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  • 1.
    Abu Hamdeh, Sami
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Enblad: Neurosurgery.
    Virhammar, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Enblad: Neurosurgery.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Alafuzoff, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Cesarini, Kristina G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Enblad: Neurosurgery.
    Marklund, Niklas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Enblad: Neurosurgery.
    Brain tissue Aβ42 levels are linked to shunt response in idiopathic normal pressure hydrocephalus2019In: Journal of Neurosurgery, ISSN 0022-3085, E-ISSN 1933-0693, Vol. 130, no 1, p. 121-129Article in journal (Refereed)
    Abstract [en]

    Objective The authors conducted a study to test if the cortical brain tissue levels of soluble amyloid beta (Aβ) reflect the propensity of cortical Aβ aggregate formation and may be an additional factor predicting surgical outcome following idiopathic normal pressure hydrocephalus (iNPH) treatment.

    Methods Highly selective ELISAs (enzyme-linked immunosorbent assays) were used to quantify soluble Aβ40, Aβ42, and neurotoxic Aβ oligomers/protofibrils, associated with Aβ aggregation, in cortical biopsy samples obtained in patients with iNPH (n = 20), sampled during ventriculoperitoneal (VP) shunt surgery. Patients underwent pre- and postoperative (3-month) clinical assessment with a modified iNPH scale. The preoperative CSF biomarkers and the levels of soluble and insoluble Aβ species in cortical biopsy samples were analyzed for their association with a favorable outcome following the VP shunt procedure, defined as a ≥ 5-point increase in the iNPH scale.

    Rrsults The brain tissue levels of Aβ42 were negatively correlated with CSF Aβ42 (Spearman's r = -0.53, p < 0.05). The Aβ40, Aβ42, and Aβ oligomer/protofibril levels in cortical biopsy samples were higher in patients with insoluble cortical Aβ aggregates (p < 0.05). The preoperative CSF Aβ42 levels were similar in patients responding (n = 11) and not responding (n = 9) to VP shunt treatment at 3 months postsurgery. In contrast, the presence of cortical Aβ aggregates and high brain tissue Aβ42 levels were associated with a poor outcome following VP shunt treatment (p < 0.05).

    Conclusions Brain tissue measurements of soluble Aβ species are feasible. Since high Aβ42 levels in cortical biopsy samples obtained in patients with iNPH indicated a poor surgical outcome, tissue levels of Aβ species may be associated with the clinical response to shunt treatment.

  • 2. Banka, Vinay
    et al.
    Kelleher, Andrew
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Sigurdsson, Einar M
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Ding, Yu-Shin
    Development of brain-penetrable antibody radioligands for in vivo PET imaging of amyloid-β and tau2023In: Frontiers in nuclear medicine, ISSN 2673-8880, Vol. 3, article id 1173693Article in journal (Refereed)
    Abstract [en]

    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 [18F]SFB as the prosthetic group, all four 18F-labeled bispecific antibody probes were then prepared by conjugation of antibody and [18F]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. [18F]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 [18F]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 [18F]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.

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  • 3.
    Beretta, Chiara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Dakhel, Abdulkhalek
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Eltom, Khalid
    Rosqvist, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Clinical Nutrition and Metabolism. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Uzoni, Simon
    Department of Chemistry and Molecular Biology, University of Gothenburg.
    Mothes, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Rostami, Jinar
    Fletcher, John S.
    Department of Chemistry and Molecular Biology, University of Gothenburg.
    Risérus, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Clinical Nutrition and Metabolism. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Michno, Wojciech
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Astrocytes with Alzheimer’s disease pathology provoke lipid droplet mediated cell-to-cell propagation of MHC II complexesManuscript (preprint) (Other academic)
    Abstract [en]

    Background. Astrocytes are critical for maintaining brain homeostasis, but are also highly involved in neuroinflammation. In the Alzheimer disease (AD) brain, reactive, inflammatory astrocytes are situated closely around amyloid β (Aβ) plaques. We have previously shown that reactive astrocytes ingest large quantities of soluble Aβ aggregates, but are unable to degrade the material, which leads to intracellular Aβ accumulation and severe cellular stress. A common response to cellular stress is the formation of lipid droplets (LDs). Novel data indicate that LDs play an important role in inflammatory processes. However, the involvement of LDs in AD inflammation and progression remains unclear.

