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.