⁶⁴Cu-Labeled Gp2 Domain for PET Imaging of Epidermal Growth Factor Receptor

This purpose of this study is to determine the efficacy of a 45-amino acid Gp2 domain, engineered to bind to epidermal growth factor receptor (EGFR), as a positron emission tomography (PET) probe of EGFR in a xenograft mouse model. The EGFR-targeted Gp2 (Gp2-EGFR) and a nonbinding control were site-specifically labeled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Binding affinity was tested toward human EGFR and mouse EGFR. Biological activity on downstream EGFR signaling was examined in cell culture. DOTA-Gp2 molecules were labeled with ⁶⁴Cu and intravenously injected (0.6–2.3 MBq) into mice bearing EGFR^high (<i>n</i> = 7) and EGFR^low (<i>n</i> = 4) xenografted tumors. PET/computed tomography (CT) images were acquired at 45 min, 2 h, and 24 h. Dynamic PET (25 min) was also acquired. Tomography results were verified with gamma counting of resected tissues. Two-tailed <i>t</i> tests with unequal variances provided statistical comparison. DOTA-Gp2-EGFR bound strongly to human (<i>K</i>_D = 7 ± 5 nM) and murine (<i>K</i>_D = 29 ± 6 nM) EGFR, and nontargeted Gp2 had no detectable binding. Gp2-EGFR did not agonize EGFR nor antagonize EGF-EGFR. ⁶⁴Cu-Gp2-EGFR tracer effectively localized to EGFR^high tumors at 45 min (3.2 ± 0.5%ID/g). High specificity was observed with significantly lower uptake in EGFR^low tumors (0.9 ± 0.3%ID/g, <i>p</i> < 0.001), high tumor-to-background ratios (11 ± 6 tumor/muscle, <i>p</i> < 0.001). Nontargeted Gp2 tracer had low uptake in EGFR^high tumors (0.5 ± 0.3%ID/g, <i>p</i> < 0.001). Similar data was observed at 2 h, and tumor signal was retained at 24 h (2.9 ± 0.3%ID/g). An engineered Gp2 PET imaging probe exhibited low background and target-specific EGFR^high tumor uptake at 45 min, with tumor signal retained at 24 h postinjection, and compared favorably with published EGFR PET probes for alternative protein scaffolds. These beneficial <i>in vivo</i> characteristics, combined with thermal stability, efficient evolution, and small size of the Gp2 domain validate its use as a future class of molecular imaging agents