There is a critical need for compounds that target cell surface
integrin receptors for applications in
cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium
trypsin inhibitor (EETI-II), a
knottin peptide from the squash family of
protease inhibitors, as a new class of
integrin-binding agents. We generated yeast-displayed libraries of EETI-II by substituting its 6-amino
acid trypsin binding loop with 11-amino
acid loops containing the
Arg-Gly-Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high-throughput manner by fluorescence-activated cell sorting to identify mutants that bound to alpha(v)
beta(3) integrin. Select
peptides were synthesized and were shown to compete for natural
ligand binding to
integrin receptors expressed on the surface of U87MG
glioblastoma cells with half-maximal inhibitory concentration values of 10-30 nM. Receptor specificity assays demonstrated that engineered
knottin peptides bind to both alpha(v)beta(3) and alpha(v)beta(5)
integrins with high affinity. Interestingly, we also discovered a
peptide that binds with high affinity to alpha(v)beta(3), alpha(v)beta(5), and alpha(5)beta(1)
integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on
tumors. In addition, we showed that engineered
knottin peptides inhibit
tumor cell adhesion to the
extracellular matrix protein vitronectin, and in some cases
fibronectin, depending on their
integrin binding specificity. Collectively, these data validate EETI-II as a scaffold for
protein engineering, and highlight the development of unique
integrin-binding
peptides with potential for translational applications in
cancer.