title: Engineering and characterization of a bispecific HER2 x EGFR-binding affibody molecule.
abstract: HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G4S)3 [(Gly4-Ser)3]-encoding gene fragment. The encoded 30 kDa affibody construct (ZHER2)2-(G4S)3-(ZEGFR)2, with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.
authors.affiliation: Division of Molecular Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden.
authors.forename: Mikaela
authors.initials: M
authors.lastname: Friedman
authors.forename: Sara
authors.initials: S
authors.lastname: Lindström
authors.forename: Lina
authors.initials: L
authors.lastname: Ekerljung
authors.forename: Helene
authors.initials: H
authors.lastname: Andersson-Svahn
authors.forename: Jörgen
authors.initials: J
authors.lastname: Carlsson
authors.forename: Hjalmar
authors.initials: H
authors.lastname: Brismar
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
mesh_terms: D018033:Antibodies, Bispecific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000918:Antibody Specificity; D015374:Biosensing Techniques; D002450:Cell Communication; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018033:Antibodies, Bispecific; D066246:ErbB Receptors; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C512478:EGFR protein, human; C508053:ERBB2 protein, human