title: Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension.
abstract: Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.
authors.affiliation: Division of Protein Technology, KTH Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Malm
authors.forename: Tarek
authors.initials: T
authors.lastname: Bass
authors.forename: Lindvi
authors.initials: L
authors.lastname: Gudmundsdotter
authors.forename: Martin
authors.initials: M
authors.lastname: Lord
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: D000418:Albumins / Q000276:immunology*; D018033:Antibodies, Bispecific / Q000276:immunology*; D057005:Bioengineering / Q000379:methods; D015374:Biosensing Techniques / Q000379:methods; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods; D006207:Half-Life; D006801:Humans; D008024:Ligands; D010766:Phosphorylation / Q000276:immunology; D011485:Protein Binding / Q000276:immunology*; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D020893:Receptor, ErbB-3 / Q000276:immunology*
chemical_list: D000418:Albumins; D018033:Antibodies, Bispecific; D008024:Ligands; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; C508053:ERBB2 protein, human; C581292:ERBB3 protein, human
keywords: Affibody molecules; Albumin-binding domain; Bispecific; HER3; Half-life extension