Improved PET imaging of uPAR expression using new (64)Cu-labeled cross-bridged peptide ligands: comparative in vitro and in vivo studies

Theranostics. 2013 Aug 3;3(9):618-32. doi: 10.7150/thno.6810. eCollection 2013.


The correlation between uPAR expression, cancer cell invasion and metastases is now well-established and has prompted the development of a number of uPAR PET imaging agents, which could potentially identify cancer patients with invasive and metastatic lesions. In the present study, we synthesized and characterized two new cross-bridged (64)Cu-labeled peptide conjugates for PET imaging of uPAR and performed a head-to-head comparison with the corresponding and more conventionally used DOTA conjugate. Based on in-source laser-induced reduction of chelated Cu(II) to Cu(I), we now demonstrate the following ranking with respect to the chemical inertness of their complexed Cu ions: DOTA-AE105 << CB-TE2A-AE105 < CB-TE2A-PA-AE105, which is correlated to their corresponding demetallation rate. No penalty in the uPAR receptor binding affinity of the targeting peptide was encountered by conjugation to either of the macrobicyclic chelators (IC50 ~ 5-10 nM) and high yields and radiochemical purities (>95%) were achieved in all cases by incubation at 95ºC. In vivo, they display identical tumor uptake after 1h, but differ significantly after 22 hrs, where the DOTA-AE105 uptake remains surprisingly high. Importantly, the more stable of the new uPAR PET tracers, (64)Cu-CB-TE2A-PA-AE105, exhibits a significantly reduced liver uptake compared to (64)Cu-DOTA-AE105 as well as (64)Cu-CB-TE2A-AE105, (p<0.0001), emphasizing that our new in vitro stability measurements by mass spectrometry predicts in vivo stability in mice. Specificity of the best performing ligand, (64)Cu-CB-TE2A-PA-AE105 was finally confirmed in vivo using a non-binding (64)Cu-labeled peptide as control ((64)Cu-CB-TE2A-PA-AE105(mut)). This control PET-tracer revealed significantly reduced tumor uptake (p<0.0001), but identical hepatic uptake compared to its active counterpart ((64)Cu-CB-TE2A-PA-AE105) after 1h. In conclusion, our new approach using in-source laser-induced reduction of Cu(II)-chelated PET-ligands provides useful information, which are predictive for the tracer stability in vivo in mice. Furthermore, the increased stability of our new macrobicyclic (64)Cu-CB-TE2A-PA-AE105 PET ligand is paralleled by an excellent imaging contrast during non-invasive PET scanning of uPAR expression in preclinical mouse cancer models. The translational promises displayed by this PET-tracer for future clinical cancer patient management remains, however, to be investigated.

Keywords: CB-TE2A; CD87; Ly-6; SPR; biomarker; positron emission tomography; tumor targeting.; urokinase-type plasminogen activator receptor.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers, Tumor / analysis*
  • Cell Line, Tumor
  • Copper / metabolism*
  • Humans
  • Ligands
  • Mice
  • Mice, Nude
  • Neoplasms / diagnosis*
  • Peptides / metabolism*
  • Positron-Emission Tomography / methods*
  • Receptors, Urokinase Plasminogen Activator / analysis*
  • Staining and Labeling / methods*


  • Biomarkers, Tumor
  • Ligands
  • Peptides
  • Receptors, Urokinase Plasminogen Activator
  • Copper