Natural and recombinant IgG type antibodies are usually glycosylated at the Asn-297 site of the Fc domain. Compelling data have shown that the structures of Fc glycans can profoundly affect the effector functions of antibodies, including the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and anti-inflammatory activity. We have recently developed a general chemoenzymatic method for Fc glycan remodeling of intact natural or recombinant antibodies with structurally well-defined glycans. The method usually involves two key steps: deglycosylation of antibodies to remove the heterogeneous Fc N-glycans using a wild-type endoglycosidase such as Endo-S and Endo-S2 from streptococcus pyogenes and subsequent enzymatic transfer of a pre-assembled N-glycan by an endoglycosynthase mutant that lacks Fc glycan hydrolytic activity but can act on a highly activated glycan oxazoline as a donor substrate for transglycosylation. This method has been successfully applied to anti-cancer monoclonal antibodies to enhance their ADCC activity and to the remodeling of intravenous immunoglobins (IVIG) to improve its anti-inflammatory activities. In this lecture, we describe the extension of this chemoenzymatic method to site-specific bioconjugation of antibodies with cytotoxic drugs to make homogeneous antibody-drug conjugates (ADCs).  We found that Endo-S and Endo-S2 could efficiently transfer azide- and other functional group-modified glycans to antibodies, which allows subsequent click reaction with a payload to make ADCs with varied drug/antibody ratios (DAR 2-12). We found that wild-type Endo-S2 could use a series of synthetic azide-tagged disaccharide oxazolines as substrates for transglycosylation without product hydrolysis, enabling a simultaneous deglycosylation and glycosylation without the need of separation of the intermediate and enzyme. Moreover, we found that Endo-S2 could also directly transfer drug-preloaded disaccharide to antibody to fulfill a truly single-step ADC production from a recombinant antibody. On the other hand, the 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 glycoengineering of antibodies for site-specific antibody-drug conjugation and beyond

ACS Spring 2022

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