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. 2010 Feb 12;285(7):4826-36.
doi: 10.1074/jbc.M109.081828. Epub 2009 Dec 14.

Cross-species binding analyses of mouse and human neonatal Fc receptor show dramatic differences in immunoglobulin G and albumin binding

Jan Terje Andersen  1 , Muluneh Bekele Daba, Gøril Berntzen, Terje E Michaelsen, Inger Sandlie
Affiliations

Cross-species binding analyses of mouse and human neonatal Fc receptor show dramatic differences in immunoglobulin G and albumin binding

Jan Terje Andersen et al. J Biol Chem. .

Abstract

The neonatal Fc receptor (FcRn) regulates the serum half-life of both IgG and albumin through a pH-dependent mechanism that involves salvage from intracellular degradation. Therapeutics and diagnostics built on IgG, Fc, and albumin fusions are frequently evaluated in rodents regarding biodistribution and pharmacokinetics. Thus, it is important to address cross-species ligand reactivity with FcRn, because in vivo testing of such molecules is done in the presence of competing murine ligands, both in wild type (WT) and human FcRn (hFcRn) transgenic mice. Here, binding studies were performed in vitro using enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant soluble forms of human (shFcRn(WT)) and mouse (smFcRn(WT)) receptors. No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed. Specifically, smFcRn(WT) bound human serum albumin with a K(D) of approximately 90 microM, whereas shFcRn(WT) bound mouse serum albumin with a K(D) of 0.8 microM. shFcRn(WT) ignored mouse IgG1, and smFcRn(WT) bound strongly to human IgG1. The latter pair also interacted at physiological pH with calculated affinity in the micromolar range. In all cases, binding of albumin and IgG from either species to both receptors were additive. Cross-species albumin binding differences could partly be explained by non-conserved amino acids found within the alpha2-domain of the receptor. Such distinct cross-species FcRn binding differences must be taken into consideration when IgG- and albumin-based therapeutics and diagnostics are evaluated in rodents for their pharmacokinetics.

