A Two-pronged Binding Mechanism of IgG to the Neonatal Fc Receptor Controls Complex Stability and IgG Serum Half-life

Abstract

The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism of the neonatal Fc receptor (FcRn). For long serum half-life of therapeutic IgGs, the highly pH-dependent interaction with FcRn needs to be balanced to allow efficient FcRn binding and release at slightly acidic pH and physiological pH, respectively. Some IgGs, like the antibody briakinumab has an unusually short half-life of ϳ8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two- (7-12) and the molecular mechanism is not clear. Limited structural information is available for the complex of full-length IgG and FcRn and much of our knowledge of the binding interface comes from X-ray crystal structures of only the Fc region bound to FcRn (3,4).We recently applied a method based on hydrogen/deuterium exchange mass spectrometry (HDX-MS) to detect local structural conformation and dynamics of IgG and FcRn upon interaction (13). HDX-MS monitors the isotopic exchange of hydrogen and deuterium of proteins in solution and reports on the protection of amide hydrogens from intra-and intermolecular hydrogen bonds and to a lesser extent solvent accessibility (14, 15). Our studies (13) revealed structural stabilization of the Fab regions upon FcRn interaction, which could be caused by a conformational link between the Fc and Fab regions or by direct interaction with FcRn.Here, we perform HDX-MS analyses of a unique set of antibodies with different FcRn binding characteristics to dissect the conformational origins of the involvement of Fab in FcRn binding. The antibodies ustekinumab (Stelara) and briakinumab (Ozespa) targeting the p40 subunit of IL-12 and From the ‡Department

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

“…Several studies have suggested that the antigen binding fragment (Fab) can contribute to FcRn binding, 11,17–19 and a recent publication supports this hypothesis by demonstrating that the Fab region of an IgG can interact directly with FcRn. 16 Our hypothesis was additionally strengthened by recent publications by other groups showing a correlation between antibody charge and the FcRn affinity or PK of IgG molecules not influenced by target-mediated CL. 5,11,18,25,26 In the current study, we set out to further understand if Fab-containing domains can influence PK.…”

Evaluation of an Fcrn Affinity Chromatographic Method for Igg1-Type Antibodies and Evaluation of Igg Variants

“…Results from Wang et al suggest that Fab charges cannot only influence the outcome of FcRn affinity chromatography but at the same time influence the outcome of SPR-based assays, depending on the chosen setup [19]. Correlations between FcRn affinity chromatography results of antibodies with differently charged Fab fragments and their in vivo PK were already established previously [4] and recent hydrogen-deuterium exchange (HDX) data supplemented by FcRn affinity chromatography data furthermore i ndicate that both interactions contribute to binding [37].…”

Extending human IgG half-life using structure-guided design

“…Further, the 250-helix residues demonstrated enhanced deuterium exchange at low pH in the absence of FcRn, suggesting that a pH-specific conformational change occurred within this region. 23,24 …”