Design, synthesis, and evaluation of Trans-cyclooctene (TCO) and cis-dioxolane fused TCO (d-TCO) probes for bioorthogonal pretargeted PET imaging
Immuno-positron emission tomography (PET) imaging using radiolabeled monoclonal antibodies (mAbs) is a powerful tool with high specificity and sensitivity for cancer diagnosis and therapy response monitoring. However, slow mAb pharmacokinetics require the use of long-lived radioisotopes, resulting in suboptimal target-to-background ratios and high radiation exposure in patients. To address this, a pretargeting strategy based on the bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine was proposed. In this approach, an mAb-conjugated bioorthogonal tag is initially administered, allowing clearance from circulation. Subsequently, a radiolabeled reaction partner is injected, which covalently binds to the tag on the mAb. This allows the use of short-lived radioisotopes such as Fluorine-18, consequently minimizing radiation exposure while enhancing in vivo imaging contrast. The TCO-tetrazine ligation is a perfect candidate and has been extensively studied for this application. In this project, we focused on the development and characterization of fluorinated TCO and cis-dioxolane-fused TCO (d-TCO) derivatives as radiolabeled reaction partners with CC49 (anti-TAG-72) mAb-conjugated tetrazine tags for in vivo pretargeted imaging in mice bearing LS174T colorectal tumor xenografts. Based on previous studies, a novel [18F]FTCO derivative with a chelator linker was developed and evaluated in a mouse model of colorectal cancer xenografts. The 18F-labeled TCO derivative showed improved in vivo stability and demonstrated clear visualization of tumor tissue in the pretargeted group when compared to the control. Furthermore, a small library of d-TCO derivatives with enhanced reaction kinetics and diverse linkers was developed. Two of these compounds demonstrated excellent in vivo ligation in a pre-targeted blocking assay. Further radiochemical development and analysis revealed higher in vivo stability and favorable clearance of the 18F-labeled d-TCO derivative containing a squaramide linker. This radiotracer also showed a significant difference in tumor uptake compared with the control group in pretargeted imaging. Additionally, d-TCO derivatives were tested in a turn-on fluorescence-based cell permeability assay to visualize internalizing or intracellular targets. A d-TCO derivative containing a cyclobutyl fluoride linker exhibited excellent intracellular ligation in this assay. Although further fine-tuning of the linkers is required to obtain improved image contrast, this project demonstrates the potential of TCO- and d-TCO-based radiotracers in pretargeted PET imaging and provides insights for future probe development.
Antwerp : University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Department of Pharmaceutical Sciences , 2024
IX, 244 p.
Supervisor: Augustyns, Koen [Supervisor]
Supervisor: Elvas, Filipe [Supervisor]
Supervisor: Stroobants, Sigrid [Supervisor]
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Research group
Project info
Development of new TCO-probes and their evaluation for a novel pre-targeted intracellular PET imaging strategy.
Publication type
Publications with a UAntwerp address
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Creation 22.01.2024
Last edited 17.06.2024
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