Engineered Cystine-Knot Peptides that Bind α vβ 3 Integrin with Antibody-Like Affinities

Abstract

The α vβ 3 integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4-kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to α vβ 3 integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a six amino acid loop of AgRP with a nine amino acid loop containing the Arg-Gly-Asp integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting to identify clones with high affinity to detergent-solubilized α vβ 3 integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing α vβ 3 integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown to bind specifically to α vβ 3 integrins and had only minimal or no binding to α vβ 5, α 5β 1, and α iibβ 3 integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally occurring ligand for α vβ 3 and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second-generation libraries by individually randomizing these loops in one of the high-affinity integrin-binding variants. Screening of these loop-randomized libraries against α vβ 3 integrins resulted in peptides that retained high affinities for α vβ 3 and had increased specificities for α vβ 3 over α iibβ 3 integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.