89 results on '"Sehlin, D"'
Search Results
2. Murine type II Abeta fibril from ARTE10 mouse
- Author
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Zielinski, M., primary, Peralta Reyes, F.S., additional, Gremer, L., additional, Schemmert, S., additional, Frieg, B., additional, Willuweit, A., additional, Donner, L., additional, Elvers, M., additional, Nilsson, L.N.G., additional, Syvanen, S., additional, Sehlin, D., additional, Ingelsson, M., additional, Willbold, D., additional, and Schroeder, G.F., additional
- Published
- 2023
- Full Text
- View/download PDF
3. DI1 Abeta fibril from tg-SwDI mouse
- Author
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Zielinski, M., primary, Peralta Reyes, F.S., additional, Gremer, L., additional, Schemmert, S., additional, Frieg, B., additional, Willuweit, A., additional, Donner, L., additional, Elvers, M., additional, Nilsson, L.N.G., additional, Syvanen, S., additional, Sehlin, D., additional, Ingelsson, M., additional, Willbold, D., additional, and Schroeder, G.F., additional
- Published
- 2023
- Full Text
- View/download PDF
4. Proximity ligation-based detection of biomarkers of protein folding disorders: 247
- Author
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Kamali-M, M., Englund, H., Schallmeiner, E., Le Hir, G., Karlsson, J., Darmanis, S., Pettersson, F. Ekholm, Comoy, E., Sehlin, D., Deslys, J-P., Lannfelt, L, and Landegren, U.
- Published
- 2007
5. Pathogenic and therapeutic studies using the ArcSwe transgenic mouse model of Alzheimerʼs disease: 217
- Author
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Nilsson, L. N. G., Lord, A., Philipsson, O., Englund, H., Sehlin, D., Ekholm-Pettersson, F., Clausen, F., Hillered, L., Portelius, E., Blennow, K., Kalimo, H., and Lannfelt, L.
- Published
- 2007
6. Engineered antibodies: new possibilities for brain PET?
- Author
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Sehlin, D, Syvanen, S, Ballanger, B, Barthel, H, Bischof, GN, Boche, D, Boecker, H, Bohn, KP, Borghammer, P, Cross, D, Di Monte, D, Drzezga, A, Endepols, H, Giehl, K, Goedert, M, Hammes, J, Hansson, O, Herholz, K, Hoeglinger, G, Hoenig, M, Jessen, F, Klockgether, T, Lafaye, P, Lammerstma, A, Mandelkow, E, Mandelkow, E-M, Maurer, A, Mollenhauer, B, Neumaier, B, Nordberg, A, Onur, O, Reetz, K, Rodriguez-Vietez, E, Rominger, A, Rowe, J, Sabri, O, Schneider, A, Strafella, A, van Eimeren, T, Vasdev, N, Villemagne, V, Willbold, D, Sehlin, D, Syvanen, S, Ballanger, B, Barthel, H, Bischof, GN, Boche, D, Boecker, H, Bohn, KP, Borghammer, P, Cross, D, Di Monte, D, Drzezga, A, Endepols, H, Giehl, K, Goedert, M, Hammes, J, Hansson, O, Herholz, K, Hoeglinger, G, Hoenig, M, Jessen, F, Klockgether, T, Lafaye, P, Lammerstma, A, Mandelkow, E, Mandelkow, E-M, Maurer, A, Mollenhauer, B, Neumaier, B, Nordberg, A, Onur, O, Reetz, K, Rodriguez-Vietez, E, Rominger, A, Rowe, J, Sabri, O, Schneider, A, Strafella, A, van Eimeren, T, Vasdev, N, Villemagne, V, and Willbold, D
- Abstract
Almost 50 million people worldwide are affected by Alzheimer's disease (AD), the most common neurodegenerative disorder. Development of disease-modifying therapies would benefit from reliable, non-invasive positron emission tomography (PET) biomarkers for early diagnosis, monitoring of disease progression, and assessment of therapeutic effects. Traditionally, PET ligands have been based on small molecules that, with the right properties, can penetrate the blood-brain barrier (BBB) and visualize targets in the brain. Recently a new class of PET ligands based on antibodies have emerged, mainly in applications related to cancer. While antibodies have advantages such as high specificity and affinity, their passage across the BBB is limited. Thus, to be used as brain PET ligands, antibodies need to be modified for active transport into the brain. Here, we review the development of radioligands based on antibodies for visualization of intrabrain targets. We focus on antibodies modified into a bispecific format, with the capacity to undergo transferrin receptor 1 (TfR1)-mediated transcytosis to enter the brain and access pathological proteins, e.g. amyloid-beta. A number of such antibody ligands have been developed, displaying differences in brain uptake, pharmacokinetics, and ability to bind and visualize the target in the brain of transgenic mice. Potential pathological changes related to neurodegeneration, e.g. misfolded proteins and neuroinflammation, are suggested as future targets for this novel type of radioligand. Challenges are also discussed, such as the temporal match of radionuclide half-life with the ligand's pharmacokinetic profile and translation to human use. In conclusion, brain PET imaging using bispecific antibodies, modified for receptor-mediated transcytosis across the BBB, is a promising method for specifically visualizing molecules in the brain that are difficult to target with traditional small molecule ligands.
- Published
- 2019
7. S.23.01 Pathogenic mechanisms in Alzheimer's disease
- Author
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Lannfelt, L., primary, Sehlin, D., additional, Englund, H., additional, Lord, A., additional, Johansson, A.S., additional, Nilsson, L., additional, and Ekholm-Pettersson, F., additional
- Published
- 2006
- Full Text
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8. Interference from heterophilic antibodies in amyloid-β oligomer ELISAs.
- Author
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Sehlin D, Söllvander S, Paulie S, Brundin R, Ingelsson M, Lannfelt L, Pettersson FE, Englund H, Sehlin, Dag, Söllvander, Sofia, Paulie, Staffan, Brundin, RoseMarie, Ingelsson, Martin, Lannfelt, Lars, Pettersson, Frida Ekholm, and Englund, Hillevi
- Abstract
Amyloid-β (Aβ) oligomers of different sizes and forms have recently been the focus formany Alzheimer's disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Aβ species in different forms of human samples using little or no sample dilutions. However, the possibility that positive results may be caused by interference from heterophilic antibodies (HA) is often overlooked. HA, which recognize immunoglobulins from other species, are present in human plasma and cerebrospinal fluid (CSF) and may cause interference in sandwich immunoassays like enzyme-linked immunosorbent assays (ELISAs) by cross-binding the capture and detection antibodies of the assay. They thus may generate a false positive signal. Here we show that when assessing the Aβ oligomer content in plasma samples from 44 individuals with a sandwich ELISA, none of the 21 positive signals remained when the assay was repeated in the presence of factors blocking HA. Similarly, in CSF samples from 104 individuals, the signals from the 22 positive samples were strongly reduced when analyzed after anti-HA treatment. Taken together, HA interference is a problem that needs to be addressed when measuring low levels of an antigen in human plasma and CSF samples. [ABSTRACT FROM AUTHOR]
- Published
- 2010
9. Sensitive detection of Aβ protofibrils by proximity ligation--relevance for Alzheimer's disease.
- Author
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Kamali-Moghaddam M, Pettersson FE, Wu D, Englund H, Darmanis S, Lord A, Tavoosidana G, Sehlin D, Gustafsdottir S, Nilsson LN, Lannfelt L, Landegren U, Kamali-Moghaddam, Masood, Pettersson, Frida Ekholm, Wu, Di, Englund, Hillevi, Darmanis, Spyros, Lord, Anna, Tavoosidana, Gholamreza, and Sehlin, Dag
- Abstract
Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.Results: For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]- Published
- 2010
- Full Text
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10. Antibody engagement with amyloid-beta does not inhibit [ 11 C]PiB binding for PET imaging.
- Author
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Xiong M, Dahlén A, Roshanbin S, Wik E, Aguilar X, Eriksson J, Sehlin D, and Syvänen S
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- Animals, Mice, Alzheimer Disease metabolism, Alzheimer Disease diagnostic imaging, Benzothiazoles pharmacology, Carbon Radioisotopes, Radiopharmaceuticals, Male, Antibodies, Monoclonal, Humanized pharmacology, Plaque, Amyloid metabolism, Plaque, Amyloid diagnostic imaging, Female, Humans, Antibodies, Monoclonal pharmacology, Positron-Emission Tomography methods, Amyloid beta-Peptides metabolism, Amyloid beta-Peptides immunology, Aniline Compounds pharmacology, Thiazoles pharmacology, Brain metabolism, Brain diagnostic imaging, Brain drug effects, Mice, Transgenic
- Abstract
The elimination of amyloid-beta (Aβ) plaques in Alzheimer's disease patients after treatment with anti-Aβ antibodies such as lecanemab and aducanumab is supported by a substantially decreased signal in amyloid positron emission tomography (PET) imaging. However, this decreased PET signal has not been matched by a similar substantial effect on cognitive function. There may be several reasons for this, including short treatment duration and advanced disease stages among the patients. However, one aspect that has not been investigated, and the subject of this study, is whether antibody engagement with amyloid plaques inhibits the binding of amyloid-PET ligands, leading to a false impression of Aβ removal from the brain. In the present study, tg-ArcSwe mice received three injections of RmAb158, the murine version of lecanemab or phosphate-buffered saline (PBS) before the administration of the amyloid-PET radioligand [
11 C]PiB, followed by isolation of brain tissue. Autoradiography showed that RmAb158- and PBS-treated mice displayed similar [11 C]PiB binding. Moreover, the total Aβ1-40 levels, representing the major Aβ species of plaques in the tg-ArcSwe model, as well as soluble triggering receptor on myeloid cells 2 (sTREM2) levels, were similar in both groups. Interestingly, the concentration of soluble Aβ aggregates was decreased in the RmAb158-treated group, along with a small but significant decrease in the total Aβ1-42 levels. In conclusion, this study indicates that the binding of [11 C]PiB to Aβ accurately mirrors the load of Aβ plaques in the brain, aligning with how amyloid-PET is interpreted in clinical studies of anti-Aβ antibodies. However, early treatment effects on soluble Aβ aggregates and Aβ1-42 levels were not detected., (© 2024 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)- Published
- 2024
- Full Text
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11. Indium-111 radiolabelling of a brain-penetrant Aβ antibody for SPECT imaging.
