title: Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3.
abstract: Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal Protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.
authors.affiliation: Division of Protein Technology, KTH Royal Institute of Technology, AlbaNova University Center, 106 91, Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Nilvebrant
authors.forename: Mikael
authors.initials: M
authors.lastname: Astrand
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.forename: Sophia
authors.initials: S
authors.lastname: Hober
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D015374:Biosensing Techniques; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D017433:Protein Structure, Secondary; D020893:Receptor, ErbB-3 / Q000235:genetics / Q000378:metabolism*; D012709:Serum Albumin / Q000378:metabolism*; D000075462:Serum Albumin, Human; D013696:Temperature
chemical_list: C422455:ALB protein, human; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D008024:Ligands; C106766:NRG2 protein, human; D009414:Nerve Growth Factors; D019151:Peptide Library; D012709:Serum Albumin; C581292:ERBB3 protein, human; D020893:Receptor, ErbB-3; D000075462:Serum Albumin, Human