title: Half-life extension of a single-chain diabody by fusion to domain B of staphylococcal protein A.
abstract: Binding of a therapeutic protein to a long-circulating plasma protein can result in a strongly extended half-life. Among these plasma proteins, albumin and immunoglobulins are of special interest because of their exceptionally long half-life, which is to a great extent determined by recycling through the neonatal Fc receptor (FcRn). Many strategies have been established employing reversible binding to albumin, e.g. using an albumin-binding domain from streptococcal protein G. We show here that the half-life of a recombinant antibody molecule can also be prolonged by fusion to a single immunoglobulin-binding domain (IgBD) from staphylococcal protein A. This domain (domain B, SpA(B)) is composed of 56 amino acid residues and was fused to the C-terminus of a bispecific single-chain diabody (scDb). The scDb-SpA(B) fusion protein was produced in HEK293 cells and retained its antigen-binding activity as shown by enzyme-linked immunosorbent assay and flow cytometry. Furthermore, the fusion protein was capable of binding to human and mouse IgG in a pH-dependent manner. In mice, the terminal half-life of the fusion protein was improved from ∼1-2 h of the unmodified scDb to 11.8 h. Although the fusion protein did not reach the long half-life seen for IgG, our results established the applicability of a single bacterial IgBD for half-life extension purposes.
authors.affiliation: Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Unverdorben
authors.forename: Aline
authors.initials: A
authors.lastname: Färber-Schwarz
authors.forename: Fabian
authors.initials: F
authors.lastname: Richter
authors.forename: Meike
authors.initials: M
authors.lastname: Hutt
authors.forename: Roland E
authors.initials: RE
authors.lastname: Kontermann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D045744:Cell Line, Tumor; D006207:Half-Life; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D013205:Staphylococcal Protein A