- Author
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Gustavsson T, Herth MM, Sehlin D, and Syvänen S
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- Animals, Mice, Tissue Distribution, Mice, Transgenic, Antibodies, Bispecific pharmacokinetics, Blood-Brain Barrier metabolism, Blood-Brain Barrier diagnostic imaging, Receptors, Transferrin metabolism, Receptors, Transferrin immunology, Radiopharmaceuticals pharmacokinetics, Alzheimer Disease diagnostic imaging, Alzheimer Disease metabolism, Indium Radioisotopes, Tomography, Emission-Computed, Single-Photon methods, Brain diagnostic imaging, Brain metabolism, Amyloid beta-Peptides metabolism
- Abstract
Background: The development of bispecific antibodies that can traverse the blood-brain barrier has paved the way for brain-directed immunotherapy and when radiolabelled, immunoPET imaging. The objective of this study was to investigate how indium-111 (
111 In) radiolabelling with compatible chelators affects the brain delivery and peripheral biodistribution of the bispecific antibody RmAb158-scFv8D3, which binds to amyloid-beta (Aβ) and the transferrin receptor (TfR), in Aβ pathology-expressing tg-ArcSwe mice and aged-matched wild-type control mice., Methods: Bispecific RmAb158-scFv8D3 (biAb) was radiolabelled with111 In using CHX-A"-DTPA, DOTA, or DOTA-tetrazine (DOTA-Tz). Affinity toward TfR and Aβ, as well as stability, was investigated in vitro . Mice were then intravenously administered with the three different radiolabelled biAb variants, and blood samples were collected for monitoring pharmacokinetics. Brain concentration was quantified after 2 and 72 h, and organ-specific retention was measured at 72 h by gamma counting. A subset of mice also underwent whole-body Single-photon emission computed tomography (SPECT) scanning at 72 h after injection. Following post-mortem isolation, the brains of tg-ArcSwe and WT mice were sectioned, and the spatial distribution of biAb was further investigated with autoradiography., Results: All three [111 In]biAb variants displayed similar blood pharmacokinetics and brain uptake at 2 h after administration. Radiolabelling did not compromise affinity, and all variants showed good stability, especially the DOTA-Tz variant. Whole-body SPECT scanning indicated high liver, spleen, and bone accumulation of all [111 In]biAb variants. Subsequent ex vivo measurement of organ retention confirmed SPECT data, with retention in the spleen, liver, and bone - with very high bone marrow retention. Ex vivo gamma measurement of brain tissue, isolated at 72 h post-injection, and ex vivo autoradiography showed that WT mice, despite the absence of Aβ, exhibited comparable brain concentrations of [111 In]biAb as those found in the tg-ArcSwe brain., Conclusions: The successful111 In-labelling of biAb with retained binding to TfR and Aβ, and retained ability to enter the brain, demonstrated that111 In can be used to generate radioligands for brain imaging. A high degree of [111 In]biAb in bone marrow and intracellular accumulation in brain tissue indicated some off-target interactions or potential interaction with intrabrain TfR resulting in a relatively high non-specific background signal., Competing Interests: The authors report no conflict of interest., (© 2024 The Author(s).)- Published
- 2024
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12. Synthesis and evaluation of fluorine-18 labelled tetrazines as pre-targeting imaging agents for PET.
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Schlein E, Rokka J, Odell LR, van den Broek SL, Herth MM, Battisti UM, Syvänen S, Sehlin D, and Eriksson J
- 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
18 F-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 [18 F]MeTz and an H-tetrazine [18 F]HTz were radiolabelled using a two-step procedure via [18 F]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 [18 F]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 [18 F]HTz potentially more beneficial for pre-targeting applications., Conclusion: This study demonstrates a significant potential of [18 F]MeTz and [18 F]HTz as agents for pre-targeted PET brain imaging due to their efficient brain uptake, swift clearance and appropriate chemical stability., (© 2024. The Author(s).)- Published
- 2024
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13. Comparing in vitro affinity measurements of antibodies to TfR1: Surface plasmon resonance versus on-cell affinity.
- Author
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Bonvicini G, Bagawath Singh S, Nygren P, Xiong M, Syvänen S, Sehlin D, Falk R, and Andersson KG
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- Rats, Mice, Humans, Animals, Blood-Brain Barrier metabolism, Brain metabolism, Receptors, Transferrin metabolism, Surface Plasmon Resonance methods, Antibodies metabolism
- Abstract
Despite years of utilizing the transferrin receptor 1 (TfR1) to transport large biomolecules into the brain, there is no consensus on how to optimally measure affinity to it. The aim of this study was to compare different methods for measuring the affinities of anti-TfR1 antibodies. Antibodies 15G11, OX26 and 8D3 are known to successfully carry large biologics across the blood-brain barrier in humans, rats, and mice, respectively. The affinity to their respective species of TfR1 was measured with different surface plasmon resonance setups in Biacore and an on-cell assay. When the antibody was captured and TfR1 was the analyte, the dissociation in Biacore was very slow. The dissociation was faster when the antibody was the analyte and TfR1 was the ligand. The Biacore setup with capture of N-terminal FLAG-tag TfR1 yielded the most similar apparent affinities as the cell assay. In conclusion, it is important to evaluate assay parameters including assay orientation, surface capture method, and antibody-format when comparing binding kinetics for TfR1 antibodies. Although it seems possible to determine relative affinities of TfR1 antibodies using the methods described here, both the FLAG-tag TfR1 capture setup and cell assays likely yield apparent affinities that are most translatable in vivo., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gillian Bonvicini, Sunitha Singh, Patrik Nygren, Ronny Falk, and Ken G. Andersson are employees of BioArctic AB, Sweden. The other authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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14. Altered amyloid-β structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion.
- Author
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Pagnon de la Vega M, Syvänen S, Giedraitis V, Hooley M, Konstantinidis E, Meier SR, Rokka J, Eriksson J, Aguilar X, Spires-Jones TL, Lannfelt L, Nilsson LNG, Erlandsson A, Hultqvist G, Ingelsson M, and Sehlin D
- Subjects
- Animals, Humans, Mice, Amyloid beta-Protein Precursor genetics, Amyloid beta-Protein Precursor metabolism, Amyloid Precursor Protein Secretases metabolism, Brain pathology, Disease Models, Animal, Gliosis pathology, Ligands, Mice, Transgenic, Alzheimer Disease pathology, Amyloid beta-Peptides metabolism
- Abstract
Deposition of amyloid beta (Aβ) into plaques is a major hallmark of Alzheimer's disease (AD). Different amyloid precursor protein (APP) mutations cause early-onset AD by altering the production or aggregation properties of Aβ. We recently identified the Uppsala APP mutation (APPUpp), which causes Aβ pathology by a triple mechanism: increased β-secretase and altered α-secretase APP cleavage, leading to increased formation of a unique Aβ conformer that rapidly aggregates and deposits in the brain. The aim of this study was to further explore the effects of APPUpp in a transgenic mouse model (tg-UppSwe), expressing human APP with the APPUpp mutation together with the APPSwe mutation. Aβ pathology was studied in tg-UppSwe brains at different ages, using ELISA and immunohistochemistry. In vivo PET imaging with three different PET radioligands was conducted in aged tg-UppSwe mice and two other mouse models; tg-ArcSwe and tg-Swe. Finally, glial responses to Aβ pathology were studied in cell culture models and mouse brain tissue, using ELISA and immunohistochemistry. Tg-UppSwe mice displayed increased β-secretase cleavage and suppressed α-secretase cleavage, resulting in AβUpp42 dominated diffuse plaque pathology appearing from the age of 5-6 months. The γ-secretase cleavage was not affected. Contrary to tg-ArcSwe and tg-Swe mice, tg-UppSwe mice were [
11 C]PiB-PET negative. Antibody-based PET with the 3D6 ligand visualized Aβ pathology in all models, whereas the Aβ protofibril selective mAb158 ligand did not give any signals in tg-UppSwe mice. Moreover, unlike the other two models, tg-UppSwe mice displayed a very faint glial response to the Aβ pathology. The tg-UppSwe mouse model thus recapitulates several pathological features of the Uppsala APP mutation carriers. The presumed unique structural features of AβUpp42 aggregates were found to affect their interaction with anti-Aβ antibodies and profoundly modify the Aβ-mediated glial response, which may be important aspects to consider for further development of AD therapies., (© 2024. The Author(s).)- Published
- 2024
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15. Single domain antibody-scFv conjugate targeting amyloid β and TfR penetrates the blood-brain barrier and interacts with amyloid β.
- Author
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Faresjö R, Sjöström EO, Dallas T, Berglund MM, Eriksson J, Sehlin D, and Syvänen S
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- Animals, Humans, Mice, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Brain metabolism, Brain immunology, Immunoconjugates immunology, Immunoconjugates pharmacology, Immunoconjugates pharmacokinetics, Blood-Brain Barrier metabolism, Amyloid beta-Peptides immunology, Amyloid beta-Peptides metabolism, Receptors, Transferrin immunology, Receptors, Transferrin metabolism, Single-Chain Antibodies immunology, Alzheimer Disease metabolism, Alzheimer Disease drug therapy, Alzheimer Disease immunology, Single-Domain Antibodies immunology
- Abstract
Neurodegenerative diseases such as Alzheimer's disease (AD) pose substantial challenges to patients and health-care systems, particularly in countries with aging populations. Immunotherapies, including the marketed antibodies lecanemab (Leqembi®) and donanemab (Kisunla
TM ), offer promise but face hurdles due to limited delivery across the blood-brain barrier (BBB). This limitation necessitates high doses, resulting in increased costs and a higher risk of side effects. This study explores transferrin receptor (TfR)-binding camelid single-domain antibodies (VHHs) for facilitated brain delivery. We developed and evaluated fusion proteins (FPs) combining VHHs with human IgG Fc domains or single-chain variable fragments (scFvs) of the anti-amyloid-beta (Aβ) antibody 3D6. In vitro assessments showed varying affinities of the FPs for TfR. In vivo evaluations indicated that specific VHH-Fc and VHH-scFv fusions reached significant brain concentrations, emphasizing the importance of optimal TfR binding affinities. The VHH-scFv fusions were further investigated in mouse models with Aβ pathology, showing higher retention compared to wild-type mice without Aβ pathology. Our findings suggest that these novel VHH-based FPs hold potential for therapeutic and diagnostic applications in AD, providing a strategy to overcome BBB limitations and enhance brain targeting of antibody-based treatments. Furthermore, our results suggest that a given bispecific TfR-binding fusion format has a window of "optimal" affinity where parenchymal delivery is adequate, while blood pharmacokinetics aligns with the desired application of the fusion protein.- Published
- 2024
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16. Reducing neonatal Fc receptor binding enhances clearance and brain-to-blood ratio of TfR-delivered bispecific amyloid-β antibody.
- Author
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Schlein E, Andersson KG, Dallas T, Syvänen S, and Sehlin D
- Subjects
- Animals, Mice, Amyloid beta-Peptides, Brain diagnostic imaging, Brain metabolism, Immunoglobulin G metabolism, Mice, Transgenic, Alzheimer Disease drug therapy, Alzheimer Disease metabolism, Antibodies, Bispecific, Histocompatibility Antigens Class I, Receptors, Fc immunology, Receptors, Fc metabolism, Receptors, Transferrin immunology, Receptors, Transferrin metabolism
- Abstract
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 ( App
NL-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.- Published
- 2024
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17. Cryo-EM of Aβ fibrils from mouse models find tg-APP ArcSwe fibrils resemble those found in patients with sporadic Alzheimer's disease.
- Author
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Zielinski M, Peralta Reyes FS, Gremer L, Schemmert S, Frieg B, Schäfer LU, Willuweit A, Donner L, Elvers M, Nilsson LNG, Syvänen S, Sehlin D, Ingelsson M, Willbold D, and Schröder GF
- Subjects
- Humans, Mice, Animals, Cryoelectron Microscopy, Amyloid beta-Peptides metabolism, Mice, Transgenic, Brain metabolism, Disease Models, Animal, Alzheimer Disease pathology
- Abstract
The use of transgenic mice displaying amyloid-β (Aβ) brain pathology has been essential for the preclinical assessment of new treatment strategies for Alzheimer's disease. However, the properties of Aβ in such mice have not been systematically compared to Aβ in the brains of patients with Alzheimer's disease. Here, we determined the structures of nine ex vivo Aβ fibrils from six different mouse models by cryogenic-electron microscopy. We found novel Aβ fibril structures in the APP/PS1, ARTE10 and tg-SwDI models, whereas the human type II filament fold was found in the ARTE10, tg-APP
Swe and APP23 models. The tg-APPArcSwe mice showed an Aβ fibril whose structure resembles the human type I filament found in patients with sporadic Alzheimer's disease. A detailed assessment of the Aβ fibril structure is key to the selection of adequate mouse models for the preclinical development of novel plaque-targeting therapeutics and positron emission tomography imaging tracers in Alzheimer's disease., (© 2023. The Author(s).)- Published
- 2023
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18. Synaptic density in aging mice measured by [ 18 F]SynVesT-1 PET.
