title: Development of a Radiolabeled Irreversible Peptide Ligand for PET Imaging of Vascular Endothelial Growth Factor.
abstract: Imaging agents based on peptide probes have desirable pharmacokinetic properties provided that they have high affinities for their target in vivo. An approach to improve a peptide ligand's affinity for its target is to make this interaction covalent and irreversible. For this purpose, we evaluated a (64)Cu-labeled affinity peptide tag, (64)Cu-L19K-(5-fluoro-2,4-dinitrobenzene) ((64)Cu-L19K-FDNB), which binds covalently and irreversibly to vascular endothelial growth factor (VEGF) as a PET imaging agent. We compared the in vivo properties of (64)Cu-L19K-FDNB in VEGF-expressing tumor xenografts with its noncovalent binding analogs, (64)Cu-L19K-(2,4-dinitrophenyl) ((64)Cu-L19K-DNP) and (64)Cu-L19K.
  
  
  METHODS
  The L19K peptide (GGNECDIARMWEWECFERK-CONH2) was constructed with 1,4,7-triazacyclononane-1,4,7-triacetic acid at the N terminus for radiolabeling with (64)Cu with a polyethylene glycol spacer between peptide and chelate. 1,5-difluoro-2,4-dinitrobenzene was conjugated at the C-terminal lysine for cross-linking to VEGF, resulting in L19K-FDNB. (64)Cu-L19K-FDNB was assayed for covalent binding to VEGF in vitro. As a control, L19K was conjugated to 1-fluoro-2,4-dinitrobenzene, resulting in L19K-DNP. PET imaging and biodistribution studies of (64)Cu-L19K-FDNB, (64)Cu-L19K-DNP, and the native (64)Cu-L19K were compared in HCT-116 xenografts. Blocking studies of (64)Cu-L19K-FDNB was performed with a coinjection of excess unlabeled L19K-FDNB.
  
  
  RESULTS
  In vitro binding studies confirmed the covalent and irreversible binding of (64)Cu-L19K-FDNB to VEGF, whereas (64)Cu-L19K-DNP and (64)Cu-L19K did not bind covalently. PET imaging showed higher tumor uptake with (64)Cu-L19K-FDNB than with (64)Cu-L19K-DNP and (64)Cu-L19K, with mean standardized uptake values of 0.62 ± 0.05, 0.18 ± 0.06, and 0.34 ± 0.14, respectively, at 24 h after injection (P < 0.05), and 0.53 ± 0.05, 0.32 ± 0.14, and 0.30 ± 0.09, respectively, at 48 h after injection (P < 0.05). Blocking studies with (64)Cu-L19K-FDNB in the presence of excess unlabeled peptide showed a 53% reduction in tumor uptake at 48 h after injection.
  
  
  CONCLUSIONS
  In this proof-of-concept study, the use of a covalent binding peptide ligand against VEGF improves tracer accumulation at the tumor site in vivo, compared with its noncovalent binding peptide analogs. This technique is a promising tool to enhance the potency of peptide probes as imaging agents.
authors.affiliation: Department of Radiology, Washington University School of Medicine, Saint Louis, Missouri.
authors.forename: Bernadette V
authors.initials: BV
authors.lastname: Marquez
authors.affiliation: Department of Radiology, Washington University School of Medicine, Saint Louis, Missouri.
authors.forename: Oluwatayo F
authors.initials: OF
authors.lastname: Ikotun
authors.affiliation: Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, Missouri; and.
authors.forename: Jesse J
authors.initials: JJ
authors.lastname: Parry
authors.affiliation: Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, Missouri; and.
authors.forename: Buck E
authors.initials: BE
authors.lastname: Rogers
authors.affiliation: Department of Chemistry, University of California, Davis, California.
authors.forename: Claude F
authors.initials: CF
authors.lastname: Meares
authors.affiliation: Department of Radiology, Washington University School of Medicine, Saint Louis, Missouri lapis@mir.wustl.edu.
authors.forename: Suzanne E
authors.initials: SE
authors.lastname: Lapi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015195:Drug Design; D004355:Drug Stability; D045325:HCT116 Cells; D006801:Humans; D007553:Isotope Labeling; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D049268:Positron-Emission Tomography / Q000379:methods*; D014018:Tissue Distribution; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D008024:Ligands; D010455:Peptides; D042461:Vascular Endothelial Growth Factor A
keywords: PET; VEGF; covalent; molecular imaging; peptide