Tay Johan C K, Wang Junjian, Du Zhicheng, Ng Yu Yang, Li Zhendong, Ren Yuefang, Zhang Chang, Zhu Jianqing, Xu Xue Hu, Wang Shu
Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.
Department of Gynaecologic Oncology, Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Hangzhou 310022, P.R. China.
Mol Ther Methods Clin Dev. 2021 Mar 3;21:107-120. doi: 10.1016/j.omtm.2021.02.023. eCollection 2021 Jun 11.
Non-viral platforms can be applied rapidly and cost-effectively for chimeric antigen receptor (CAR)-T cell manufacturing. In the present paper, we describe in detail a clinically relevant manufacturing process for NKG2D CAR-T cells through electroporation of CAR-encoding piggyBac transposon plasmids and expansion with K562 artificial antigen-presenting cells. With an optimized protocol, we generated the final cell therapy products with 89.2% ± 10.2% NKG2D CAR-positive cells and achieved the corresponding antigen-dependent expansion between 50,000 and 60,000 folds within 4 weeks. To facilitate repeated CAR-T cell infusions, we evaluated the practicality of cryopreservation followed by post-thaw expansion and an extended manufacturing process for up to 9 rounds of weekly K562 cell stimulation. We found that neither compromised the anti-tumor activity of NKG2D CAR-T cells. Interestingly, the expression of T cell exhaustion markers TIGIT, TIM3, and LAG3 was reduced with extended manufacturing. To enhance the safety profile of the NKG2D CAR-T cells, we incorporated a full-length CD20 transgene in tandem with the CAR construct and demonstrated that autologous NK cells could mediate efficient antibody-dependent cell-mediated cytotoxicity to remove these CAR-T cells. Collectively, our study illustrates a protocol that generates large numbers of efficacious NKG2D CAR-T cells suitable for multiple rounds of infusions.
非病毒平台可快速且经济高效地应用于嵌合抗原受体（CAR）-T细胞的制造。在本文中，我们详细描述了一种临床相关的NKG2D CAR-T细胞制造工艺，该工艺通过电穿孔导入编码CAR的猪尾巴转座子质粒，并与K562人工抗原呈递细胞一起进行扩增。通过优化方案，我们获得了最终的细胞治疗产品，其中NKG2D CAR阳性细胞的比例为89.2%±10.2%，并在4周内实现了50000至60000倍的相应抗原依赖性扩增。为便于重复输注CAR-T细胞，我们评估了冷冻保存后解冻扩增以及长达9轮每周K562细胞刺激的扩展制造工艺的实用性。我们发现这两种方法均未损害NKG2D CAR-T细胞的抗肿瘤活性。有趣的是，随着制造过程的延长，T细胞耗竭标志物TIGIT、TIM3和LAG3的表达降低。为提高NKG2D CAR-T细胞的安全性，我们在CAR构建体串联中加入了全长CD20转基因，并证明自体NK细胞可介导有效的抗体依赖性细胞介导的细胞毒性以去除这些CAR-T细胞。总的来说，我们的研究阐述了一种可产生大量适用于多轮输注的有效NKG2D CAR-T细胞的方案。
