HAI Book 2025 - Flipbook - Page 444
Honer, Michael
115
Advancing antibody-based pretargeted PET imaging of protein
aggregates in the brain
Tobias Gustavsson1, Michael Honer5, Lars Hvass2, Vladimir Shalgunov3, Anne Skovsbo
Clausen2, Sophie Stotz1, Thomas Wünsche1, Sara Lopes van den Broek1, Gitte Moos Knudsen4,
Blanca Aldana1, Jens Niewoehner5, Luca Gobbi5, Thomas Kustermann5, Umberto Battisti1,
Andreas Kjær2,3, Matthias M. Herth1,3
1
Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, DK
Cluster for Molecular Imaging, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, DK
3
Department of Clinical Physiology, Nuclear Medicine & PET, Rigshospitalet, Copenhagen, DK
4
Neurobiology Research Unit and Center for Integrated Molecular Brain Imaging, Rigshospitalet, Copenhagen, DK
5
Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, CH
2
The discovery of small molecule PET tracers for certain forms of protein aggregates has proven challenging.
Monoclonal antibodies (mAbs) with their excellent specificity along with improved blood-brain barrier (BBB)
penetration upon modification may offer an alternative approach. In particular, the pretargeted imaging strategy,
uncoupling the long pharmacokinetic half-life of mAbs from the shorter physical half-life of the radionuclide, may
result in higher imaging contrast and lower radiation burden. Several pretargeting approaches are based on
bioorthogonal reactions, the tetrazine (Tz) ligation between a trans-cyclooctene (TCO) and a Tz being the most
prominent one.
We evaluated whether an in-house developed BBB-penetrable Tz ([18F]BBB-Tz) with rapid TCO ligation kinetics
can be used for imaging A´ pathology in the 5xFAD mouse model using a TCO-modified bispecific construct of the
antibody BS-mAb31 (TCO-bsAb), which targets both A´ fibrils and the mouse transferrin receptor (mTfR).
TCO-bsAb (10 TCOs per bsAb on average) exhibited minor affinity alteration to A´ and mTfR, and no change in
antibody integrity. On-section Tz-TCO ligation on 5xFAD brain sections generated three days after TCO-bsAb
injection revealed a [18F]BBB-Tz binding pattern colocalizing with A´ pathology and yielded cortical/thalamic-tocerebellar ratios of 3-6. Conversely, in wildtype mice, no region-specific binding of [18F]BBB-Tz was detected,
with a cortical/thalamic-to-cerebellar ratio of 1. This data indicated that TCOs on BS-mAb remained reactive three
days post injection.
Intravenous injection of [18F]BBB-Tz in 5xFAD mice three days after administration of TCO-bsAb showed
successful Tz-TCO ligation in vivo and specific visualization of A´-bound TCO-bsAb by revealing a [18F]BBB-Tz
binding pattern colocalizing with A´ pathology and demonstrating a ~1.4-higher cortical and hippocampal tracer
retention in 5xFAD mice compared to wildtypes.
Pre-treatment with a peripheral, non-BBB-penetrable Tz reduced blood plasma retention of [18F]BBB-Tz by 77%
highlighting the possibility of using masking agents to improve target-to-blood ratios.
Keywords: Pretargeting, tetrazine, TCO, 5xFAD
HAI2025 - 444
