Adamantane/Cucurbituril: A Potential Pretargeted Imaging Strategy in Immuno-PET

Abstract

Positron emission tomography (PET) imaging with biological macromolecules greatly expands the possibilities of molecular imaging. There are, however, practical aspects limiting the potential of the approach, including the dosimetric consequences of the slow kinetics of radiolabeled biomacromolecules. Pretargeting strategies have led to impactful improvements in the field but are themselves limited by shortcomings of available bioconjugation methodology. We report our initial findings concerning the suitability of the adamantane/cucurbit[7]uril system for pretargeted immuno-PET imaging and provide proof-of-concept PET/computed tomography imaging experiments to establish the stability and rapid formation of host-guest complexes in vivo. The adamantane/cucurbit[7]uril system itself without antibody conjugation has shown remarkably fast association kinetics and clearance in vivo. We further demonstrate the modulation of biodistribution achievable by cucurbituril complexation with relevance for pharmaceutical formulation as well as the radiosynthetic access to relevant reporter molecules labeled with ¹¹C or ¹⁸F. This work, an early proof-of-concept, supports the notion that the adamantane/cucurbit[7]uril system warrants further exploration in pretargeted PET imaging applications.

Keywords: PET; antibody; bioconjugation; cucurbituril; imaging.

Figure 1.

Strategies in immuno-PET imaging. 1, Classic immuno-PET imaging employs antibodies bearing long-lived radioisotopes that match slow antibody kinetics, limiting its application due to poor dosimetry.^, 2, Pretargeting temporally and spatially decouples the antibody distribution and the radiovisualization steps. Different strategies can be employed, for example 2a. Biotinylated antibodies that are visualized after initial clearance with radiolabeled streptavidin or 2b. Trans-cyclooctene-tagged antibodies that are visualized with radiolabeled tetrazine derivatives via inverse electron demand diels alder reactions. 2c, We propose that cucurbituril-tagged antibodies can be visualized with radiolabeled adamantane derivatives, which could circumvent limitations inherent to other bioconjugation strategies. PET indicates positron emission tomography.

Figure 2.

Changes in brain penetrance of martinostat (1) in the presence of CB7. 1 Exhibits high brain uptake. When complexed by CB7 prior to administration (“precomplexed”), practically no radioactivity is observed in the brain. The effect can be reproduced by “pretreating” the animal with systemic CB7 prior to radiotracer administration. CB7 indicates cucurbit[7]uril.

Figure 3.

Biodistribution of 2 to 4 in the absence and presence of CB7. Three different ¹¹C-labeled molecules show distinct biodistribution in mice when administered individually. The left image column represents sagittal views of a 3-dimensional rendering of dynamic PET data averaged from 30 to 60 minutes postradiotracer injection overlaid on a CT of the same animal. 1 mg/mL of CB7 was used for precomplexed experiments. The transverse view to the right shows the same animal from a different perspective for clarity. In the right column of images, the distribution of the same ¹¹C-labeled molecules is shown when precomplexed with CB7. CB7 indicates cucurbit[7]uril; PET, positron emission tomography; CT, computed tomography.

Figure 4.

Fluorinated adamantane is a suitable reporter moiety. (A) Scheme of the radiosynthesis of fluoroadamantylamine 5 used for imaging; (B) change in HPLC retention behavior upon addition of CB7 to the tracer; (C) ratio of average radioactivity retained in rat kidneys over liver (averaged 30-60 minutes), which increases 4-fold when 5 is complexed by CB7 or when the animal is pretreated with CB7. HPLC indicates high-performance liquid chromatography; CB7, cucurbit[7]uril.