Biotechnol. Appl. Biochem. (2009) 54, 121–131 (Printed in Great Britain) doi:10.1042/BA20090096 121 Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule Mikaela Friedman* 1 , Sara Lindstr ¨ om† 1 , Lina Ekerljung§, Helene Andersson-Svahn†, J¨ orgen Carlsson§, Hjalmar Brismar‡, Lars Gedda§, Fredrik Y. Frejd§¶ and Stefan St˚ ahl* 2 *Division of Molecular Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden, †Division of Nanobiotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden, ‡Department of Cell Physics, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden, §Unit of Biomedical Radiations Sciences, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden, and ¶ Affibody AB, PO Box 20137, SE-161 02 Bromma, Sweden 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 (G 4 S) 3 [(Gly 4 -Ser) 3 ]-encoding gene fragment. The encoded 30 kDa affibody construct (Z HER2 ) 2 –(G 4 S) 3 – (Z EGFR ) 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, simultan- eous 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) im- munofluorescence 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. Introduction An increasing number of clinically approved treatments for cancer have been developed on the basis of knowledge about molecular targets that are specific for, or overexpressed in, the tumour and their molecular function. The use of mAbs (monoclonal antibodies) is a well-established approach for targeting of tumour-associated antigens both in in vivo diagnostics and in therapy. Today, nine mAbs are approved by US Food and Drug Administration (FDA) for therapeutic use in oncology and several more are in preclinical and clinical development. Efficient tumour targeting for therapy or molecular imaging is dependent on sufficient tumour uptake and selectivity. Furthermore, fast clearance to achieve good contrast is particularly important in molecular imaging. Advances in protein engineering has led to the generation of a number of antibody derivatives [e.g. scFvs (single-chain variable fragments), Fabs (Fab fragments), diabodies (small recombinant bispecific antibodies), minibodies (recombinant antibodies in which the heavy- and light-chain variable regions are part of the same polypeptide chain, which also includes the heavy-chain hinge region and one heavy-chain constant domain) and dAbs (single-domain antibodies)] and more recently also to other scaffold proteins [e.g. Kunitz domains, knottins, Adnectins TM , anticalins, DARPins (designed ankyrin-repeat proteins) and Key words: bispecific (bs) affibody molecule, human epidermal-growth-factor receptor-3 (ErbB), microwell array, protein engineering, tumour targeting. Abbreviations used: ABD, albumin-binding domain; BiTE, bispecific T-cell engager; bs, bispecific; DTT, dithiothreitol; EGFR, epidermal-growth-factor receptor; ECD, extracellular domain; FBS, fetal bovine serum; (G 4 S) 3 , (Gly 4 -Ser) 3 ; hEGFR, human EGFR-1; hEGFR-Fc, hEGFR Fc chimaera; HER2/ErbB2, human EGFR-2; HER3/ErbB3, human EGFR-3; hHER2- Fc, HER2 Fc chimaera; His 6 , hexahistidine; IMAC, immobilized-metal-ion affinity chromatography; mAb, monoclonal antibody; scFv, single-chain variable fragment. 1 These authors contributed equally to the work. 2 To whom correspondence should be addressed (email stefan.stahl@biotech.kth.se) C  2009 Portland Press Ltd