Abstract

At present, numerous studies have been reported to remodel the N-glycans of therapeutic antibodies for the gain of functions. Among the ways of remodeling antibody N-glycans, the chemoenzymatic glycoengineering approach by endoglycosidase (ENGase) has been deeply investigated and provided a significant tool for IgG glycoengineering. Among these cases, the transglycosylation activity of Endo-F3, compared to Endo-S and S2, is insufficient and limits its power in remodeling IgG glycosylation. Herein, we chemically conjugated the Endo-F3 mutant D165A with an Fc binding peptide (FcBP), aiming to improve the affinity of Endo-F3 D165A to IgGs, and therefore enhance the transglycosylation activity of D165A. In this report, we investigated the conjugation site of FcBP to D165A and the linkers between them and found that the conjugation indeed significantly increases the transglycosylation activity of D165A. Meanwhile, we optimized the FcBP-D165A catalyzed transglycosylation process, including the enzyme quantity, oxazoline concentration, and so on. Finally, by this method, we remodeled the N-glycans of rituximab and trastuzumab into homogeneous S2G2F, G2F, GN2M3, and M3 types with decreased enzyme quantity, oxazoline ratio, and catalyzing time. This method not only provides an enhanced ENGase for IgG glycoengineering but also suggests that ligand-directed localization of enzymes is a potential strategy to enhance the activity of enzymes towards the targeted substrate.