Co-editing of NKG2A and FAS increases long-term cytotoxic capacity and persistence of CAR NK cells

Natural killer (NK) cells have the intrinsic ability to kill cancer cells and are thus targeted in immunotherapy. Anti-CD19 CAR NK cells have shown efficacy in clinical studies in treating B cell malignancies; however, the full cytotoxic capacities of CAR NK cells may be limited by the expression of inhibitory receptors on NK cells. The NKG2A/HLA-E axis has been identified as an important negative regulator of NK cell activity, and different strategies on genome and protein levels are evaluated to block NKG2A-mediated inhibition. In this study, we engineered CD19 CAR NK cells harboring genetic disruption of <i>KLRC1</i>, the NKG2A-encoding gene, including NKG2A-promoter-driven CD19 CAR NK cells, and show that editing of NKG2A improves the killing capacity of CAR NK cells, especially after repeated challenge. Furthermore, we find that NKG2A-edited CAR NK cells exhibited significantly enhanced cytotoxicity against CD19-negative target cells, including cancer cells from a relapsed CD19-negative PDX. Co-editing the death receptor FAS increases the persistence of NKG2A-edited CAR NK cells both <i>in vitro</i> and <i>in vivo</i>. Together, our findings present a novel approach to enhance both cytotoxicity and persistence of CAR NK cell in cancer immunotherapy and reduce the risk of antigen-negative relapse.