Generation of tumour‐necrosis‐factor‐α‐specific affibody¹ 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.

Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

“…The three-helix Z-domain α-peptide scaffold has proven to be very versatile, with customized variants reported for over 20 protein targets of biomedical interest (12,14,(19)(20)(21). The resulting miniproteins are attractive in terms of development of therapeutic antagonists of deleterious protein-protein associations and as starting points for highly specific imaging agents (11)(12)(13)(14)(19)(20)(21)(22)(23).…”

“…The resulting miniproteins are attractive in terms of development of therapeutic antagonists of deleterious protein-protein associations and as starting points for highly specific imaging agents (11)(12)(13)(14)(19)(20)(21)(22)(23). Selection of new protein-binding molecules from Z-domain …”

“…Z-TNFα presumably adopts the characteristic three-helix bundle tertiary structure and contacts TNFα via the surface defined by helices 1 and 2. Z-TNFα has been reported to bind TNFα with an apparent dissociation constant (K D ) of 0.1-0.5 nM, as measured by surface plasmon resonance, and can block binding of TNFα to the receptor TNFR2 in vitro, as indicated by experiments with etanercept (a TNFR2+IgG1 Fc fusion protein) (20). We designed α/β-TNFα-1 and α/β-TNFα-2 by removing helix 3 of Z-TNFα, adding a disulfide, and introducing α→β and Aib replacements at positions corresponding to those in the VEGF-and IgG-targeted α/β-peptides described above ( Fig.…”

“…Engineered proteins that bind to TNFα and inhibit association with its receptors (TNFRs) are currently used to treat rheumatoid arthritis, inflammatory bowel disease, and other illnesses (40). Using phage display, Jonsson et al (20) previously developed the 58-mer Z-TNFα (Fig. 1A), based on the Z-domain scaffold.…”

“…1 A and B) (19), tumor necrosis factor-α (TNFα) (peptide Z-TNFα; Fig. 1A) (20), and human epidermal growth factor receptor 2 (HER2) (21). Such Z-domain analogs might represent alternatives to antibodies for selective detection of disease marker proteins or for blocking deleterious signal transduction (11)(12)(13)(14).…”