Bispecific NK Cell Engagers Activating NK Cells via NKp30 to Improve Tumor Cell Lysis and Pro-Inflammatory Cytokine Release

Katja Klausz

Katja Klausz

Activating Natural Killer (NK) cell receptors represent promising target structures to elicit potent anti-tumor immune responses. Novel Fc comprising NK cell engagers (NKCEs) were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format. The Fab arm was derived from Cetuximab and either yeast surface display-derived affinity-optimized N-terminal IgV domains of the natural ligand B7-H6 (ΔB7-H6) or NKp30 binding camelid VHH single domain antibodies were fused to activate NK cells via NKp30. Biochemical and functional characterization of ΔB7-H6-derived NKCEs revealed an up to 45-fold enhanced affinity for NKp30 and up to 87-fold improved NK cell-mediated, EGFR-dependent killing of tumor cells compared to the NKCEs based on the wild-type ΔB7-H6 domain. Using NKp30-specific VHHs against different epitopes on NKp30 revealed that those NKCEs built with VHHs that compete with B7-H6’s natural ligand binding site were significantly more potent in eliciting tumor cell lysis of EGFR-positive tumor cells than NKCEs harboring VHHs that target different epitopes on NKp30. Nevertheless, these non-competing NKCEs were capable to leave physiological processes between NKp30 and membrane-bound B7-H6 unaffected and still elicited tumor cell killing at low picomolar concentrations. Furthermore, release of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) was vastly increased by all NKCEs – irrespective of using ΔB7- H6 or NKp30 VHH. Importantly, all NKCEs could be further improved by concomitant engagement of Fcγ receptor IIIa (FcγRIIIa) and NKp30 on NK cells. These NKCEs carrying an active Fc were even more potent than the clinically approved antibody Cetuximab in terms of tumor cell killing and pro-inflammatory cytokine release, both features which might be beneficial for anti-tumor therapy. Thus, NKCEs activating NK cells via NKp30 show unique modes of action and might represent an effective strategy for cancer immunotherapy.

Go back