- Author
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Xiong M, Roshanbin S, Sehlin D, Hansen HD, Knudsen GM, Rokka J, Eriksson J, and Syvänen S
- Subjects
- Humans, Mice, Animals, Infant, Mice, Inbred C57BL, Pyridines pharmacokinetics, Brain diagnostic imaging, Brain metabolism, Positron-Emission Tomography methods, Pyrrolidines pharmacokinetics
- Abstract
Synaptic alterations in certain brain structures are related to cognitive decline in neurodegeneration and in aging. Synaptic loss in many neurodegenerative diseases can be visualized by positron emission tomography (PET) imaging of synaptic vesicle glycoprotein 2A (SV2A). However, the use of SV2A PET for studying synaptic changes during aging is not particularly explored. Thus, in the present study, PET ligand [
18 F]SynVesT-1, which binds to SV2A, was used to investigate synaptic density at different ages in healthy mice. Wild type C57BL/6 mice divided into three age groups (4-5 months (n = 7), 12-14 months (n = 11), 17-19 months (n = 7)) were PET scanned with [18 F]SynVesT-1. Brain retention of [18 F]SynVesT-1 expressed as the volume of distribution (VIDIF ) was calculated using an image-derived input function. Estimates of VIDIF were derived using either a one-tissue compartment model (1TCM), a two-tissue compartment model (2TCM), or the Logan plot with blood input to find the best-fit model for [18 F]SynVesT-1. After the PET scans, tissue sections were immunostained for the detection of SV2A and neuronal markers. We found that [18 F]SynVesT-1 data acquired 60 min post intravenously injection and analyzed with 1TCM described the brain pharmacokinetics of the radioligand in mice well. [18 F]SynVesT-1 brain retention was lower in the oldest group of mice, indicating a decrease in synaptic density in this age group. However, no gradual age-dependent decrease in synaptic density at a region-specific level was observed. Immunostaining indicated that SV2A expression and neuron numbers were similar across all three age groups. In general, these data obtained in healthy aging mice are consistent with previous findings in humans where synaptic density appeared stable during aging up to a certain age, after which a small decrease is observed., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)- Published
- 2023
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19. Age, dose, and binding to TfR on blood cells influence brain delivery of a TfR-transported antibody.
- Author
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Faresjö R, Sehlin D, and Syvänen S
- Subjects
- Mice, Animals, Amyloid beta-Peptides metabolism, Mice, Transgenic, Brain metabolism, Blood-Brain Barrier metabolism, Receptors, Transferrin metabolism, Blood Cells metabolism, Alzheimer Disease metabolism, Antibodies, Bispecific
- Abstract
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 Aβ-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
125 I-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 [125 I]mAb3D6-scFv8D3 and higher concentrations in the blood cell fraction compared to young mice. For [125 I]mAb3D6, no significant differences in blood or brain delivery were observed between young and aged mice or between genotypes. A low dose of [125 I]mAb3D6-scFv8D3 was associated with increased relative parenchymal delivery, as well as increased blood cell distribution. Brain concentrations and relative parenchymal distribution of [125 I]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 [125 I]mAb3D6., Conclusion: Age-dependent differences in blood and brain concentrations were observed for the bispecific antibody mAb3D6-scFv8D3 but not for the conventional Aβ antibody mAb3D6, indicating an age-related effect on TfR1-mediated brain delivery. The lowest dose of [125 I]mAb3D6-scFv8D3 was associated with higher relative BBB penetration but, at the same time, a higher distribution to blood cells. Overall, Aβ-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., (© 2023. The Author(s).)- Published
- 2023
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20. Long-term effects of immunotherapy with a brain penetrating Aβ antibody in a mouse model of Alzheimer's disease.
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Gustavsson T, Metzendorf NG, Wik E, Roshanbin S, Julku U, Chourlia A, Nilsson P, Andersson KG, Laudon H, Hultqvist G, Syvänen S, and Sehlin D
- Subjects
- Mice, Animals, Mice, Transgenic, Amyloid beta-Peptides metabolism, Brain metabolism, Antibodies therapeutic use, Antibodies pharmacology, Immunotherapy, Disease Models, Animal, Alzheimer Disease genetics
- Abstract
Background: Brain-directed immunotherapy is a promising strategy to target amyloid-β (Aβ) deposits in Alzheimer's disease (AD). In the present study, we compared the therapeutic efficacy of the Aβ protofibril targeting antibody RmAb158 with its bispecific variant RmAb158-scFv8D3, which enters the brain by transferrin receptor-mediated transcytosis., Methods: App
NL-G-F knock-in mice received RmAb158, RmAb158-scFv8D3, or PBS in three treatment regimens. First, to assess the acute therapeutic effect, a single antibody dose was given to 5 months old AppNL-G-F mice, with evaluation after 3 days. Second, to assess the antibodies' ability to halt the progression of Aβ pathology, 3 months old AppNL-G-F mice received three doses during a week, with evaluation after 2 months. Reduction of RmAb158-scFv8D3 immunogenicity was explored by introducing mutations in the antibody or by depletion of CD4+ T cells. Third, to study the effects of chronic treatment, 7-month-old AppNL-G-F mice were CD4+ T cell depleted and treated with weekly antibody injections for 8 weeks, including a final diagnostic dose of [125 I]RmAb158-scFv8D3, to determine its brain uptake ex vivo. Soluble Aβ aggregates and total Aβ42 were quantified with ELISA and immunostaining., Results: Neither RmAb158-scFv8D3 nor RmAb158 reduced soluble Aβ protofibrils or insoluble Aβ1-42 after a single injection treatment. After three successive injections, Aβ1-42 was reduced in mice treated with RmAb158, with a similar trend in RmAb158-scFv8D3-treated mice. Bispecific antibody immunogenicity was somewhat reduced by directed mutations, but CD4+ T cell depletion was used for long-term therapy. CD4+ T cell-depleted mice, chronically treated with RmAb158-scFv8D3, showed a dose-dependent increase in blood concentration of the diagnostic [125 I]RmAb158-scFv8D3, while concentration was low in plasma and brain. Chronic treatment did not affect soluble Aβ aggregates, but a reduction in total Aβ42 was seen in the cortex of mice treated with both antibodies., Conclusions: Both RmAb158 and its bispecific variant RmAb158-scFv8D3 achieved positive effects of long-term treatment. Despite its ability to efficiently enter the brain, the benefit of using the bispecific antibody in chronic treatment was limited by its reduced plasma exposure, which may be a result of interactions with TfR or the immune system. Future research will focus in new antibody formats to further improve Aβ immunotherapy., (© 2023. The Author(s).)- Published
- 2023
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21. Development of brain-penetrable antibody radioligands for in vivo PET imaging of amyloid-β and tau.
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Banka V, Kelleher A, Sehlin D, Hultqvist G, Sigurdsson EM, Syvänen S, and Ding YS
- Abstract
Introduction: Alzheimer's disease (AD) is characterized by the misfolding and aggregation of two major proteins: amyloid-beta (Aβ) and tau. Antibody-based PET radioligands are desirable due to their high specificity and affinity; however, antibody uptake in the brain is limited by the blood-brain barrier (BBB). Previously, we demonstrated that antibody transport across the BBB can be facilitated through interaction with the transferrin receptor (TfR), and the bispecific antibody-based PET ligands were capable of detecting Aβ aggregates via ex vivo imaging. Since tau accumulation in the brain is more closely correlated with neuronal death and cognition, we report here our strategies to prepare four F-18-labeled specifically engineered bispecific antibody probes for the selective detection of tau and Aβ aggregates to evaluate their feasibility and specificity, particularly for in vivo PET imaging., Methods: We first created and evaluated (via both in vitro and ex vivo studies) four specifically engineered bispecific antibodies, by fusion of single-chain variable fragments (scFv) of a TfR antibody with either a full-size IgG antibody of Aβ or tau or with their respective scFv. Using [
18 F]SFB as the prosthetic group, all four18 F-labeled bispecific antibody probes were then prepared by conjugation of antibody and [18 F]SFB in acetonitrile/0.1 M borate buffer solution (final pH ~ 8.5) with an incubation of 20 min at room temperature, followed by purification on a PD MiniTrap G-25 size exclusion gravity column., Results: Based on both in vitro and ex vivo evaluation, the bispecific antibodies displayed much higher brain concentrations than the unmodified antibody, supporting our subsequent F18-radiolabeling. [18 F]SFB was produced in high yields in 60 min (decay-corrected radiochemical yield (RCY) 46.7 ± 5.4) with radiochemical purities of >95%, confirmed by analytical high performance liquid chromatography (HPLC) and radio-TLC. Conjugation of [18 F]SFB and bispecific antibodies showed a 65%-83% conversion efficiency with radiochemical purities of 95%-99% by radio-TLC., Conclusions: We successfully labeled four novel and specifically engineered bispecific antibodies with [18 F]SFB under mild conditions with a high RCY and purities. This study provides strategies to create brain-penetrable F-18 radiolabeled antibody probes for the selective detection of tau and Aβ aggregates in the brain of transgenic AD mice via in vivo PET imaging., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.- Published
- 2023
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22. ImmunoPET imaging of amyloid-beta in a rat model of Alzheimer's disease with a bispecific, brain-penetrating fusion protein.
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Bonvicini G, Syvänen S, Andersson KG, Haaparanta-Solin M, López-Picón F, and Sehlin D
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- Animals, Mice, Rats, Brain diagnostic imaging, Mice, Transgenic, Disease Models, Animal, Positron-Emission Tomography, Alzheimer Disease diagnostic imaging, Amyloid beta-Peptides metabolism
- Abstract
Background: Hijacking the transferrin receptor (TfR) is an effective strategy to transport amyloid-beta (Aβ) immuno-positron emission tomography (immunoPET) ligands across the blood-brain barrier (BBB). Such ligands are more sensitive and specific than small-molecule ligands at detecting Aβ pathology in mouse models of Alzheimer's disease (AD). This study aimed to determine if this strategy would be as sensitive in rats and to assess how TfR affinity affects BBB transport of bispecific immunoPET radioligands., Methods: Two affinity variants of the rat TfR antibody, OX26, were chemically conjugated to a F(ab')
2 fragment of the anti-Aβ antibody, bapineuzumab (Bapi), to generate two bispecific fusion proteins: OX265 -F(ab')2 -Bapi and OX2676 -F(ab')2 -Bapi. Pharmacokinetic analyses were performed 4 h and 70 h post-injection of radioiodinated fusion proteins in wild-type (WT) rats. [124 I]I-OX265 -F(ab')2 -Bapi was administered to TgF344-AD and WT rats for in vivo PET imaging. Ex vivo distribution of injected [124 I]I-OX265 -F(ab')2 -Bapi and Aβ pathology were assessed., Results: More [125 I]I-OX265 -F(ab')2 -Bapi was taken up into the brain 4 h post-administration than [124 I]I-OX2676 -F(ab')2 -Bapi. [124 I]I-OX265 -F(ab')2 -Bapi PET visualized Aβ pathology with significantly higher signals in the TgF344-AD rats than in the WT littermates without Aβ pathology. The PET signals significantly correlated with Aβ levels in AD animals., Conclusion: Affinity to TfR affects how efficiently a TfR-targeting bispecific fusion protein will cross the BBB, such that the higher-affinity bispecific fusion protein crossed the BBB more efficiently. Furthermore, bispecific immunoPET imaging of brain Aβ pathology using TfR-mediated transport provides good imaging contrast between TgF344-AD and WT rats, suggesting that this immunoPET strategy has the potential to be translated to higher species., (© 2022. The Author(s).)- Published
- 2022
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23. Brain pharmacokinetics of mono- and bispecific amyloid-β antibodies in wild-type and Alzheimer's disease mice measured by high cut-off microdialysis.
