells
86Viral vectors
C12N 2500/42
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
2500Specific components of cell culture medium
30Organic components
42Organic phosphate, e.g. beta glycerophosphate
C12N 2501/2302
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
2501Active agents used in cell culture processes, e.g. differentation
20Cytokines; Chemokines
23Interleukins [IL]
2302Interleukin-2 (IL-2)
C12N 2501/2315
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
2501Active agents used in cell culture processes, e.g. differentation
20Cytokines; Chemokines
23Interleukins [IL]
2315Interleukin-15 (IL-15)
Déposants BEIJING DOING TRANSLATIONAL MEDICAL RESEARCH CENTER
北京多赢时代转化医学研究院
Inventeurs GE LIYUN
盖丽云
LI XIANGQUN
李香群
LI GANGYI
李刚毅
Mandataires 北京中誉威圣知识产权代理有限公司 11279
北京中誉威圣知识产权代理有限公司 11279
Titre
(EN) Method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells
(ZH) 联合制备CAR-Vγ9Vδ2T细胞和CAR-NKT细胞的方法
Abrégé
(EN)
The invention discloses a method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells, comprising the steps of 1) adding peripheral blood mononuclear cells to a cell medium, pre-activating VGamma9VDelta2T cells and NKT cells therein, and amplifying the VGamma9VDelta2T cells and NKT cells by selective in-vitro activation; 2) transducing the mixed cells of step 1) by using a lentiviral vector carrying chimeric antigen receptor's gene sequence. It is possible to carry out standard and batch preparation herein by using GammaDeltaT of a healthy donor; the purities of the VGamma9VDelta2T cells and NKT cells in the application can reach 60% and 30% and above respectively without purification, and therefore pre-purification can be omitted; in-vivo activation proliferation level and existence time of CAR-VGamma9VDelta2T cells and CAR-NKT cells jointly prepared by using the method can be controlled through clinical medication.

(ZH)
本发明公开了一种联合制备CAR‑Vγ9Vδ2T细胞和CAR‑NKT细胞的方法,该方法包括以下步骤:1)将外周血单个核细胞加入细胞培养基中,预活化其中的Vγ9Vδ2T细胞和NKT细胞,再选择性体外活化扩增Vγ9Vδ2T细胞和NKT细胞;2)用携带有嵌合抗原受体的基因序列的慢病毒载体转导步骤1)所得混合细胞。该方法使用健康供者的γδT可以进行标准化和批量化制备;另外本申请中Vγ9Vδ2T细胞和NKT细胞的纯度无需纯化即可分别达到60%和30%以上,因此可以避免预先纯化;使用该方法联合制备的CAR‑Vγ9Vδ2T细胞和CAR‑NKT细胞可通过临床用药来控制其在体内的活化增殖程度及存续时间。