Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-Aß-Affibodies.

Affibodies targeting amyloid-beta (Aß) could potentially be used as therapeutic and diagnostic agents in Alzheimer's disease (AD). Affibodies display suitable characteristics for imaging applications such as high stability and a short biological half-life. The aim of this study was to explore brain delivery and retention of Aß protofibril-targeted affibodies in wild-type (WT) and AD transgenic mice and to evaluate their potential as imaging agents. Two affibodies, Z5 and Z1, were fused with the blood-brain barrier (BBB) shuttle single-chain variable fragment scFv8D3. In vitro binding of 125I-labeled affibodies with and without scFv8D3 was evaluated by ELISA and autoradiography. Brain uptake and retention of the affibodies at 2 h and 24 h post injection was studied ex vivo in WT and transgenic (tg-Swe and tg-ArcSwe) mice. At 2 h post injection, [125I]I-Z5 and [125I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 showed increased brain concentrations of 0.53 ± 0.16% and 1.20 ± 0.35%ID/g brain. At 24 h post injection, brain retention of [125I]I-Z1 and [125I]I-Z5 was low, while [125I]I-scFv8D3-Z1 and [125I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [125I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around Aß deposits. Affibody-scFv8D3 fusions displayed increased brain and parenchymal delivery compared with the non-fused affibodies. However, fast brain washout and a suboptimal balance between Aß and mTfR1 affinity resulted in low intrabrain retention around Aß deposits.

PET Imaging in Preclinical Anti-Aß Drug Development.

Positron emission tomography (PET), a medical imaging technique allowing for studies of the living human brain, has gained an important role in clinical trials of novel drugs against Alzheimer's disease (AD). For example, PET data contributed to the conditional approval in 2021 of aducanumab, an antibody directed towards amyloid-beta (Aß) aggregates, by showing a dose-dependent reduction in brain amyloid after treatment. In parallel to clinical studies, preclinical studies in animal models of Aß pathology may also benefit from PET as a tool to detect target engagement and treatment effects of anti-Aß drug candidates. PET is associated with a high level of translatability between species as similar, non-invasive protocols allow for longitudinal rather than cross-sectional studies and can be used both in a preclinical and clinical setting. This review focuses on the use of preclinical PET imaging in genetically modified animals that express human Aß, and its present and potential future role in the development of drugs aimed at reducing brain Aß levels as a therapeutic strategy to halt disease progression in AD.

Age, dose, and binding to TfR on blood cells influence brain delivery of a TfR-transported antibody.

BACKGROUND: Transferrin receptor 1 (TfR1) mediated brain delivery of antibodies could become important for increasing the efficacy of emerging immunotherapies in Alzheimer's disease (AD). However, age, dose, binding to TfR1 on blood cells, and pathology could influence the TfR1-mediated transcytosis of TfR1-binders across the blood-brain barrier (BBB). The aim of the study was, therefore, to investigate the impact of these factors on the brain delivery of a bispecific TfR1-transported Aß-antibody, mAb3D6-scFv8D3, in comparison with the conventional antibody mAb3D6. METHODS: Young (3-5 months) and aged (17-20 months) WT and tg-ArcSwe mice (AD model) were injected with 125I-labeled mAb3D6-scFv8D3 or mAb3D6. Three different doses were used in the study, 0.05 mg/kg (low dose), 1 mg/kg (high dose), and 10 mg/kg (therapeutic dose), with equimolar doses for mAb3D6. The dose-corrected antibody concentrations in whole blood, blood cells, plasma, spleen, and brain were evaluated at 2 h post-administration. Furthermore, isolated brains were studied by autoradiography, nuclear track emulsion, and capillary depletion to investigate the intrabrain distribution of the antibodies, while binding to blood cells was studied in vitro using blood isolated from young and aged mice. RESULTS: The aged WT and tg-ArcSwe mice showed significantly lower brain concentrations of TfR-binding [125I]mAb3D6-scFv8D3 and higher concentrations in the blood cell fraction compared to young mice. For [125I]mAb3D6, no significant differences in blood or brain delivery were observed between young and aged mice or between genotypes. A low dose of [125I]mAb3D6-scFv8D3 was associated with increased relative parenchymal delivery, as well as increased blood cell distribution. Brain concentrations and relative parenchymal distribution of [125I]mAb3D6-scFv8D6 did not differ between tg-ArcSwe and WT mice at this early time point but were considerably increased compared to those observed for [125I]mAb3D6. CONCLUSION: Age-dependent differences in blood and brain concentrations were observed for the bispecific antibody mAb3D6-scFv8D3 but not for the conventional Aß antibody mAb3D6, indicating an age-related effect on TfR1-mediated brain delivery. The lowest dose of [125I]mAb3D6-scFv8D3 was associated with higher relative BBB penetration but, at the same time, a higher distribution to blood cells. Overall, Aß-pathology did not influence the early brain distribution of the bispecific antibody. In summary, age and bispecific antibody dose were important factors determining brain delivery, while genotype was not.

