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Abstract
Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.
Highlights
The neonatal Fc receptor (FcRn) is a heterodimeric protein consisting of a transmembrane MHC class I-like heavy chain (FcRn α-chain) and the β2-microglobulin (β2m) light chain, of which the latter is a part of MHC class I receptors [1,2].FcRn is predominantly located in endosomes and is able to bind to serum albumin (SA) and immunoglobulin G (IgG) at pH≤6.5 and release them at pH≥7.0 [3,4,5]
Vector design for expression of FcRnECD Genes encoding the extracellular domain of the α-chain of human FcRn and human β2m were inserted into the lentiviral transfer plasmids 2K7bsd and 2K7neo
Studies on FcRn have revealed many important functions based on its interactions with IgG and HSA, including half-life extension of both proteins and trancytosis of IgG across the upper airway epitelium, the small proximal intestines and the placenta [12]
Summary
FcRn is predominantly located in endosomes and is able to bind to serum albumin (SA) and immunoglobulin G (IgG) at pH≤6.5 and release them at pH≥7.0 [3,4,5]. When IgG, SA and other serum proteins are passively endocytosed by cells in contact with blood, the pH becomes gradually lower in the formed endosomes permitting binding of IgG and SA to FcRn. The receptor is together with its bound ligand, transported back via recycling endosomes to the plasma membrane, and rescues the ligand from lysosomal degradation. The IgG and SA sites of interaction on FcRn are distinct and it has been shown that the three proteins may form a ternary complex [10]. FcRn is involved in transmission of IgG from the mother to the fetus via the placenta [11] and in rodents for transmission of IgG from the mother’s milk to the newborn via the proximal small intestine [1]
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