Pharmacokinetic Optimization of Radiocopper-Based Theranostic Pretargeting

In vivo pretargeting offers a strategy to improve nuclear imaging and radiopharmaceutical therapy by increasing tumor-to-background activity concentration ratios and decreasing radiation burden to healthy tissues. One particularly promising approach to in vivo pretargeting is predicated on the inverse electron-demand Diels-Alder (IEDDA) ligation between tetrazine (Tz)-based radioligands and <i>trans</i>-cyclooctene (TCO)-bearing immunoconjugates. Not surprisingly, the performance of such systems is highly dependent upon the pharmacokinetic profiles of the small molecule radioligands. Herein, we report the synthesis and characterization of a trio of sarcophagine-bearing tetrazines─SarAr-Tz, SarAr-PEG₅-Tz, and SarAr-PEG₁₀-Tz─as well as their radiolabeling with copper-64 (⁶⁴Cu, <i>t</i>_1/2 ∼ 12.7 h), a positron-emitting radioisotope of copper. These radioligands were paired with a TCO-bearing variant of the A33 antigen-targeting antibody huA33 (i.e., huA33-TCO) for pretargeted immunoPET in a murine model of colorectal cancer, revealing that all three produced images with excellent tumor-to-background contrast, but [⁶⁴Cu]Cu-SarAr-PEG₁₀-Tz yielded the best tumor-to-tissue activity concentration ratios. In light of its superior performance, SarAr-PEG₁₀-Tz was subsequently radiolabeled with copper-67 (⁶⁷Cu, <i>t</i>_1/2 ∼ 61.8 h), a β^--emitting radioisotope of copper, to produce [⁶⁷Cu]Cu-SarAr-PEG₁₀-Tz. This radioligand was then paired with huA33-TCO for in vivo biodistribution and longitudinal therapy studies, ultimately revealing that pretargeted radioimmunotherapy with [⁶⁷Cu]Cu-SarAr-PEG₁₀-Tz exhibits promising efficacy and safety in a murine model of colorectal cancer.