Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

Abstract

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 (RGD) 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 αvβ3 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 (IC50) values of 10-30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both αvβ3 and αvβ5 integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to αvβ3, αvβ5, and α5β1 integrins. This finding has important clinical implications since all three of these receptors can be co-expressed 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.

Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma

“…Integrin-binding specificities were determined in previous studies (20,23,32,33). Ex vivo contrast is measured as the ratio of tumor signal to normal cerebellum signal in the same mouse.…”

“…Knottin peptides were prepared as previously described in detail (23,25). Briefly, the linear peptide sequences were made by solid-phase peptide synthesis on a CS Bio (Menlo Park, CA) instrument using standard 9-fluorenylmethyloxycarbonyl chemistry.…”

“…We engineered a small (∼3.5 kDa), conformationally constrained cystine knot peptide, termed EETI 2.5F, that binds with high affinity and unique specificity to α v β 3 , α v β 5 , and α 5 β 1 integrins (23). The cystine knot structural family, also known as knottins, consists of small polypeptides (30-50 amino acids) linked by at least three interwoven disulfide bonds, creating a rigid molecular "knot" that confers high chemical, thermal, and proteolytic stability (24).…”

“…EETI 2.5F, which is based on the Ecballium elaterium trypsin inhibitor-II (EETI-II, Fig. 1 A and B), was identified via high-throughput screening of yeast-displayed knottin libraries (23). Radiolabeled versions of EETI 2.5F and other engineered integrin-binding knottin peptides were previously shown to be promising molecular probes for PET imaging in s.c. mouse xenograft models due to their high tumor contrast and low imaging signals in nondiseased tissue, including the liver and kidneys (25,26).…”

Pacifastin-derived Peptides Target Tumors for Use in In Vivo Imaging

“…LCMI-II is highly tolerant to mutation, as has been seen in cystine-knotted peptides such as EETI, MCoTI, AgRP, and others (29)(30)(31), with some residues more tolerant of mutation than others. We have identified the sensitivity to mutagenesis of a number of residues in the THP1 scaffold.…”