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 protofbril-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 afbodies with and without scFv8D3 was evaluated by ELISA and autoradiography. Brain uptake and retention of the afbodies 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 afbodies without scFv8D3, but could not confrm specifc 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.