A Chemically Cross-Linked Knottin Dimer Binds Integrins with Picomolar Affinity and Inhibits Tumor Cell Migration and Proliferation

Abstract

Molecules that target and inhibit αvβ3, αvβ5, and α5β1 integrins have generated great interest because of the role of these receptors in mediating angiogenesis and metastasis. Attempts to increase the binding affinity and hence the efficacy of integrin inhibitors by dimerization have been marginally effective. In the present work, we achieved this goal by using oxime-based chemical conjugation to synthesize dimers of integrin-binding cystine knot (knottin) miniproteins with low-picomolar binding affinity to tumor cells. A non-natural amino acid containing an aminooxy side chain was introduced at different locations within a knottin monomer and reacted with dialdehyde-containing cross-linkers of different lengths to create knottin dimers with varying molecular topologies. Dimers cross-linked through an aminooxy functional group located near the middle of the protein exhibited higher apparent binding affinity to integrin-expressing tumor cells compared with dimers cross-linked through an aminooxy group near the C-terminus. In contrast, the cross-linker length had no effect on the integrin binding affinity. A chemical-based dimerization strategy was critical, as knottin dimers created through genetic fusion to a bivalent antibody domain exhibited only modest improvement (less than 5-fold) in tumor cell binding relative to the knottin monomer. The best oxime-conjugated knottin dimer achieved an unprecedented 150-fold increase in apparent binding affinity over the knottin monomer. Also, this dimer bound 3650-fold stronger and inhibited tumor cell migration and proliferation compared with cilengitide, an integrin-targeting peptidomimetic that performed poorly in recent clinical trials, suggesting promise for further therapeutic development.

Targeted Drug Delivery with an Integrin-Binding Knottin–Fc–MMAF Conjugate Produced by Cell-Free Protein Synthesis

“…The cytotoxicity of 2.5F-Fc at high concentrations, particularly in A2780 cells, and to a lesser degree MB-468 cells, is likely due to the ability of integrintargeting molecules to disrupt cell adhesion. Similar results were seen when tumor cells were treated with high concentrations of 2.5F peptide in a previous study, in contrast with U87MG cells which are more resistant to detachment-induced apoptosis (16). Linker-modified MMAF treatment alone inhibits cell proliferation to some degree; a similar level of inhibition was observed upon coadministration of 2.5F-Fc plus linker-modified MMAF, indicating the lack of synergistic effects from combination treatment.…”

“…To measure the relative binding affinities of knottin-Fc fusion proteins, competition binding assays were performed as described previously with some modifications (16). AlexaFluor 488-conjugated EETI 2.5F (AF488-2.5F) was used as a competitor to compare the relative binding of 2.5F-Fc, 2.5F-Fc-MMAF, and CTRL-Fc-MMAF.…”

Amide Rotation Hindrance Predicts Proteolytic Resistance of Cystine-Knot Peptides

“…1–5 Different from some other classes of peptide molecules, one most attractive feature of cystine-knot peptides is their remarkable proteolytic resistance. In fact, some cystine-knot peptides, such as BPTI and MCTI-A, 6–8 are known as very potent canonical serine proteinases inhibitors, which bind to the enzyme active site in a substrate manner but can resist proteolysis ranging from hours to even years.…”

Scaffolding of Cystine‐Stabilized Miniproteins

“…This allows the possibility of developing proteins that can distinguish between targets from healthy and infectious cells . Cochran and co‐workers have worked extensively on developing such proteins that can simultaneously bind, for example, integrins with picomolar affinities or integrin and VEGFR2 receptors simultaneously . Advantages of using such proteins have been further detailed in a perspective article and book chapter written by them.…”