Piggybat transposase for the generation of CD19 specific chimeric antigen receptor T cells

Abstract

Background & Aim Introduction DNA transposon systems offer an alternative to viral vectors for genetic modification of eukaryotic cells. The Piggybac system is being used in CAR T-cell clinical trials presently, but its widespread use may be limited by licensing restrictions. Transposon mining of the little brown bat (Myotis lucifugus) genome has revealed the presence of an active DNA transposase named Piggybat. Aims To optimise manufacturing conditions and evaluate the genomic integration profile of the Piggybat transposase in the generation of CD19 specific CAR T-cells (CAR19-T) compared to Piggybac transposase. Methods, Results & Conclusion Methods CAR19-T were generated from healthy donors (n=3) by co-electroporation of two plasmids encoding the transposase and a CAR19 transposon. T-cells were co-cultured with irradiated feeder cells and cytokines. CAR19-T expansion, CAR expression, memory phenotype, CD19 specific activation and cytotoxicity were assessed after a 2-week culture period. CAR gene integration copy number was determined by droplet digital polymerase chain reaction and integration site analysis was conducted by next generation sequencing of PCR amplicons spanning genomic DNA/CAR gene intersection sites. Results Over 2 weeks, Piggybat CAR19-T expanded a mean of 26 fold with 52% CAR expression, and Piggybac CAR19-T expanded a mean of 11 fold with 67% CAR expression. Piggybat and Piggybac CAR19-T had a mean 59% and 48% CD8+ T-cells respectively, with a similar CAR19-T memory phenotype. Using 50ug/ml concentration of each transposon and transposase plasmids, Piggybat had fewer genomic integrants than Piggybac (mean 3.4 and 10.1 copies per cell respectively). Integrant copy number could be titrated by altering transposon plasmid concentration. Peak CAR19-T expansion, expression and MFI were achieved using 1ug Piggybat transposase plasmid and 5ug transposon plasmid. Piggybat and Piggybac CAR19-T demonstrated similar CD19 specificity and cytotoxicity. Piggybat CAR19-T showed 1920 unique genomic integration sites, 11.13% within 2.5kb of transcription start sites (TSS) and 43.72% being intragenic, compared to Piggybac with 2697 unique integration sites, 20.33% TSS localised and 47.77% intragenic. Conclusions Compared to Piggybac, Piggybat transposase can generate CD19 specific CAR T-cells with similar CAR expression, function and insertion site profile, but fewer integrants per cell. Thus, Piggybat is a new DNA transposon that may allow cost-effective CAR T-cell production for clinical use.