Design of an Optimized Scaffold for Affibody Molecules

Joachim Feldwisch, Vladimir Tolmachev, Christofer Lendel, Nina Herne, Anna Sjöberg, Barbro Larsson, Daniel Rosik, Eva Lindqvist, Gunilla Fant, Ingmarie Höidén-Guthenberg, Joakim Galli, Per Jonasson, Lars Abrahmsén

Research output: Contribution to journalArticlepeer-review

95 Citations (Scopus)


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 ZHER2:342. Replacements 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]-ZHER2:2891-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, KD, 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 γ, 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.

Original languageEnglish
Pages (from-to)232-247
Number of pages16
JournalJournal of Molecular Biology
Issue number2
Publication statusPublished - Apr 2010
Externally publishedYes


  • Affibody molecules
  • HER2
  • Peptide synthesis
  • Protein engineering
  • Scaffold

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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