New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

Abstract

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.

Subunit disassembly and inhibition of TNFα by a semi-synthetic bicyclic peptide

“…Despite their success in the clinic, anti-TNF antibodies suffer from drawbacks, including high manufacturing costs and poor tissue penetration spurring the development of alternative TNF antagonist agents. Examples include the small-molecule binder SPD304 ( He et al , 2005 ), the peptide antagonists WP9QY ( Takasaki et al , 1997 ), the human ubiquitin derived F10 ( Hoffmann et al , 2012 ) or the DNA aptamer VR11 ( Orava et al , 2012 ).…”

Emerging drug development technologies targeting ubiquitination for cancer therapeutics

“…Engineered UbVs have also been developed to target proteins that are not known to associate with Ub. Hoffmann et al used ribosome display (Zahnd, Amstutz, & Pluckthun, 2007) to develop a UbV that bound to and inhibited the activity of tumor necrosis factor alpha (Hoffmann et al, 2012). Similarly, we recently isolated UbVs that bound specifically to the human epidermal growth factor receptor 3 (HER3) and the growth factor receptor-bound protein 2 (Grb2) and showed that UbVs acted as potent antagonists of Grb2-mediated cell signalling (Leung, Jarvik, & Sidhu, 2017).…”

Peptide Aptamers: Development and Applications

“…Changes introduced by diversifying those positions do not cause any significant loss of stability. In fact, some of the binders identified in library screenings demonstrated remarkable stability to pH changes, thermal denaturation and high concentrations of denaturing agents [102, 117-121]. …”