Chemoenzymatic Synthesis and Fcγ Receptor Binding of Homogeneous Glycoforms of Antibody Fc Domain. Presence of a Bisecting Sugar Moiety Enhances the Affinity of Fc to FcγIIIa Receptor

Abstract

Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on antibody’s effector functions. We report in this paper chemoenzymatic synthesis and Fcγ receptor binding of an array of homogeneous IgG-Fc glycoforms. The chemoenzymatic approach consists of the chemical synthesis of defined N-glycan oxazolines as donor substratess, the expression of the Fc domain in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines. The enzyme from Arthrobacter protophormiae (Endo-A) was found to be remarkably efficient to take various modified N-glycan core oxazolines, including the bisecting sugar-containing derivatives, for Fc glycosylation remodeling, resulting in the formation of the corresponding homogeneous Fc glycoforms. Nevertheless, neither Endo-A, nor the Mucor hiemalis endoglycosidase mutants (EndoM-N175A and EndoM-N175Q), was able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. SPR binding studies with the synthetic IgG-Fc glycoforms unambiguously proved that the presence of a bisecting GlcNAc moiety could significantly enhance the binding of Fc to FcγRIIIa, the activating Fcγ receptor, independent of Fc core-fucosylation. Interestingly, the Fc glycoforms carrying an unusual bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced affinity to FcγRIIIa. On the orther hand, the presence of a bisecting GlcNAc or core fucosylation had little effect on the affinity of Fc to the inhibitory Fcγ receptor, FcγRIIb. Our experimental data also showed that the α-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high-affinity of Fc to both FcγRIIIa and FcγRIIb. The synthetic homogeneous Fc glycoforms thus provide a useful tool for elucidating how a fine Fc N-glycan structure precisely affects the function of the Fc domain.

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

“…Thus, these Endo-S2-derived mutants represent the first glycosynthases that can efficiently transfer high-mannose and hybrid type N-glycans to core-fucosylated GlcNAc acceptor in an intact antibody. It should be mentioned that the Endo-A mutant (N171A and N171Q) could transfer high-mannose type N-glycan to the GlcNAc-Fc domain, but they were unable to use core-fucosylated GlcNAc-Fc as acceptor (22,23,36). These studies also show that the combined use of wild type Endo-S2 and Endo-S2 glycosynthase mutants provides a particularly efficient glycosylation remodeling approach to various homogeneous glycoforms of antibodies starting from a single precursor.…”

“…In parallel to the attempt to control glycosylation through host glycosylation pathway engineering (14 -20), a chemoenzymatic glycosylation remodeling method, which involves endoglycosidase-catalyzed deglycosylation and subsequent transglycosylation of intact antibodies, has been emerging as a promising approach to obtain homogeneous antibody glycoforms (21). We have initially shown that the Fc glycans of recombinant IgG-Fc domain could be remodeled through the enzymatic deglycosylation-transglycosylation steps under the catalysis of an appropriate endoglycosidase, including Endo-A 2 and Endo-D, without the need of denaturing the proteins (22)(23)(24). In 2012, we reported the first example of glycosylation remodeling of an intact therapeutic monoclonal antibody and intravenous immunoglobulin, which was enabled by the discovery of glycosynthase mutants of Endo-S, an endoglycosidase from Streptococcus pyogenes (25).…”

A common glycan structure on immunoglobulin G for enhancement of effector functions

“…During this study, it was discovered that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimal structure that is able to enhance the activities of antibodies against cancer, influenza, and inflammatory diseases. (22) and later applied to antibody glycoengineering (25)(26)(27). The strategy starts with the use of exoglycosidases or endoglycosidases to cleave most of the N-glycans to form a homogeneous glycoform containing a well-defined glycan, followed by extension of the glycan using glycosyltransfer enzymes.…”

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

“…Depending on the host cell line, the potential carbohydrate residues will have specific features such as bisecting GlcNAc residues or core fucosylation. Studies into the effect of bisecting structures present in antibody glycan moieties have shown that bisecting GlcNAc sugar residues lead to increased ADCC [33]. However, the presence (or absence) of other monosaccharides has been shown to have a more profound effect on in vivo modes of action.…”

“…ERp57, calnexin/calreticulin, and/or protein disulfide isomerise) exhibited position effects in Productivity. Disulfide isomerase in particular increased specific mAb productivity by 55% in transient gene expression, but there were no/negative effects in stable gene expression [33]. In addition, targeting the unfolded protein response (UPR) pathway is another approach to enhancing recombinant protein yield.…”