Enhanced NK-cell cytotoxicity by combining genome editing and RIG-I activation

Background

Natural killer (NK) cells can recognize and eliminate cellular targets without MHC-restriction, rendering them particularly amenable to off-the-shelf cellular immunotherapy. Nonetheless, potential target cells employ diverse molecular signals to limit NK-cell recruitment and evade cytotoxicity. Both innate immune activation and checkpoint inhibition on NK cells are promising approaches to improving the infiltration and cytotoxicity of NK-cell immunotherapy.

Methods

We examined the effect of the modulation of the immune checkpoint axis NKG2A/HLA-E on NK-cell tumor toxicity both alone and in combination with targeted activation of the innate immune receptor Retinoic-acid Inducible Gene I (RIG-I). Using CRISPR/Cas genome editing, we ablated the NKG2A-ligand HLA-E in diverse human tumor cell lines. Moreover, we developed a robust protocol for the targeted genome editing of primary and immortalized NK cells, allowing us to reduce NKG2A levels in effector cells. We then analyzed the activity and cytotoxic potential of these NKG2A-/- NK cells in vitro both in the presence and absence of HLA-E expression on tumor cells as well as alone and in combination with stimulation with a specific RIG-I agonist (3pRNA).

Results

All tumor cell lines tested demonstrated enhanced susceptibility to NK-cell killing after ablation of HLA-E on their targets. In line with this, NKG2A-/- NK cells also demonstrated greater degranulation, cytotoxicity towards target cells and cytokine induction, all of which occurred in an HLA-E dependent manner. Activation of RIG-I in tumor cells also enhanced NK cell activity overall but concurrently induced the expression of HLA-E in target cells. However, combining RIG-I activation with NKG2A-/- counteracted the effect of HLA-E induction and thus resulted in the highest cytotoxicity against target cells.

Conclusion

Altogether, our data demonstrate that combining NKG2A-/- NK cells with RIG-I activation is an effective approach to boosting allogeneic antitumor responses in vitro. Moreover, by establishing an efficient system for genome editing of primary NK cells, we provide an approach to targeting other promising candidate genes during NK-cell based therapies. Further studies will be needed to examine further NK checkpoints in this context and to investigate NKG2A-/- /RIG-I combination therapy in vivo.

Offenlegung Interessenkonflikt:

G.H. is an inventor on a patent covering synthetic RIG-I ligand and was a co-founder of Rigontec GmbH. All of others have no conflict of interest.

Invited talks abstract/summary