title: 64Cu-Labeled Gp2 Domain for PET Imaging of Epidermal Growth Factor Receptor.
abstract: 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 with64Cu and intravenously injected (0.6-2.3 MBq) into mice bearing EGFRhigh(n = 7) and EGFRlow(n = 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 t tests with unequal variances provided statistical comparison. DOTA-Gp2-EGFR bound strongly to human (KD= 7 ± 5 nM) and murine (KD= 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 EGFRhightumors at 45 min (3.2 ± 0.5%ID/g). High specificity was observed with significantly lower uptake in EGFRlowtumors (0.9 ± 0.3%ID/g, p < 0.001), high tumor-to-background ratios (11 ± 6 tumor/muscle, p < 0.001). Nontargeted Gp2 tracer had low uptake in EGFRhightumors (0.5 ± 0.3%ID/g, p < 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 EGFRhightumor 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 in vivo 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.
authors.affiliation: Department of Chemical Engineering and Materials Science, ‡Department of Pharmacology, §Department of Medicine, and ∥Masonic Cancer Center, University of Minnesota-Twin Cities , 421 16th Avenue SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Max A
authors.initials: MA
authors.lastname: Kruziki
authors.affiliation: Department of Chemical Engineering and Materials Science, ‡Department of Pharmacology, §Department of Medicine, and ∥Masonic Cancer Center, University of Minnesota-Twin Cities , 421 16th Avenue SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Brett A
authors.initials: BA
authors.lastname: Case
authors.affiliation: Department of Chemical Engineering and Materials Science, ‡Department of Pharmacology, §Department of Medicine, and ∥Masonic Cancer Center, University of Minnesota-Twin Cities , 421 16th Avenue SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Jie Y
authors.initials: JY
authors.lastname: Chan
authors.affiliation: Department of Chemical Engineering and Materials Science, ‡Department of Pharmacology, §Department of Medicine, and ∥Masonic Cancer Center, University of Minnesota-Twin Cities , 421 16th Avenue SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Zudock
authors.affiliation: Department of Chemical Engineering and Materials Science, ‡Department of Pharmacology, §Department of Medicine, and ∥Masonic Cancer Center, University of Minnesota-Twin Cities , 421 16th Avenue SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Woldring
authors.affiliation: Department of Chemical Engineering and Materials Science, ‡Department of Pharmacology, §Department of Medicine, and ∥Masonic Cancer Center, University of Minnesota-Twin Cities , 421 16th Avenue SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Douglas
authors.initials: D
authors.lastname: Yee
authors.affiliation: Department of Chemical Engineering and Materials Science, ‡Department of Pharmacology, §Department of Medicine, and ∥Masonic Cancer Center, University of Minnesota-Twin Cities , 421 16th Avenue SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hackel
mesh_terms: D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D003301:Copper Radioisotopes / Q000737:chemistry*; D066246:ErbB Receptors / Q000737:chemistry*; D005260:Female; D005434:Flow Cytometry; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006801:Humans; D051379:Mice; D049268:Positron-Emission Tomography / Q000379:methods*; D014183:Transplantation, Heterologous
chemical_list: D003301:Copper Radioisotopes; D006573:Heterocyclic Compounds, 1-Ring; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D066246:ErbB Receptors
keywords: Gp2 domain protein scaffold; epidermal growth factor receptor imaging; micropositron emission tomography; murine model