CRISPR/Cas9 Gene Editing of Immune Checkpoint Receptor NKG2A Improves the Efficacy of Primary CD33-CAR-NK Cells Against AML

BACKGROUND: CD33-targeting chimeric antigen receptor (CAR)-T cells already showed utility for the treatment of AML. Yet clinical application of CD33-CAR-T cells remains challenging due to potential side effects and its restriction to autologous cell preparations. In contrast, natural killer (NK) cells can be safely administered to HLA-mismatched recipients without severe side effects. Recently, we reported on the successful generation of primary CD33-CAR-NK cells, which are highly effective against AML in vitro and in vivo in AML-xenograft models (Albinger et al., Blood Cancer J 2022). However, CAR-NK cell function can be impaired by high levels of the inhibitory immune checkpoint receptor NKG2A (natural killer group 2A) expressed on NK cells (Bexte et al., Oncoimmunology 2022). By applying a CRISPR/Cas9 gene editing for knockout (KO) of NKG2A, we significantly improved CD33-CAR-NK cell functionality in vitro and in vivo.

METHODS: CD33-targeting CAR-NK cells were generated by lentiviral transduction. KO of the NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus was performed using CRISPR-Cas9 technology. The CD33-CAR- and NKG2A-expression as well as cytotoxicity were analysed using flow cytometry after feeder cell-free, IL-15/IL-2-based expansion. The in vivo-efficacy was evaluated in OCI-AML2 (GFP⁺, Luc⁺) xenografted NSG-SGM3 mouse models.

RESULTS: Lentiviral transduction resulted in up to 60% CD33-CAR-positive NK cells, while KLRC1 gene disruption resulted in 50% reduction of NKG2A expression. Cite-Seq and qPCR analysis revealed a distinct gene regulation pattern in CD33-CAR- and CD33-CAR-NKG2A-KO-NK cells and CAR-KO-NK cells showed significantly higher elimination of CD33⁺/HLA-E⁺ OCI-AML2 cells in in vitro cytotoxicity assays compared to NKG2A-KO- or CD33-CAR-NK cells. Furthermore, a reduction of leukemic burden was observed in vivo following a single injection of a low dose (3x10⁶ cells) of CAR-KO-NK cells compared to NKG2A-KO or CD33-CAR-NK cell treatment in an AML-xenografted mouse model. A double injection led to a complete elimination of AML and leukemia-initiating cells in the bone marrow of mice treated with CAR-KO NK cells, which was confirmed by bone marrow re-engraftment analysis.

CONCLUSION: Removing an inhibitory receptor in CAR-NK cells showed a highly beneficial effect for the treatment of AML. This double genetic modification has the potential to enable NK cells to bypass the suppressive effect not only in the tumor microenvironment in context of AML, but also in a broad range of malignant diseases.

Penack:Equillium Bio: Membership on an entity's Board of Directors or advisory committees; SOBI: Membership on an entity's Board of Directors or advisory committees; Shionogi: Membership on an entity's Board of Directors or advisory committees; Priothera: Membership on an entity's Board of Directors or advisory committees, Research Funding; Therakos: Honoraria; Pfizer: Honoraria; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Omeros: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Klusmann:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees. Möker:Miltenyi Biotec: Current Employment. Ullrich:BMS: Honoraria; Phialogics: Honoraria.