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

Abstract

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 (KD) of 760 ± 60 nM (N = 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.

Keywords: CFCA, calibration-free concentration analysis; FcRn; FcRn, neonatal Fc receptor; IgG; RU, response units; Rmax, maximum binding response; SPR; SPR, surface plasmon resonance; WT, wild-type; anti-Id, anti-idiotypic; cyFcRn, cynomolgus monkey FcRn; cyIgG, cynomolgus monkey IgG; hErbB2, human ErbB2; hFcRn, human FcRn; hIgG, human IgG; label-free biosensor; mAb, monoclonal antibody; mFcRn, mouse FcRn; neonatal Fc receptor; pI, isoelectric point; rFcRn, rat FcRn; rIgG, rat IgG.

Figure 1.

Affinity determination of the rFcRn/rIgG2a interaction at pH 5.8 using various assay orientations. Kinetic analysis of rIgG2a flowed over biotinylated rFcRn that was captured at (A) high or (B) low capacities via amine-coupled neutravidin on a Biacore C1 chip; kinetic analysis of rFcRn flowed over (C) antigen-captured rIgG2a on a ProteOn NLC chip or (D) amine-coupled rIgG2a on a Biacore CM5 chip; and (E) solution affinity using a high capacity of amine-coupled rIgG2a on a Biacore CM5 chip to probe for free rFcRn in equilibrated mixtures with titrating levels of rIgG2a. Analytes were injected as a 3-fold dilution series with top at 500 nM (A and B) or as both a 3-fold (green) and 5-fold (red) dilution series with top at 1000 nM (C) or 3000 nM (D); the 3000 nM curves have been excluded from D. Panel E shows the titration of 17 nM rFcRn with 2 unrelated mAbs of subtype rIgG2a (distinguished by the solid or open symbols). All samples were analyzed in replicate binding cycles. Each panel shows an example data set (N of 1) where the measured data (colored lines) were fit globally to a simple model (black lines). The K_D values are the mean ± SD for N independent measurements. See Table 1.

Figure 2.

Kinetic analysis at pH 5.8 of a multi-species panel of FcRn proteins binding as analytes to trastuzumab hIgG1-N434Y that was first captured via (A) biotinylated mFcRn or (B) biotinylated hErbB2 on Biacore C1 chips to which neutravidin was amine-coupled. Analytes were injected in duplicate binding cycles as a 3-fold dilution series with top at 180, 300, 60, or 100 nM for hFcRn, cyFcRn, mFcRn, and rFcRn respectively. Each overlay plot shows a representative example of the measured data (colored lines) and the global fit to a simple model (black lines) for a typical experiment (N of 1) and the K_D values are the mean ± SD of N = 6.

Figure 3.

One-shot kinetic analysis at pH 5.8 of mFcRn binding as analyte to (A) trastuzumab homodimers and (B) their corresponding heterodimers that were captured at similar levels via ProteOn NLC or GLC chips coated with biotinylated hErbB2. Mouse FcRn was injected as a 3-fold or 5-fold dilution series with top concentrations of 180 nM (WT and N434H) or 85 nM (N434Y). Each overlay plot shows a representative example of the measured data (noisy lines) and the global fit (smooth lines) for a subset of the constructs tested. Multiple constructs per variants were fit simultaneously to give global apparent mean K_D ± SD (N = 4) values of 58 ± 10 nM (WT-hIgG1, WT:WT, WT E arm, WT R arm, I253A:WT and AAA:WT), 5 ± 1 nM (N434H, N434H R arm, I253A:N434H and AAA:N434H), and 0.47 ± 0.07 nM (N434Y, N434Y R arm, AAA:N434Y). For a summary of all interactions tested in this way, see Supplemental Material (Table S2).

Figure 4.

Solution affinity determinations of mFcRn binding to trastuzumab hIgG1 variants. (A) WT and AAA:WT at pH 5.8, (B) N434H and AAA:N434H at pH 5.8, (C) N434Y and AAA:N434Y at pH 5.8 and (D) N434Y and AAA:N434Y at pH 7.4. Each overlay plot shows an example of the K_D-controlled curves obtained for titrations of a hIgG1 homodimer (blue symbols) and its corresponding heterodimer (red symbols) into a fixed concentration of mFcRn (4.5, 2.7, 0.9, and 36 nM for panels A-D respectively). See Table 2.

Figure 5.

In vivo clearance of a panel of trastuzumab hIgG1 homodimers and heterodimers in mice and rats. Overlay plot of WT-hIgG1, WT:WT and AAA:WT in (A) mice and (B) rats. Overlay plot of N434H:N434H and AAA:N434H in (C) mice and (D) rats. (E) Overlay plot of WT:WT, AAA:WT, and AAA:N434Y in mice and rats. Data points represent the mean ± SD of 3 or 4 animals per group.