Design of an Optimized Scaffold for Affibody Molecules Joachim Feldwisch 1,2 ⁎, Vladimir Tolmachev 1,2 , Christofer Lendel 1 , Nina Herne 1 , Anna Sjöberg 1 , Barbro Larsson 1 , Daniel Rosik 1 , Eva Lindqvist 1 , Gunilla Fant 1 , Ingmarie Höidén-Guthenberg 1 , Joakim Galli 1 , Per Jonasson 1 and Lars Abrahmsén 1 1 Affibody AB, Lindhagensgatan 133, SE-112 51 Stockholm, Sweden 2 Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden Received 2 December 2009; received in revised form 26 February 2010; accepted 3 March 2010 Available online 10 March 2010 Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z HER2:342 . Replace- ments in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 °C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]- Z HER2:2891 -Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY- 025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K D , of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 °C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor α, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor β) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface. © 2010 Elsevier Ltd. All rights reserved. Edited by I. Wilson Keywords: Affibody molecules; scaffold; protein engineering; HER2; peptide synthesis *Corresponding author. Affibody AB, Lindhagensgatan 133, SE-112 51 Stockholm, Sweden. E-mail address: joachim.feldwisch@affibody.com. Present address: C. Lendel, Department of Molecular Biology, SLU, Box 590, SE-751 24 Uppsala, Sweden. Abbreviations used: ACN, acetonitrile; DIPEA, N, N′-diisopropylethylamine; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; His 6 , hexahistidine; HOBt, hydroxybenzotriazole; HRP, horseradish peroxidase; MMA-DOTA, maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; NEM, N- ethylmaleimide; PBS, phosphate-buffered saline; PDB, Protein Data Bank; PDGFRβ, platelet-derived growth factor receptor β; SPA, Staphylococcus aureus protein A; TFA, trifluoroacetic acid; TMB, tetramethylbenzidine; TNFα, tumor necrosis factor α; VTM, variable temperature measurement. doi:10.1016/j.jmb.2010.03.002 J. Mol. Biol. (2010) 398, 232–247 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.