title: Robust recombinant FcRn production in mammalian cells enabling oriented immobilization for IgG binding studies.
abstract: The MHC class-I related receptor or neonatal Fc receptor (FcRn) protects IgG and albumin from degradation by rescuing them in endothelial cells in a pH dependent fashion and consequently increases their respective half-lives. Monoclonal antibody-based therapies are of increasing interest and characterizing the interaction with FcRn is important for the development of an antibody candidate. In order to facilitate the production of soluble FcRn suitable for interaction studies, we generated semi-stable pools co-expressing FcRn α-chain, β2-microglobulin, biotin ligase and EGFP using a dual promoter, multi-cistronic vector. Human and mouse FcRn were purified in the mg/L range of culture medium and a single purification step was sufficient to reach a high level of purity. The receptors were characterized by ELISA, flow cytometry and surface plasmon resonance and shown to be functional. The single site biotinylation facilitated the directional immobilization of FcRn on the sensor chip and significantly increased the response level of the surface compared to amine coupling used in previous studies. Using this system, the affinity constants of seven IgGs, from various species and isotypes, were determined for human and mouse FcRn, including two hamster isotypes. These results confirm the higher selectivity of the human receptor and the promiscuous binding of mFcRn to IgGs from different species.
authors.affiliation: NovImmune SA, 14 Chemin des Aulx, 1228 Plan-Les-Ouates, Switzerland. gmagistrelli@novimmune.com
authors.forename: Giovanni
authors.initials: G
authors.lastname: Magistrelli
authors.forename: Pauline
authors.initials: P
authors.lastname: Malinge
authors.forename: Nadia
authors.initials: N
authors.lastname: Anceriz
authors.forename: Marjorie
authors.initials: M
authors.lastname: Desmurs
authors.forename: Sophie
authors.initials: S
authors.lastname: Venet
authors.forename: Sébastien
authors.initials: S
authors.lastname: Calloud
authors.forename: Bruno
authors.initials: B
authors.lastname: Daubeuf
authors.forename: Marie
authors.initials: M
authors.lastname: Kosco-Vilbois
authors.forename: Nicolas
authors.initials: N
authors.lastname: Fischer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D019921:Biotinylation / Q000379:methods; D002478:Cells, Cultured; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D011961:Receptors, Fc / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D011961:Receptors, Fc; D011994:Recombinant Proteins; C100688:Fc receptor, neonatal