Engineering NKG2D ligand affinity transforms EGFR-targeted NK cell engagers into high-potency effectors against pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) often escapes T cell–mediated immunity through impaired major histocompatibility complex class I (MHC-I) antigen presentation, contributing to its limited responsiveness to T cell–based immunotherapies. Because natural killer (NK) cells are capable of eliminating MHC-I–low tumor cells, redirecting NK cytotoxicity represents a promising strategy for these immune-cold tumors such as PDAC. Among activating NK receptors, NKG2D has been widely exploited in NK cell–engaging platforms through the incorporation of NKG2D ligands (MICA/B and ULBP family members). However, the impact of NKG2D ligand (NKG2DL) identity and binding affinity on NK cell engager potency has not been quantitatively defined. We engineered a panel of epidermal growth factor receptor (EGFR)–targeted, heterodimeric IgG1 Fc-based αEGFR×NKG2DL immune cell engagers (ICEs), each pairing a panitumumab-derived EGFR arm with one of six human NKG2DL extracellular domains (MICA, MICB, ULBP1, ULBP2, and the ULBP6 allelic variants ULBP0601 and ULBP0602). All ICEs bound specifically to EGFR and NKG2D, and NKG2D binding affinity correlated directly with NK cell cytotoxic potency across PDAC cell lines expressing different EGFR densities. Among native ligands, ULBP0602 exhibited the strongest NKG2D binding and highest killing activity. Yeast display–based affinity improvement of ULBP0602 yielded the variant ULBP6#2, which showed approximately a 13-fold improvement in affinity due primarily to slower dissociation kinetics. The affinity-improved αEGFR×ULBP6#2 induced up to an 11-fold reduction in EC₅₀ and greater maximal lysis than the parental construct, along with enhanced IFN-γ and TNF-α secretion by NK cells. In NK-humanized NSG mice bearing PDAC xenografts, αEGFR×ULBP6#2 achieved markedly greater tumor growth inhibition compared with αEGFR×ULBP0602 in both PANC-1 (89% versus 57%) and BxPC-3 (60% versus 25%) models, without observable toxicity or weight loss. These results support an affinity–activity relationship for NKG2D-engaging ICEs within the EGFR-targeted, Fc-competent format evaluated here. Notably, improving ULBP6 affinity enhanced NK cell effector function and increased antitumor efficacy in NK-reconstituted PDAC models. Collectively, our findings provide a design framework for engineering high-potency, tumor-tethered NKG2D engagers for the immunotherapy of immune-cold tumors such as PDAC.