A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life

Abstract

Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of a similar size. It is wellknown that this phenomenon is due to IgG's ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG. Using a high-throughput biolayer interferometry (BLI) platform, the human FcRn association rate at acidic pH and subsequent dissociation rate at physiological pH was determined for 5 human IgG1 mAbs. Comparing the combined FcRn association and dissociation rates to the Phase 1 clinical study half-lives of the mAbs resulted in a strong correlation. The correlation was also verified in vivo using mice transgenic for human FcRn. The model was used to characterize various factors that may influence FcRn-mAb binding, including mAb variable region sequence differences and constant region glycosylation patterns. Results indicated that the complementarity-determining regions of the heavy chain significantly influence the mAb's FcRn binding properties, while the absence of glycosylation does not alter mAb-FcRn binding. Development of this high-throughput FcRn binding model could potentially predict the half-life of therapeutic IgGs and aid in selection of lead candidates while also serving as a screening tool for the development of mAbs with desired pharmacokinetic properties.

Pharmacokinetic properties of IgG and various Fc fusion proteins in mice

“…However, further studies are required to unravel the molecular mechanisms responsible for these differences, e.g., performing a mutational screening study. A recent study also considered, in addition to the dissociation rate at pH 7.5 the association rate at pH 6.0 to determine FcRn binding rates predictive for in vivo half-life, 19 which might be further investigated for Fc fusion proteins. Furthermore, studies might be extended evaluating the pharmacokinetic properties in transgenic mice endowed with the human FcRn.…”

“…However, analyzing an scDb-Fc and scFv-scCLCH1-Fc fusion protein with a size and hydrodynamic radius similar to that of IgG molecules did not improve the pharmacokinetic properties compared to scFv-Fc fusion protein, which was reflected by a faster initial and terminal half-life. Here, 19 This includes differences in interaction with FcRn at acidic and neutral pH, steric hindrance between the Fc-domain and the fusion partner influencing protein stability and interaction with FcRn, alternative clearance pathways dependent on the fusion partner, and a contribution of the Fab arms or the molecular architecture to FcRn binding and recycling. 4,20,21 Furthermore, differences in glycosylation, e.g., caused by using different expression systems, might influence half-life.…”

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

“…Several studies have investigated the effect of Fc-glycans on FcRn binding and FcRn-dependent clearance [23,[26][27][28][29][30]. Whereas a difference in FcRn binding could be …”

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

“…Schlothauer et al (6) recently demonstrated that intact antibodies can dissociate from FcRn at different pH values than their corresponding Fc fragments strongly suggesting a contribution of the antibody Fab in FcRn binding. However, the role of the Fab regions in FcRn binding is still a matter of discussion in the literature (7)(8)(9)(10)(11)(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).…”