Structural Determinants of Unique Properties of Human IgG4-Fc

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 2.35 Å, 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.

The Solution Structures of Two Human IgG1 Antibodies Show Conformational Stability and Accommodate Their C1q and FcγR Ligands

“…2). IgG4 also has Ser 330 and Ser 331 substitutions compared with the other IgG subclasses (61,68). These substitutions in IgG2 and IgG4 lead to weaker Fc␥R binding in comparison with IgG1 and IgG3, which bind to all Fc␥R classes.…”

Acid-induced aggregation propensity of nivolumab is dependent on the Fc

“…These findings are generally consistent with previous reports that IgG 4 is prone to aggregation in acidic condition, 27,28 and IgG 4 aggregation is dependent on the Fc region, which has some hydrophobic aggregation motifs. 29 To provide context for the results of our experiments with nivolumab, we examined the pH-dependent stability of 3 other IgG subclass antibodies and their Fcs. The pH range investigated in our study is wider (pH2.0-4.0) than that typically used in industrial manufacturing processes.…”

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“…A recent review by Karsten and Kohl provides valuable insights into structural features of Igs that facilitate C1q binding 23 . Accordingly, IgG4 hinge regions may potentially attain greater flexibility through dynamic Fab arm exchange mechanism allowing greater accessibility of C1q to binding sites within the C H 2 (Fc) domain to activate complements [24][25][26] . The effect size of this phenomenon is uncertain as little is known about the proportion of IgG4 molecules that actually conflict probably requires the reevaluation of historical data in the light of newer understanding of the properties of IgG4.…”