Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen presenting cells

Abstract Non-viral platforms can be applied rapidly and cost-effectively for CAR-T cell manufacturing. In the present paper, we described in details a clinically relevant manufacturing process for NKG2D CAR-T cells through electroporation of CAR-encoding piggyBac transposon plasmids and in vitro 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 to 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 in vitro anti-tumour 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.