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

Abstract

Pretargeting is a promising nuclear imaging technique that allows for the usage of antibodies (Abs) with enhanced imaging contrast and reduced patient radiation burden. It is based on bioorthogonal chemistry with the tetrazine ligation—a reaction between trans-cyclooctenes (TCOs) and tetrazines (Tzs)—currently being the most popular reaction due to its high selectivity and reactivity. As Abs can be designed to bind specifically to currently ‘undruggable’ targets such as protein isoforms or oligomers, which play a crucial role in neurodegenerative diseases, pretargeted imaging beyond the BBB is highly sought after, but has not been achieved yet. A challenge in this respect is that large molecules such as Abs show poor brain uptake. Uptake can be increased by receptor mediated transcytosis; however, it is largely unknown if the achieved brain concentrations are sufficient for pretargeted imaging. In this study, we investigated whether the required concentrations are feasible to reach. As a model Ab, we used the bispecific anti-amyloid beta (Aβ) anti-transferrin receptor (TfR) Ab 3D6scFv8D3 and conjugated it to a different amount of TCOs per Ab and tested different concentrations in vitro. With this model in hand, we estimated the minimum required TCO concentration to achieve a suitable contrast between the high and low binding regions. The estimation was carried out using pretargeted autoradiography on brain sections of an Alzheimer’s disease mouse model. Biodistribution studies in wild-type (WT) mice were used to correlate how different TCO/Ab ratios alter the brain uptake. Pretargeted autoradiography showed that increasing the number of TCOs as well as increasing the TCO-Ab concentration increased the imaging contrast. A minimum brain concentration of TCOs for pretargeting purposes was determined to be 10.7 pmol/g in vitro. Biodistribution studies in WT mice showed a brain uptake of 1.1% ID/g using TCO-3D6scFv8D3 with 6.8 TCO/Ab. According to our estimations using the optimal parameters, pretargeted imaging beyond the BBB is not a utopia. Necessary brain TCO concentrations can be reached and are in the same order of magnitude as required to achieve sufficient contrast. This work gives a first estimate that pretargeted imaging is indeed possible with antibodies. This could allow the imaging of currently ‘undruggable’ targets and therefore be crucial to monitor (e.g., therapies for intractable neurodegenerative diseases).

Recent Advances in Bioorthogonal Click Chemistry for Enhanced PET and SPECT Radiochemistry

“…A 18 F-labeled bispyridyl tetrazine with a satisfactory RCY (16%), molar activity (57 GBq/μmol), and high radiochemical purity (RCP, over 98%) was developed and chosen by Garca-Vázquez and co-workers via aliphatic nucleophilic substitution, displaying a favorable target-to-background ratio compared with the previously published radiotracers . In addition, Herth’s group revealed that a radiolabeled orthogonal tetrazine [ 18 F]­F-Tz with slow metabolism and high lipophilicity was chosen from a series of 18 F-labeled tetracyclines based on the basis of their structure–activity connection . The authors also examined the possibility of pretargeted imaging using 18 F-labeled tetrazine by optimizing labeling parameters, such as elution conditions, precursor and catalyst amounts, reaction time, and temperature …”

“…75 In addition, Herth's group revealed that a radiolabeled orthogonal tetrazine [ 18 F]F-Tz with slow metabolism and high lipophilicity was chosen from a series of 18 F-labeled tetracyclines based on the basis of their structure−activity connection. 76 The authors also examined the possibility of pretargeted imaging using 18 Flabeled tetrazine by optimizing labeling parameters, such as elution conditions, precursor and catalyst amounts, reaction time, and temperature. 77 In conclusion, the selection of radiolabeled reactive scaffolds may facilitate the creation of peptide-or protein-based radiotracers.…”

The inverse electron demand Diels–Alder cycloaddition with carbon‐11 and fluorine‐18: A gateway to pretargeted imaging across the blood–brain barrier

“…Shalgunov et al 55 have recently reported tetrazine [ 18 F] 56 , which shows many of these ideal properties. The same group has also recently reported the determination of the minimum concentration of TCO‐labelled antibodies required for successful pretargeting in the brain and showed that this concentration can be achieved using antibodies transported across the BBB using the transferrin receptor 65 . The community eagerly awaits further validation of their system in a more complex, disease relevant pretargeting model.…”

Applications of radiolabeled antibodies in neuroscience and neuro‐oncology

“…28,[97][98][99] This approach has been used preclinically, including for PET imaging of amyloid-β in the brains of transgenic mice. 99,100 Interestingly, a related pre-targeting strategy has also recently been used with 64 Cu-labeled tetrazines and TCO-functionalized antibodies, even though copper-64 has a longer half-life (12.7 h). 101 However, to the best of our knowledge, the tetrazine/TCO pre-targeting approach has not yet been used clinically for CNS targets, and general questions remain about whether separate pharmacology/toxicology studies are going to be required for each component of a pre-targeting cocktail prior to clinical translation.…”

“…There are numerous ways pre‐targeting has been accomplished, such as biorthogonal click reactions between 18 F‐labeleled tetrazines and trans‐cyclooctyne (TCO)‐functionalized antibodies 28,97–99 . This approach has been used preclinically, including for PET imaging of amyloid‐β in the brains of transgenic mice 99,100 . Interestingly, a related pre‐targeting strategy has also recently been used with 64 Cu‐labeled tetrazines and TCO‐functionalized antibodies, even though copper‐64 has a longer half‐life (12.7 h) 101 .…”