CRISPR/Cas9 editing of NKG2A improves the efficacy of primary CD33-directed chimeric antigen receptor natural killer cells

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells show antileukemic activity against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by the interaction between human leukocyte antigen (HLA)-E and the inhibitory receptor, NKG2A. Here, we describe a strategy that overcomes CAR-NK cell inhibition mediated by the HLA-E-NKG2A immune checkpoint. We generate CD33-specific, AML-targeted CAR-NK cells (CAR33) combined with CRISPR/Cas9-based gene disruption of the NKG2A-encoding KLRC1 gene. Using single-cell multi-omics analyses, we identified transcriptional features of activation and maturation in CAR33-KLRC1ko-NK cells, which are preserved following exposure to AML cells. Moreover, CAR33-KLRC1ko-NK cells demonstrate potent antileukemic killing activity against AML cell lines and primary blasts in vitro and in vivo. We thus conclude that NKG2A-deficient CAR-NK cells have the potential to bypass immune suppression in AML. NK cell-based therapy can kill acute myeloid leukemia (AML), but immune suppression may occur. Here the authors overcome the immunosuppression of AML-targeted CAR33-NK cells via non-viral CRISPR-editing of the immune checkpoint NKG2A, leading to an enhanced potency of the CAR-NK cell product with sustained anti-tumor efficacy.