SITE-SPECIFIC GLYCOSYLATION ANALYSIS OF IgG3 AND THE EFFECT OF GLYCOSYLATION ON THE STRUCTURE, PHYSICAL STABILITY AND Fc RECEPTOR INTERACTIONS OF IgG3 Fc

Abstract

Therapeutic monoclonal antibodies based on the IgG class of antibodies are glycoproteins that contain glycosylation sites within their Fc region. Glycosylation is important for Fc-mediated effector functions such as Antibody Dependent Cellular Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC). IgG has four subclasses: IgG1, IgG2, IgG3 and IgG4. All IgG subclasses contain a consensus glycosylation site in the CH2 domain at N297, and glycosylation at that site is considered essential for optimal Fc function and stability. IgG3 is the only subclass that has an additional glycosylation site in the CH3 domain at N392. This site has nearly gone unmentioned and was only recently shown to be partially glycosylated in serum IgG3; however, the biological role of N392 glycosylation remains uninvestigated in the literature. The primary focus of this dissertation work is to study the effect of N-glycosylation on Fc receptor interactions, structure and physical stability of IgG3. LC-MS based glycopeptide mapping studies revealed that the N392 site is partially glycosylated in human serum IgG3 and the glycans at the N392 site showed significant differences compared to the N297 site in levels of core fucosylation, bisecting GlcNAc and mannosylation. The presence of glycosylation at the N392 site in the naturally occurring IgG3 establishes its biological relevance and makes a case for its characterization. For structural and biochemical characterization studies, the Fc fragment of IgG3 was expressed in a glycosylation-deficient strain of the yeast Pichia pastoris. Two variants of IgG3 Fc were produced, one with both sites glycosylated and another with only the N297 site glycosylated. These two forms were compared to investigate the effect of N392 glycosylation on the Fc receptor binding activity and physical stability of IgG3 Fc. Our results indicate that presence of the additional N392 glycan does not affect the binding of IgG3 Fc with the FcRIIIA and FcRn receptors but does affect the conformational stability and aggregation propensity of IgG3 Fc. Additionally, crystal structures of both the IgG3 Fc variants were determined with a resolution of 1.8-2.0 Å and were compared with the published structures of Fc fragments from the other IgG subclasses. Structural analysis of key amino acid differences between IgG3 and other IgG subclasses provided insight into IgG3-specific properties, especially related to binding with protein A, protein G, and the FcRn receptor. The importance of N297 glycans for IgG3 Fc- FcRIIIA interaction was also demonstrated from a structural perspective. The location and orientation of the N392 glycans in the IgG3 Fc structure provided a probable explanation for the observed effect of these glycans on Fc receptor binding activity and physical stability of IgG3 Fc.