(47d) Engineered Bispecific Proteins That Simultaneously Bind to and Inhibit Both VEGFR2 and Alphav Beta3 Integrin for Cancer Therapy and Diagnosis

Vascular endothelial growth factor receptor-2 (VEGFR2) and alphav beta3 integrin are overexpressed on the tumor vasculature and play a strong role in tumor proliferation, angiogenesis, and metastasis. Bispecific proteins that target both receptors offer promise for improved therapeutic efficacy compared to single agents, as there is significant crosstalk between the cell signaling pathways mediated by these receptors. Moreover, non-invasive molecular imaging agents are critically needed for cancer staging and disease management. We used a natural growth factor ligand as a scaffold to engineer bispecific proteins that bound to both VEGFR2 and alphav beta3 integrin with low nanomolar affinities. Mutations were introduced into a single-chain VEGF ligand (scVEGF) that was previously shown to retain VEGFR2 binding, but prevent receptor dimerization and activation. We substituted an entire loop of this scVEGF antagonist with randomized loop sequences using degenerate codons. Thirty-two different libraries of varying loop lengths and positions were analyzed to determine which loop substitutions were least disruptive to VEGFR2 binding. scVEGF mutant libraries (~10⁷ clones each) were displayed on the yeast cell surface and high-throughput flow cytometric screening identified several variants with high affinity to both VEGFR2 and alphav beta3 integrin. The scVEGF mutants were solubly expressed in yeast, purified using affinity and size exclusion chromatography, and extensively characterized for their binding and biological properties. Engineered scVEGF mutants were specific for alphav beta3 integrin, and did not bind to the related integrins alphav beta5, alphaiib beta3, or alpha5 beta1. In addition, we used surface plasmon resonance to show that bispecific proteins can simultaneously bind to both VEGFR2 and alphav beta3 integrin. To measure their biological activity, assays were performed with cells expressing VEGFR2 and alphav beta3 integrin, in the presence of vitronectin (a natural integrin ligand) and/or VEGF. The bispecific scVEGF proteins strongly inhibited cell adhesion and ligand-mediated receptor phosphorylation and cell proliferation compared to scVEGF protein variants that bound only VEGFR2, particularly on vitronectin-coated surfaces. Moreover, the bispecific proteins strongly inhibited angiogenesis in a matrigel sprouting assay. We are currently evaluating the promise of these engineered proteins for use as cancer therapeutics and diagnostics in murine tumor models. Funding: American Cancer Society, NRSA NIH 5T32 CA09302, and EMBO Fellowship Program