CD19 specific chimeric antigen receptor expression optimisation in human T-cells using the piggyBac transposase system

Abstract

Chimeric antigen receptors (CARs) can redirect T-cells against tumour antigens. The optimal techniques to express CARs are not clear, with lentiviral, retroviral and transposase systems being the most widely employed methods. Transposase systems may have advantages over viral systems, including improved safety and scalability. The primary aim was to establish the optimal electroporation settings to express a CD19-specific CAR in primary T-cells using the piggyBac transposase system. The second aim was to expand CAR-transfected T-cells in tissue culture in a reproducible way. The third aim was to repeat the results for CAR-expression in cytomegalovirus specific T-cell cultures. The highest CAR expression was seen at the electroporation voltage of 2400V (26%±12%). There was a dose-response between increasing voltage and CAR expression (P< 0.0001). Absolute CAR T-cell numbers expanded 457-fold (±275-fold) after two weeks by co-culturing with irradiated autologous PBMC, in the presence of cytokines interleukin-2 (IL2) 50U/ml, IL7 (10ng/ml) and IL15 (10ng/ml). Results in cytomegalovirus-specific cultures were poor, with CAR-expression 10% at best, and with poor cell recovery (~5%). In summary, using piggyBac transposase and tissue culture we generated useful numbers of CAR-redirected T-cells, results which will serve as a platform for future lab studies and clinical trials.