Improved Immuno-PET Imaging of HER2-Positive Tumors in Mice: Urokinase Injection-Triggered Clearance Enhancement of ⁶⁴Cu-Trastuzumab

Immuno-positron emission tomography (immuno-PET) is expected to improve the specificity of small chemical tracers such as ¹⁸F-fluorodeoxyglucose. Whole antibodies significantly accumulate in target molecule-expressing tumors but frequently persist too long in the blood circulation for imaging purposes. We investigated the utility of whole antibodies, ⁶⁴Cu-labeled via a urokinase-substrate linker, and their exogenous urokinase-responsive cleavage to enhance clearance of immuno-PET probes from the blood and shorten the time required to develop adequate imaging contrast. Specifically, we used ⁶⁴Cu-labeled trastuzumab in human epidermal growth factor receptor 2 (HER2)-positive tumor-bearing mice. ⁶⁴Cu-labeled trastuzumab with a urokinase-cleavage site (⁶⁴Cu-CB-TE1A1P-USL-trastuzumab) was synthesized using a bifunctional chelator incorporating an urokinase substrate peptide. Urokinase cleavage was analyzed in vitro by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and radio-gel permeation–high-performance liquid chromatography. Improvements in radioisotope clearance and HER2-imaging by urokinase injection were evaluated by PET imaging and ex vivo biodistribution studies in A431 tumor-bearing mice. ⁶⁴Cu-CB-TE1A1P-USL-trastuzumab was cleaved into smaller radioactive fragments by 20 000 IU/mL urokinase treatment in vitro at an efficacy of ∼95%. The probe targeted HER2 in A431 tumors in mice within 24 h post-injection, and approximately two-thirds of the probe in the blood circulation was eliminated via renal clearance of radioactive fragments after three urokinase injections. Therefore, the tumor/blood ratio increased 3.0-fold. Without urokinase injection, the tumor accumulation of ⁶⁴Cu-CB-TE1A1P-USL-trastuzumab slowly increased, and the blood radioactivity decreased over 72 h. However, the tumor/blood ratios in mice after three urokinase injections were higher at 24 h than those in mice without injections at 72 h. The results indicate that our approach shortened the time required to develop adequate imaging contrast of immuno-PET by >2 days. Therefore, this approach can benefit high-sensitivity imaging under lower radioactive decay conditions and can decrease patient radiation exposure. In addition, it could reduce other adverse effects of radioimmunotherapy.