Mouse Fc-FcγRIV structure guides Fc engineering for cross-species FcγR recognition

Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action for many FDA-approved therapeutic antibodies that is driven by interactions between the antibody Fc and Fcγ receptors (FcγRs) on immune effector cells. Murine models used for preclinical antibody evaluation currently have limited predictive value for clinical ADCC performance due to interspecies differences in Fc-FcγR interactions. The molecular determinants governing Fc-FcγR engagement in mice remain poorly defined, complicating the interpretation of murine ADCC data and its clinical relevance. To address this, we present the high-resolution crystal structure of the receptor that regulates Fc-mediated cytotoxicity in mice, mouse FcγRIV, alone and in complex with mouse IgG2a Fc. This complex preserves key features of the human IgG1 Fc-human FcγRIIIa interface which mediates ADCC in humans including salt bridges, hydrogen bonds, and a proline sandwich. However, subtle variations in receptor orientation, Fc-FcγR electrostatics, and glycan positions reduce human IgG1 Fc- mouse FcγRIV binding affinity, resulting in species-restricted Fc-FcγR mediated immune responses. Modeling of human IgG1 Fc interactions with mouse FcγRIV predicted steric clashes, suggesting opportunities to modulate the interaction. One structure-guided substitution variant of human IgG1, Fc_humo, maintains comparable human FcγRIIIa engagement with enhanced binding to and activation of mouse FcγRIV, relative to human IgG1 Fc. This study provides proof-of-concept for engineering human Fc domains for cross-species FcγR recognition and provides a strategic framework to improve the predictive power of in vivo preclinical models.