Development of a Radiolabeled Irreversible Peptide Ligand for PET Imaging of Vascular Endothelial Growth Factor

J Nucl Med. 2014 Jun;55(6):1029-34. doi: 10.2967/jnumed.113.130898. Epub 2014 Apr 14.


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.

Conclusion: 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.

Keywords: PET; VEGF; covalent; molecular imaging; peptide.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Drug Design*
  • Drug Stability
  • HCT116 Cells
  • Humans
  • Isotope Labeling
  • Ligands
  • Mice
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / metabolism*
  • Peptides / pharmacokinetics
  • Positron-Emission Tomography / methods*
  • Tissue Distribution
  • Vascular Endothelial Growth Factor A / metabolism*


  • Ligands
  • Peptides
  • Vascular Endothelial Growth Factor A