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.