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

Abstract

Affibodies targeting amyloid-beta (Aβ) 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 Aβ 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 Aβ 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 Aβ and mTfR1 affinity resulted in low intrabrain retention around Aβ deposits.

Development and Preclinical Evaluation of a Copper-64-Labeled Antibody Targeting Glycine-Alanine Dipeptides for PET Imaging of C9orf72-Associated Amyotrophic Lateral Sclerosis/Frontotemporal Dementia

“…Transferrin receptor ligands promote the active transport of large cargo via transcytosis across brain endothelial cells. As a result, high-resolution PET/CT images can be obtained with radiolabeled bispecific antibodies, e.g., targeting Aβ. , A similar strategy would likely improve the signal-to-noise ratio for our tracer.…”

Hopping the Hurdle: Strategies to Enhance the Molecular Delivery to the Brain through the Blood–Brain Barrier

“…One of the main uncertainties in this context, however, is how internalized molecules can be sorted and determined to be transported into the brain. For example, even with a similar molecular structure, antibodies binding to the transferrin receptor showed varying capabilities to cross the BBB [46], an indication that specific molecular structure is a key determinant for the fate of a molecule. Subsequently, this fate occurring through the endosomal trafficking pathway delivery is determined by sorting tubules, demonstrating that the interaction with proteins in the cytoplasm may determine the capability of a molecule to go through transcytosis [34].…”

IgG Fusion Proteins for Brain Delivery of Biologics via Blood–Brain Barrier Receptor-Mediated Transport

“…The administration of 1 mg/kg BW TfRMAb-Aβ-ScFv IV three times per week or 5 mg/kg BW SC daily for 12 weeks to B6C3-Tg(APPswe, PSEN1dE9)85 Dbo/J (PSAPP) double transgenic mice produced a 40–61% reduction in the brain concentration of Aβ 1−42 [ 95 , 96 ] without brain microhemorrhage [ 97 ], a common adverse side effect seen in the immune therapy of AD. A reverse construct wherein the transport domain is a form of an ScFv fused to the C-terminus of the light chain of a therapeutic MAb has also been reported [ 98 ]. These constructs present the advantage of re-engineering any therapeutic MAb into a brain-penetrating tetravalent bispecific MAb targeting either the BBB-TfR [ 98 ] or the BBB-HIR [ 99 ].…”

“…A reverse construct wherein the transport domain is a form of an ScFv fused to the C-terminus of the light chain of a therapeutic MAb has also been reported [98]. These constructs present the advantage of reengineering any therapeutic MAb into a brain-penetrating tetravalent bispecific MAb targeting either the BBB-TfR [98] or the BBB-HIR [99]. In order to validate the bifunctional HIRMAb-Aβ-ScFv in a mouse model of AD, a surrogate molecule targeting the mouse TfR was engineered (Table 2) [38].…”