Characterization of Structurally Diverse 18F-Labeled d-TCO Derivatives as a PET Probe for Bioorthogonal Pretargeted Imaging

Abstract

Background: The pretargeted imaging strategy using inverse electron demand Diels-Alder (IEDDA) cycloaddition between a trans-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 ¹⁸F-labeled cis-dioxolane-fused TCO (d-TCO) derivatives to further expand the bioorthogonal toolbox for in vivo ligation and evaluate their potential for positron emission tomography (PET) pretargeted imaging. Results: 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 in vivo ligation of these compounds without labor-intensive prior radiochemical development. Two compounds showed excellent in vivo ligation results with blocking efficiencies of 95 and 97%. Two novel ¹⁸F-labeled d-TCO radiotracers were developed, from which [¹⁸F]MICA-214 showed good in vitro stability, favorable pharmacokinetics, and moderate in vivo stability. Micro-PET pretargeted imaging with [¹⁸F]MICA-214 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). Conclusions: 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 ¹⁸F-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 ¹⁸F-labeled d-TCO will find use in studies where increased hydrophilicity and fast bioconjugation are required.

Scheme 1. Synthesis of d-TCO Derivatives

Figure 1

(A) Procedure followed for the in vivo pretargeted blocking approach. (B) In vivo representative fluorescence image of TCO-Cy5 uptake in LS174T tumor-bearing mice. Images were acquired 24 h post-TCO-Cy5 injection, and the fluorescence signal was quantified as total efficiency. The white dashed line encircles the tumor. (C) Tumor uptake of TCO-Cy5 in vivo and ex vivo was expressed as total efficiency in the blocked group. Data were normalized to TCO-Cy5 uptake in positive and negative control groups. (mean ± SD, n = 4/group).

Scheme 2. Precursor Synthesis and Radiosynthesis of [¹⁸F]MICA-214 and [¹⁸F]MICA-215

Figure 2

Biodistribution of (A) [¹⁸F]MICA-214 and (B) [¹⁸F]MICA-215 in naïve BALB/c mice (mean ± SD, n = 3/group).

Figure 3

(A) In vivo representative coronal μPET/CT image of LS174T tumor-bearing mice injected with [¹⁸F]MICA-214 72 h post-CC49 (control) or CC49-Tz (pretargeted) injection. PET images represent the averaged tracer distribution from the dynamic scans (0–60 min). The white dashed lines encircle the tumor region, and the arrows represent the bladder (B), intestines (I), and liver (L). (B) Time–activity curve of tumor uptake of [¹⁸F]MICA-214 in groups injected with CC49-Tz and control group injected with CC49 (0 to 60 min) (mean ± SD, n = 4/group). (C) Ex vivo biodistribution of LS174T tumor-bearing mice 60 min post tracer injection.