    Methods. The aim of this study was to investigate how astrocytic Aβ pathology affects lipid metabolism and antigen presentation. For this purpose, human induced pluripotent stem cell (iPSC) derived astrocytes were exposed to soluble Aβ42 aggregates and analyzed over time, using a battery of experimental approaches.

    Results. Our results show that Aβ exposure induces LD accumulation in astrocytes, although the overall lipid composition remains unchanged. Moreover, astrocytes transfer LDs to neighboring cells via tunneling nanotubes (TNTs) and extracellular vesicle (EVs). Interestingly, we found that the antigen presenting protein major histocompatibility complex II (MHCII) is present inside LDs, suggesting an active role of LDs in astrocytic antigen presentation. Immunohistochemical analysis of human brain tissue verified the presence of LD-loaded MHCII+ astrocytes in AD individuals. Moreover, we found infiltrated CD4+ T cells to be in close contact with astrocytes, confirming an astrocyte T cell cross-talk in the AD brain

    Conclusions. Taken together, our data show that Aβ pathology drastically affects lipid storage in astrocytes, which in turn modulates the astrocytic antigen presentation, indicating a role for astrocytic LDs in T cell responses in the AD brain.

  • 4.
    Beretta, Chiara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Nikitidou, Elisabeth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Gallasch, Linn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Extracellular vesicles from amyloid-beta exposed cell cultures induce severe dysfunction in cortical neurons2020In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, article id 19656Article in journal (Refereed)
    Abstract [en]

    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-beta (A beta) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of A beta (42) protofibrils by astrocytes results in the release of EVs containing neurotoxic A beta. Here, we describe the cellular mechanisms behind EV-associated neurotoxicity in detail. EVs were isolated from untreated and A beta (42) 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 A beta (42) 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 A beta exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of A beta -induced pathology.

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  • 5.
    Beretta, Chiara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Svensson, Elina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics. Department of Neuroinflammation, UCL Queen Square Institute of Neurology, 1 Wakefield Street, WC1N 1PJ London, United Kingdom of Great Britain and Northern Ireland.
    Dakhel, Abdulkhalek
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Zyśk, Marlena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Hanrieder, J.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Michno, Wojciech
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Amyloid-β deposits in human astrocytes contain truncated and highly resistant proteoforms2024In: Molecular and Cellular Neuroscience, ISSN 1044-7431, E-ISSN 1095-9327, Vol. 128, article id 103916Article in journal (Refereed)
    Abstract [en]

    Alzheimer's disease (AD) is a neurodegenerative disorder that develops over decades. Glial cells, including astrocytes are tightly connected to the AD pathogenesis, but their impact on disease progression is still unclear. Our previous data show that astrocytes take up large amounts of aggregated amyloid-beta (Aβ) but are unable to successfully degrade the material, which is instead stored intracellularly. The aim of the present study was to analyze the astrocytic Aβ deposits composition in detail in order to understand their role in AD propagation. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aβ42 fibrils and magnetic beads. Live cell imaging and immunocytochemistry confirmed that the ingested Aβ aggregates and beads were transported to the same lysosomal compartments in the perinuclear region, which allowed us to successfully isolate the Aβ deposits from the astrocytes. Using a battery of experimental techniques, including mass spectrometry, western blot, ELISA and electron microscopy we demonstrate that human astrocytes truncate and pack the Aβ aggregates in a way that makes them highly resistant. Moreover, the astrocytes release specifically truncated forms of Aβ via different routes and thereby expose neighboring cells to pathogenic proteins. Taken together, our study establishes a role for astrocytes in mediating Aβ pathology, which could be of relevance for identifying novel treatment targets for AD.

