title: Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions.
abstract: The fine structures of Fc N-glycans can modulate the effector functions of IgG antibodies. It has been demonstrated that lack of the core fucose on the Fc N-glycans leads to drastic enhancement of antibody-dependent cellular cytotoxicity (ADCC), while terminal α2,6-sialylation of Fc glycan plays a critical role for the anti-inflammatory activity of human intravenous immunoglobulin (IVIG). We describe in this paper a highly efficient chemoenzymatic method for site-selective Fc glycoengineering of intact monoclonal antibody and IVIG. Two new glycosynthase mutants (EndoS-D233A and D233Q) were generated by site-directed mutagenesis of EndoS (an endoglycosidase from  Streptococcus pyogenes ) and were found to be capable of efficiently transferring predefined N-glycans from corresponding glycan oxazolines to the Fc-deglycosylated intact IgGs without product hydrolysis. As a model study, rituximab (a therapeutic monoclonal antibody) was successfully transformed from mixtures of G0F, G1F, and G2F glycoforms to well-defined homogeneous glycoforms, including a fully sialylated (S2G2F) glycoform that may gain anti-inflammatory activity, a nonfucosylated G2 glycoform that showed significantly enhanced FcγIIIa receptor-binding activity, and an azido-tagged glycoform that can be further transformed into other glycoforms. We also found that EndoS could selectively remove the Fc N-glycans in the presence of FAB glycosylation. This finding, coupled with the remarkable transglycosylation activity of the EndoS glycosynthase mutants, permitted a highly selective glycoengineering of the IVIG's Fc glycans into a fully sialylated Fc glycoform, which may possess significantly enhanced anti-inflammatory activity. The glycoengineering approach described here provides a general platform to modulate the effector functions of IgG antibodies, enabling the optimization of therapeutic efficacy and gain of new functions of monoclonal antibodies and IVIG.
authors: Institute of Human Virology and Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors: Wei
authors: W
authors: Huang
authors: John
authors: J
authors: Giddens
authors: Shu-Quan
authors: SQ
authors: Fan
authors: Christian
authors: C
authors: Toonstra
authors: Lai-Xi
authors: LX
authors: Wang
mesh_terms: D000595:Amino Acid Sequence; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry / Q000378:metabolism; D005690:Galactose / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000737:chemistry; D017452:Receptors, IgG / Q000378:metabolism; D000069283:Rituximab; D013297:Streptococcus pyogenes / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D007074:Immunoglobulin G; D011134:Polysaccharides; D017452:Receptors, IgG; D000069283:Rituximab; D006026:Glycoside Hydrolases; D005690:Galactose