Comparative analysis and optimization of protocols for producing recombinant lentivirus carrying the anti-Her2 chimeric antigen receptor gene
Comparative analysis and optimization of protocols for producing recombinant lentivirus carrying the anti-Her2 chimeric antigen receptor gene
复制标题
携带抗Her2嵌合抗原受体基因的重组慢病毒生产方案的比较分析与优化
DOI：
10.1002/jgm.3027
复制
发表时间：
2018-07-01
影响因子：
3.5
通讯作者：
Li, Hongzhi
中科院分区：
文献类型：
--
作者：
Yuan, Weihua;Chen, Jie;Li, Hongzhi
背景抗Her 2嵌合抗原受体（CAR）T细胞的生产需要优化以使其成为一种可靠的疗法。方法将表达抗Her 2 CAR的三种类型的慢病毒载体与包装质粒一起共转染到293 T-17细胞中。筛选出包装效率最高的载体，并对包装细胞培养体系和包装质粒体系进行了优化。针对慢病毒储备液的浓度优化离心速度。所用的各种纯化方法包括膜过滤、蔗糖垫离心和新颖设计的瞬时高速离心。将重组慢病毒以优化的感染复数（MOI）转导到人外周T细胞中。结果共转染时，悬浮包装细胞优于常用的贴壁培养条件，包装系统psPAX 2/pMD 2. G优于pCMV-dR 8.91/pVSV-G。离心浓缩的最佳转速为20 000 g，而不是通常使用的超速。重要的是，增加瞬时离心纯化显著提高了人外周T细胞活力（从13.25%提高到62.80%），这是CAR-T细胞制备的技术突破。转导人外周血T细胞的最佳MOI值为40。结论优化了能在T细胞中表达第三代抗Her 2 CAR的重组慢病毒的制备方法，为进一步提高CAR-T细胞杀伤靶细胞的效率奠定了基础。
BackgroundThe production of anti-Her2 chimeric antigen receptor (CAR) T cells needs to be optimized to make it a reliable therapy.MethodsThree types of lentiviral vectors expressing anti-Her2 CAR together with packaging plasmids were co-transfected into 293T-17 cells. The vector with the best packaging efficiency was selected, and the packaging cell culture system and packaging plasmid system were optimized. Centrifugation speed was optimized for the concentration of lentivirus stock. The various purification methods used included membrane filtration, centrifugation with a sucrose cushion and the novelly-designed instantaneous high-speed centrifugation. The recombinant lentiviruses were transduced into human peripheral T cells with an optimized multiplicity of infection (MOI). CAR expression levels by three vectors and the efficacy of CAR-T cells were compared.ResultsWhen co-transfected, packaging cells in suspension were better than the commonly used adherent culture condition, with the packaging system psPAX2/pMD2.G being better than pCMV-dR8.91/pVSV-G. The optimal centrifugation speed for concentration was 20 000g, rather than the generally used ultra-speed. Importantly, adding instantaneous centrifugation for purification significantly increased human peripheral T cell viability (from 13.25% to 62.80%), which is a technical breakthrough for CAR-T cell preparation. The best MOI value for transducing human peripheral T cells was 40. pLVX-EF1a-CAR-IRES-ZsGreen1 expressed the highest level of CAR in human peripheral T cells and the cytotoxicity of CAR-T cells reached 63.56%.ConclusionsWe optimized the preparation of recombinant lentivirus that can express third-generation anti-Her2 CAR in T cells, which should lay the foundation for improving the efficacy of CAR-T cells with respect to killing target cells.