Fluorine-18-Labeled Antibody Ligands for PET Imaging of Amyloid-β in Brain

Antibodies are attractive as radioligands due to their outstanding specificity and high affinity, but their inability to cross the blood-brain barrier (BBB) limits their use for CNS targets. To enhance brain distribution, amyloid-β (Aβ) antibodies were fused to a transferrin receptor (TfR) antibody fragment, enabling receptor mediated transport across the BBB. The aim of this study was to label these bispecific antibodies with fluorine-18 and use them for Aβ PET imaging. Bispecific antibody ligands RmAb158-scFv8D3 and Tribody A2, both targeting Aβ and TfR, were functionalized with trans-cyclooctene (TCO) groups and conjugated with ¹⁸F-labeled tetrazines through an inverse electron demand Diels-Alder reaction performed at ambient temperature. ¹⁸F-labeling did not affect antibody binding in vitro, and initial brain uptake was high. Conjugates with the first tetrazine variant ([¹⁸F]T1) displayed high uptake in bone, indicating extensive defluorination, a problem that was resolved with the second and third tetrazine variants ([¹⁸F]T2 and [¹⁸F]T3). Although the antibody ligands' half-life in blood was too long to optimally match the physical half-life of fluorine-18 (t_1/2 = 110 min), [¹⁸F]T3-Tribody A2 PET seemed to discriminate transgenic mice (tg-ArcSwe) with Aβ deposits from wild-type mice 12 h after injection. This study demonstrates that ¹⁸F-labeling of bispecific, brain penetrating antibodies is feasible and, with further optimization, could be used for CNS PET imaging.