Engineering of potent CAR NK cells using non-viral Sleeping Beautytransposition from minimalistic DNA vectors

Natural Killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target-specificity, with these cells applicable as ‘off-the-shelf’ products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty(SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into ‘genomic safe harbors’ than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared to non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia (ALL) cells and patient-derived primary B-ALL samples as targets in vitroand in vivoin a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies.