Precision Fc remodeling via glycosylation-competent CHO display enables ultra-selective FcγRIIIa targeting and enhanced antitumor activity

Improving tumor cell clearance by therapeutic antibodies remains a translational bottleneck because wild-type IgG1 Fc typically elicits suboptimal NK cell–mediated antibody-dependent cellular cytotoxicity (ADCC), necessitating Fc engineering to enhance activating FcγR engagement while preserving antigen specificity and manufacturability. FcγRIIIa (CD16A) is the principal activating receptor on NK cells, and its clinical relevance is underscored by the FCGR3A-158V/F polymorphism, which modulates IgG1 Fc affinity and therapeutic response. Because FcγRIIIa engagement critically depends on Fc glycosylation, microbial display platforms lacking mammalian glycan processing are limited in capturing Fc–FcγRIIIa energetics and selectivity. We developed a glycosylation-integrated Fc engineering platform using CHO surface display to screen glycosylated Fc libraries in a post-translationally accurate context. Multiparameter flow-cytometric selection with FcγRIIIa binding and FcγRIIb counter-screening enabled iterative isolation of PS-series Fc variants with ultra-selective, allotype-compatible FcγRIIIa recognition. Lead variants achieved 262-fold (158V) and 497-fold (158F) FcγRIIIa affinity gains while reducing FcγRIIb binding by up to 4.2-fold, expanding activating-to-inhibitory selectivity up to 2,096. This exceeded clinically deployed FcγRIIIa-enhancing Fc variants by 525-fold and 108-fold, respectively, in activating-to-inhibitory selectivity, compared with DE (S239D/I332E; used in tafasitamab) and VLPLL (L235V/F243L/R292P/Y300L/P396L; used in margetuximab). When grafted onto trastuzumab, PS variants enhanced NK cytotoxicity and improved tumor control in a trastuzumab-refractory xenograft model. Modular transfer to cetuximab and rituximab also increased cytotoxic activity. This study establishes a glycosylation-integrated CHO display platform for precision FcγRIIIa-targeted Fc engineering, generating modular effector domains with broad allotype compatibility, minimal FcγRIIb binding, and robust therapeutic potential in cancer immunotherapy.