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

Authors

  • Richard H. Kimura,

    1. Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, California 94305
    2. Department of Radiology and Molecular Imaging Program, Stanford University, Stanford, California 94305
    Search for more papers by this author
    • Richard H. Kimura and Aron M. Levin contributed equally to this work

  • Aron M. Levin,

    1. Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, California 94305
    Search for more papers by this author
    • Richard H. Kimura and Aron M. Levin contributed equally to this work

  • Frank V. Cochran,

    1. Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, California 94305
    Search for more papers by this author
  • Jennifer R. Cochran

    Corresponding author
    1. Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, California 94305
    • Department of Bioengineering, Cancer Center, Bio-X Program, 318 Campus Dr. West, MC5439, The James H. Clark Center W250, Stanford, CA 94305-5439
    Search for more papers by this author

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 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 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 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. Proteins 2009. © 2009 Wiley-Liss, Inc.

Ancillary