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Figures

FIGURE 1.
FIGURE 1.
SDS-PAGE analyses of soluble receptor preparations. Secreted GST-tagged smFcRnWT and shFcRnWT molecules were purified from supernatants harvested from transiently transfected HEK 293E cells and analyses by 12% SDS-PAGE. Lane 1 shows protein standard. Lanes 2 and 3 show non-reduced (NR) and reduced (R) samples of shFcRnWT, respectively. Lanes 4 and 5 show NR and R samples of smFcRnWT. The bands corresponding to GST fused HCs and hβ2m are indicated by arrows.
FIGURE 2.
FIGURE 2.
pH-dependent binding of shFcRnWT and smFcRnWT to IgG variants in ELISA. Binding of shFcRnWT (A) and smFcRnWT (C) to hIgG1, hIgG1H435A, mIgG1, and mIgG2b at pH 6.0. Binding of shFcRnWT (B) and smFcRnWT (D) to hIgG1, hIgG1H435A, mIgG1, and mIgG2b at pH 7.4. The numbers given represent the mean of triplicates.
FIGURE 3.
FIGURE 3.
pH-dependent binding of shFcRnWT and smFcRnWT to albumin variants in ELISA. Binding of shFcRnWT to HSA (A) and MSA (B) at pH 6.0 and 7.4. Binding of smFcRnWT to MSA (C) and HSA (D) at pH 6.0 and 7.4. Numbers given represent the mean of triplicates.
FIGURE 4.
FIGURE 4.
SPR analyses of the smFcRnWT interaction with hIgG1 and mIgG1. Representative sensorgrams of serial dilutions of mIgG1 over immobilized smFcRnWT at pH 6.0 (A) and 7.4 (B), serial dilutions of hIgG1 over immobilized smFcRnWT at pH 6.0 (C) and 7.4 (D). In all experiments smFcRnWT was immobilized by amine coupling to ∼100–200 RU. Dilutions of mIgG1 and hIgG1 were injected over an immobilized smFcRnWT at 25 °C. The flow rate was 50 μl/min.
FIGURE 5.
FIGURE 5.
SPR analyses of the shFcRnWT and smFcRnWT interaction with HSA and MSA. Representative sensorgrams of serial dilutions of MSA injected over immobilized smFcRnWT at pH 6.0 (A) and 7.4 (B), serial dilutions of HSA injected over immobilized smFcRnWT at pH 6.0 (C), and serial dilutions of MSA injected to immobilized shFcRnWT at pH 6.0 (D). In all experiments shFcRnWT and smFcRnWT were immobilized by amine coupling to ∼500–800 RU. Dilutions of MSA and HSA were injected over an immobilized receptor at 25 °C. The flow rate was 50 μl/min.
FIGURE 6.
FIGURE 6.
Competitive FcRn-albumin binding across species. A, serial dilutions of HSA (0.5–0.05 μm) and MSA (0.5–0.05 μm) were preincubated with shFcRnWT (0.05 μm) and injected over immobilized HSA (∼2600 RU). B, serial dilutions of HSA (10.0–0.1 μm) and MSA (1.0–0.1 μm) were preincubated with smFcRnWT (0.10 μm) and injected over immobilized MSA (∼2000 RU). The representative binding data are presented as percent inhibition of the FcRn binding to immobilized albumin. Injections were performed at 25 °C, and the flow rate was 50 μl/min.
FIGURE 7.
FIGURE 7.
The crystal structure of shFcRn. A, amino acids flanking His-166 (hFcRn) and His-168 (mFcRn) located within the heavy chain α2-domain are shown. His-166 and His-168 are shown in bold. The non-conserved Arg-164 and Glu-165 of hFcRn, and Leu-167 and Gly-168 of mFcRn are shown in italic. B, the crystal structure of shFcRn shown in two orientations. The localization of amino acids essential for IgG (Glu-115 and Glu-116) and albumin (His-166) binding are highlighted as blue and red spherical balls. The non-conserved Arg-164 and Glu-165 (human) are highlighted with yellow and gray spherical balls, respectively. The FcRn heavy chains are shown in green and the β2m in orange. The figures were designed using PyMOL (DeLano Scientific) with the crystallographic data of shFcRn (37).
FIGURE 8.
FIGURE 8.
SPR analyses of albumin binding to rodentized and humanized FcRn variants. Representative sensorgrams of serial dilutions of HSA (A) and MSA (B) injected over immobilized rodentized shFcRnR164L/E165G at pH 6.0. Serial dilutions of MSA (C) and HSA (D) injected over smFcRnL166R/G167E. In all experiments the receptor variants were immobilized by amine coupling to ∼1000–2000 RU. Dilutions of MSA and HSA were injected over immobilized receptors at 25 °C. The flow rate was 50 μl/min. E, binding of shFcRnWT, shFcRnH166A, shFcRnR164L/E165G, and shFcRnE115A/E116A to hIgG1 at pH 6.0 in ELISA. F, binding of smFcRnWT, smFcRnH166A, and smFcRnL166R/G167E to hIgG1 at pH 6.0 in ELISA. The numbers given represent the mean of triplicates.
FIGURE 9.
FIGURE 9.
SPR analyses of the interaction of shFcRnWT and smFcRnWT with human and mouse ligands. A, HSA, hIgG1, MSA, and mIgG1 injected over shFcRnWT at pH 6.0. B, MSA, mIgG1, HSA, and hIgG1 injected over shFcRnWT at pH 6.0. HSA was injected at 10 μm, MSA at 5 μm, and both IgG variants at 100 nm. In all experiments shFcRnWT and smFcRnWT were immobilized by amine coupling to ∼600 RU. Injections were performed at 25 °C and the flow rate was 50 μl/min.
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