Abstract by Sara Irene Lopes van den Broek

Nanomedicines for nuclear molecular imaging has gained great popularity over the last decades. This is due to the unique properties that nanomedicines provide, such as high target selectivity and affinity. This allows for high quality nuclear imaging, which can be used for diagnosis, disease monitoring and treatment. However, the application of nanomedicines as radioligands is hampered by their slow pharmacokinetics. Next to this, the poor blood-brain barrier (BBB) permeability of these large molecules makes their use as imaging agents for neurological conditions challenging. Several strategies have been investigated to increase the BBB uptake of nanomedicines into the brain. Transport mechanisms, mainly receptor-mediated transcytosis (RMT), have been exploited, and the first bispecific antibody is currently investigated in the clinic. More recently, temporary disruption of the BBB have been investigated. Effectiveness of focused ultrasound (FUS) for BBB disruption in patients was published in 2021.

The slow pharmacokinetics of nanomedicines results in slow target accumulation and blood clearance. Long-lived radioisotopes are therefore needed when conventional imaging techniques are used. This reduces the imaging quality and increases the patient radiation burden. Pretargeting is a novel strategy where a tagged nanomedicine is administered first and allowed to accumulate at its target, a process which typically takes days. Next, a radiolabeled small molecule is administered which reacts bioorthogonally to the tagged nanomedicine and rapidly clears from the blood. This allows the use of short-lived radioisotopes which results in higher target-to-background ratios and reduced radiation burden. The most attractive bioorthogonal reaction for pretargeting is the tetrazine ligation. This reaction between trans-cyclooctenes (TCOs) and tetrazines (Tzs) exhibits fast reaction kinetics, selectivity and has already been successfully applied in vivo.

This first part of this thesis focused on pretargeting beyond the BBB. BBB-permeable tetrazines radiolabeled with fluorine-18 – the most commonly applied radionuclide for positron emission tomography (PET) imaging – were developed and a lead candidate for brain pretargeting was developed. TCO-modified antibodies (Abs) targeting amyloid beta for Alzheimer’s disease diagnosis were successfully visualized with pretargeted autoradiography. Properties - e.g. TCO-loading and required Ab concentration - of these TCO-Abs were assessed in vitro. Ex vivo biodistribution showed 1.1% brain uptake of these TCO-Abs which allows to obtain higher TCO concentrations in the brain than was needed for in vitro pretargeted autoradiography. The pretargeted autoradiography methodology was also applied to compare three different animal models: APP/PS1, 5xFAD, and Tg-ArcSwe. All models showed high cortex uptake, but APP/PS1 also showed uptake in other tissues, such as the cerebellum. 5xFAD and Tg-ArcSwe mice are therefore considered more suitable for pretargeting of amyloid beta.

The tetrazine ligation can also be exploited as an in vitro radiolabeling strategy for nanomedicines. This was investigated in the second part of this thesis. Nanobody W25 and Fc-fused W25 (W25Fc) for neutralization of COVID-19 were developed and successfully radiolabeled using tetrazine ligation based click chemistry. Ex vivo biodistribution with ¹¹¹In-labeled W25Fc was conducted. W25 and W25Fc were radiolabeled with fluorine-18 to serve as PET radioligands. Finally, a new method for site-specific TCO-modifications using the free cysteine residue of human serum albumin was developed. Successful conjugation was achieved and reactivity of the TCO-moiety after conjugation was confirmed with cold Tz and ¹¹¹In-labeled Tz.

In summary, BBB-permeable ¹⁸F-Tzs were developed and TCO-Abs and mouse models for AD were evaluated in this thesis which should bring pretargeting within the brain closer to the clinic. Additionally, we have showed successful in vitro radiolabeling of nanomedicines by tetrazine ligation and applied this in vivo.