Effects of Glycan Structure on the Stability and Receptor Binding of an IgG4-Fc

Abstract

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc g receptor IIIA (FcgRIIIA). High mannose (HM, GlcNAc 2 Man (8þn) [n ¼ 0-4]), Man 5 (GlcNAc 2 Man 5), GlcNAc 1 , and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (l max ¼ 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn 297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc 1 > Man 5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man 5 glycoforms display increased affinity for FcgRIIIA by at least 14.7-fold compared with GlcNAc 1 IgG4-Fc. The affinities measured for the HM and Man 5 IgG4-Fc (0.39-0.52 mM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcgRIIIA.

Understanding the structure–property relationship of bispecific monoclonal antibodies with Fc site-specific substitutions

“…The observed values of T m 1 (~65.0°C) and T m 2 (~68.0°C) are similar to the literature reported values for the Fc domain of the IgG4 subclass. 23 The same trend is also observed with intact HC/HC* heterodimers and two intact homodimers. The heterodimer shows two thermal transitions that can correspond to the T m from single HC and single HC* respectively (Figure S5).…”

Using atomistic solution scattering modelling to elucidate the role of the Fc glycans in human IgG4

“…FcγR interaction [11]. The lower binding affinity of IgG4 can start to be rationalised by our finding in the present study that the deglycosylated IgG4-Fc subunit is more conformationally labile than the seemingly more restricted glycosylated IgG4-Fc subunit [18].…”

“…Functional studies of these IgG-FcγR complexes are limited by the availability of crystal structures for only IgG1-Fc complexes, but not for IgG4-Fc in complex with the FcγRs. Functional studies of deglycosylated IgG4 demonstrated abrogated binding to FcγRIIIa, which implicated a role for the glycans to assist in the FcγR interaction [ 11 ]. The lower binding affinity of IgG4 can start to be rationalised by our finding in the present study that the deglycosylated IgG4-Fc subunit is more conformationally labile than the seemingly more restricted glycosylated IgG4-Fc subunit [ 18 ].…”

“…For example, afucosylated IgG4 antibodies are able to elicit a stronger antibody-dependent cytotoxicity response through their binding to FcγRIIIa receptors [ 10 ]. Deglycosylated IgG4 was unable to bind to FcγRIIIa, indicating the importance of the glycans for FcγR binding [ 11 ]. Structures for IgG4 are essential to understand how it binds to FcγRs.…”

Affinity Purification Coupled to Stable Isotope Dilution LC-MS/MS Analysis to Discover IgG4 Glycosylation Profiles for Autoimmune Pancreatitis

“…There is evidence that endogenous IgG with more fucosylation and sialylation has anti-inflammation biological functions [ 23 ]. However, effector analyses or biological studies regarding IgG4 were mostly focused on IgG4 monoclonal antibodies (mAbs) [ 24 ]. For example, Gong Q et al found that the afucosylated percentage of IgG4 monoclonal antibodies was related to their activity of antibody-dependent cell-mediated cytotoxicity (ADCC) [ 25 ].…”