Transiently redirected Tcells for adoptive transfer
Background aims. T cells can be redirected to reject cancer by retroviral transductionwith a chimeric antigen recepto (CAR) or by administration of a bispeciﬁ c Tcell engager (BiTE). We demonstrate that transfection of T cells with messenger(m) RNA coding for CAR is an alternative strategy. Methods . We describe the pre-clinical evaluation of a methobased on transientmodiﬁcation of expanded T cells with a CD19 CAR directed against B-cellmalignancies. CAR mRNA was generated under cell-free conditions in a scalableprocess using recombinant RNA polymerase. Efﬁcient and non-toxisquare-waveelectroporation was used to load the mRNA into the cytoplasm of T cells with norisk of insertional mutagenesis. Results . After transfection 80% of T cells were viable, with 94% CARexpression. Transfected T cells were cytolytic to CD19 targets and produced interferon (IFN)- γ inresponse. Killing of CD19 target cellswas demonstrated eve at day 8 with undetectable CAR expression. Increasing theconcentration of mRNA resulted in higher surface CAR expression, better killingand more IFN- γ release but at the expense of increasedactivation-induced cell death. Finally, we demonstrated that a second transgenecould be introduced by co-electroporation of CXCR4 or CCR7 with CAR to alsmodify chemotactic responses. Conclusions . We advocate thetransient redirection approach as well suited to meet safet aspects for earlyphase studies, prior to trials using stably transduced cells once CAR has beenproven safe. The simplicitof this methodology also facilitates rapid screeningof candidate targets and novel receptors in pre-clinical studies.
转染编码编嵌合抗原受体的mRNA是构建CAR-T的一种可实现的策略。作者描述一种将已扩增的T细胞瞬间转化为携带有抗B-细胞恶性肿瘤的CD19 CAR-Ｔ细胞的临床前评估。嵌合抗原受体的mRNA可在无细胞的条件下使用RNA聚合酶生产，通过电转染的方式转染mRNA到Ｔ细胞的细胞质以避免插入染色体中风险。转染后80％的T细胞存活下来，这些Ｔ细胞中有94％的CAR表达。转染的T细胞对CD19阳性细胞产生细胞毒性并表达了干扰素（IFN） - γ。 对CD19靶细胞的杀伤能力即使在转染后第８天检测不到CAR的表达时也能被证明。增加mRNA的浓度可使细胞表面CAR表达更高，也可让CAR-T获得更细胞杀伤和IFN-γ释放能。最后，我们证明了其他基因如CXCR4和CCR7的可以和CAR一起通过电转染的方式转染细胞并引发趋化反应。作者认为这种方式安全简单， 适合早期的安全性的研究，并适合新抗原受体在临床前研究中的快速筛选。