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

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 (K_D 9.6 nM). The ability to present various peptides through substitution of the hCpn10 mobile loop demonstrates its utility as a novel protein scaffold.