Background Natural killer (NK) cell therapies have shown a great promise for solid and liquid tumors in initial clinical trials. NK cells are innate immune cells with distinct potential safety and efficacy advantages compared to adoptive T-cell therapies. However, there are limitations with the persistence and immunosuppression of these cells in the tumor microenvironment. Furthermore, multiple sources of NK cells have been used in clinical trials and there are challenges with manufacturing homogeneous and high doses of these cells. Induced Pluripotent Stem Cell (iPSC)-derived NK cells offer an opportunity to generate unlimited and homogenous NK cells for allogeneic off-the-shelf therapies. We combined Cytovia’s iNK cell platform with Cellectis TALEN^® gene editing technology to improve potency and the manufacturing process. Clonally edited iPSC lines were generated by knocking in IL-15 and knocking out TGFβR2 to improve the persistence and antitumor activity, respectively. Edited iPSCs were differentiated into iNK cells with high efficiency using Cytovia’s proprietary platform.

Methods iPSCs were edited at the B2M locus with TALEN^® pairs along with a template to knock in IL-15 by electroporation. Another TALEN^® pair was sequentially used to knock out TGFβR2. Edited single cell iPSCs were printed, expanded, and screened for clone selection. Selected clones were sequence verified by NGS and samples were submitted for off-target assessment by GUIDE-seq. Expression of IL-15 and TGFβR2 was measured by ELISA and western blot, respectively. iPSCs were differentiated into iNK cells and analyzed by surface staining and flow cytometry. Cytotoxicity assay was performed against K562 tumor cells.

Results Several single and double gene edited iPSC lines were generated with high efficiency to produce NK cells: iPSC-IL-15 (+/+), iPSC-TGFβR2 (-/-) and iPSC-IL-15 (+/+)-TGFβR2 (-/-). These TALEN^®-edited iPSCs kept their pluripotency, exhibited normal morphology and karyotype with no detected off-target effect. Edited iPSCs were differentiated into NK cells with high efficiency (CD56+/CD45+>95%) and enhanced cytotoxicity against K562 tumor cells compared to unedited iNK cells. Moreover, TGFβR2 protein was not detected, while the expression of secreted IL-15 was observed in the edited iNK cells confirming the phenotype of these cells.

Conclusions Cytovia’s iPSC-NK platform combined with TALEN^® gene editing robustly and reliably generated single cell edited iPSC clones, which were expanded and differentiated into functionally improved iNK cells. The data indicated iNK cells edited with an IL-15 knock in and TGFβR2 knock out resulted in enhanced antitumor activity. The editing and manufacturing process will enable clinical evaluation of these product candidates.