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

Abstract

A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics.

Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics

“…Human FcRn binding and protection is one of the dominant factors governing mAb PK in vivo, but the literature around the use of in vitro hFcRn binding assays for selecting or deselecting mAbs has provided conflicting results that often depend on assay format. [33][34][35][36][37] Piche-Nicholas et al 27 have recently observed a correlation of positive charge in the mAb variable region with affinity to immobilized human hFcRn using the same SPR format for which we have observed a strong correlation with hFcRn Tg32-projected human CL. A combined interaction with both hFcRn and the supporting matrix is likely responsible for the behavior we and others 13,27 have observed in immobilized hFcRn affinity chromatography, as well as in SPR.…”

“…A Biacore TM T100 or T200 (GE Healthcare) was used to measure the steady-state binding affinities of the interaction between IgG molecules and matrix-immobilized FcRn as previously described. 27,39 Briefly, 20-70 resonance units (RU) of biotinylated human FcRn-b2m complex were affinity captured on a carboxymethylated dextran chip pre-immobilized with streptavidin (SA) (GE Healthcare, #BR100531). A series of dilutions of IgG analytes (»100 mg total mAb) were prepared in sample buffer (20 mM MES, 150 nM NaCl, 3 mM EDTA, 0.5% Polysorbate 20, pH 6.0) and sequentially flowed over the FcRn coated SA chip.…”

“…Measurements of mAb binding to immobilized hFcRn in affinity chromatography and SPR assays have been reported to show correlation between column retention time or binding affinity and CL in hFcRn Tg276 hemizygous 13 and Tg32 homozygous mice. 27 We evaluated the mAb sets in these two matriximmobilized hFcRn binding formats, which require 50-200 mg of each mAb.…”

Fc Glycan-Mediated Regulation of Placental Antibody Transfer

“…Pregnancy-specific changes in glycosylation have been observed across both the Fc and the Fab domain of antibodies (Bondt et al, 2014;Jansen et al, 2016a;Sonneveld et al, 2016). However, glycosylated Fabs do not have the capacity to interact with FcRn, which binds to antibodies in the CH3 domain (Jensen et al, 2017;Piche-Nicholas et al, 2018), suggesting that only changes in Fc-glycosylation are likely to regulate placental selection. The potentially critical role of galactosylation for FcRn binding has been previously noted, likewise demonstrating enhanced FcRn binding to digalactosylated antibodies (Dashivets et al, 2015).…”

Looking for the Optimal PD-1/PD-L1 Inhibitor in Cancer Treatment: A Comparison in Basic Structure, Function, and Clinical Practice

“…Notably, elimination appears to play a dominant role in PK and is under the influence of various regulation such as neonatal Fc receptor (FcRn)-dependent recycling, target-mediated drug disposition (TMDD), and non-specific or off-target bindingmediated clearance, among which FcRn-dependent recycling plays a major role (50,51). It has been demonstrated that TMDD leads to rapid clearance by inducing internalization and downstream degradation (50).…”