title: Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules.
abstract: Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.
authors.affiliation: Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm.
authors.forename: Nina
authors.initials: N
authors.lastname: Kronqvist
authors.forename: Magdalena
authors.initials: M
authors.lastname: Malm
authors.forename: Lovisa
authors.initials: L
authors.lastname: Göstring
authors.forename: Elin
authors.initials: E
authors.lastname: Gunneriusson
authors.forename: Martin
authors.initials: M
authors.lastname: Nilsson
authors.forename: Ingmarie
authors.initials: I
authors.lastname: Höidén Guthenberg
authors.forename: Lars
authors.initials: L
authors.lastname: Gedda
authors.forename: Fredrik Y
authors.initials: FY
authors.lastname: Frejd
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D020893:Receptor, ErbB-3 / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013210:Staphylococcus
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D020893:Receptor, ErbB-3