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Julku U, Xiong M, Wik E, Roshanbin S, Sehlin D, and Syvänen S
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- Animals, Mice, Tissue Distribution, Mice, Transgenic, Amyloid beta-Peptides metabolism, Brain metabolism, Disease Models, Animal, Alzheimer Disease, Antibodies, Bispecific pharmacokinetics
- Abstract
Background: Treatment with amyloid-β (Aβ) targeting antibodies is a promising approach to remove Aβ brain pathology in Alzheimer's disease (AD) and possibly even slow down or stop progression of the disease. One of the main challenges of brain immunotherapy is the restricted delivery of antibodies to the brain. However, bispecific antibodies that utilize the transferrin receptor (TfR) as a shuttle for transport across the blood-brain barrier (BBB) can access the brain better than traditional monospecific antibodies. Previous studies have shown that bispecific Aβ targeting antibodies have higher brain distribution, and can remove Aβ pathology more efficiently than monospecific antibodies. Yet, there is only limited information available on brain pharmacokinetics, especially regarding differences between mono- and bispecific antibodies., Methods: The aim of the study was to compare brain pharmacokinetics of Aβ-targeting monospecific mAb3D6 and its bispecific version mAb3D6-scFv8D3 that also targets TfR. High cut-off microdialysis was used to measure intravenously injected radiolabelled mAb3D6 and mAb3D6-scFv8D3 antibodies in the interstitial fluid (ISF) of hippocampus in wild-type mice and the App
NL-G-F mouse model of AD. Distribution of the antibodies in the brain and the peripheral tissue was examined by ex vivo autoradiography and biodistribution studies., Results: Brain concentrations of the bispecific antibody were elevated compared to the monospecific antibody in the hippocampal ISF measured by microdialysis and in the brain tissue at 4-6 h after an intravenous injection. The concentration of the bispecific antibody was approximately twofold higher in the ISF dialysate compared to the concentration of monospecific antibody and eightfold higher in brain tissue 6 h post-injection. The ISF dialysate concentrations for both antibodies were similar in both wild-type and AppNL-G-F mice 24 h post-injection, although the total brain tissue concentration of the bispecific antibody was higher than that of the monospecific antibody at this time point. Some accumulation of radioactivity around the probe area was observed especially for the monospecific antibody indicating that the probe compromised the BBB to some extent at the probe insertion site., Conclusion: The BBB-penetrating bispecific antibody displayed higher ISF concentrations than the monospecific antibody. The concentration difference between the two antibodies was even larger in the whole brain than in the ISF. Further, the bispecific antibody, but not the monospecific antibody, displayed higher total brain concentrations than ISF concentrations, indicating association to brain tissue., (© 2022. The Author(s).)- Published
- 2022
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24. Blood-brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer's disease.
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Rofo F, Metzendorf NG, Saubi C, Suominen L, Godec A, Sehlin D, Syvänen S, and Hultqvist G
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- Animals, Mice, Disease Models, Animal, Mice, Transgenic, Proteolysis, Alzheimer Disease pathology, Amyloid beta-Peptides metabolism, Blood-Brain Barrier metabolism, Neprilysin metabolism
- Abstract
Background: Aggregation of the amyloid-β (Aβ) peptide in the brain is one of the key pathological events in Alzheimer's disease (AD). Reducing Aβ levels in the brain by enhancing its degradation is one possible strategy to develop new therapies for AD. Neprilysin (NEP) is a membrane-bound metallopeptidase and one of the major Aβ-degrading enzymes. The secreted soluble form of NEP (sNEP) has been previously suggested as a potential protein-therapy degrading Aβ in AD. However, similar to other large molecules, peripherally administered sNEP is unable to reach the brain due to the presence of the blood-brain barrier (BBB)., Methods: To provide transcytosis across the BBB, we recombinantly fused the TfR binding moiety (scFv8D3) to either sNEP or a previously described variant of NEP (muNEP) suggested to have higher degradation efficiency of Aβ compared to other NEP substrates, but not per se to degrade Aβ more efficiently. To provide long blood half-life, an Fc-based antibody fragment (scFc) was added to the designs, forming sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3. The ability of the mentioned recombinant proteins to degrade Aβ was first evaluated in vitro using synthetic Aβ peptides followed by sandwich ELISA. For the in vivo studies, a single injection of 125-iodine-labelled sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3 was intravenously administered to a tg-ArcSwe mouse model of AD, using scFc-scFv8D3 protein that lacks NEP as a negative control. Different ELISA setups were applied to quantify Aβ concentration of different conformations, both in brain tissues and blood samples., Results: When tested in vitro, sNEP-scFc-scFv8D3 retained sNEP enzymatic activity in degrading Aβ and both constructs efficiently degraded arctic Aβ. When intravenously injected, sNEP-scFc-scFv8D3 demonstrated 20 times higher brain uptake compared to sNEP. Both scFv8D3-fused NEP proteins significantly reduced aggregated Aβ levels in the blood of tg-ArcSwe mice, a transgenic mouse model of AD, following a single intravenous injection. In the brain, monomeric and oligomeric Aβ were significantly reduced. Both scFv8D3-fused NEP proteins displayed a fast clearance from the brain., Conclusion: A one-time injection of a BBB-penetrating NEP shows the potential to reduce, the likely most toxic, Aβ oligomers in the brain in addition to monomers. Also, Aβ aggregates in the blood were reduced., (© 2022. The Author(s).)
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- 2022
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25. Development of the First Tritiated Tetrazine: Facilitating Tritiation of Proteins.
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Radjani Bidesi NS, Battisti UM, Lopes van de Broek S, Shalgunov V, Dall AM, Bøggild Kristensen J, Sehlin D, Syvänen S, Moos Knudsen G, and Herth MM
- Subjects
- Cell Line, Tumor, Cyclooctanes chemistry, Radiopharmaceuticals chemistry, Heterocyclic Compounds
- Abstract
Tetrazine (Tz)-trans-cyclooctene (TCO) ligation is an ultra-fast and highly selective reaction and it is particularly suited to label biomolecules under physiological conditions. As such, a
3 H-Tz based synthon would have wide applications for in vitro/ex vivo assays. In this study, we developed a3 H-labeled Tz and characterized its potential for application to pretargeted autoradiography. Several strategies were explored to synthesize such a Tz. However, classical approaches such as reductive halogenation failed. For this reason, we designed a Tz containing an aldehyde and explored the possibility of reducing this group with NaBT4 . This approach was successful and resulted in [3 H]-(4-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)phenyl)methan-t-ol with a radiochemical yield of 22 %, a radiochemical purity of 96 % and a molar activity of 0.437 GBq/μmol (11.8 Ci/mmol). The compound was successfully applied to pretargeted autoradiography. Thus, we report the synthesis of the first3 H-labeled Tz and its successful application as a labeling building block., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)- Published
- 2022
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26. Functionalization of Radiolabeled Antibodies to Enhance Peripheral Clearance for High Contrast Brain Imaging.
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Schlein E, Syvänen S, Rokka J, Gustavsson T, Rossin R, Robillard M, Eriksson J, and Sehlin D
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- Animals, Mice, Mannose, Amyloid beta-Peptides metabolism, Brain diagnostic imaging, Brain metabolism, Mice, Transgenic, Amyloid metabolism, Antibodies metabolism, Disease Progression, Neuroimaging, Positron-Emission Tomography methods, Immunoconjugates pharmacology, Alzheimer Disease diagnostic imaging, Alzheimer Disease metabolism
- Abstract
Small molecule imaging agents such as [
11 C]PiB, which bind to the core of insoluble amyloid-β (Aβ) fibrils, are useful tools in Alzheimer's disease (AD) research, diagnostics, and drug development. However, the [11 C]PiB PET signal saturates early in the disease progression and does not detect soluble or diffuse Aβ pathology which are believed to play important roles in the disease progression. Antibodies, modified into a bispecific format to enter the brain via receptor-mediated transcytosis, could be a suitable alternative because of their diversity and high specificity for their target. However, the circulation time of these antibodies is long, resulting in an extended exposure to radiation and low imaging contrast. Here, we explore two alternative strategies to enhance imaging contrast by increasing clearance of the antibody ligand from blood. The bispecific Aβ targeting antibody RmAb158-scFv8D3 and the monospecific RmAb158 were radiolabeled and functionalized with either α-d-mannopyranosylphenyl isothiocyanate (mannose) or with trans -cyclooctene (TCO). While mannose can directly mediate antibody clearance via the liver, TCO-modified antibody clearance was induced by injection of a tetrazine-functionalized, liver-targeting clearing agent (CA). In vivo experiments in wild type and AD transgenic mice demonstrated the ability of both strategies to drastically shorten the circulation time of RmAb158, while they had limited effect on the bispecific variant RmAb158-8D3. Furthermore, single photon emission computed tomography imaging with TCO-[125 I]I-RmAb158 in AD mice showed higher contrast 1 day after injection of the tetrazine-functionalized CA. In conclusion, strategies to enhance the clearance of antibody-based imaging ligands could allow imaging at earlier time points and thereby open the possibility to combine antibodies with short-lived radionuclides such as fluorine-18.- Published
- 2022
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27. Passive and receptor mediated brain delivery of an anti-GFAP nanobody.
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Meier SR, Sehlin D, and Syvänen S
- Subjects
- Animals, Mice, Glial Fibrillary Acidic Protein metabolism, Neuroinflammatory Diseases, Positron-Emission Tomography methods, Brain diagnostic imaging, Brain metabolism, Amyloid beta-Peptides metabolism, Mice, Transgenic, Alzheimer Disease metabolism
- Abstract
Purpose: Antibody-based constructs, engineered to enter the brain using transferrin receptor (TfR) mediated transcytosis, have been successfully used as PET radioligands for imaging of amyloid-beta (Aβ) in preclinical studies. However, these radioligands have been large and associated with long circulation times, i.e. non-optimal properties for neuroPET radioligands. The aim of this study was to investigate the in vivo brain delivery of the radiolabeled nanobody VHH-E9 that binds to glial fibrillary acidic protein (GFAP) expressed by reactive astrocytes, without and with fusion to a TfR binding moiety, as potential tools to detect neuroinflammation., Methods: Three protein constructs were recombinantly expressed: 1) The GFAP specific nanobody VHH-E9, 2) VHH-E9 fused to a single chain variable fragment of the TfR binding antibody 8D3 (scFv8D3) and 3) scFv8D3 alone. Brain delivery of the constructs was investigated at 2 h post injection. Binding to GFAP was studied with autoradiography while in vivo brain retention of [
125 I]VHH-E9 and [125 I]VHH-E9-scFv8D3 was further investigated at 8 h, 24 h and 48 h in wild-type (WT), and at the same time points in transgenic mice (ArcSwe) that in addition to Aβ pathology also display neuroinflammation., Results: At 2 h after administration, [125 I]VHH-E9-scFv8D3 and [125 I]scFv8D3 displayed 3-fold higher brain concentrations than [125 I]VHH-E9. In vitro autoradiography showed distinct binding of both [125 I]VHH-E9-scFv8D3 and [125 I]VHH-E9 to regions with abundant GFAP in ArcSwe mice. However, in vivo, there was no difference in brain concentrations between WT and ArcSwe at any of the studied time points., Conclusions: Fused to scFv8D3, VHH-E9 displayed increased brain delivery. When radiolabeled and applied on brain sections, the bispecific construct was able to discriminate between WT and ArcSwe mice, but in vivo brain uptake and retention over time did not differ between WT and ArcSwe mice., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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28. Pretargeted Imaging beyond the Blood-Brain Barrier-Utopia or Feasible?