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  • 6.
    Bidesi, Natasha Shalina Radjani
    et al.
    Univ Copenhagen, Fac Hlth & Med Sci, Dept Drug Design & Pharmacol, Jagtvej 160, DK-2100 Copenhagen, Denmark..
    Battisti, Umberto Maria
    Univ Copenhagen, Fac Hlth & Med Sci, Dept Drug Design & Pharmacol, Jagtvej 160, DK-2100 Copenhagen, Denmark..
    van de Broek, Sara Lopes
    Univ Copenhagen, Fac Hlth & Med Sci, Dept Drug Design & Pharmacol, Jagtvej 160, DK-2100 Copenhagen, Denmark..
    Shalgunov, Vladimir
    Univ Copenhagen, Fac Hlth & Med Sci, Dept Drug Design & Pharmacol, Jagtvej 160, DK-2100 Copenhagen, Denmark.;Rigshosp, Dept Clin Physiol, Nucl Med, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.;Rigshosp, PET, Blegdamsvej 9, DK-2100 Copenhagen, Denmark..
    Dall, Anne-Mette
    Novo Nordisk AS, Smormosevej 17-19, DK-2880 Copenhagen, Denmark..
    Kristensen, Jesper Boggild
    Novo Nordisk AS, Smormosevej 17-19, DK-2880 Copenhagen, Denmark..
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Knudsen, Gitte Moos
    Rigshosp, Neurobiol Res Unit, Blegdamsvej 9, DK-2100 Copenhagen, Denmark..
    Herth, Matthias Manfred
    Univ Copenhagen, Fac Hlth & Med Sci, Dept Drug Design & Pharmacol, Jagtvej 160, DK-2100 Copenhagen, Denmark.;Rigshosp, Dept Clin Physiol, Nucl Med, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.;Rigshosp, PET, Blegdamsvej 9, DK-2100 Copenhagen, Denmark..
    Development of the First Tritiated Tetrazine: Facilitating Tritiation of Proteins2022In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 23, no 23Article in journal (Refereed)
    Abstract [en]

    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 H-3-Tz based synthon would have wide applications for in vitro/ex vivo assays. In this study, we developed a H-3-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 [H-3]-(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/mu mol (11.8 Ci/mmol). The compound was successfully applied to pretargeted autoradiography. Thus, we report the synthesis of the first H-3-labeled Tz and its successful application as a labeling building block.

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  • 7.
    Bonvicini, Gillian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. BioArctic AB, Warfvinges väg 35, SE-112 51, Stockholm, Sweden..
    Singh, Sunitha
    BioArctic AB, Warfvinges väg 35, SE-112 51, Stockholm, Sweden..
    Nygren, Patrik
    BioArctic AB, Warfvinges väg 35, SE-112 51, Stockholm, Sweden..
    Xiong, Mengfei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Falk, Ronny
    BioArctic AB, Warfvinges väg 35, SE-112 51, Stockholm, Sweden..
    Andersson, Ken G
    BioArctic AB, Warfvinges väg 35, SE-112 51, Stockholm, Sweden..
    Comparing in vitro affinity measurements of antibodies to TfR1: Surface plasmon resonance versus on-cell affinity2024In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 686, article id 115406Article in journal (Refereed)
    Abstract [en]

    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.

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  • 8.
    Bonvicini, Gillian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Singh, Sunitha
    BioArctic AB.
    Sandersjöö, Lisa
    BioArctic AB.
    Dallas, Tiffany
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Schlein, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Andersson, Ken G.
    BioArctic AB.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Stronger affinity to Transferrin receptor enhances detection of amyloid-β pathology with bispecific antibody radioligands at a tracer doseManuscript (preprint) (Other academic)
    Abstract [en]

    A popular method for delivering biologic therapeutics and diagnostics to the brain is by hijacking transferrin receptor (TfR)-mediated transcytosis. Moderate affinity towards TfR is beneficial for TfR-mediated brain delivery at therapeutic doses while a few studies have indicated that high TfR affinity may be more beneficial at tracer doses. With the development of antibody-based PET radioligands for neurodegenerative diseases, such as Alzheimer’s disease, a better understanding of the pharmacokinetics of TfR-binders at tracer dose is essential. Thus the aim of this study was to evaluate the effect of TfR affinity on brain uptake at a tracer dose in both wild-type (WT) mice and in a mouse model of Aβ pathology.

