Beyond Antibodies: Development of a Novel Protein Scaffold Based on Human Chaperonin 10

Abstract

Human Chaperonin 10 (hCpn10) was utilised as a novel scaffold for presenting peptides of therapeutic and diagnostic significance. Molecular dynamic simulations and protein sizing analyses identified a peptide linker (P1) optimal for the formation of the quarternary hCpn10 heptamer structure. hCpn10 scaffold displaying peptides targeting Factor VIIa (CE76-P1) and CD44 (CP7) were expressed in E. coli. Functional studies of CE76-P1 indicated nanomolar affinity for Factor VIIa (3 nM) similar to the E-76 peptide (6 nM), with undetectable binding to Factor X. CE76-P1 was a potent inhibitor of FX activity (via inhibition of Factor VIIa) and prolonged clot formation 4 times longer than achieved by E-76 peptide as determined by prothrombin time (PT) assays. This improvement in clotting function by CE76-P1, highlights the advantages of a heptamer-based scaffold for improving avidity by multiple peptide presentation. In another example of hCPn10 utility as a scaffold, CP7 bound to native CD44 overexpressed on cancer cells and bound rCD44 with high affinity (KD 9.6 nM). The ability to present various peptides through substitution of the hCpn10 mobile loop demonstrates its utility as a novel protein scaffold.

Protein Binder (ProBi) as a New Class of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution

“…Therefore, we are convinced that there is ample space to expand the current portfolio of protein scaffolds so that they can complement antibodies and interact with new target molecules. Therefore, a systematic method to streamline the development of new scaffolds with desired properties would be beneficial but relatively few attempts can be found in the literature [ 18 , 28 , 29 ]. In general, a good small protein scaffold should display a high stability, together with flexibility of binding.…”

Protein scaffolds in human clinics

“…The human chaperonin 10 (hCpn10) is an intracellular homooligomeric protein composed by 7 subunits that assist protein folding or re-folding. It is a stable beta-barrel core that does not undergo posttranslational modifications, shows low immunogenicity and it has been safely tested in clinical trials (Alsultan et al, 2016;Broadley et al, 2009). The loop responsible for the natural interactivity of Cpn60 has been successfully substituted for different target-specific ligands, thus preventing the native binding and conferring multivalent ligand display with different clinical applications.…”

“…The loop responsible for the natural interactivity of Cpn60 has been successfully substituted for different target-specific ligands, thus preventing the native binding and conferring multivalent ligand display with different clinical applications. Molecular dynamic modelling was used to design and insert linkers at the junctures of the mobile loop to prevent interferences of inserted ligands with the subunit interface and to maintain the heptameric structure that allows multivalent peptide presentation (Alsultan et al, 2016).…”

MutDock: A computational docking approach for fixed-backbone protein scaffold design

“…However, binding for a subclass of these alternative scaffolds is governed by point mutations to surface residues in highly stable secondary structures, and these mutations do not alter the proteins’ structures. Examples of such binding proteins include affibodies and designed ankyrin repeats (DARPins) ( Alsultan et al, 2016 ). An affibody consists of 58 amino acids and is arranged in a three alpha–helix bundle framework.…”