Development of a radiolabeled irreversible peptide ligand for PET imaging of vascular endothelial growth factor

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 ⁶⁴Cu-labeled affinity peptide tag, ⁶⁴Cu- L19K-(5-fluoro-2,4-dinitrobenzene) (⁶⁴Cu-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 ⁶⁴Cu-L19K- FDNB in VEGF-expressing tumor xenografts with its noncovalent binding analogs, ⁶⁴Cu-L19K-(2,4-dinitrophenyl) (⁶⁴Cu-L19K-DNP) and ⁶⁴Cu-L19K. Methods: The L19K peptide (GGNECDIARMWEWECFERK-CONH ₂) was constructed with 1,4,7-triazacyclononane-1,4,7-triacetic acid at the N terminus for radiolabeling with ⁶⁴Cu 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. ⁶⁴Cu-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 ⁶⁴Cu-L19K-FDNB, ⁶⁴Cu-L19K-DNP, and the native ⁶⁴Cu-L19K were compared in HCT-116 xenografts. Blocking studies of ⁶⁴Cu-L19K-FDNB was performed with a coinjection of excess unlabeled L19K-FDNB. Results: In vitro binding studies confirmed the covalent and irreversible binding of ⁶⁴Cu-L19K-FDNB to VEGF, whereas ⁶⁴Cu-L19K-DNP and ⁶⁴Cu-L19K did not bind covalently. PET imaging showed higher tumor uptake with ⁶⁴Cu-L19K-FDNB than with ⁶⁴Cu-L19K-DNP and ⁶⁴Cu-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 ⁶⁴Cu-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.