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8. Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-A beta-Affibodies

Author

Faresjö, Rebecca, Lindberg, Hanna, Stahl, Stefan, Lofblom, John, Syvänen, Stina, Sehlin, Dag, Faresjö, Rebecca, Lindberg, Hanna, Stahl, Stefan, Lofblom, John, Syvänen, Stina, and Sehlin, Dag

Abstract

Affibodies targeting amyloid-beta (A beta) 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 A beta 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 I-125-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, [I-125]I-Z5 and [I-125]I-Z1 displayed brain concentrations of 0.37 +/- 0.09% and 0.46 +/- 0.08% ID/g brain, respectively. [I-125]I-scFv8D3-Z5 and [I-125]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 [I-125]I-Z1 and [I-125]I-Z5 was low, while [I-125]I-scFv8D3-Z1 and [I-125]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [I-125]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [I-125]I-scFv8D3-Z5 and [I-125]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around A beta 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 A beta and mTfR1 affinity resulted in low intrabrain retention around A beta deposits.

Published
2022
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9. Additional file 1 of Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different size

Author

Faresjö, Rebecca, Bonvicini, Gillian, Fang, Xiaotian T., Aguilar, Ximena, Sehlin, Dag, and Syvänen, Stina

Subjects
Pharmacology, Ecology, Physiology, FOS: Clinical medicine, Immunology, 111714 Mental Health, FOS: Health sciences, Biochemistry, Space Science, FOS: Biological sciences, Medicine, 110309 Infectious Diseases, Molecular Biology, Neuroscience, Cancer
Abstract

Additional file 1: Fig. S1 a Non-reducing SDS-PAGE (4-12% Bis-Tris gel) of purified di-scFv3D6-8D3 and mAb3D6-scFv8D3. Lane 1 is protein ladder (Chameleon® Duo Pre-stained Protein ladder): lanes 2-4 is di-scFv3D6-8D3 purified by a HisTrap column; lanes 5-7 is di-scFv3D6-8D3 after HiTrap ion exchange chromatography; and lanes 8-10 is mAb3D6-scFv8D3 after purification by a Protein G column. For all proteins, 2, 1 and 0.5 μg was added in lanes 2-4, 5-7 and 8-10, respectively b Quantification of %Purity of lane 2 and 5, HMW = High molecular weight (>58 kDa) and LMW = low molecular weight (< 58 kDa). Fig. S2 a Aβ42 (50 nM) indirect ELISA of mAb3D6-scFv8D3 and [125I]mAb3D6-scFv8D3 b EC50 (mean±SD) and paired t-test of n=3 repetitions Aβ (50 nM) indirect ELISA of mAb3D6-scFv8D3 and [125I]mAb3D6-scFv8D3 c mTfR1 (13.3 nM) indirect ELISA of mAb3D6-scFv8D3 and [125I]mAb3D6-scFv8D3 c EC50 (mean±SD) and paired t-test of n=3 repetitions mTfR1 (13.3 nM) indirect ELISA of mAb3D6-scFv8D3 and [125I]mAb3D6-scFv8D3. Fig. S3 a (50 nM) indirect ELISA of di-scFv3D6-8D3 and [125I]di-scFv3D6-8D3 b EC50 (mean±SD) and paired t-test of n=3 repetitions Aβ (50 nM) indirect ELISA for di-scFv3D6-8D3 and [125I]di-scFv3D6-8D3 c mTfR1 competition ELISA of di-scFv3D6-8D3 and [125I]di-scFv3D6-8D3 d IC50 (mean ± SD) and paired t-test of n=3 repetitions mTfR1 competition ELISAs for di-scFv3D6-8D3 and [125I]di-scFv3D6-8D3. Fig. S4 125I Standards mean intensity ± SD of the plates used in the brain autoradiography experiments (1000 Bq n=7; 333 Bq n=7; 111 Bq n = 11). Fig. S5 Example overlay image. The accuracy of the thresholding in the NTE-image quantification was evaluated visually by applying the respective ROI outlines (NTE and CD31) as overlays with 50% opacity on the original composite image. Fig. S6 Color-inverted version of Fig. 7a-b, showing NTE (white puncta) detecting i.v. injected a [125I]mAb3D6-scFv8D3 or b [125I]di-scFv3D6-8D3 and CD31-flourescent staining (red) in mouse brain sections.

