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Recent development of amyloid-β (Aβ)-targeted immunotherapies for Alzheimer’s disease (AD) have highlighted the need for accurate diagnostic methods. Antibody-based positron emission tomography (PET) ligands are well suited for this purpose as they can be directed toward the same target as the therapeutic antibody. Bispecific, brain-penetrating antibodies can achieve sufficient brain concentrations, but their slow blood clearance remains a challenge, since it prolongs the time required to achieve a target-specific PET signal. Here, two antibodies were designed based on the Aβ antibody bapineuzumab (Bapi) – one monospecific IgG (Bapi) and one bispecific antibody with an antigen binding fragment (Fab) of the transferrin receptor (TfR) antibody 8D3 fused to one of the heavy chains (Bapi-Fab8D3) for active, TfR-mediated transport into the brain. A variant of each antibody was designed to harbor a mutation to the neonatal Fc receptor (FcRn) binding domain, to increase clearance. Blood and brain pharmacokinetics of radiolabeled antibodies were studied in wildtype (WT) and AD mice (AppNL-G-F). The FcRn mutation substantially reduced blood half-life of both Bapi and Bapi-Fab8D3. Bapi-Fab8D3 showed high brain uptake and the brain-to-blood ratio of its FcRn mutated form was significantly higher in AppNL-G-F mice than in WT mice 12 h after injection and increased further up to 168 h. Ex vivo autoradiography showed specific antibody retention in areas with abundant Aβ pathology. Taken together, these results suggest that reducing FcRn binding of a full-sized bispecific antibody increases the systemic elimination and could thereby drastically reduce the time from injection to in vivo imaging.

Abstract Background The brain is a challenging target for antibody-based positron emission tomography (immunoPET) imaging due to the restricted access of antibody-based ligands through the blood–brain barrier (BBB). To overcome this physiological obstacle, we have previously developed bispecific antibody ligands that pass through the BBB via receptor-mediated transcytosis. While these radiolabelled ligands have high affinity and specificity, their long residence time in the blood and brain, typical for large molecules, poses another challenge for PET imaging. A viable solution could be a two-step pre-targeting approach which involves the administration of a tagged antibody that accumulates at the target site in the brain and then clears from the blood, followed by administration of a small radiolabelled molecule with fast kinetics. This radiolabelled molecule can couple to the tagged antibody and thereby make the antibody localisation visible by PET imaging. The in vivo linkage can be achieved by using the inverse electron demand Diels–Alder reaction (IEDDA), with trans-cyclooctene (TCO) and tetrazine groups participating as reactants. In this study, two novel 18F-labelled tetrazines were synthesized and evaluated for their potential use as pre-targeting imaging agents, i.e., for their ability to rapidly enter the brain and, if unbound, to be efficiently cleared with minimal background retention. Results The two compounds, a methyl tetrazine [18F]MeTz and an H-tetrazine [18F]HTz were radiolabelled using a two-step procedure via [18F]F-Py-TFP synthesized on solid support followed by amidation with amine-bearing tetrazines, resulting in radiochemical yields of 24% and 22%, respectively, and a radiochemical purity of > 96%. In vivo PET imaging was performed to assess their suitability for in vivo pre-targeting. Time-activity curves from PET-scans showed [18F]MeTz to be the more pharmacokinetically suitable agent, given its fast and homogenous distribution in the brain and rapid clearance. However, in terms of rection kinetics, H-tetrazines are advantageous, exhibiting faster reaction rates in IEDDA reactions with dienophiles like trans-cyclooctenes, making [18F]HTz potentially more beneficial for pre-targeting applications. Conclusion This study demonstrates a significant potential of [18F]MeTz and [18F]HTz as agents for pre-targeted PET brain imaging due to their efficient brain uptake, swift clearance and appropriate chemical stability.

The positron emission tomography (PET) radioligand [11C]UCB-J binds to synaptic vesicle protein 2A (SV2A) and is used to investigate synaptic density in the living brain. Clinical studies have indicated reduced [11C]UCB-J binding in Alzheimer's disease (AD) and Parkinson's disease (PD) brains compared to healthy controls. Still, it is unknown whether [11C]UCB-J PET can visualise synaptic loss in mouse models of these disorders. Such models are essential for understanding disease pathology and for evaluating the effects of novel disease-modifying drug candidates. In the present study, synaptic density in transgenic models of AD (ArcSwe) and PD (L61) was studied using [11C]UCB-J PET. Data were acquired during 60 min after injection, and time-activity curves (TACs) in different brain regions and the left ventricle of the heart were generated based on the dynamic PET images. The [11C]UCB-J brain concentrations were expressed as standardised uptake value (SUV) over time. The area under the SUV curve (AUC), the ratio of AUC in the brain to that in the heart (AUCbrain/blood), and the volume of distribution (VT) obtained by kinetic modelling using the heart TAC as input were compared between transgenic and age-matched wild type (WT) mice. The L61 mice displayed 11–13% lower AUCbrain/blood ratio and brain VT generated by kinetic modeling compared to the control WT mice. In general, also transgenic ArcSwe mice tended to show lower [11C]UCB-J brain exposure than age-matched WT controls, but variation within the different animal groups was high. Older WT mice (18–20 months) showed lower [11C]UCB-J brain exposure than younger WT mice (8–9 months). Together, these data imply that [11C]UCB-J PET reflects synaptic density in mouse models of neurodegeneration and that inter-subject variation is large. In addition, the study suggested that model-independent AUCbrain/blood ratio can be used to evaluate [11C]UCB-J binding as an alternative to full pharmacokinetic modelling.

Abstract Introduction [11C]UCB-J is a tracer developed for PET (positron emission tomography) that has high affinity towards synaptic vesicle glycoprotein 2A (SV2A), a protein believed to participate in the regulation of neurotransmitter release in neurons and endocrine cells. The localisation of SV2A in the synaptic terminals makes it a viable target for in vivo imaging of synaptic density in the brain. Several SV2A targeting compounds have been evaluated as PET tracers, including [11C]UCB-J, with the aim to facilitate studies of synaptic density in neurological diseases. The original two-step synthesis method failed in our hands to produce sufficient amounts of [11C]UCB-J, but served as an excellent starting point for further optimizations towards a high yielding and simplified one-step method. [11C]Methyl iodide was trapped in a clear THF-water solution containing the trifluoroborate substituted precursor, potassium carbonate and palladium complex. The resulting reaction mixture was heated at 70 °C for 4 min to produce [11C]UCB-J. Results After semi-preparative HPLC purification and reformulation in 10% ethanol/phosphate buffered saline, the product was obtained in 39 ± 5% radiochemical yield based on [11C]methyl iodide, corresponding to 1.8 ± 0.5 GBq at EOS. The radiochemical purity was > 99% and the molar activity was 390 ± 180 GBq/μmol at EOS. The product solution contained