Characterization of Structurally Diverse F-Labeled d-TCO Derivatives as a PET Probe for Bioorthogonal Pretargeted Imaging.

The pretargeted imaging strategy using inverse electron demand Diels-Alder (IEDDA) cycloaddition between a -cyclooctene (TCO) and tetrazine (Tz) has emerged and rapidly grown as a promising concept to improve radionuclide imaging and therapy in oncology. This strategy has mostly relied on the use of radiolabeled Tz together with TCO-modified targeting vectors leading to a rapid growth of the number of available radiolabeled tetrazines, while only a few radiolabeled TCOs are currently reported. Here, we aim to develop novel and structurally diverse F-labeled cis-dioxolane-fused TCO (d-TCO) derivatives to further expand the bioorthogonal toolbox for ligation and evaluate their potential for positron emission tomography (PET) pretargeted imaging. A small series of d-TCO derivatives were synthesized and tested for their reactivity against tetrazines, with all compounds showing fast reaction kinetics with tetrazines. A fluorescence-based pretargeted blocking study was developed to investigate the ligation of these compounds without labor-intensive prior radiochemical development. Two compounds showed excellent ligation results with blocking efficiencies of 95 and 97%. Two novel F-labeled d-TCO radiotracers were developed, from which [] showed good stability, favorable pharmacokinetics, and moderate stability. Micro-PET pretargeted imaging with [] in mice bearing LS174T tumors treated with tetrazine-modified CC49 monoclonal antibody (mAb) (CC49-Tz) showed significantly higher uptake in tumor tissue in the pretargeted group (CC49-Tz 2.16 ± 0.08% ID/mL) when compared to the control group with nonmodified mAb (CC49 1.34 ± 0.07% ID/mL). A diverse series of fast-reacting fluorinated d-TCOs were synthesized. A pretargeted blocking approach in tumor-bearing mice allowed the choice of a lead compound with fast reaction kinetics with Tz. A novel F-labeled d-TCO tracer was developed and used in a pretargeted PET imaging approach, allowing specific tumor visualization in a mouse model of colorectal cancer. Although further optimization of the radiotracer is needed to enhance the tumor-to-background ratios for pretargeted imaging, we anticipate that the F-labeled d-TCO will find use in studies where increased hydrophilicity and fast bioconjugation are required.