Selection and characterization of affibody ligands binding to Alzheimer amyloid β peptides

Affibody (R) (Affibody) ligands specific for human amyloid beta (A beta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified forranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to A beta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of A beta peptides from human plasma and serum. It was found that both ligands could efficiently capture A beta that were spiked (100 mu g ml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of A beta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of A beta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed A beta peptide, which we speculate to be dimeric A beta. In conclusion, we have successfully selected Affibody ligands that efficiently capture A beta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of A beta peptides in order to slow down or reduce amyloid plaque formation, is discussed. (c) 2006 Elsevier B.V. All rights reserved.