    Three different affinity variants of 8D3, produced by alanine point mutations, were selected. Bispecific antibodies were designed with knob-into-hole technology where one arm was the anti-mouse TfR antibody, 8D3, and the other arm was the anti-human Aβ antibody, bapineuzumab (Bapi). Antibody affinities were measured in an in vitro cell assay. In vivo pharmacokinetic analyses of radioiodinated bispecific antibodies and Bapi in brain, blood and peripheral organs were performed over 7 days post-injection in WT and Aβ mice.

    The affinities of the three bispecific antibodies to TfR were 10 nM, 20 nM and 240 nM. Independent of genotype, stronger TfR-affinity resulted in higher brain uptake. The two bispecific antibodies with stronger affinity behaved similarly and differentiated between WT and Aβ model mice at an earlier time point than the low affinity variant.

    This study supports the hypothesis that stronger TfR affinity yields better brain uptake at a tracer dose. With the better detection of Aβ pathology, stronger affinity to TfR is a critical feature for the design of future bispecific immunoPET radioligands for intrabrain targets using TfR-mediated transcytosis.

  • 9.
    Bonvicini, Gillian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Singh, Sunitha
    BioArctic AB.
    Sandersjöö, Lisa
    BioArctic AB.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Andersson, Ken G.
    BioArctic AB.
    Evaluation of valency effects on TfR-mediated brain delivery in vivoManuscript (preprint) (Other academic)
    Abstract [en]

    Monovalent binding to the transferrin receptor (TfR) is assumed to be the most efficient binding mode for avoiding lysosomal degradation of the protein constructs that utilise TfR-mediated transcytosis to cross the blood-brain barrier. However, past studies evaluating the effects of valency to TfR on brain uptake generally had shortcomings in the protein design. This led to protein constructs that differed in valency but also in affinity and/or protein size. Therefore, the aim here was to evaluate the effect of valency on TfR-mediated brain delivery.

    Affinity variants of antibody 8D3 were produced by introducing alanine point mutations into the complementarity-determining regions. Eleven Fab fragments and 29 IgGs were screened for affinity against murine TfR. Six of each were chosen to be produced with a knob-in-hole design to have monovalent and bivalent TfR binders in full-length antibody format. These 12 antibodies were tested in a cell assay and 2 pairs of monovalent and bivalent antibodies were determined based on the apparent affinity. The stronger apparent affinity pair was radiolabelled with iodine-125 and injected into WT mice at a tracer dose. The biodistribution was measured in brain, blood and peripheral organs at 4 h post-injection.

    The antibodies from the stronger apparent affinity pair had similar blood pharmacokinetics and peripheral distribution suggesting that the apparent affinities were indeed similar. The monovalent antibody had significantly higher brain uptake than the bivalent antibody; supporting that monovalent binding yields better brain delivery than bivalent binding to TfR. It further suggests that the effect of valency on brain delivery is apparent affinity independent.

  • 10.
    Bonvicini, Gillian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. BioArctic AB, S-11251 Stockholm, Sweden..
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Andersson, Ken G. G.
    BioArctic AB, S-11251 Stockholm, Sweden..
    Haaparanta-Solin, Merja
    Univ Turku, Turku PET Ctr, Preclin Imaging Lab, Turku 20520, Finland.;Univ Turku, MediCity Res Lab, Turku 20520, Finland..
    Lopez-Picon, Francisco
    Univ Turku, Turku PET Ctr, Preclin Imaging Lab, Turku 20520, Finland.;Univ Turku, MediCity Res Lab, Turku 20520, Finland..
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    ImmunoPET imaging of amyloid-beta in a rat model of Alzheimer's disease with a bispecific, brain-penetrating fusion protein2022In: Translational Neurodegeneration, ISSN 2047-9158, Vol. 11, article id 55Article in journal (Refereed)
    Abstract [en]

