Head-to-Head Comparison of the in Vivo Performance of Highly Reactive and Polar 18F-Labeled Tetrazines

Abstract

Pretargeted imaging harnessing tetrazine ligation has gained increased interest over recent years. Targeting vectors with slow pharmacokinetics may be visualized using short-lived radionuclides, such as fluorine-18 (¹⁸F) for positron emission tomography (PET), and result in improved target-to-background ratios compared to conventionally radiolabeled slowly accumulating vectors. We recently developed different radiochemical protocols enabling the direct radiofluorination of various tetrazine scaffolds, resulting in the development of various highly reactive and polar ¹⁸F-labeled tetrazines as lead candidates for pretargeted imaging. Here, we performed a direct head-to-head-comparison of our lead candidates to evaluate the most promising for future clinical translation. For that, all ¹⁸F-labeled tetrazine-scaffolds were synthesized in similar molar activity for improved comparability of their in vivo pretargeting performance. Intriguingly, previously reported dicarboxylic acid lead candidates with a net charge of -1 were outperformed by respective monocarboxylic acid derivatives bearing a net charge of 0, warranting further evaluation of such scaffolds prior to their clinical translation.

Keywords: bioorthogonal chemistry; fluorine-18; phenyl-tetrazines; pretargeting; tetrazine ligation.

Figure 1

(A) The tetrazine–TCO ligation. (B) Chemical structures of the ¹⁸F-tetrazines evaluated in this study.

Figure 2

Radiosynthesis of [¹⁸F]1–5 prior to their injection into LS174T xenograft nude mice. (A) Radiosynthesis of [¹⁸F]1–5. (B) Analytical HPLC of reference compounds 1–5 (UV/vis 254 nm; R_t = 1, 4.910 min; 2, 4.650 min; 3, 4.720 min; 4, 4.927; 5: 6.223 min) (dashed graphs) and radio-HPLC of the formulated [¹⁸F]1–5 (R_t(RCP) = [¹⁸F]1, 5.020 min (>97%); [¹⁸F]2, 4.767 min (>90%^a); [¹⁸F]3, 4.853 min (>96%); [¹⁸F]4, 5.057 min (>96%); [¹⁸F]5, 6.337 min (>97%)) (black graphs). (C) Radio-HPLC of the formulated [¹⁸F]1–5 before (dashed graph) and after (black graph) the addition of TCO-PNB ester (performed 20–30 min after formulation). ^aRCP of [¹⁸F]2 lower than observed for previous syntheses and purifications. HPLC conditions: Luna 5 μm C18(2) 100 Å, 150 mm × 4.6 mm eluted with a gradient of ACN with 0.1% v/v TFA (solvent B) in water with 0.1% v/v TFA (solvent B) at 2 mL/min. Gradient: 0–1 min, 0% B; 1–11 min, 0–100% B; 11–12 min, 100% B; 12–13 min, 100–0% B; 13–15 min, 0% B.

Figure 3

PET/CT scan of CC49-TCO pretargeted [¹⁸F]1–5 in nude mice bearing LS174T xenografts. (A) Structures investigated. (B) PET-image derived mean tumor uptake in CC49 and CC49-TCO pretargeted. (C) Representative PET images 3 h after injection. Arrows indicate tumors. (D) Tumor-to-muscle (T/M), tumor-to-abdomen (T/A), and tumor-to-blood (T/B) ratios.

Figure 4

Ex vivo tumor biodistribution conducted after final scan (3 h after injection). For each compound, scanned animals were either dissected immediately or perfused with saline prior to dissection.