Introducing or removing heparan sulfate binding sites does not alter brain uptake of the blood-brain barrier shuttle scFv8D3.

The blood-brain barrier (BBB) greatly limits the delivery of protein-based drugs into the brain and is a major obstacle for the treatment of brain disorders. Targeting the transferrin receptor (TfR) is a strategy for transporting protein-based drugs into the brain, which can be utilized by using TfR-binding BBB transporters, such as the TfR-binding antibody 8D3. In this current study, we investigated if binding to heparan sulfate (HS) contributes to the brain uptake of a single chain fragment variable of 8D3 (scFv8D3). We designed and produced a scFv8D3 mutant, engineered with additional HS binding sites, HS(+)scFv8D3, to assess whether increased HS binding would improve brain uptake. Additionally, a mutant with a reduced number of HS binding sites, HS(-)scFv8D3, was also engineered to see if reducing the HS binding sites could also affect brain uptake. Heparin column chromatography showed that only the HS(+)scFv8D3 mutant bound HS in the experimental conditions. Ex vivo results showed that the brain uptake was unaffected by the introduction or removal of HS binding sites, which indicates that scFv8D3 is not dependent on the HS binding sites for brain uptake. Conversely, introducing HS binding sites to scFv8D3 decreased its renal excretion while removing them had the opposite effect.

Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different size.

BACKGROUND: Transferrin receptor (TfR1) mediated enhanced brain delivery of antibodies have been studied extensively in preclinical settings. However, the brain pharmacokinetics, i.e. brain entry, distribution and elimination are still not fully understood for this class of antibodies. The overall aim of the study was to compare the brain pharmacokinetics of two BBB-penetrating bispecific antibodies of different size (210 vs 58 kDa). Specifically, we wanted to investigate if the faster systemic clearance of the smaller non-IgG antibody di-scFv3D6-8D3, in comparison with the IgG-based bispecific antibody mAb3D6-scFv8D3, was also reflected in the brain. METHODS: Wild-type (C57/Bl6) mice were injected with 125I-iodinated ([125I]) mAb3D6-scFv8D3 (n = 46) or [125I]di-scFv3D6-8D3 (n = 32) and euthanized 2, 4, 6, 8, 10, 12, 16, or 24 h post injection. Ex vivo radioactivity in whole blood, peripheral organs and brain was measured by γ-counting. Ex vivo autoradiography and nuclear track emulsion were performed on brain sections to investigate brain and parenchymal distribution. Capillary depletion was carried out at 2, 6, and 24 h after injection of [125I]mAb3D6-scFv8D3 (n = 12) or [125I]di-scFv3D6-8D3 (n = 12), to estimate the relative levels of radiolabelled antibody in brain capillaries versus brain parenchyma. In vitro binding kinetics for [125I]mAb3D6-scFv8D3 or [125I]di-scFv3D6-8D3 to murine TfR were determined by LigandTracer. RESULTS: [125I]di-scFv3D6-8D3 showed faster elimination from blood, lower brain Cmax, and Tmax, a larger parenchymal-to-capillary concentration ratio, and a net elimination from brain at an earlier time point after injection compared with the larger [125I]mAb3D6-scFv8D3. However, the elimination rate from brain did not differ between the antibodies. The study also indicated that [125I]di-scFv3D6-8D3 displayed lower avidity than [125I]mAb3D6-scFv8D3 towards TfR1 in vitro and potentially in vivo, at least at the BBB. CONCLUSION: A smaller size and lower TfR1 avidity are likely important for fast parenchymal delivery, while elimination of brain-associated bispecific antibodies may not be dependent on these characteristics.

Fluorine-18-Labeled Antibody Ligands for PET Imaging of Amyloid-ß 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-ß (Aß) 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 Aß PET imaging. Bispecific antibody ligands RmAb158-scFv8D3 and Tribody A2, both targeting Aß 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 (t1/2 = 110 min), [18F]T3-Tribody A2 PET seemed to discriminate transgenic mice (tg-ArcSwe) with Aß 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.