Engineered Monospecific and Bispecific Proteins That Target Tumor Vascular Receptors As In Vivo Imaging Agents

Engineered proteins that bind to tumor vascular receptors have great potential as non-invasive in vivo molecular imaging agents to diagnose and stage disease, to monitor response to therapy, and to identify which patients are most likely to respond to a particular treatment regimen.  We used three distinct cystine knot scaffolds naturally found in plants, spiders, and humans to engineer peptides that bound to tumor-associated integrin receptors with high affinity and specificity. Tumor uptake and tissue biodistribution of these integrin-binding cystine knot peptides in mouse xenograft models were compared to the wild-type versions of the peptides after conjugation to a near-infrared dye. We found that peptides based on the Ecbalium elaterium cystine knot scaffold had the most desirable properties in terms of high tumor uptake and low uptake in non-target organs such as the liver and kidney. Another strategy to potentially increase tumor versus normal tissue specificity is to engineer proteins that target two tumor-associated receptors. We previously used a growth factor-based antagonist to engineer a bispecific tumor-targeting agent that bound with low nanomolar affinity to both alphav beta3 integrin and vascular endothelial growth factor receptor-2 (VEGFR2). Comparisons of tumor uptake and tissue biodistribution observed with this bispecific protein and its monospecific counterparts will be discussed. Funding: NIH NCI P50 CA114747 and American Cancer Society.