使用病人来源的材料和piggy转座子基因移植构建 CD19 CAR-T细胞
Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials. BACKGROUND AIMS: Protocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification.METHODS: We varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period.RESULTS: Using optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19(+) cell lines and efficiently lysed both CD19(+) cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer.DISCUSSION: We have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies. 使用病人来源的材料和piggy转座子基因移植构建 CD19 CAR-T细胞背景目标：生产CD19 CAR-T细胞常常比较负责和昂贵，因为要使用到逆转录病毒和慢病毒载体，而且需要一个适合CAR19 T细胞扩增的环境。我们目标是利用基因修饰的piggyBac转座子系统简化正常供者和病人外周血中CAR19T细胞的生产。方法：我们通过不同的电击电压、细胞活素和刺激环境，持续三周，来生产和扩增CAR19T细胞。结果：采用优化的电击电压，白细胞介素15，和外周血单个核细胞单独或共同培养，我们能够将供者来源的CAR19 T细胞在三周内扩增到765倍,B细胞恶性肿瘤来源的CAR19T细胞扩增180倍最终，在捐献者外周血中，72%的CAR19有表达，在急性淋巴细胞白血病、慢性淋巴细胞白血病、非霍奇金淋巴瘤患者中有81%的CAR19有表达。CAR19T细胞在CD19阳性细胞环境内产生干扰素γ，能够有效的溶解CD19阳性细胞和原代白血病细胞。结论：我们已经生产了一个简单、并不昂贵而且容易操作的CAR 19 T细胞，使用piggyBac转座子系统适合在临床中使用。这提供了一个强大的平台，为进一步提高T细胞产品和测试新的CAR技术做基础。出自爱康得生物技术
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