Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties

Abstract

Background: Targeting molecules to tumor cells is a promising mode of action for cancer therapy.Results: Ubiquitin-based high affinity, specific, and stable binding molecules for extradomain B are accumulated in the tumor.Conclusion: Ubiquitin may be engineered for high affinity target binding and modified with half-life extension technologies.Significance: Ubiquitin qualifies as a well suited scaffold protein adaptable to specific tasks.

Peptide Aptamers: Development and Applications

“…The introduction of binding sites into relatively rigid secondary structure elements is considered advantageous because of the reduced entropy cost for structural rearrangement of the binding domain upon target interaction relative to those that may occur with the flexible binding structures generated in hypervariable or single loops [102]. The following examples provide the most recent developments of this scaffold concept.…”

“…β-sheets within both structures provide the binding surfaces, of which eight near-surface residues in γ-B Crystallin and six in Ubiquitin are suitable for modification [63]. Reevaluation of the platform revealed the benefits of using dimeric, head-to-tail, Ubiquitin units in the library, and a more extensive randomization of residues, five in the ²-sheet and four in the ²-turn [102]. Changes introduced by diversifying those positions do not cause any significant loss of stability.…”

“…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]. …”

“…This is a domain of a Fibronectin isoform normally absent in most adult tissues, but specifically expressed during wound healing, inflammation and in most cancers. Primary screening of a phage display library composed of Ubiquitin dimers was followed by ribosome display affinity maturation, with evolved versions displaying affinities in the range of 30-200 pM [102]. …”

The future of protein scaffolds as affinity reagents for purification

“…This is a structure with 176 aa that fold into several b-sheets, and is highly stable-for example, the bovine derived protein is stable in a wide range of pH (1-9), at high temperatures (75 C), and even in strong denaturation conditions (7 M urea). Affilins were selected to bind the Fc fragment of IgG (10 À8 M, 10 nM), estradiol and testosterone (100-200 nM) (Ebersbach et al, 2007), and also to extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues (30 and 200 pM) (Lorey et al, 2014). The human g-B-crystallin structure was randomized by phage display at the N-terminal and in eight amino acid positions, with solvent-exposed side chains in b-sheets, against recombinant pro-form of nerve growth factor (proNGF).…”