Positron emission tomography (PET) enables identification, localization, and characterization of biomarkers in primary tumors and metastases, while being non-invasive, quantitative, and sensitive to picomolar concentrations. Clinical PET of epidermal growth factor receptor (EGFR) would allow prediction of treatment response in wild-type KRAS metastatic colorectal cancer patients, tumor staging, and treatment monitoring while assessing spatiotemporal heterogeneities that immunohistochemistry cannot. Efficient evolution of small protein scaffolds provides imaging agents that benefit from increased extravasation, tissue penetration, and clearance compared to larger proteins or particles. Systematic evaluation of the Protein Data Bank identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2). De novo ligand discovery and directed evolution towards four targets yielded target-specific binders with affinities as strong as 200 ±100 pM and high thermal stability (Figure 1A). For cancer targeting, a Gp2 domain for EGFR was evolved with 18 ±8 nM affinity and receptor-specific binding (Kruziki et al, 2015). Binders to hepatocyte growth factor receptor and insulin receptor have been evolved and results of ongoing characterization will be presented. Gp2 domains were site-specifically conjugated with a chelator and radiolabeled with 64Cu for PET. Mice, xenografted with A431 (EGFRhigh) or MDA-MB-435 (EGFRlow) tumors, were intravenously injected with 64Cu-Gp2 and imaged at 1 h and 2 h post-injection. Dissected tissues and fluids were also analyzed via gamma counting to corroborate imaging results. Gp2EGFR localized to xenografted EGFRhigh tumors with 11 ±1 tumor-to-blood ratio and 22 ±3 tumor-to-muscle (Figure 1B) and a non-binding Gp2 showed significantly lower tumor signal. However, similar to other small scaffolds, high kidney and modest liver signal were observed. To modify physiological distribution, the Gp2EGFR framework was rationally mutated to modulate charge and increase hydrophilicity, without comprising affinity (Figure 1C).

PET imaging of growth factor receptor expression in cancer using the 45-amino acid Gp2 scaffold

251st National Meeting (2016)

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