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Lopes van den Broek S, Shalgunov V, García Vázquez R, Beschorner N, Bidesi NSR, Nedergaard M, Knudsen GM, Sehlin D, Syvänen S, and Herth MM
- Abstract
Pretargeting is a promising nuclear imaging technique that allows for the usage of antibodies (Abs) with enhanced imaging contrast and reduced patient radiation burden. It is based on bioorthogonal chemistry with the tetrazine ligation-a reaction between trans -cyclooctenes (TCOs) and tetrazines (Tzs)-currently being the most popular reaction due to its high selectivity and reactivity. As Abs can be designed to bind specifically to currently 'undruggable' targets such as protein isoforms or oligomers, which play a crucial role in neurodegenerative diseases, pretargeted imaging beyond the BBB is highly sought after, but has not been achieved yet. A challenge in this respect is that large molecules such as Abs show poor brain uptake. Uptake can be increased by receptor mediated transcytosis; however, it is largely unknown if the achieved brain concentrations are sufficient for pretargeted imaging. In this study, we investigated whether the required concentrations are feasible to reach. As a model Ab, we used the bispecific anti-amyloid beta (Aβ) anti-transferrin receptor (TfR) Ab 3D6scFv8D3 and conjugated it to a different amount of TCOs per Ab and tested different concentrations in vitro. With this model in hand, we estimated the minimum required TCO concentration to achieve a suitable contrast between the high and low binding regions. The estimation was carried out using pretargeted autoradiography on brain sections of an Alzheimer's disease mouse model. Biodistribution studies in wild-type (WT) mice were used to correlate how different TCO/Ab ratios alter the brain uptake. Pretargeted autoradiography showed that increasing the number of TCOs as well as increasing the TCO-Ab concentration increased the imaging contrast. A minimum brain concentration of TCOs for pretargeting purposes was determined to be 10.7 pmol/g in vitro. Biodistribution studies in WT mice showed a brain uptake of 1.1% ID/g using TCO-3D6scFv8D3 with 6.8 TCO/Ab. According to our estimations using the optimal parameters, pretargeted imaging beyond the BBB is not a utopia. Necessary brain TCO concentrations can be reached and are in the same order of magnitude as required to achieve sufficient contrast. This work gives a first estimate that pretargeted imaging is indeed possible with antibodies. This could allow the imaging of currently 'undruggable' targets and therefore be crucial to monitor (e.g., therapies for intractable neurodegenerative diseases).
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- 2022
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29. The Alzheimer's disease 5xFAD mouse model is best suited to investigate pretargeted imaging approaches beyond the blood-brain barrier.
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Lopes van den Broek S, Sehlin D, Andersen JV, Aldana BI, Beschörner N, Nedergaard M, Knudsen GM, Syvänen S, and Herth MM
- Abstract
Alzheimer's disease (AD) is the most common neurodegenerative disease, with an increasing prevalence. Currently, there is no ideal diagnostic molecular imaging agent for diagnosing AD. Antibodies (Abs) have been proposed to close this gap as they can bind selectively and with high affinity to amyloid β (Aβ)-one of the molecular hallmarks of AD. Abs can even be designed to selectively bind Aβ oligomers or isoforms, which are difficult to target with small imaging agents. Conventionally, Abs must be labeled with long-lived radionuclides which typically results in in high radiation burden to healthy tissue. Pretargeted imaging could solve this challenge as it allows for the use of short-lived radionuclides. To develop pretargeted imaging tools that can enter the brain, AD mouse models are useful as they allow testing of the imaging approach in a relevant animal model that could predict its clinical applicability. Several mouse models for AD have been developed with different characteristics. Commonly used models are: 5xFAD, APP/PS1 and tg-ArcSwe transgenic mice. In this study, we aimed to identify which of these models were best suited to investigate pretargeted imaging approaches beyond the blood brain barrier. We evaluated this by pretargeted autoradiography using the Aβ-targeting antibody 3D6 and an
111 In-labeled Tz. Evaluation criteria were target-to-background ratios and accessibility. APP/PS1 mice showed Aβ accumulation in high and low binding brain regions and is as such less suitable for pretargeted purposes. 5xFAD and tg-ArcSwe mice showed similar uptake in high binding regions whereas low uptake in low binding regions and are better suited to evaluate pretargeted imaging approaches. 5xFAD mice are advantaged over tg-ArcSwe mice as pathology can be traced early (6 months compared to 18 months of age) and as 5xFAD mice are commercially available., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Lopes van den Broek, Sehlin, Andersen, Aldana, Beschörner, Nedergaard, Knudsen, Syvänen and Herth.)- Published
- 2022
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30. Development of process analytical tools for rapid monitoring of live virus vaccines in manufacturing.
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Yi S, McCracken R, Davide J, Salovich DR, Whitmer T, Bhat A, Vlasak J, Ha S, Sehlin D, Califano J, Ploeger K, and Mukherjee M
- Subjects
- Bioreactors, Nucleic Acids, Vaccines
- Abstract
In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry (or nanoscale flow cytometry) to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between "good" and "damaged" virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs., (© 2022. The Author(s).)
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- 2022
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31. A Brain-Targeting Bispecific-Multivalent Antibody Clears Soluble Amyloid-Beta Aggregates in Alzheimer's Disease Mice.
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Rofo F, Meier SR, Metzendorf NG, Morrison JI, Petrovic A, Syvänen S, Sehlin D, and Hultqvist G
- Subjects
- Animals, Mice, Mice, Transgenic, Amyloid beta-Peptides metabolism, Brain metabolism, Receptors, Transferrin metabolism, Receptors, Transferrin therapeutic use, Immunoglobulin G therapeutic use, Alzheimer Disease drug therapy
- Abstract
Amyloid-β (Aβ) oligomers and protofibrils are suggested to be the most neurotoxic Aβ species in Alzheimer's disease (AD). Hence, antibodies with strong and selective binding to these soluble Aβ aggregates are of therapeutic potential. We have recently introduced HexaRmAb158, a multivalent antibody with additional Aβ-binding sites in the form of single-chain fragment variables (scFv) on the N-terminal ends of Aβ protofibril selective antibody (RmAb158). Due to the additional binding sites and the short distance between them, HexaRmAb158 displayed a slow dissociation from protofibrils and strong binding to oligomers in vitro. In the current study, we aimed at investigating the therapeutic potential of this antibody format in vivo using mouse models of AD. To enhance BBB delivery, the transferrin receptor (TfR) binding moiety (scFv8D3) was added, forming the bispecific-multivalent antibody (HexaRmAb158-scFv8D3). The new antibody displayed a weaker TfR binding compared to the previously developed RmAb158-scFv8D3 and was less efficiently transcytosed in a cell-based BBB model. HexaRmAb158 detected soluble Aβ aggregates derived from brains of tg-ArcSwe and App
NL-G-F mice more efficiently compared to RmAb158. When intravenously injected, HexaRmAb158-scFv8D3 was actively transported over the BBB into the brain in vivo. Brain uptake was marginally lower than that of RmAb158-scFv8D3, but significantly higher than observed for conventional IgG antibodies. Both antibody formats displayed similar brain retention (72 h post injection) and equal capacity in clearing soluble Aβ aggregates in tg-ArcSwe mice. In conclusion, we demonstrate a bispecific-multivalent antibody format capable of passing the BBB and targeting a wide-range of sizes of soluble Aβ aggregates., (© 2022. The Author(s).)- Published
- 2022
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32. Reduction of αSYN Pathology in a Mouse Model of PD Using a Brain-Penetrating Bispecific Antibody.
- Author
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Roshanbin S, Julku U, Xiong M, Eriksson J, Masliah E, Hultqvist G, Bergström J, Ingelsson M, Syvänen S, and Sehlin D
- Abstract
Immunotherapy targeting aggregated alpha-synuclein (αSYN) is a promising approach for the treatment of Parkinson's disease. However, brain penetration of antibodies is hampered by their large size. Here, RmAbSynO2-scFv8D3, a modified bispecific antibody that targets aggregated αSYN and binds to the transferrin receptor for facilitated brain uptake, was investigated to treat αSYN pathology in transgenic mice. Ex vivo analyses of the blood and brain distribution of RmAbSynO2-scFv8D3 and the unmodified variant RmAbSynO2, as well as in vivo analyses with microdialysis and PET, confirmed fast and efficient brain uptake of the bispecific format. In addition, intravenous administration was shown to be superior to intraperitoneal injections in terms of brain uptake and distribution. Next, aged female αSYN transgenic mice (L61) were administered either RmAbSynO2-scFv8D3, RmAbSynO2, or PBS intravenously three times over five days. Levels of TBS-T soluble aggregated αSYN in the brain following treatment with RmAbSynO2-scFv8D3 were decreased in the cortex and midbrain compared to RmAbSynO2 or PBS controls. Taken together, our results indicate that facilitated brain uptake of αSYN antibodies can improve treatment of αSYN pathology.
- Published
- 2022
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33. Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-Aβ-Affibodies.
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Faresjö R, Lindberg H, Ståhl S, Löfblom J, Syvänen S, and Sehlin D
- Subjects
- Amyloid beta-Peptides metabolism, Animals, Brain metabolism, Disease Models, Animal, Mice, Mice, Transgenic, Positron-Emission Tomography methods, Receptors, Transferrin metabolism, Alzheimer Disease drug therapy, Blood-Brain Barrier metabolism
- 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
125 I-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, [125 I]I-Z5 and [125 I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [125 I]I-scFv8D3-Z5 and [125 I]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 [125 I]I-Z1 and [125 I]I-Z5 was low, while [125 I]I-scFv8D3-Z1 and [125 I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [125 I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [125 I]I-scFv8D3-Z5 and [125 I]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., (© 2022. The Author(s).)- Published
- 2022
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34. PET Imaging in Preclinical Anti-Aβ Drug Development.