Published
2021
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11. Fluorine-18-Labeled Antibody Ligands for PET Imaging of Amyloid-β in Brain

Author

Syvänen, Stina, Fang, Xiaotian T., Faresjö, Rebecca, Rokka, Johanna, Lannfelt, Lars, Olberg, Dag E., Eriksson, Jonas, Sehlin, Dag, Syvänen, Stina, Fang, Xiaotian T., Faresjö, Rebecca, Rokka, Johanna, Lannfelt, Lars, Olberg, Dag E., Eriksson, Jonas, and Sehlin, Dag

Abstract

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 (t1/2 = 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.

Published
2020
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12. Journal of Labelled Compounds and Pharmaceuticals : Neurosciences session

Author

Rokka, Johanna, Fang, Xiaotian T., Hultqvist, Greta, Faresjö, Rebecca, Olberg, Dag, Antoni, Gunnar, Lannfelt, Lars, Sehlin, Dag, Syvänen, Stina, Eriksson, Jonas, Rokka, Johanna, Fang, Xiaotian T., Hultqvist, Greta, Faresjö, Rebecca, Olberg, Dag, Antoni, Gunnar, Lannfelt, Lars, Sehlin, Dag, Syvänen, Stina, and Eriksson, Jonas

Published
2019
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13. Additional file 1 of Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different size

Author

Faresjö, Rebecca, Bonvicini, Gillian, Fang, Xiaotian T., Aguilar, Ximena, Sehlin, Dag, and Syvänen, Stina

Subjects
3. Good health
Abstract

Additional file 1: Fig. S1 a Non-reducing SDS-PAGE (4-12% Bis-Tris gel) of purified di-scFv3D6-8D3 and mAb3D6-scFv8D3. Lane 1 is protein ladder (Chameleon® Duo Pre-stained Protein ladder): lanes 2-4 is di-scFv3D6-8D3 purified by a HisTrap column; lanes 5-7 is di-scFv3D6-8D3 after HiTrap ion exchange chromatography; and lanes 8-10 is mAb3D6-scFv8D3 after purification by a Protein G column. For all proteins, 2, 1 and 0.5 μg was added in lanes 2-4, 5-7 and 8-10, respectively b Quantification of %Purity of lane 2 and 5, HMW = High molecular weight (>58 kDa) and LMW = low molecular weight (< 58 kDa). Fig. S2 a Aβ42 (50 nM) indirect ELISA of mAb3D6-scFv8D3 and [125I]mAb3D6-scFv8D3 b EC50 (mean±SD) and paired t-test of n=3 repetitions Aβ (50 nM) indirect ELISA of mAb3D6-scFv8D3 and [125I]mAb3D6-scFv8D3 c mTfR1 (13.3 nM) indirect ELISA of mAb3D6-scFv8D3 and [125I]mAb3D6-scFv8D3 c EC50 (mean±SD) and paired t-test of n=3 repetitions mTfR1 (13.3 nM) indirect ELISA of mAb3D6-scFv8D3 and [125I]mAb3D6-scFv8D3. Fig. S3 a (50 nM) indirect ELISA of di-scFv3D6-8D3 and [125I]di-scFv3D6-8D3 b EC50 (mean±SD) and paired t-test of n=3 repetitions Aβ (50 nM) indirect ELISA for di-scFv3D6-8D3 and [125I]di-scFv3D6-8D3 c mTfR1 competition ELISA of di-scFv3D6-8D3 and [125I]di-scFv3D6-8D3 d IC50 (mean ± SD) and paired t-test of n=3 repetitions mTfR1 competition ELISAs for di-scFv3D6-8D3 and [125I]di-scFv3D6-8D3. Fig. S4 125I Standards mean intensity ± SD of the plates used in the brain autoradiography experiments (1000 Bq n=7; 333 Bq n=7; 111 Bq n = 11). Fig. S5 Example overlay image. The accuracy of the thresholding in the NTE-image quantification was evaluated visually by applying the respective ROI outlines (NTE and CD31) as overlays with 50% opacity on the original composite image. Fig. S6 Color-inverted version of Fig. 7a-b, showing NTE (white puncta) detecting i.v. injected a [125I]mAb3D6-scFv8D3 or b [125I]di-scFv3D6-8D3 and CD31-flourescent staining (red) in mouse brain sections.

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