A Bioorthogonal TCO–Tetrazine-Based Pretargeted PET/NIRF Platform Enabling High-Contrast Tumor Imaging

Objectives: Pretargeting strategies enhance the specificity and safety of radiopharmaceuticals by separating tumor targeting from radionuclide delivery. To address the rapid clearance and systemic exposure of directly labeled small-molecule agents, a DZ-1–based pretargeting system was developed, utilizing its broad-spectrum tumor-targeting characteristics. Methods: Three DZ-TCO precursors (DZ-1-TCO, DZ-Lys-TCO, and DZ-Lys-PEG4-TCO) were synthesized and evaluated by near-infrared fluorescence imaging in HeLa and U87MG tumor-bearing mice. Two tetrazine probes (methyl-tetrazine and mono-substituted tetrazine) were labeled with 68Ga to yield 68Ga-DOTA-Me-Tz and 68Ga-DOTA-H-Tz, whose stability was assessed in PBS and serum. Pretargeted PET imaging was performed using different precursor/probe combinations and pretargeting intervals (24, 48, and 72 h). Results: All precursors exhibited tumor accumulation peaking at 24 h and signal retention up to 96 h. Both 68Ga-DOTA-Me-Tz and 68Ga-DOTA-H-Tz maintained >85% radiochemical stability after 4 h. PET imaging identified DZ-Lys-TCO as the most effective precursor (1.98 ± 0.72 %ID/g, T/M 3.86 ± 0.91). Using 68Ga-DOTA-H-Tz, the 48 h interval achieved optimal uptake (3.24 ± 0.95 %ID/g) with the highest tumor-to-muscle ratio (8.30 ± 3.39). Biodistribution confirmed rapid renal clearance, low off-target accumulation, and peak tumor uptake of 3.53 ± 1.76 %ID/g (T/M 10.9 ± 0.3 at 30 min). Conclusions: The DZ-TCO/68Ga-DOTA-Tz pretargeting system enables high-contrast tumor imaging with low background. The combination of DZ-Lys-TCO and 68Ga-DOTA-H-Tz at a 48 h interval provides optimal performance, representing a promising platform for precise and safe radiopharmaceutical imaging.