Natural and recombinant IgG type antibodies have a conserved N-glycan at the Asn-297 site in the Fc domain. The Fc glycans play an important role in modulating an antibody’s effector functions such as the antibody-dependent cellular cytotoxicity (ADCC).  We have developed a general chemoenzymatic method for Fc glycan remodeling of intact natural or recombinant antibodies that involves deglycosylation with a wild-type endoglycosidase and subsequent glycosylation with a structurally well-defined glycan using a glycosynthase mutant. Recently, we have extended this chemoenzymatic method to site-specific bioconjugation of antibodies with cytotoxic drugs to make homogeneous antibody-drug conjugates (ADCs).  We found that the glycosynthase mutants of the endoglycosidase from Streptococcus pyogenes (Endo-S and Endo-S2) could efficiently transfer azide-modified glycans to antibodies, which allows subsequent click reaction with a payload to make ADCs with varied drug/antibody ratios (DAR 2-12). Moreover, we further discovered that wild-type endoglycosidase from the serotype 49 of Streptococcus group A (Endo-S2) could perform deglycosylation and simultaneous glycosylation with a series of synthetic azide-tagged disaccharide oxazolines as the substrates without product hydrolysis, enabling a single enzyme, one-pot, and site-specific modification of antibodies. In addition to ADC production, this chemoenzymatic Fc glycan remodeling strategy was also applied for site-specific one-pot conjugation of high-affinity ligands of cellular receptors to antibodies, enabling the construction of homogeneous lysosome-targeting chimeras (LYTACs) for targeted degradation of extracellular proteins.

Chemoenzymatic glycan remodeling for antibody-drug conjugation and beyond

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