Asymmetrical Glyco-Engineering of Monoclonal Antibodies Provides Insights into α-Gal Immunogenicity

Glycosylation heterogeneity of therapeutic monoclonal antibodies (mAbs) necessitates precise glycoengineering to optimize effector functions, such as those mediated by Fcgamma-receptors (FcgammaRs). However, current enzymatic remodeling methods are limited in scope, producing only a narrow spectrum of glycoforms and unable to achieve complete control over defined asymmetrical structures. Here, we report a multi-step, fully enzymatic platform for the solution-phase synthesis of a comprehensive library of biantennary mAb glycoforms, offering complete control over the alpha1,3- and alpha1,6-mannose arm architecture. This strategy uses a single domain of the beta-N-acetylglucosaminidase StrH (GH20-2) to achieve regioselective hydrolysis of the GlcNAc residue on the alpha1,3-mannose arm of the intact antibody, thereby introducing asymmetry. Next, the remaining terminal GlcNAc residue at the alpha1,6-antenna can be enzymatically extended with a beta1,4-galactoside and sialoside or alpha1,3-galactoside. Remarkably, after this extension, recombinant GnT-I can reintroduce the GlcNAc at the alpha1,3-mannosyl antenna, allowing precise control over glycan branching and composition. This robust, two-stage enzymatic remodeling methodology, demonstrated on infliximab and cetuximab, unlocks a broad chemical space of biantennary Fc glycans for comprehensive functional studies. We employed these well-defined glycovariants to determine the minimum structural requirements for anti-alpha-Gal IgE binding and to systematically evaluate binding affinities toward FcgammaRs, providing new insights for the rational design of next-generation immune-therapeutics.