Cross-species binding analyses of mouse and human neonatal Fc receptor show dramatic differences in immunoglobulin G and albumin binding.
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.
Andersen
Jan Terje
JT
Department of Molecular Biosciences and Centre for Immune Regulation, University of Oslo, P.O. Box 1041, N-0371 Oslo, Norway. janta@imbv.uio.no
Daba
Muluneh Bekele
MB
Berntzen
Gøril
G
Michaelsen
Terje E
TE
Sandlie
Inger
I
D000418:Albumins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D011961:Receptors, Fc / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D000418:Albumins; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D011961:Receptors, Fc; C100688:Fc receptor, neonatal