title: Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.
abstract: Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.
authors: Division of Molecular Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden.
authors: Andreas
authors: A
authors: Jonsson
authors: Helena
authors: H
authors: Wållberg
authors: Nina
authors: N
authors: Herne
authors: Stefan
authors: S
authors: Ståhl
authors: Fredrik Y
authors: FY
authors: Frejd
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics*; D015374:Biosensing Techniques; D016000:Cluster Analysis; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding / Q000235:genetics*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D019151:Peptide Library; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha