Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands 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 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 (DB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel DB7-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 with the NK cell engager based on the wild-type DB7-H6 domain. In this regard, potencies (EC 50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-g and TNF-a was significantly increased. Importantly, equipment of the DB7-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 with molecules either triggering FcgRIIIa or NKp30. Additionally, INF-g and TNF-a release was increased compared with molecules solely triggering FcgRIIIa, including the clinically approved Ab 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.

In this work, we have generated novel Fc-comprising NK cell engagers (NKCEs) that bridge human NKp30 on NK cells to human epidermal growth factor receptor (EGFR) on tumor cells. Camelid-derived VHH single-domain Abs specific for human NKp30 and a humanized Fab derived from the EGFR-specific therapeutic Ab cetuximab were used as binding arms. By combining camelid immunization with yeast surface display, we were able to isolate a diverse panel of NKp30-specific VHHs against different epitopes on NKp30. Intriguingly, NKCEs built with VHHs that compete for binding to NKp30 with B7-H6, the natural ligand of NKp30, were significantly more potent in eliciting tumor cell lysis of EGFR-positive tumor cells than NKCEs harboring VHHs that target different epitopes on NKp30 from B7-H6. We demonstrate that the NKCEs can be further improved with respect to killing capabilities by concomitant engagement of FcγRIIIa and that soluble B7-H6 does not impede cytolytic capacities of all scrutinized NKCEs at significantly higher B7-H6 concentrations than observed in cancer patients. Moreover, we show that physiological processes requiring interactions between membrane-bound B7-H6 and NKp30 on NK cells are unaffected by noncompeting NKCEs still eliciting tumor cell killing at low picomolar concentrations. Ultimately, the NKCEs generated in this study were significantly more potent in eliciting NK cell–mediated tumor cell lysis than cetuximab and elicited a robust release of proinflammatory cytokines, both features which might be beneficial for antitumor therapy.

Summary Natural killer (NK) cells exert an important role in cancer immune surveillance. Recognition of malignant cells and controlled activation of effector functions are facilitated by the expression of activating and inhibitory receptors, which is a complex interplay that allows NK cells to discriminate malignant cells from healthy tissues. Due to their unique profile of effector functions, the recruitment of NK cells is attractive in cancer treatment and a key function of NK cells in antibody therapy is widely appreciated. In recent years, besides the low-affinity fragment crystallizable receptor for immunoglobulin G (FcγRIIIA), the activating natural killer receptors p30 (NKp30) and p46 (NKp46), as well as natural killer group 2 member D (NKG2D), have gained increasing attention as potential targets for bispecific antibody-derivatives to redirect NK cell cytotoxicity against tumors. Beyond modulation of the receptor activity on NK cells, therapeutic targeting of the respective ligands represents an attractive approach. Here, novel therapeutic approaches to unleash NK cells by engagement of activating NK-cell receptors and alternative strategies targeting their tumor-expressed ligands in cancer therapy are summarized.