Structural Determinants of Unique Properties of Human IgG4-Fc

Anna M Davies,Theo Rispens,Pleuni Ooijevaar-De Heer,Hannah J Gould,Roy Jefferis,Rob C Aalberse,Brian J Sutton

doi:10.1016/j.jmb.2013.10.039

Abstract

Human IgG4, normally the least abundant of the four subclasses of IgG in serum, displays a number of unique biological properties. It can undergo heavy-chain exchange, also known as Fab-arm exchange, leading to the formation of monovalent but bispecific antibodies, and it interacts poorly with FcγRII and FcγRIII, and complement. These properties render IgG4 relatively “non-inflammatory” and have made it a suitable format for therapeutic monoclonal antibody production. However, IgG4 is also known to undergo Fc-mediated aggregation and has been implicated in auto-immune disease pathology. We report here the high-resolution crystal structures, at 1.9 and 2.35Å, respectively, of human recombinant and serum-derived IgG4-Fc. These structures reveal conformational variability at the CH3–CH3 interface that may promote Fab-arm exchange, and a unique conformation for the FG loop in the CH2 domain that would explain the poor FcγRII, FcγRIII and C1q binding properties of IgG4 compared with IgG1 and -3. In contrast to other IgG subclasses, this unique conformation folds the FG loop away from the CH2 domain, precluding any interaction with the lower hinge region, which may further facilitate Fab-arm exchange by destabilisation of the hinge. The crystals of IgG4-Fc also display Fc–Fc packing contacts with very extensive interaction surfaces, involving both a consensus binding site in IgG-Fc at the CH2–CH3 interface and known hydrophobic aggregation motifs. These Fc–Fc interactions are compatible with intact IgG4 molecules and may provide a model for the formation of aggregates of IgG4 that can cause disease pathology in the absence of antigen.

Highlights

The four subclasses of human IgG display a high degree of sequence homology in their constant regions, yet they play distinct roles and exhibit different patterns of receptor interactions
These structures reveal conformational variability at the CH3–CH3 interface that may promote Fab-arm exchange, and a unique conformation for the FG loop in the CH2 domain that would explain the poor FcγRII, FcγRIII and C1q binding properties of IgG4 compared with IgG1 and -3
The core sequence of the IgG4 hinge, which promotes formation of intra- rather than inter-H chain disulfide bonds, and residue Arg409 at the CH3–CH3 interface, which weakens the non-covalent association between these domains, are requirements for Fab-arm exchange (FAE) to occur, and dissociation of the CH3 domains is a rate-limiting step in the exchange mechanism [3,14,15,16,17]

Summary

Introduction

The four subclasses of human IgG display a high degree of sequence homology in their constant regions, yet they play distinct roles and exhibit different patterns of receptor interactions. While IgG4 is normally the least represented IgG subclass in serum, levels are elevated in rheumatoid arthritis [6], IgG4-related disease (IgG4-RD) and auto-immune pancreatitis [7,8], as well as under conditions of chronic exposure to an antigen, or after allergen-specific immunotherapy [9,10] It does exhibit a unique property, namely the ability for the two heavy (H) chains to disengage, forming a “half-molecule”, and re-assemble with H chains of another IgG4 antibody, perhaps with different specificity, to form a bispecific antibody that is monovalent with respect to each specificity [9,11,12]. The core sequence of the IgG4 hinge (residues 226–230), which promotes formation of intra- rather than inter-H chain disulfide bonds, and residue Arg409 at the CH3–CH3 interface, which weakens the non-covalent association between these domains, are requirements for FAE to occur, and dissociation of the CH3 domains is a rate-limiting step in the exchange mechanism [3,14,15,16,17]

Methods

Results

Discussion

Conclusion