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.
Highlights
Monoclonal antibodies are widely used in the life sciences as diagnostic reagents and biologic medicines. mAbs are natural protein scaffolds, which display multiple peptide loops, forming an extended interface that enables high affinity recognition of target antigens
We report the development of a novel protein scaffold based on human Chaperonin 10, an essential oligomeric protein that assists in folding of translated polypeptides or refolding of denatured eukaryotic proteins7. hCpn[10] is a homo-oligomer composed of seven subunits (Fig. 1e)
Each monomer consists of β-barrel core structure that is flanked by two flexible peptide loops, known as the β-hairpin and mobile loop
Summary
Monoclonal antibodies (mAbs) are widely used in the life sciences as diagnostic reagents and biologic medicines. mAbs are natural protein scaffolds, which display multiple peptide loops (i.e. complementarity determining regions or CDRs), forming an extended interface that enables high affinity recognition of target antigens. There is a plethora of other molecular entities in nature with differing protein backbones that display loops with various functionalities, and there is global interest in the development of these non-antibody binding entities, through presentation of peptides within these molecular scaffolds (Fig. 1a–d). These include scaffolds used for cancer therapy such as the Affibody[1] targeting EGFR, DARPin[2] targeting HER2, Monobody[3] (of fibronectin type III) targeting VEGFR-2 for Non-Hodgkin’s lymphoma and an Anticalin[4] (derived from lipocalins) targeting VEGF in solid tumours. The binding of the hCpn10-P7 (named CP7) scaffold to cancer cells was evaluated as a diagnostic tool
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