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Syvänen S, Meier SR, Roshanbin S, Xiong M, Faresjö R, Gustavsson T, Bonvicini G, Schlein E, Aguilar X, Julku U, Eriksson J, and Sehlin D
- Subjects
- Amyloid metabolism, Animals, Brain metabolism, Drug Development, Positron-Emission Tomography methods, Alzheimer Disease diagnostic imaging, Alzheimer Disease drug therapy, Alzheimer Disease pathology, Amyloid beta-Peptides metabolism
- Abstract
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 (Aβ) aggregates, by showing a dose-dependent reduction in brain amyloid after treatment. In parallel to clinical studies, preclinical studies in animal models of Aβ pathology may also benefit from PET as a tool to detect target engagement and treatment effects of anti-Aβ 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 Aβ, and its present and potential future role in the development of drugs aimed at reducing brain Aβ levels as a therapeutic strategy to halt disease progression in AD., (© 2022. The Author(s).)
- Published
- 2022
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35. In vivo imaging of alpha-synuclein with antibody-based PET.
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Roshanbin S, Xiong M, Hultqvist G, Söderberg L, Zachrisson O, Meier S, Ekmark-Lewén S, Bergström J, Ingelsson M, Sehlin D, and Syvänen S
- Subjects
- Amyloid beta-Peptides metabolism, Animals, Brain metabolism, Humans, Mice, Positron-Emission Tomography methods, alpha-Synuclein metabolism, Antibodies, Bispecific metabolism, Multiple System Atrophy metabolism, Parkinson Disease metabolism, Synucleinopathies
- Abstract
The protein alpha-synuclein (αSYN) plays a central role in synucleinopathies such as Parkinsons's disease (PD) and multiple system atrophy (MSA). Presently, there are no selective αSYN positron emission tomography (PET) radioligands that do not also show affinity to amyloid-beta (Aβ). We have previously shown that radiolabeled antibodies, engineered to enter the brain via the transferrin receptor (TfR), is a promising approach for PET imaging of intrabrain targets. In this study, we used this strategy to visualize αSYN in the living mouse brain. Five bispecific antibodies, binding to both the murine TfR and αSYN were generated and radiolabeled with iodine-125 or iodine-124. All bispecific antibodies bound to αSYN and mTfR before and after radiolabelling in an ELISA assay, and bound to brain sections prepared from αSYN overexpressing mice as well as human PD- and MSA subjects, but not control tissues in autoradiography. Brain concentrations of the bispecific antibodies were between 26 and 63 times higher than the unmodified IgG format 2 h post-injection, corresponding to about 1.5% of the injected dose per gram brain tissue. Additionally, intrastriatal αSYN fibrils were visualized with PET in an αSYN deposition mouse model with one of the bispecific antibodies, [
124 I]RmAbSynO2-scFv8D3. However, PET images acquired in αSYN transgenic mice with verified brain pathology injected with [124 I]RmAbSynO2-scFv8D3 and [124 I]RmAb48-scFv8D3 showed no increase in antibody retention compared to WT mice. Despite successful imaging of deposited extracellular αSYN using a brain-penetrating antibody-based radioligand with no cross-specificity towards Aβ, this proof-of-concept study demonstrates challenges in imaging intracellular αSYN inclusions present in synucleinopathies., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)- Published
- 2022
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36. 11 C-PiB and 124 I-Antibody PET Provide Differing Estimates of Brain Amyloid-β After Therapeutic Intervention.
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Meier SR, Sehlin D, Roshanbin S, Falk VL, Saito T, Saido TC, Neumann U, Rokka J, Eriksson J, and Syvänen S
- Subjects
- Amyloid beta-Peptides metabolism, Aniline Compounds metabolism, Animals, Antibodies metabolism, Brain metabolism, Disease Models, Animal, Iodine Radioisotopes, Mice, Mice, Transgenic, Plaque, Amyloid metabolism, Positron-Emission Tomography methods, Alzheimer Disease metabolism
- Abstract
PET imaging of amyloid-β (Aβ) has become an important component of Alzheimer disease diagnosis.
11 C-Pittsburgh compound B (11 C-PiB) and analogs bind to fibrillar Aβ. However, levels of nonfibrillar, soluble, aggregates of Aβ appear more dynamic during disease progression and more affected by Aβ-reducing treatments. The aim of this study was to compare an antibody-based PET ligand targeting nonfibrillar Aβ with11 C-PiB after β-secretase (BACE-1) inhibition in 2 Alzheimer disease mouse models at an advanced stage of Aβ pathology. Methods: Transgenic ArcSwe mice (16 mo old) were treated with the BACE-1 inhibitor NB-360 for 2 mo, whereas another group was kept as controls. A third group was analyzed at the age of 16 mo as a baseline. Mice were PET-scanned with11 C-PiB to measure Aβ plaque load followed by a scan with the bispecific radioligand124 I-RmAb158-scFv8D3 to investigate nonfibrillar aggregates of Aβ. The same study design was then applied to another mouse model, AppNL-G-F In this case, NB-360 treatment was initiated at the age of 8 mo and animals were scanned with11 C-PiB-PET and125 I-RmAb158-scFv8D3 SPECT. Brain tissue was isolated after scanning, and Aβ levels were assessed. Results:124 I-RmAb158-scFv8D3 concentrations measured with PET in hippocampus and thalamus of NB-360-treated ArcSwe mice were similar to those observed in baseline animals and significantly lower than concentrations observed in same-age untreated controls. Reduced125 I-RmAb158-scFv8D3 retention was also observed with SPECT in hippocampus, cortex, and cerebellum of NB-360-treated AppNL-G-F mice. Radioligand in vivo concentrations corresponded to postmortem brain tissue analysis of soluble Aβ aggregates. For both models, mice treated with NB-360 did not display a reduced11 C-PiB signal compared with untreated controls, and further, both NB-360 and control mice tended, although not reaching significance, to show higher11 C-PiB signal than the baseline groups. Conclusion: This study demonstrated the ability of an antibody-based radioligand to detect changes in brain Aβ levels after anti-Aβ therapy in ArcSwe and AppNL-G-F mice with pronounced Aβ pathology. In contrast, the decreased Aβ levels could not be quantified with11 C-PiB PET, suggesting that these ligands detect different pools of Aβ., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)- Published
- 2022
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37. Pinpointing Brain TREM2 Levels in Two Mouse Models of Alzheimer's Disease.
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Meier SR, Sehlin D, Hultqvist G, and Syvänen S
- Subjects
- Animals, Brain diagnostic imaging, Brain Chemistry physiology, Disease Models, Animal, Mice, Neuroinflammatory Diseases metabolism, Positron-Emission Tomography, Alzheimer Disease metabolism, Antibodies, Bispecific analysis, Antibodies, Bispecific chemistry, Antibodies, Bispecific metabolism, Brain metabolism, Membrane Glycoproteins analysis, Membrane Glycoproteins metabolism, Molecular Imaging methods, Receptors, Immunologic analysis, Receptors, Immunologic metabolism
- Abstract
Purpose: The triggering receptor expressed on myeloid cells 2 (TREM2) is expressed by brain microglia. Microglial activation, as observed in Alzheimer's disease (AD) as well as in transgenic mice expressing human amyloid-beta, appears to increase soluble TREM2 (sTREM2) levels in CSF and brain. In this study, we used two different transgenic mouse models of AD pathology and investigated the potential of TREM2 to serve as an in vivo biomarker for microglial activation in AD., Procedures: We designed and generated a bispecific antibody based on the TREM2-specific monoclonal antibody mAb1729, fused to a single-chain variable fragment of the transferrin receptor binding antibody 8D3. The 8D3-moiety enabled transcytosis of the whole bispecific antibody across the blood-brain barrier. The bispecific antibody was radiolabeled with I-125 (ex vivo) or I-124 (PET) and administered to transgenic AD and wild-type (WT) control mice. Radioligand retention in the brain of transgenic animals was compared to WT mice by isolation of brain tissue at 24 h or 72 h, or with in vivo PET at 24 h, 48 h, and 72 h. Intrabrain distribution of radiolabeled mAb1729-scFv8D3
CL was further studied by autoradiography, while ELISA was used to determine TREM2 brain concentrations., Results: Transgenic animals displayed higher total exposure, calculated as the AUC based on SUV determined at 24h, 48h, and 72h post injection, of PET radioligand [124 I]mAb1729-scFv8D3CL than WT mice. However, differences were not evident in single time point PET images or SUVs. Ex vivo autoradiography confirmed higher radioligand concentrations in cortex and thalamus in transgenic mice compared to WT, and TREM2 levels in brain homogenates were considerably higher in transgenic mice compared to WT., Conclusion: Antibody-based radioligands, engineered to enter the brain, may serve as PET radioligands to follow changes of TREM2 in vivo, but antibody formats with faster systemic clearance to increase the specific signal in relation to that from blood in combination with antibodies showing higher affinity for TREM2 must be developed to further progress this technique for in vivo use., (© 2021. The Author(s).)- Published
- 2021
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38. In vivo imaging of synaptic density with [ 11 C]UCB-J PET in two mouse models of neurodegenerative disease.
- Author
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Xiong M, Roshanbin S, Rokka J, Schlein E, Ingelsson M, Sehlin D, Eriksson J, and Syvänen S
- Subjects
- Aging, Alzheimer Disease, Amyloid beta-Peptides genetics, Animals, Area Under Curve, Brain ultrastructure, Mice, Mice, Inbred C57BL, Mice, Transgenic, Organ Specificity, Parkinson Disease, Peptide Fragments genetics, Amyloid beta-Peptides analysis, Brain diagnostic imaging, Carbon Radioisotopes pharmacokinetics, Disease Models, Animal, Membrane Glycoproteins analysis, Nerve Tissue Proteins analysis, Peptide Fragments analysis, Positron-Emission Tomography methods, Pyridines pharmacokinetics, Pyrrolidinones pharmacokinetics, Radiopharmaceuticals pharmacokinetics, Synaptic Vesicles ultrastructure, Synucleinopathies diagnostic imaging
- Abstract
The positron emission tomography (PET) radioligand [
11 C]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 [11 C]UCB-J binding in Alzheimer's disease (AD) and Parkinson's disease (PD) brains compared to healthy controls. Still, it is unknown whether [11 C]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 [11 C]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 [11 C]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 [11 C]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 [11 C]UCB-J brain exposure than younger WT mice (8-9 months). Together, these data imply that [11 C]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 [11 C]UCB-J binding as an alternative to full pharmacokinetic modelling., (Copyright © 2021. Published by Elsevier Inc.)- Published
- 2021
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39. Novel multivalent design of a monoclonal antibody improves binding strength to soluble aggregates of amyloid beta.
- Author
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Rofo F, Buijs J, Falk R, Honek K, Lannfelt L, Lilja AM, Metzendorf NG, Gustavsson T, Sehlin D, Söderberg L, and Hultqvist G
- Subjects
- Amyloid, Animals, Antibodies, Monoclonal, Mice, Mice, Transgenic, Alzheimer Disease drug therapy, Alzheimer Disease metabolism, Amyloid beta-Peptides metabolism
- Abstract
Background: Amyloid-β (Aβ) immunotherapy is a promising therapeutic strategy in the fight against Alzheimer's disease (AD). A number of monoclonal antibodies have entered clinical trials for AD. Some of them have failed due to the lack of efficacy or side-effects, two antibodies are currently in phase 3, and one has been approved by FDA. The soluble intermediate aggregated species of Aβ, termed oligomers and protofibrils, are believed to be key pathogenic forms, responsible for synaptic and neuronal degeneration in AD. Therefore, antibodies that can strongly and selectively bind to these soluble intermediate aggregates are of great diagnostic and therapeutic interest., Methods: We designed and recombinantly produced a hexavalent antibody based on mAb158, an Aβ protofibril-selective antibody. The humanized version of mAb158, lecanemab (BAN2401), is currently in phase 3 clinical trials for the treatment of AD. The new designs involved recombinantly fusing single-chain fragment variables to the N-terminal ends of mAb158 antibody. Real-time interaction analysis with LigandTracer and surface plasmon resonance were used to evaluate the kinetic binding properties of the generated antibodies to Aβ protofibrils. Different ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to Aβ aggregates of different sizes. Finally, the ability of the antibodies to protect cells from Aβ-induced effects was evaluated by MTT assay., Results: Using real-time interaction analysis with LigandTracer, the hexavalent design promoted a 40-times enhanced binding with avidity to protofibrils, and most of the added binding strength was attributed to the reduced rate of dissociation. Furthermore, ELISA experiments demonstrated that the hexavalent design also had strong binding to small oligomers, while retaining weak and intermediate binding to monomers and insoluble fibrils. The hexavalent antibody also reduced cell death induced by a mixture of soluble Aβ aggregates., Conclusion: We provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of Aβ aggregates. This approach should be general and work for any aggregated protein or repetitive target., (© 2021. The Author(s).)
