Radiolabelling and Evaluation of a ⁴⁴Sc-labelled DOTA Tetrazine for Pretargeted PET Imaging

Objectives: Pretargeted imaging of slow clearing nanomedicines allows for the use of short lived isotopes thus reducing the absorbed radiation dose in healthy tissues when compared to direct targeting methods. The inverse electron demand Diels-Alder (iEDDA) reaction between a radiolabelled tetrazine (Tz) and a trans-cyclooctene (TCO) functionalized targeting vector has been established as a promising tool for pretargeted imaging. The first example of this strategy used an ¹¹¹In-labelled DOTA-Tz to image TCO functionalized antibodies in murine tumour models. This DOTA-Tz has since been applied in pretargeted experiments using TCO functionalized affibodies and bisphosphonates (BPs). A ¹⁷⁷Lu labelled analogue has also been developed and used to demonstrate the reduced radiation dose in a preclinical model. A PET based analogue is desired as compliment to these already established SPECT and therapeutic agents. Herein we report ⁴⁴Sc-labelling of the DOTA-Tz and its use as a pretargeting imaging agent with TCO-functionalized BPs in healthy rodents.

Methods: ⁴⁴Sc was obtained from an in house ⁴⁴Ti/⁴⁴Sc generator. The 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) functionalized tetrazine was radiolabelled with ⁴⁴Sc giving [⁴⁴Sc]Sc-DOTA-Tz in RCYs of 25-27% (n = 2). One group of healthy Wister rats (n = 3) were administered i.v. with 0.6 mg (3 mg/kg) of TCO functionalized alendronic acid (BP-TCO). 1 h later 10 MBq of [⁴⁴Sc]Sc-DOTA-Tz was administered. As a negative control one rat was pre-treated with saline prior to the administration of [⁴⁴Sc]Sc-DOTA-Tz. A dynamic PET/CT scan focusing on the shoulder region was performed for 90 min p.i. followed by whole body PET/CT at 90 min p.i. Ex vivo biodistribution studies were performed 2 h p.i.

Results: The dynamic PET scan revealed that the [⁴⁴Sc]Sc-DOTA-Tz accumulated in the shoulders within the first minutes of the study and remained stable throughout the time course (SUV_Humerus = 0.2). Despite high accumulation in the kidneys (5% ID/g) and the urine (22% ID/g) visualization of the uptake in the shoulder and knees was still possible with PET. Ex vivo analysis revealed higher radioactivity accumulation in the left femur (0.26 ± 0.05% ID/g) and left humerus (0.29 ± 0.03% ID/g) in the rats pre-treated with BP-TCO when compared to the untreated rat (0.02% ID/g in both left humerus and femur).

Conclusions: In this pilot study we have prepared a ⁴⁴Sc labelled-DOTA-Tz and used it in pretargeted PET imaging. The BP-TCO allowed for the evaluation of the iEDDA reaction with a radiolabelled Tz in a healthy rats. Although the overall uptake in the targeted tissue was lower than the previously reported direct targeting method (e.g. 3% ID/g) the pretargeted method resulted in good tumour visualization. Non-specific binding was low as there was a 100-fold higher uptake in the pre-treated rats compared to the non-treated control. Since the iEDDA reaction product forms early and remains stable over the course of the experiment, the imaging time can be optimized to match the clearance profile of the unreacted tetrazine, making scandium-44 (t_1/2 = 3.97 h) is suitable PET isotope for this endeavour.