Activating Natural Killer (NK) cell receptors represent promising target structures to elicit potent anti-tumor immune responses. We have generated novel immunoligands 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 based on affinity-optimized versions of B7-H6 and the Fab arm derived from Cetuximab. To enhance NKp30 binding, the solitary N terminal IgV domain of B7-H6 was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel B7-H6-derived NK cell engagers revealed an up to 45-fold enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared to the NK cell engagers based on the wild-type B7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) was significantly increased. Importantly, equipment of the B7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared to molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared to molecules solely triggering FcγRIIIa including the clinically approved antibody Cetuximab. Thus, incorporating affinity-matured ligands for NK cell activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.