    Background: Hijacking the transferrin receptor (TfR) is an effective strategy to transport amyloid-beta (A beta) 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 A beta 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-A beta antibody, bapineuzumab (Bapi), to generate two bispecific fusion proteins: OX26(5)-F(ab')(2)-Bapi and OX26(76)-F(ab')(2)-Bapi. Pharmacokinetic analyses were performed 4 h and 70 h post-injection of radioiodinated fusion proteins in wild-type (WT) rats. [I-124]I-OX26(5)-F(ab')(2)-Bapi was administered to TgF344-AD and WT rats for in vivo PET imaging. Ex vivo distribution of injected [I-124]I-OX26(5)-F(ab')(2)-Bapi and A beta pathology were assessed.

    Results: More [I-125]I-OX26(5)-F(ab')(2)-Bapi was taken up into the brain 4 h post-administration than [I-124]I-OX26(76)-F(ab')(2)-Bapi. [I-124]I-OX26(5)-F(ab')(2)-Bapi PET visualized A beta pathology with significantly higher signals in the TgF344-AD rats than in the WT littermates without A beta pathology. The PET signals significantly correlated with A beta 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 A beta 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.

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  • 11.
    de la Vega, Maria Pagnon
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Giedraitis, Vilmantas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Michno, Wojciech
    Univ Gothenburg, Dept Psychiat & Neurochem, S-43180 Gothenburg, Sweden.;UCL, Dept Neurosci, London WC1E 6BT, England..
    Kilander, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Guener, Goekhan
    Tech Univ Munich, German Ctr Neurodegenerat Dis DZNE & Neuroprote, Sch Med, Klinikum Rechts Isar, D-81377 Munich, Germany..
    Zielinski, Mara
    Forschungszentrum Julich, Inst Biol Informat Proc, Julich Ctr Struct Biol, Struct Biochem IBI 7, D-52425 Julich, Germany.;Forschungszentrum Julich, JuStruct, Julich Ctr Struct Biol, D-52425 Julich, Germany..
    Degerman Gunnarsson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Brundin, RoseMarie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Danfors, Torsten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Soderberg, Linda
    BioArctic AB, S-11251 Stockholm, Sweden..
    Alafuzoff, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Nilsson, Lars N. G.
    Univ Oslo, Dept Pharmacol, N-0316 Oslo, Norway.;Oslo Univ Hosp, N-0316 Oslo, Norway..
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Willbold, Dieter
    Forschungszentrum Julich, Inst Biol Informat Proc, Julich Ctr Struct Biol, Struct Biochem IBI 7, D-52425 Julich, Germany.;Forschungszentrum Julich, JuStruct, Julich Ctr Struct Biol, D-52425 Julich, Germany.;Heinrich Heine Univ Dusseldorf, Inst Phys Biol, D-40225 Dusseldorf, Germany.;State Univ, Res Ctr Mol Mech Aging & Age Related Dis, Moscow Inst Phys & Technol, Dolgoprudnyi 141701, Russia..
    Mueller, Stephan A.
    Tech Univ Munich, German Ctr Neurodegenerat Dis DZNE & Neuroprote, Sch Med, Klinikum Rechts Isar, D-81377 Munich, Germany..
    Schroeder, Gunnar F.
    Forschungszentrum Julich, Inst Biol Informat Proc, Julich Ctr Struct Biol, Struct Biochem IBI 7, D-52425 Julich, Germany.;Forschungszentrum Julich, JuStruct, Julich Ctr Struct Biol, D-52425 Julich, Germany.;Heinrich Heine Univ Dusseldorf, Phys Dept, D-40225 Dusseldorf, Germany..
    Hanrieder, Jorg
    Univ Gothenburg, Dept Psychiat & Neurochem, S-43180 Gothenburg, Sweden.;UCL, Dept Neurodegenerat Dis, Queen Sq Inst Neurol, London WC1N 3BG, England..
    Lichtenthaler, Stefan F.
    Tech Univ Munich, German Ctr Neurodegenerat Dis DZNE & Neuroprote, Sch Med, Klinikum Rechts Isar, D-81377 Munich, Germany.;Munich Cluster Syst Neurol SyNergy, D-81377 Munich, Germany..
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    The Uppsala APP deletion causes early onset autosomal dominant Alzheimer's disease by altering APP processing and increasing amyloid beta fibril formation2021In: Science Translational Medicine, ISSN 1946-6234, E-ISSN 1946-6242, Vol. 13, no 606, article id eabc6184Article in journal (Refereed)
    Abstract [en]