- Published
- 2021
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40. The Uppsala APP deletion causes early onset autosomal dominant Alzheimer's disease by altering APP processing and increasing amyloid β fibril formation.
- Author
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Pagnon de la Vega M, Giedraitis V, Michno W, Kilander L, Güner G, Zielinski M, Löwenmark M, Brundin R, Danfors T, Söderberg L, Alafuzoff I, Nilsson LNG, Erlandsson A, Willbold D, Müller SA, Schröder GF, Hanrieder J, Lichtenthaler SF, Lannfelt L, Sehlin D, and Ingelsson M
- Subjects
- Amyloid Precursor Protein Secretases metabolism, Amyloid beta-Protein Precursor genetics, Amyloid beta-Protein Precursor metabolism, Brain metabolism, Humans, Alzheimer Disease genetics, Amyloid beta-Peptides metabolism
- Abstract
Point mutations in the amyloid precursor protein gene ( APP ) cause familial Alzheimer's disease (AD) by increasing generation or altering conformation of amyloid β (Aβ). Here, we describe the Uppsala APP mutation (Δ690-695), the first reported deletion causing autosomal dominant AD. Affected individuals have an age at symptom onset in their early forties and suffer from a rapidly progressing disease course. Symptoms and biomarkers are typical of AD, with the exception of normal cerebrospinal fluid (CSF) Aβ42 and only slightly pathological amyloid-positron emission tomography signals. Mass spectrometry and Western blot analyses of patient CSF and media from experimental cell cultures indicate that the Uppsala APP mutation alters APP processing by increasing β-secretase cleavage and affecting α-secretase cleavage. Furthermore, in vitro aggregation studies and analyses of patient brain tissue samples indicate that the longer form of mutated Aβ, AβUpp1-42
Δ19-24 , accelerates the formation of fibrils with unique polymorphs and their deposition into amyloid plaques in the affected brain., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)- Published
- 2021
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41. Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different size.
- Author
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Faresjö R, Bonvicini G, Fang XT, Aguilar X, Sehlin D, and Syvänen S
- Subjects
- Animals, Antibodies, Bispecific administration & dosage, Blood-Brain Barrier drug effects, Brain drug effects, Female, Injections, Intravenous, Iodine Radioisotopes administration & dosage, Male, Mice, Mice, Inbred C57BL, Particle Size, Protein Binding physiology, Antibodies, Bispecific pharmacokinetics, Blood-Brain Barrier metabolism, Brain metabolism, Iodine Radioisotopes metabolism
- Abstract
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
125 I-iodinated ([125 I]) mAb3D6-scFv8D3 (n = 46) or [125 I]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 [125 I]mAb3D6-scFv8D3 (n = 12) or [125 I]di-scFv3D6-8D3 (n = 12), to estimate the relative levels of radiolabelled antibody in brain capillaries versus brain parenchyma. In vitro binding kinetics for [125 I]mAb3D6-scFv8D3 or [125 I]di-scFv3D6-8D3 to murine TfR were determined by LigandTracer., Results: [125 I]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 [125 I]mAb3D6-scFv8D3. However, the elimination rate from brain did not differ between the antibodies. The study also indicated that [125 I]di-scFv3D6-8D3 displayed lower avidity than [125 I]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.- Published
- 2021
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42. Flow virometry for process monitoring of live virus vaccines-lessons learned from ERVEBO.
- Author
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Ricci G, Minsker K, Kapish A, Osborn J, Ha S, Davide J, Califano JP, Sehlin D, Rustandi RR, Dick LW Jr, Vlasak J, Culp TD, Baudy A, Bell E, and Mukherjee M
- Subjects
- Animals, Chlorocebus aethiops, Ebola Vaccines standards, Humans, Temperature, Vaccines, Synthetic standards, Vero Cells, Virion ultrastructure, Flow Cytometry methods, Vaccines, Attenuated standards, Vaccinology methods, Vaccinology standards, Viral Vaccines standards
- Abstract
Direct at line monitoring of live virus particles in commercial manufacturing of vaccines is challenging due to their small size. Detection of malformed or damaged virions with reduced potency is rate-limited by release potency assays with long turnaround times. Thus, preempting batch failures caused by out of specification potency results is almost impossible. Much needed are in-process tools that can monitor and detect compromised viral particles in live-virus vaccines (LVVs) manufacturing based on changes in their biophysical properties to provide timely measures to rectify process stresses leading to such damage. Using ERVEBO, MSD's Ebola virus vaccine as an example, here we describe a flow virometry assay that can quickly detect damaged virus particles and provide mechanistic insight into process parameters contributing to the damage. Furthermore, we describe a 24-h high throughput infectivity assay that can be used to correlate damaged particles directly to loss in viral infectivity (potency) in-process. Collectively, we provide a set of innovative tools to enable rapid process development, process monitoring, and control strategy implementation in large scale LVV manufacturing.
- Published
- 2021
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43. Enhanced neprilysin-mediated degradation of hippocampal Aβ42 with a somatostatin peptide that enters the brain.
- Author
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Rofo F, Ugur Yilmaz C, Metzendorf N, Gustavsson T, Beretta C, Erlandsson A, Sehlin D, Syvänen S, Nilsson P, and Hultqvist G
- Subjects
- Alzheimer Disease metabolism, Alzheimer Disease pathology, Animals, Blood-Brain Barrier metabolism, Brain metabolism, Female, Hormones pharmacology, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Alzheimer Disease drug therapy, Amyloid beta-Peptides metabolism, Blood-Brain Barrier drug effects, Brain drug effects, Hippocampus metabolism, Neprilysin pharmacology, Peptide Fragments metabolism, Somatostatin pharmacology
- Abstract
Background: Aggregation of the amyloid-beta (Aβ) peptide is one of the main neuropathological events in Alzheimer's disease (AD). Neprilysin is the major enzyme degrading Aβ, with its activity enhanced by the neuropeptide somatostatin (SST). SST levels are decreased in the brains of AD patients. The poor delivery of SST over the blood-brain barrier (BBB) and its extremely short half-life of only 3 min limit its therapeutic significance. Methods: We recombinantly fused SST to a BBB transporter binding to the transferrin receptor. Using primary neuronal cultures and neuroblastoma cell lines, the ability of the formed fusion protein to activate neprilysin was studied. SST-scFv8D3 was administered to mice overexpressing the Aβ-precursor protein (AβPP) with the Swedish mutation (APPswe) as a single injection or as a course of three injections over a 72 h period. Levels of neprilysin and Aβ were quantified using an Enzyme-linked immunosorbent assay (ELISA). Distribution of SST-scFv8D3 in the brain, blood and peripheral organs was studied by radiolabeling with iodine-125. Results: The construct, SST-scFv8D3, exhibited 120 times longer half-life than SST alone, reached the brain in high amounts when injected intravenously and significantly increased the brain concentration of neprilysin in APPswe mice. A significant decrease in the levels of membrane-bound Aβ42 was detected in the hippocampus and the adjacent cortical area after only three injections. Conclusion: With intravenous injections of our BBB permeable SST peptide, we were able to significantly increase the levels neprilysin, an effect that was followed by a significant and selective degradation of membrane-bound Aβ42 in the hippocampus. Being that membrane-bound Aβ triggers neuronal toxicity and the hippocampus is the central brain area in the progression of AD, the study has illuminated a new potential treatment paradigm with a promising safety profile targeting only the disease affected areas., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)
- Published
- 2021
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44. Fluorine-18-Labeled Antibody Ligands for PET Imaging of Amyloid-β in Brain.
- Author
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Syvänen S, Fang XT, Faresjö R, Rokka J, Lannfelt L, Olberg DE, Eriksson J, and Sehlin D
- Subjects
- Animals, Brain diagnostic imaging, Ligands, Mice, Fluorine Radioisotopes, Positron-Emission Tomography
- 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
18 F-labeled tetrazines through an inverse electron demand Diels-Alder reaction performed at ambient temperature.18 F-labeling did not affect antibody binding in vitro , and initial brain uptake was high. Conjugates with the first tetrazine variant ([18 F]T1) displayed high uptake in bone, indicating extensive defluorination, a problem that was resolved with the second and third tetrazine variants ([18 F]T2 and [18 F]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), [18 F]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 that18 F-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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45. Extracellular vesicles from amyloid-β exposed cell cultures induce severe dysfunction in cortical neurons.
- Author
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Beretta C, Nikitidou E, Streubel-Gallasch L, Ingelsson M, Sehlin D, and Erlandsson A
- Subjects
- Alzheimer Disease etiology, Alzheimer Disease metabolism, Animals, Astrocytes drug effects, Cells, Cultured, Cerebral Cortex drug effects, Coculture Techniques, Extracellular Vesicles drug effects, Mice, Mice, Inbred C57BL, Neurons drug effects, Alzheimer Disease pathology, Amyloid beta-Peptides toxicity, Astrocytes pathology, Cerebral Cortex pathology, Extracellular Vesicles pathology, Microscopy, Electron, Transmission methods, Neurons pathology
- Abstract
Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-β (Aβ) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of Aβ
42 protofibrils by astrocytes results in the release of EVs containing neurotoxic Aβ. Here, we describe the cellular mechanisms behind EV-associated neurotoxicity in detail. EVs were isolated from untreated and Aβ42 protofibril exposed neuroglial co-cultures, consisting mainly of astrocytes. The EVs were added to cortical neurons for 2 or 4 days and the neurodegenerative processes were followed with immunocytochemistry, time-lapse imaging and transmission electron microscopy (TEM). Addition of EVs from Aβ42 protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM analysis demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathological lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from Aβ exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of Aβ-induced pathology.- Published
- 2020
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46. Brain delivery of biologics using a cross-species reactive transferrin receptor 1 VNAR shuttle.
