The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

Abstract

(2015) The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity, mAbs, 7:2, 331-343, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (K D ) of 760 § 60 nM (N D 14) at 25 C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37 C than at 25 C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.

Target-independent variable region mediated effects on antibody clearance can be FcRn independent

“…TMDD effects may be ameliorated by engineering pH-dependent binding, 44 and nonspecific binding 32 and FcRn affinity [9][10][11][12][13][14] can be modulated by antibody engineering. The somewhat limited success of engineering efforts to improve antibody clearance PK by modulating FcRn affinity 5 may be in many instances attributable to dominant nonspecific tissue clearance mechanisms that operate independently of FcRn recycling, as demonstrated for the antibodies in this report.…”

“…17,19,20 Affinity measurements in many of these experiments use surface plasmon resonance (SPR) or biolayer interferometry (BLI), conjugating FcRn to the sensor surface. As has been noted in previous studies, measurements for equivalent antibodies can vary significantly if great care is not taken, 14,26 and additionally the presence of a very small amount of aggregate can have a noticeable effect on apparent affinity measurement. 27 In this study, we aimed to establish whether Fv region nonspecificity can accelerate systemic clearance in vivo by entirely FcRn-independent mechanisms.…”

“…5 The residues of the IgG1 Fc portion that interact with FcRn are well established, [6][7][8] and as such many efforts have focused on improving FcRn affinity to improve PK. [9][10][11][12][13][14] In addition to Fc engineering efforts, multiple studies have reported disparate PK parameters in antibodies with identical Fc regions but different variable regions. [15][16][17][18][19][20] In particular, clearance rates are increased in antibodies with polyreactive [21][22][23] or cross-reactive 24,25 profiles.…”

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

“…As a consequence, they may have extended serum half-lifes by endocytic salvage via neonatal FcR (FcRn), which may compensate for slow tissue penetration. [30][31][32][33][34][35] Furthermore, they may elicit antibody-dependent cell-mediated cytotoxicity or phagocytosis (ADCC or ADCP, respectively) via Fcg receptors (FcgRs), which is important for cancer biotherapeutics. 3,[36][37][38][39] Fully functional Fc domains may also confer the potential to trigger the classical pathway of complementdependent humoral response, and thereby elicit complementdependent cytotoxicity (CDC).…”

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

“…18,19,20 Technologies used for evaluating FcRn-rhumAb interactions vary extensively. Some of the methods used to assay FcRn-rhumAb interactions include surface plasmon resonance (SPR) (Biacore, ProteOn), 13,17,21,22,23,24,25,26 biolayer interferometry (Octet), 26 isothermal titration calorimetry, 27 enzyme-linked immunosorbent assays (ELISA), 28,29 cellbased assays, 30,31 AlphaScreen, 32 affinity chromatography, 33 and asymmetrical flow field flow fractionation. 34 Among these technologies, SPR-based biosensor assays are the most commonly used, likely due to the fact that they can provide real-time quality data on binding specificity and kinetics over a wide range of binding affinities.…”