The development of chimeric antigen receptor (CAR) T cell products with optimal safety and efficacy requires insights how distinct gene-transfer strategies affect CAR expression and ensuing T cell function.

We established a test platform that allows us to characterize and compare CAR-T cells engineered by either, lentiviral transduction (LV), Sleeping Beauty (SB) transposon-based gene transfer or CRISPR-Cas-mediated targeted CAR insertion (KI), to express CAR constructs that cover a range of different target and epitope specificities in graded affinities.

We characterized the CAR-T cells phenotypically by flow cytometry, genomically by CAR copy number variation (CNV) determination via droplet digital (dd)PCR, and analysed CAR surface expression and organization by dSTORM super-resolution microscopy.

Further, we employed a library of target cells with distinct antigen densities to functionally characterize the CAR-T cells regarding short vs. long-term cytolytic activity, cytokine secretion, proliferation capacity, as well as the rate of antigen-induced cell death (AICD) in vitro.

We found that charachteristic CNV from highest (SB) to lowest (KI) manifested in pronounced differences in spatiotemporal CAR expression that translated to distinct anti-tumor functionality depending on the target-density. LV/SB CAR-T cells showed higher cytokine production and conferred stronger cytolytic activity compared to KI CAR-T cells in short-term (> 8 hours) in short term lysis assays. 

LV/SB CAR-T cells showed higher cytokine production and stronger cytolytic activity compared to KI CAR-T cells in short-term assays. All CAR-T cells were equally effective with longer follow-up. We observed less AICD for KI CAR-T cells.

These results highlight the relevance of gene-transfer strategy for CAR-T cell function.