Antagonistic VEGF Variants Engineered to Simultaneously Bind to and Inhibit Both VEGFR2 and Alphav Beta3 Integrin

Significant crosstalk exists between receptors that regulate angiogenesis, such as vascular endothelial growth factor receptor-2 (VEGFR2) and alphav beta3 integrin. Here, we developed dual-specific proteins that antagonize both of these receptors and effectively inhibit angiogenic processes in vitro and in vivo, compared to a protein variant that targets only VEGFR2. The vast majority of currently-available bispecific protein therapeutics are antibodies or antibody fragments which are assembled through associating domains or a flexible tether. We created a novel dual-specific therapeutic protein that does not rely on an antibody framework or physical linkage. For this goal, we used an antagonistic VEGF ligand as a scaffold to engineer dual-specific proteins that bound to both VEGFR2 and alphav beta3 integrin with low nanomolar affinities. Mutations were first introduced into a single-chain VEGF ligand (scVEGF) that retained VEGFR2 binding, but prevented receptor dimerization and activation. Next, we tested the tolerance of this scVEGF scaffold to introduction of randomized loops at locations distinct from the VEGFR2 binding site. Thirty-two different scVEGF 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 that bound with high affinity to both VEGFR2 and alphav beta3 integrin. 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 and cell binding assays to show that the dual-specific proteins simultaneously engaged both VEGFR2 and alphav beta3 integrin receptors. Compared to a VEGFR2-targeting agent, these dual-specific proteins significantly inhibited adhesion and VEGF-mediated receptor phosphorylation and proliferation of endothelial cells cultured on vitronectin-coated surfaces. Moreover, dual-specificity conferred nearly complete inhibition of VEGF-mediated blood vessel formation in vivo, while a protein variant that binds only VEGFR2 was marginally effective. We are currently evaluating the promise of these engineered proteins for use as cancer therapeutics and diagnostics in murine tumor models. This work highlights an approach where a natural ligand can be used a scaffold to engineer high affinity binding against an additional therapeutic target of interest.

Funding: American Cancer Society, NRSA NIH 5T32 CA09302, and EMBO Fellowship Program