Asymmetrical glycoengineering of monoclonal antibodies: new insights in ɑ-gal immunogenicity

Glycosylation heterogeneity in therapeutic monoclonal antibodies (mAbs) necessitates precise glycoengineering to optimize effector functions, such as those mediated by Fcγ-receptors (FcγRs). Current enzymatic remodeling methods are limited in scope, producing only a narrow spectrum of glycoforms and struggling to achieve complete control over the synthesis of defined asymmetrical structures, which are often critical for enhanced mAb efficacy. We report a multi-step, fully enzymatic platform for the solution-phase synthesis of a comprehensive library of biantennary mAb glycoforms with complete control over the α1,3- and α1,6-mannose arm architecture. The strategy relies on two key catalytic steps: the use of the single domain of the β-N-acetylglucosaminidase StrH (GH20-2) to achieve the regioselective hydrolysis of the GlcNAc residue on the α1,3-mannose arm of the intact antibody, thereby introducing asymmetry; and exploiting the intrinsic, broad acceptor flexibility of GnT-I to reinstall the α1,3-mannose arm GlcNAc even after the α1,6-mannose arm has been selectively extended and terminally capped with Neu5Ac or the α-gal epitope. This robust, two-stage enzymatic remodeling methodology, demonstrated on infliximab and cetuximab, unlocks the complete chemical space of biantennary Fc-glycans for comprehensive, functional studies. We employed these well-defined glycovariants to determine the minimum structural requirements for anti-α-gal IgE binding and systematically evaluate binding affinities toward FcγRs, providing essential insights for the rational design of next-generation immunotherapeutics.