Enhanced NK-cell Cytotoxicity by Combining CRISPR/Cas9 Genome Editing and RIG-I Activation. — ASN Events
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Enhanced NK-cell Cytotoxicity by Combining CRISPR/Cas9 Genome Editing and RIG-I Activation. (#183)

Sofía Beatriz Soler 1 , Thomas Zillinger 2 , Racha Hosni 3 , Ria Sharma 1 , Julia Bremser 1 , Rebecca Barker 1 , Anna-Maria Schmidt 1 , Regina Kemp 1 , Saskia Schmitz 1 , Christian Hagen 1 , Katrin Reiners 2 , Johannes Oldenburg 1 , Marieta Toma 3 , Behnaz Pezeshkpoor 1 , Gunther Hartmann 2 , Eva Bartok 1
  1. Institute of Experimental Haematology and Transfusion Medicine , University Hospital Bonn, Bonn, Germany
  2. Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
  3. Institute of Pathology, University Hospital Bonn, Bonn, Germany

Targeted innate immune activation and NK cell-based immunotherapies have both proven effective tools in cancer immunotherapy. Activation of the antiviral innate immune receptor Retinoic acid-Inducible Gene I (RIG-I) has been shown to improve NK cell toxicity, yet its effectivity varies across tumor entities. We investigated potential mediators of NK cell inhibition after RIG-I activation in tumor cells and identified the role of increased expression of classical HLA-I molecules and the non-classical HLA-I, HLA-E.

We observed that while RIG-I stimulated tumor cells are more efficiently killed by NK cells, this effect is significantly augmented in HLA-E-/- tumor cells. Therefore, we selected the HLA-E receptor NKG2A as target for CRISPR/Cas-mediated deletion in NK cells and developed a novel 2-gRNA protocol for the efficient generation of NKG2A-/- primary NK cells and immortalized NK-92 cells for use in in vitro cytotoxicity models. Cocultures of diverse tumor cell lines and cholangiocarcinoma-derived tumor organoids with primary NK cells revealed enhanced degranulation, IFNg production and cytotoxicity, indicating increased activation on NKG2A-/- cells.

Additionally, the KIR and HLA/KIR education status of NK cell donors were determined using sequence-specific PCR and high-resolution Sequencing from genomic DNA and matched and mismatched donors were examined in cytotoxicity assays.

As a combination therapy, NKG2A-/- NK cells and RIG-I activation in tumor cells worked synergistically in all lines tested, as well as in a cholangiocarcinoma organoid model, regardless of the KIR match or mismatch status of donor NK and tumor cells.

Altogether, this study establishes a rationale for approaches combining NKG2A-deletion on NK cells and tumor immunotherapies in vivo, in particular in combination with RIG-I agonists, with broad application to autologous and allogeneic NK cell immunotherapy. Moreover, by establishing a robust genome-editing platform in primary NK cells, we also open avenues for targeting other promising candidate genes for antitumoral therapy.