- Author
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Sehlin D, Stocki P, Gustavsson T, Hultqvist G, Walsh FS, Rutkowski JL, and Syvänen S
- Subjects
- Animals, Antibodies, Monoclonal, Humanized administration & dosage, Antibodies, Monoclonal, Humanized genetics, Biological Products pharmacokinetics, Blood-Brain Barrier diagnostic imaging, Drug Delivery Systems, Humans, Immunoglobulin Fc Fragments genetics, Mice, Mice, Inbred C57BL, Recombinant Fusion Proteins genetics, Thromboxane B2 genetics, Transcytosis, Antigens, CD metabolism, Biological Products administration & dosage, Blood-Brain Barrier metabolism, Receptors, Transferrin metabolism, Thromboxane B2 metabolism
- Abstract
Transferrin receptor 1 (TfR1) mediated transcytosis is an attractive strategy to enhance brain uptake of protein drugs, but translation remains a challenge. Here, a single domain shark antibody VNAR fragment (TXB2) with similar affinity to murine and human TfR1 was used to shuttle protein cargo into the brain. TXB2 was fused to a human IgG1 Fc domain (hFc) or to the amyloid-β (Aβ) antibody bapineuzumab (Bapi). TXB2-hFc displayed 20-fold higher brain concentrations compared with a control VNAR-hFc at 18 hours post-injection in wt mice. At the same time point, brain concentrations of Bapi-TXB2 was threefold higher than Bapi. In transgenic mice overexpressing human Aβ, the brain-to-blood concentration ratio increased with time due to interaction with intracerebral Aβ deposits. The relatively stable threefold difference between Bapi-TXB2 and Bapi was observed for up to 6 days after injection. PET imaging and ex vivo autoradiography revealed more parenchymal distribution of Bapi-TXB2 compared with Bapi. In conclusion, the TXB2 VNAR shuttle markedly increased brain uptake of protein cargo and increased brain concentrations of the Aβ binding antibody Bapi., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)
- Published
- 2020
- Full Text
- View/download PDF
47. SPECT imaging of distribution and retention of a brain-penetrating bispecific amyloid-β antibody in a mouse model of Alzheimer's disease.
- Author
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Gustavsson T, Syvänen S, O'Callaghan P, and Sehlin D
- Subjects
- Alzheimer Disease diagnostic imaging, Alzheimer Disease genetics, Amyloid beta-Protein Precursor genetics, Animals, Antibodies, Bispecific genetics, Antibodies, Monoclonal genetics, Antibodies, Monoclonal, Humanized administration & dosage, Antibodies, Monoclonal, Humanized metabolism, Brain diagnostic imaging, Brain drug effects, Disease Models, Animal, Female, Humans, Iodine Radioisotopes administration & dosage, Iodine Radioisotopes metabolism, Male, Mice, Mice, Transgenic, Tissue Distribution drug effects, Tissue Distribution physiology, Alzheimer Disease metabolism, Amyloid beta-Protein Precursor metabolism, Antibodies, Bispecific metabolism, Antibodies, Monoclonal metabolism, Brain metabolism, Tomography, Emission-Computed, Single-Photon methods
- Abstract
Background: Alzheimer's disease (AD) immunotherapy with antibodies targeting amyloid-β (Aβ) has been extensively explored in clinical trials. The aim of this study was to study the long-term brain distribution of two radiolabeled monoclonal Aβ antibody variants - RmAb158, the recombinant murine version of BAN2401, which has recently demonstrated amyloid removal and reduced cognitive decline in AD patients, and the bispecific RmAb158-scFv8D3, which has been engineered for enhanced brain uptake via transferrin receptor-mediated transcytosis., Methods: A single intravenous injection of iodine-125 (
125 I)-labeled RmAb158-scFv8D3 or RmAb158 was administered to AD transgenic mice (tg-ArcSwe). In vivo single-photon emission computed tomography was used to investigate brain retention and intrabrain distribution of the antibodies over a period of 4 weeks. Activity in blood and brain tissue was measured ex vivo and autoradiography was performed in combination with Aβ and CD31 immunostaining to investigate the intrabrain distribution of the antibodies and their interactions with Aβ., Results: Despite faster blood clearance, [125 I]RmAb158-scFv8D3 displayed higher brain exposure than [125 I]RmAb158 throughout the study. The brain distribution of [125 I]RmAb158-scFv8D3 was more uniform and coincided with parenchymal Aβ pathology, while [125 I]RmAb158 displayed a more scattered distribution pattern and accumulated in central parts of the brain at later times. Ex vivo autoradiography indicated greater vascular escape and parenchymal Aβ interactions for [125 I]RmAb158-scFv8D3, whereas [125 I]RmAb158 displayed retention and Aβ interactions in lateral ventricles., Conclusions: The high brain uptake and uniform intrabrain distribution of RmAb158-scFv8D3 highlight the benefits of receptor-mediated transcytosis for antibody-based brain imaging. Moreover, it suggests that the alternative transport route of the bispecific antibody contributes to improved efficacy of brain-directed immunotherapy.- Published
- 2020
- Full Text
- View/download PDF
48. Chemical imaging of evolving amyloid plaque pathology and associated Aβ peptide aggregation in a transgenic mouse model of Alzheimer's disease.
- Author
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Michno W, Wehrli P, Meier SR, Sehlin D, Syvänen S, Zetterberg H, Blennow K, and Hanrieder J
- Subjects
- Animals, Disease Models, Animal, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Alzheimer Disease pathology, Amyloid beta-Peptides, Brain pathology, Plaque, Amyloid pathology, Protein Aggregation, Pathological pathology
- Abstract
One of the major hallmarks of Alzheimer's disease (AD) pathology is the formation of extracellular amyloid β (Aβ) plaques. While Aβ has been suggested to be critical in inducing and, potentially, driving the disease, the molecular basis of AD pathogenesis is still under debate. Extracellular Aβ plaque pathology manifests itself upon aggregation of distinct Aβ peptides, resulting in morphologically different plaque morphotypes, including mainly diffuse and cored senile plaques. As plaque pathology precipitates long before any clinical symptoms occur, targeting the Aβ aggregation processes provides a promising target for early interventions. However, the chain of events of when, where and what Aβ species aggregate and form plaques remains unclear. The aim of this study was to investigate the potential of matrix-assisted laser desorption/ionization imaging mass spectrometry as a tool to study the evolving pathology in transgenic mouse models for AD. To that end, we used an emerging, chemical imaging modality - matrix-assisted laser desorption/ionization imaging mass spectrometry - that allows for delineating Aβ aggregation with specificity at the single plaque level. We identified that plaque formation occurs first in cortical regions and that these younger plaques contain higher levels of 42 amino acid-long Aβ (Aβ1-42). Plaque maturation was found to be characterized by a relative increase in deposition of Aβ1-40, which was associated with the appearance of a cored morphology for those plaques. Finally, other C-terminally truncated Aβ species (Aβ1-38 and Aβ1-39) exhibited a similar aggregation pattern as Aβ1-40, suggesting that these species have similar aggregation characteristics. These results suggest that initial plaque formation is seeded by Aβ1-42; a process that is followed by plaque maturation upon deposition of Aβ1-40 as well as deposition of other C-terminally modified Aβ species., (© 2019 International Society for Neurochemistry.)
- Published
- 2020
- Full Text
- View/download PDF
49. Engineered antibodies: new possibilities for brain PET?
- Author
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Sehlin D and Syvänen S
- Subjects
- Animals, Antibodies metabolism, Blood-Brain Barrier metabolism, Brain metabolism, Humans, Protein Transport, Antibodies genetics, Brain diagnostic imaging, Positron-Emission Tomography methods, Protein Engineering
- Abstract
Almost 50 million people worldwide are affected by Alzheimer's disease (AD), the most common neurodegenerative disorder. Development of disease-modifying therapies would benefit from reliable, non-invasive positron emission tomography (PET) biomarkers for early diagnosis, monitoring of disease progression, and assessment of therapeutic effects. Traditionally, PET ligands have been based on small molecules that, with the right properties, can penetrate the blood-brain barrier (BBB) and visualize targets in the brain. Recently a new class of PET ligands based on antibodies have emerged, mainly in applications related to cancer. While antibodies have advantages such as high specificity and affinity, their passage across the BBB is limited. Thus, to be used as brain PET ligands, antibodies need to be modified for active transport into the brain. Here, we review the development of radioligands based on antibodies for visualization of intrabrain targets. We focus on antibodies modified into a bispecific format, with the capacity to undergo transferrin receptor 1 (TfR1)-mediated transcytosis to enter the brain and access pathological proteins, e.g. amyloid-beta. A number of such antibody ligands have been developed, displaying differences in brain uptake, pharmacokinetics, and ability to bind and visualize the target in the brain of transgenic mice. Potential pathological changes related to neurodegeneration, e.g. misfolded proteins and neuroinflammation, are suggested as future targets for this novel type of radioligand. Challenges are also discussed, such as the temporal match of radionuclide half-life with the ligand's pharmacokinetic profile and translation to human use. In conclusion, brain PET imaging using bispecific antibodies, modified for receptor-mediated transcytosis across the BBB, is a promising method for specifically visualizing molecules in the brain that are difficult to target with traditional small molecule ligands.
- Published
- 2019
- Full Text
- View/download PDF
50. Pyroglutamation of amyloid-βx-42 (Aβx-42) followed by Aβ1-40 deposition underlies plaque polymorphism in progressing Alzheimer's disease pathology.
- Author
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Michno W, Nyström S, Wehrli P, Lashley T, Brinkmalm G, Guerard L, Syvänen S, Sehlin D, Kaya I, Brinet D, Nilsson KPR, Hammarström P, Blennow K, Zetterberg H, and Hanrieder J
- Subjects
- Alzheimer Disease metabolism, Amyloid beta-Peptides chemistry, Amyloid beta-Peptides genetics, Animals, Disease Progression, Humans, Male, Mice, Mice, Transgenic, Models, Animal, Peptide Fragments chemistry, Peptide Fragments genetics, Protein Conformation, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Alzheimer Disease pathology, Amyloid beta-Peptides metabolism, Peptide Fragments metabolism, Plaque, Amyloid metabolism, Pyrrolidonecarboxylic Acid metabolism
- Abstract
Amyloid-β (Aβ) pathology in Alzheimer's disease (AD) is characterized by the formation of polymorphic deposits comprising diffuse and cored plaques. Because diffuse plaques are predominantly observed in cognitively unaffected, amyloid-positive (CU-AP) individuals, pathogenic conversion into cored plaques appears to be critical to AD pathogenesis. Herein, we identified the distinct Aβ species associated with amyloid polymorphism in brain tissue from individuals with sporadic AD (s-AD) and CU-AP. To this end, we interrogated Aβ polymorphism with amyloid conformation-sensitive dyes and a novel in situ MS paradigm for chemical characterization of hyperspectrally delineated plaque morphotypes. We found that maturation of diffuse into cored plaques correlated with increased Aβ1-40 deposition. Using spatial in situ delineation with imaging MS (IMS), we show that Aβ1-40 aggregates at the core structure of mature plaques, whereas Aβ1-42 localizes to diffuse amyloid aggregates. Moreover, we observed that diffuse plaques have increased pyroglutamated Aβx-42 levels in s-AD but not CU-AP, suggesting an AD pathology-related, hydrophobic functionalization of diffuse plaques facilitating Aβ1-40 deposition. Experiments in tgAPP
Swe mice verified that, similar to what has been observed in human brain pathology, diffuse deposits display higher levels of Aβ1-42 and that Aβ plaque maturation over time is associated with increases in Aβ1-40. Finally, we found that Aβ1-40 deposition is characteristic for cerebral amyloid angiopathy deposition and maturation in both humans and mice. These results indicate that N-terminal Aβx-42 pyroglutamation and Aβ1-40 deposition are critical events in priming and maturation of pathogenic Aβ from diffuse into cored plaques, underlying neurotoxic plaque development in AD., (© 2019 Michno et al.)- Published
- 2019
- Full Text
- View/download PDF
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