Affinity Maturation of B7-H6 Translates into Enhanced NK Cell-Mediated Tumor Cell Lysis and Improved Proinflammatory Cytokine Release of Bispecific Immunoligands via NKp30 Engagement

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 (Delta B7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel Delta 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 with the NK cell engager based on the wild-type Delta B7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-gamma and TNF-alpha was significantly increased. Importantly, equipment of the Delta 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 with molecules either triggering Fc gamma RIIIa or NKp30. Additionally, INF-gamma and TNF-alpha release was increased compared with molecules solely triggering FcyRIII alpha, 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.

Automated summary

Novel immunoligands targeting activating NK cell receptors were generated and showed enhanced killing of tumor cells by NK cells. Incorporating affinity-matured ligands for NK cell-activating receptors may represent an effective strategy for potent cancer immunotherapy agents.