PH-dependent Binding Engineering Reveals an FcRn Affinity Threshold That Governs IgG Recycling

M Jack Borrok,Yanli Wu,Nurten Beyaz,Xiang-Qing Yu,Vaheh Oganesyan,William F Dall'Acqua,Ping Tsui

doi:10.1074/jbc.m114.603712

Abstract

The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.

Highlights

FcRn controls the serum persistence of antibodies
We sought to understand how increased neutral pH binding can limit in vivo half-life improvements and to better define at what point gains in serum persistence are hindered by excess FcRn affinity at the neutral cell surface
These findings are exemplified by Y31-YTE and N3E motavizumab, which have neutral pH KD values of 1160 and 860 nM, respectively (KD of 84 and 41 nM at pH 6.0) and exhibit 2–3-fold slower clearance compared with motavizumab in human FcRn (hFcRn) mice

Summary

Background

FcRn controls the serum persistence of antibodies. Results: A panel of novel Fc mutations reveals sites controlling pH dependence and FcRn affinity. The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pHdependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Understanding the parameters that govern pH-dependent binding and IgG recycling will aid in the design of Fc containing biologics and expand their application

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION