title: Use of (64)Cu-labeled fibronectin domain with EGFR-overexpressing tumor xenograft: molecular imaging.
abstract: OBJECTIVE
  To assess the ability of an engineered epidermal growth factor receptor (EGFR)-binding fibronectin domain to serve as a positron emission tomographic (PET) probe for molecular imaging of EGFR in a xenograft mouse model.
  
  
  METHODS
  An EGFR-binding fibronectin domain (fibronectin abbreviated to Fn when bound) was site-specifically labeled with copper 64 ((64)Cu) (8 MBq/nmol). Copper 64-Fn binding was tested in cell cultures with varying EGFR expression. Stability in human and mouse serum was measured in vitro. Animal experiments were approved by the Stanford University Institutional Animal Care and Use Committee. Copper 64-Fn (approximately 2 MBq) was used for PET in mice (n = 5) bearing EGFR-overexpressing xenografted tumors (approximately 5-10 mm in diameter). Results of tomography were compared with those of ex vivo gamma counting of dissected tissues. Statistical analysis was performed with t tests and adjustment for multiple comparisons.
  
  
  RESULTS
  Copper 64-Fn exhibited EGFR-dependent binding to multiple cell lines in culture. The tracer was stable for 24 hours in human and mouse serum at 37°C. The tracer exhibited good tumor localization (3.4% injected dose [ID]/g ± 1.0 [standard deviation] at 1 hour), retention (2.7% ID/g ± 0.6 at 24 hours), and specificity (8.6 ± 3.0 tumor-to-muscle ratio, 8.9 ± 4.7 tumor-to-blood ratio at 1 hour). Specific targeting was verified with low localization to low-expressing MDA-MB-435 tumors (0.7% ID/g ± 0.8 at 1 hour, P = .018); specificity was further demonstrated, as a nonbinding control fibronectin had low localization to EGFR-overexpressing xenografts (0.8% ID/g ± 0.2 at 1 hour, P = .013).
  
  
  CONCLUSIONS
  The stability, low background, and target-specific tumor uptake and retention of the engineered fibronectin domain make it a promising EGFR molecular imaging agent. More broadly, it validates the fibronectin domain as a potential scaffold for a generation of various molecular imaging agents.
authors.affiliation: Department of Radiology and Molecular Imaging Program, Stanford University, Stanford, CA 94305, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hackel
authors.forename: Richard H
authors.initials: RH
authors.lastname: Kimura
authors.forename: Sanjiv S
authors.initials: SS
authors.lastname: Gambhir
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000493:pharmacokinetics; D002277:Carcinoma / Q000000981:diagnostic imaging*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003301:Copper Radioisotopes / Q000737:chemistry / Q000493:pharmacokinetics; D004355:Drug Stability; D066246:ErbB Receptors / Q000276:immunology*; D005260:Female; D005353:Fibronectins; D005434:Flow Cytometry; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000000981:diagnostic imaging*; D051379:Mice; D008819:Mice, Nude; D010051:Ovarian Neoplasms / Q000000981:diagnostic imaging*; D049268:Positron-Emission Tomography; D011829:Radiation Dosage; D018709:Statistics, Nonparametric; D014018:Tissue Distribution; D014183:Transplantation, Heterologous; D055114:X-Ray Microtomography
chemical_list: D000911:Antibodies, Monoclonal; D003301:Copper Radioisotopes; D005353:Fibronectins; D066246:ErbB Receptors