Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity and can be applied as ‘off-the-shelf’therapy. However, NK cell function can be impaired by upregulation of inhibitory receptors, such as immune checkpoint NKG2A (natural killer group 2A).

To mitigate immune checkpoint inhibition, we established CRISPR-Cas9 based knockout of NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus in primary NK cells. Genotype analyses confirmed disruption of 85.6% of KLRC1 alleles by next-generation targeted amplicon sequencing (NGS) with minor off-target activity at one site (~0.1%). On the phenotypic level, KLRC1 disruption reduced the fraction of NKG2A-positive cells by ~50%. In functional assays, NKG2A-knockout (KO) cells showed significantly enhanced tumor cell lysis compared to the already high intrinsic killing capacity of NK cells against different multiple myeloma (MM) tumor cell lines. No further enhancement of tumor lysis was shown for selected (99% purity) NKG2A–KO NK cells. To address the increased killing capacity in a preclinical setting, we isolated primary tumor cells from bone marrow aspirates of differently treated MM patients. Allogenic NKG2A-KO NK cells showed significantly higher MM tumor cell lysis as compared to non-edited NK cells.

In conclusion, our protocol for CRISPR-Cas9 gene editing of primary NK cells provides a robust platform to overcome verity immune checkpoint inhibition against different tumor entities.