According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-␤ (A␤) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar A␤ assemblies is accompanied by a conformational change toward a high content of ␤-structure. Here, we report the solution structure of A␤(1-40) in complex with the phage-display selected affibody protein ZA␤3, a binding protein of nanomolar affinity. Bound A␤(1-40) features a ␤-hairpin comprising residues 17-36, providing the first highresolution structure of A␤ in ␤ conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar A␤. ZA␤3 stabilizes the ␤-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the A␤ hairpin within a large hydrophobic tunnel-like cavity. Consequently, Z A␤3 acts as a stoichiometric inhibitor of A␤ fibrillation. The selected A␤ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates.A␤-peptide ͉ engineered binding protein ͉ molecular recognition ͉ protein structure ͉ nuclear magnetic resonance

a b s t r a c tAffibody molecules are a class of engineered affinity proteins with proven potential for therapeutic, diagnostic and biotechnological applications. Affibody molecules are small (6.5 kDa) single domain proteins that can be isolated for high affinity and specificity to any given protein target. Fifteen years after its discovery, the Affibody technology is gaining use in many groups as a tool for creating molecular specificity wherever a small, engineering compatible tool is warranted. Here we summarize recent results using this technology, propose an Affibody nomenclature and give an overview of different HER2-specific Affibody molecules. Cumulative evidence suggests that the three helical scaffold domain used as basis for these molecules is highly suited to create a molecular affinity handle for vastly different applications.

The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. Here, we describe a HER2-specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z HER2:342 showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging. (Cancer Res 2006; 66(8): 4339-48)