Efficient, fratricide free non-viral engineering of CD70-targeted CAR NK cells for hematologic and solid tumor immunotherapy.

Natural killer (NK) cells are a component of the innate immune system with potential safety advantages over T cells for adoptive cell therapy (ACT). However, NK cells lack the potency and persistence needed for tumor clearance. Here, we report an efficient and cost-effective non-viral method for engineering CD70 targeted CAR NK cells using a hyperactive TcBuster DNA transposon system while simultaneously knocking out CD70 via CRISPR-Cas9 to prevent fratricide. CD70 CAR NK cells engineered in this manner display improved efficacy in killing acute myeloid lymphoma (AML) cells and renal cell carcinoma (RCC) cells in vitro and in vivo. Incorporating soluble IL-15 cytokine armoring into the transposon cassette further improves survival and expansion in vivo. Our findings demonstrate a novel application of non-viral transposon-based cell engineering combined with CRISPR-Cas9 knockout to produce a clinically scalable product for ACT against multiple cancer types.