Abstract 1536: Functional characterization of EDIT-201, a multiplexed CRISPR-Cas12a gene edited healthy donor derived NK cell therapy, reveals increased granzyme B and degranulation supporting improved serial killing capacity

We have previously described EDIT-201, a healthy donor derived NK cell therapy with a CISH and TGFBR2 knockout mediated by CRISPR-Cas12a that results in resistance to TGF-β inhibition and increased effector function. EDIT-201 kills tumor cells more efficiently and for a longer duration than control NK cells in the presence of TGF-β. Based on these data, we hypothesized that EDIT-201 has increased serial killing capacity. First, we explored the transcriptional changes contributed by each gene edit with a focus on transcripts critical for NK cell effector function and metabolism using Nanostring analysis. Unedited, mock electroporated, and control edited (targeting a biologically irrelevant site) NK cells were included as controls in addition to CISH and TGFBR2 single and double gene knockout (KO) NK cells to interrogate the potential impact of electroporation and double-stranded DNA breaks on NK cell function. All samples included in the analysis were cultured for 3 days in IL-15 post-electroporation. No significant transcriptional changes were detected in all control conditions, while samples that contained CISH editing clearly upregulated transcripts relevant for NK cell effector function, including contents of cytolytic granules (GZMB, GZMA and GZMH). EDIT-201 expressed an average of 22 fold more GZMB transcript than control NK cells as measured by RT-qPCR in four unique NK cell donors. Furthermore, EDIT-201 showed significantly higher levels of CD107a, a marker of degranulation, after 14hrs of co-culture with SKOV-3 tumor cells, suggesting that EDIT-201 had an increased capacity to degranulate relative to control NK cells. To determine the presence of granzyme proteins within tumor cells following engagement with NK cells, we developed a novel GzmB reporter gene and used lentiviral vectors to introduce this reporter into tumor cell lines. Using SK-OV-3 tumor cells transduced with our reporter, we demonstrated that upon coculture of EDIT-201 with tumor cells, intratumoral GzmB activity was identified 4 hours sooner than when the target cells were cultured with control NK cells. In addition, EDIT-201 affected 80% more SK-OV-3 tumor cells with granzyme B compared to control NK cells over a 20 hour period. Most significantly, our data demonstrated that EDIT-201 not only discharged GzmB more rapidly than control NK cells, but also the amount of GzmB degranulated is greater as well, confirming that enhanced degranulation is a key mechanism by which EDIT-201 h