Generation of efficient mutants of endoglycosidase from Streptococcus pyogenes and their application in a novel one-pot transglycosylation reaction for antibody modification

Abstract

The fine structures of Fc N-glycan modulate the biological functions and physicochemical properties of antibodies. By remodeling N-glycan to obtain a homogeneous glycoform or chemically modified glycan, antibody characteristics can be controlled or modified. Such remodeling can be achieved by transglycosylation reactions using a mutant of endoglycosidase from Streptococcus pyogenes (Endo-S) and glycan oxazoline. In this study, we generated improved mutants of Endo-S by introducing additional mutations to the D233Q mutant. Notably, Endo-S D233Q/Q303L, D233Q/E350Q, and several other mutations resulted in transglycosylation efficiencies exceeding 90%, with a single-digit donor-to-substrate ratio of five, and D233Q/Y402F/D405A and several other mutations resulted in slightly reduced transglycosylation efficiencies accompanied by no detectable hydrolysis activity for 48 h. We further demonstrated that the combined use of mutants of Endo-S with Endo-M or Endo-CC, endoglycosidases from Mucor hiemalis and Coprinopsis cinerea, enables one-pot transglycosylation from sialoglycopeptide to antibodies. This novel reaction enables glycosylation remodeling of antibodies, without the chemical synthesis of oxazoline in advance or in situ.

Sialylated immunoglobulin G: a promising diagnostic and therapeutic strategy for autoimmune diseases

“…Contrary to the narrow substrate specificity of other endoglycosidases, Endo-S secreted by Streptococcus pyogenes shows high specificity for IgG and can act only on biantennary complex glycans 158 , 159 . The pair of Endo-S and its mutant (Endo-S-D233Q) showed great efficiency in generating bisialylated mAbs, including rituximab and trastuzumab 25 , 160 , 161 . A glycosylase mutant (Endo-S2-D184M) from Endo-S2 was developed by Wang et al 162 , 163 , with improved efficiency of transglycosylation.…”

Site-selective modification strategies in antibody–drug conjugates

“…Several studies have used directed evolution to optimise endoglycosidase transglycosidation of antibodies. [355][356][357][358][359][360][361] Huang and co-workers have recently described the use of two of these enzymes (Endo-M and Endo-S-D233Q) to facilitate the efficient attachment of azide-containing sialic acid residues (Fig. 23).…”

Antibody Glycoengineering and Homogeneous Antibody‐Drug Conjugate Preparation

“…Another approach to avoid the side‐reaction consists in combining two ENGase mutants and SGP 1 (Scheme 4). [14c] Among the two ENGase mutants, Endo M N175Q was used for generating the active glycan intermediate, which was presumably oxazoline, although the exact structure was not identified. The generated active glycan intermediate was used for glycan transfer in situ using the ENGase mutant EndoS D233Q.…”