(205d) Engineered Mono-Specific Proteins As In Vivo Imaging Agents That Target Tumor Vasculature

Angiogenesis, the formation of neovasculature, is a key step in tumor growth and metastasis. Engineered proteins that bind to tumor vasculature receptors have great potential as non-invasive in vivo molecular imaging agents to diagnose disease, to predict response to particular treatment regimens, and to monitor response to therapy. Vascular endothelial growth factor receptor 2 (VEGFR-2) is the principal mediator of angiogenesis and consequently agents based on its activating ligand (vascular endothelial growth factor, VEGF) have attracted attention as in vivo imaging agents. However, these VEGF-based agents are agonists and thus are unlikely to be suitable for disease diagnosis and repeated serial imaging necessary for disease management and staging.

To address this short-coming we utilized protein engineering to develop a VEGF-based antagonist that displayed high-affinity for VEGFR2. We started with a single chain variant of wild-type VEGF (VEGFwt) that was previously shown to retain VEGFR2 binding, but prevented receptor dimerization and activation (VEGFmut). This was achieved by mutating one face of the VEGF ligand that converted it from a bivalent to a monovalent binder. However, this resulted in a significant loss in apparent affinity due to the loss in avidity and rendering the molecule unsuitable for in vivo targeting. Using rational and combinatorial approaches we affinity matured the parent (VEGFmut) resulting in the isolation of monovalent variants (VEGFa-m) that exhibited VEGFR2 affinities comparable to the bivalent parent (VEGFwt) of ~400 pM but retained antagonistic activity. In addition, these agents exhibited a marked shift in the preference for VEGFR2 versus VEGFR1 (a VEGFR family member that is not functionally implicated in active angiogenesis and is responsible for high renal uptake) compared to VEGFwt based agents.

In this presentation biochemical and biophysical characterization of the engineered mono-specific variants will be described. The performance of these agents with respect to the currently utilized VEGFwt based probes in in vivo tumor models will also be discussed.