title: An engineered affibody molecule with pH-dependent binding to FcRn mediates extended circulatory half-life of a fusion protein.
abstract: Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased half-life, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden;
authors.forename: Johan
authors.initials: J
authors.lastname: Seijsing
authors.affiliation: Affibody AB, SE-171 63 Solna, Sweden;
authors.forename: Malin
authors.initials: M
authors.lastname: Lindborg
authors.affiliation: Affibody AB, SE-171 63 Solna, Sweden;
authors.forename: Ingmarie
authors.initials: I
authors.lastname: Höidén-Guthenberg
authors.affiliation: Affibody AB, SE-171 63 Solna, Sweden;
authors.forename: Heiko
authors.initials: H
authors.lastname: Bönisch
authors.affiliation: Affibody AB, SE-171 63 Solna, Sweden;
authors.forename: Elin
authors.initials: E
authors.lastname: Guneriusson
authors.affiliation: Affibody AB, SE-171 63 Solna, Sweden; Biomedical Radiation Sciences, Department of Radiology, Oncology, and Radiation Sciences, Uppsala University, SE-75185 Uppsala, Sweden; and.
authors.forename: Fredrik Y
authors.initials: FY
authors.lastname: Frejd
authors.affiliation: Affibody AB, SE-171 63 Solna, Sweden;
authors.forename: Lars
authors.initials: L
authors.lastname: Abrahmsén
authors.affiliation: Affibody AB, SE-171 63 Solna, Sweden;
authors.forename: Caroline
authors.initials: C
authors.lastname: Ekblad
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden;
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.affiliation: Division of Proteomics and Nanobiotechnology, School of Biotechnology, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden.
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden; torbjorn@biotech.kth.se.
authors.forename: Torbjörn
authors.initials: T
authors.lastname: Gräslund
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006207:Half-Life; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008815:Mice, Inbred Strains; D019151:Peptide Library; D011485:Protein Binding; D011961:Receptors, Fc / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000097:blood / Q000378:metabolism* / Q000493:pharmacokinetics*
chemical_list: D002352:Carrier Proteins; D015395:Histocompatibility Antigens Class I; D019151:Peptide Library; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; C100688:Fc receptor, neonatal
keywords: ABD; affibody molecule; albumin-binding domain; neonatal Fc receptor; pharmacokinetics