Glycan pairing in therapeutic IgG orchestrates Fcγ receptor engagement and ADCC: an integrated structure-function approach for thorough evaluation of Fc N-glycans as critical quality attributes

Monoclonal antibodies (mAbs) can mediate their therapeutic activity through antigen binding and by engaging Fc receptors (FcRs) or the complement system. FcRs are expressed on immune cells and can initiate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis. The conserved Fc N-glycans of immunoglobulin G (IgG) at N297 modulate the strength and stability of Fc-FcγR interactions and the resulting effector responses. Conventional analysis typically considers individual glycans in isolation and neglects the fact that each Fc part carries two N-glycans, that may be paired symmetrically or asymmetrically. Recent studies with IgG Fc regions have demonstrated that glycan pairing can significantly shape Fc-FcR interactions. Here, the effect of glycan pairing was evaluated with full-length IgG using rituximab as a model. A set of pure, homogeneous, symmetrical, and asymmetrical Fc glyco-pair variants was generated by controlled chemoenzymatic remodeling and purification. Surface plasmon resonance binding studies across a comprehensive panel of human FcγRs revealed receptor- and allotype-specific effects: FcγRI binding was essentially unaffected, FcγRIIa showed allotype dependence, with FcγRIIa-H131 predominantly modulated by galactosylation and FcγRIIa-R131 by afucosylation, whereas FcγRIIIa required only a single afucosylation to reach maximal binding. Galactosylation exerted modest and receptor-dependent effects, most pronounced for FcγRIIIb. High-mannose species reduced binding to FcγRI and FcγRII, but displayed intermediate affinity to FcγRIIIa, which was lower than afucosylated complex types yet higher than fully fucosylated variants. To evaluate the biological relevance, cell-based ADCC reporter gene assays confirmed that single afucosylation was sufficient to drive the increase in ADCC potency, with galactosylation and high-mannose contributing only modest or context-dependent effects. Together, these findings establish glycan pairing as a critical quality attribute of therapeutic antibodies and provide a framework for precise evaluation of Fc N-glycan criticality and for tailoring mAbs with defined effector function profiles.