Precision off-the-shelf natural killer cell therapies for oncology with logic-gated gene circuits

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis (5-year survival rate of 30.5% in the United States). Designing cell therapies to target AML is challenging because no single tumor-associated antigen (TAA) is highly expressed on all cancer subpopulations. Furthermore, TAAs are also expressed on healthy cells, leading to toxicity risk. To address these targeting challenges, we engineer natural killer (NK) cells with a multi-input gene circuit consisting of chimeric antigen receptors (CARs) controlled by OR and NOT logic gates. The OR gate kills a range of AML cells from leukemic stem cells to blasts using a bivalent CAR targeting FLT3 and/or CD33. The NOT gate protects healthy hematopoietic stem cells (HSCs) using an inhibitory CAR targeting endomucin, a protective antigen unique to healthy HSCs. NK cells with the combined OR-NOT gene circuit kill multiple AML subtypes and protect primary HSCs, and the circuit also works in vivo. [Display omitted] •A three-input logic gate with activating and inhibitory CARs is optimized in NK cells•Logic-gated NK cells kill leukemic stem cells and blasts, not healthy stem cells•Selective killing of tumor cells is validated in vivo in a mixed target cell model Treating acute myeloid leukemia with conventional, single-target CAR cell therapies is challenging due to tumor heterogeneity and because tumor antigens are found on hematopoietic stem cells. Frankel et al. design logic-gated CAR-natural killer cells that simultaneously detect three antigens on prospective target cells to safely decide whether to kill them.