EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans

Abstract

Enzymes that affect glycoproteins of the human immune system, and thereby modulate defense responses, are abundant among bacterial pathogens. Two endoglycosidases from the human pathogen Streptococcus pyogenes, EndoS and EndoS2, have recently been shown to hydrolyze N-linked glycans of human immunoglobulin G. However, detailed characterization and comparison of the hydrolyzing activities have not been performed. In the present study, we set out to characterize the enzymes by comparing the activities of EndoS and EndoS2 on a selection of therapeutic monoclonal antibodies (mAbs), cetuximab, adalimumab, panitumumab and denosumab. By analyzing the glycans hydrolyzed by EndoS and EndoS2 from the antibodies using matrix-assisted laser desorption ionization time of flight, we found that both the enzymes cleaved complex glycans and that EndoS2 hydrolyzed hybrid and oligomannose structures to a greater extent compared with EndoS. A comparison of ultra-high-performance liquid chromatography (LC) profiles of the glycan pool of cetuximab hydrolyzed with EndoS and EndoS2 showed that EndoS2 hydrolyzed hybrid and oligomannose glycans, whereas these peaks were missing in the EndoS chromatogram. We utilized this difference in glycoform selectivity, in combination with the IdeS protease, and developed a LC separation method to quantify high mannose content in the Fc fragments of the selected mAbs. We conclude that EndoS and EndoS2 hydrolyze different glycoforms from the Fc-glycosylation site on therapeutic mAbs and that this can be used for rapid quantification of high mannose content.

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

“…These studies have demonstrated that the glycosynthases so far available still have limitations due to substrate specificity and also vary in transglycosylation efficiency. As an effort to expand the scope of the glycosylation remodeling strategy, we turned our attention to Endo-S2, an endoglycosidase from S. pyogenes of serotype M49 (33,34). Endo-S2 shows only 37% sequence identity to Endo-S from the same bacteria and demonstrates a broader glycan substrate specificity in Fc deglycosylation than Endo-S (34).…”

EndoSd: An IgG Glycan Hydrolyzing Enzyme in Streptococcus Dysgalactiae Subspecies Dysgalactiae

“…With one notable exception, in contrast to EndoS [16], EndoSd only released minute amount of glycans modified with a bisecting GlcNAc (Figure 6). Previous studies have shown that EndoS lacking its carbohydrate binding domain was considerably slower in releasing structures with bisecting GlcNAc as compared with other glycoforms, indicating that these structures might be more resistant to endoglycosidases [18].…”

“…It exhibits a specificity for biantennary complex type N-glycans and does neither cleave larger complex type glycans nor oligomannose and nor hybrid-type glycans to any major extent [15,16]. Protein-protein interactions are important for the activity of EndoS on glycans since it hydrolyzes the N-linked glycan only on native but not denatured IgG [17].…”

“…The ndoS2 gene also encodes an endo-β-N-acetylglucosaminidase, denoted EndoS2 with activity on all glycoforms of IgG, including oligomannose hybrid and bisected type glycans [11,16].…”

Optimal Synthetic Glycosylation of a Therapeutic Antibody

“…Alternatively, remodeling at the nonreducing termini36c,36d (“tip sugars”), although in principle capable of partially reducing heterogeneity, is limited to certain residues (i.e. Gal); it also does not remove the variation arising from bisecting sugars38 and/or hybrid/triantennary/other branching glycans 39. The use of unnatural AAs does not avoid the desire for or solve the problem of defined glycosylation.…”