Pretargeted Imaging Beyond the Blood-Brain-Barrier

Abstract

Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines. It reduces the radiation burden to healthy tissue. Pretargeting is based on bioorthogonal chemistry. The most attractive reaction for this purpose is currently the tetrazine ligation, which occurs between trans-cyclooctene (TCO) tags and tetrazines (Tzs). Pretargeted imaging beyond the blood-brain-barrier (BBB) has not been reported thus far. In this study, we developed Tz imaging agents that are capable to ligate in vivo to targets beyond the BBB. We chose to develop 18F-labeled Tzs as they can be applied to positron emission tomography (PET) - the most powerful molecular imaging technology. Fluorine-18 is an ideal radionuclide for PET due to its almost ideal decay properties. Fluorine-18 also allows - as a non-metal radionuclide - to develop Tzs with physicochemical properties enabling passive brain diffusion. In order to develop these imaging agents, we applied a rational drug design approach. This approach was based on estimated and experimental determined parameters such as the BBB score, pretargeted autoradiography contrast, in vivo input and washout curves as well as on metabolism studies. From initially 18 developed structures, five Tzs were selected to be tested on their in vivo click performance. Whereas all selected structures clicked in vivo into the brain, [18F]18 displayed the most favorable characteristics with respect to brain pretargeting. [18F]18 is our lead compound for future pretargeted imaging studies based on BBB-penetrant monoclonal antibodies. Pretargeting beyond the BBB will allow us to image targets beyond the BBB that are currently not imageable. For example, soluble protein isoforms could be imaged. These proteins are valuable drug targets for several neurodegenerative diseases and can currently not be imaged. Imaging would allow for diagnosis of these diseases, identifying responders from non-responders or to monitor treatment. Consequently, imaging will provide valuable information to accelerate drug development and greatly benefit patient care.

Pretargeted Imaging beyond the Blood–Brain Barrier—Utopia or Feasible?

“…Autoradiography is an imaging technique that allows for the visualization of radioligands on tissues of interest and thus to determine the imaging contrast of novel CNS radioligands, for example, on brain sections [ 60 , 61 ]. In order to determine the imaging contrast that is feasible to reach with pretargeting, we performed pretargeted autoradiography experiments according to our previously published pretargeted autoradiography methodology [ 52 ]. Brain sections from Tg-ArcSwe mice were used for these studies.…”

“…However, pretargeting beyond the BBB is not only dependent on the TCO concentration that can be reached within the brain, but also on the ability of the tetrazine to reach its target and be rapidly cleared from the brain tissue and the rest of the body. This is of special importance for Tzs labeled with fluorine-18 with a half-life of approximately 110 min, which we have developed previously [ 52 ]. Finally, the imaging contrast of pretargeted imaging is dependent on the efficient clearance of unbound TCO-modified antibody still circulating in the blood.…”

“…111 In-labeled Tz 2 was used for the titration of the TCO-modified antibodies ( Section S2 ) and for the preparation of [ 111 In]3D6scFv8D3 (next section). The labeling was performed as previously described (Scheme S3) [ 52 ]. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-PEG 11 -tetrazine ( 2 ) was dissolved (2 mg/mL) in metal-free water and stored at −80 °C before use.…”

“…Pretargeted autoradiography was performed according to the previously published procedure [ 52 ]. A schematic overview is provided in Section S5 .…”

“…For brain pretargeted imaging, the Tz must also enter the brain. Such Tzs have also been developed by us [ 52 ]. Usually, TCOs are conjugated to the Ab as this increases the TCO stability by reducing the isomerization rate to its unreactive cis-cyclooctene (CCO) form [ 45 , 53 , 54 ].…”