    Point mutations in the amyloid precursor protein gene (APP) cause familial Alzheimer's disease (AD) by increasing generation or altering conformation of amyloid beta (A beta). Here, we describe the Uppsala APP mutation (Delta 690-695), 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) A beta 42 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 beta-secretase cleavage and affecting alpha-secretase cleavage. Furthermore, in vitro aggregation studies and analyses of patient brain tissue samples indicate that the longer form of mutated A beta, A beta Upp1-42(Delta 19-24), accelerates the formation of fibrils with unique polymorphs and their deposition into amyloid plaques in the affected brain.

  • 12.
    Englund, Hillevi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Johansson, Ann-Sofi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Nilsson, Lars N.G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Gellerfors, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Paulie, Staffan
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ekholm Pettersson, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sensitive ELISA detection of amyloid-β protofibrils in biological samples2007In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 103, no 1, p. 334-345Article in journal (Refereed)
    Abstract [en]

    Amyloid-β (Aβ) protofibrils are known intermediates of the in vitro Aβ aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Aβ protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Aβ protofibril immunoassay, Aβ conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Aβ protofibrils without interference from Aβ monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Aβ protofibrils in both cell and animal models, proving that Aβ protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Aβ protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Aβ protofibrils in AD and has the potential of becoming an important diagnostic assay.

  • 13.
    Eriksson, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Fang, Xiaotian T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Olberg, D. E.
    Univ Oslo, Dept Pharm, Oslo, Norway.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    [F-18]Tetrazine-trans-cyclooctene mediated labelling of antibodies for PET imaging of amyloid-beta2018In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, p. S643-S643Article in journal (Other academic)
  • 14.
    Fang, Xiaotian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Meier, Silvio
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    A small bispecific antibody-based construct based on bapineuzumab as a PET tracer for amyloid beta pathology in brain2017In: Meeting abstractArticle in journal (Other academic)
  • 15.
    Fang, Xiaotian T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Eriksson, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET-MRI Platform. PET Centre, Uppsala University Hospital, 751 85 Uppsala, Sweden.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET-MRI Platform. Uppsala University Hospital, 751 85 Uppsala, SwedenUppsala University Hospital, 751 85 Uppsala, Sweden.
    Yngve, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET-MRI Platform. Uppsala University Hospital, 751 85 Uppsala, SwedenUppsala University Hospital, 751 85 Uppsala, Sweden.
    Cato, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Brain mGluR5 in mice with amyloid beta pathology studied with in vivo [(11)C]ABP688 PET imaging and ex vivo immunoblotting2017In: Neuropharmacology, ISSN 0028-3908, E-ISSN 1873-7064, Vol. 113, no Pt A, p. 293-300, article id S0028-3908(16)30459-2Article in journal (Refereed)
    Abstract [en]

    Alzheimer's disease (AD) is characterized by aggregation of amyloid beta (Aβ) into insoluble plaques. Intermediates, Aβ oligomers (Aβo), appear to be the mechanistic cause of disease. The de facto PET AD ligand, [(11)C]PIB, binds and visualizes Aβ plaque load, which does not correlate well with disease severity. Therefore, finding a dynamic target that changes with pathology progression in AD is of great interest. Aβo alter synaptic plasticity, inhibit long-term potentiation, and facilitate long-term depression; key mechanisms involved in memory and learning. In order to convey these neurotoxic effects, Aβo requires interaction with the metabotropic glutamate 5 receptor (mGluR5). The aim was to investigate in vivo mGluR5 changes in an Aβ pathology model using PET. Wild type C57/BL6 (wt) and AβPP transgenic mice (tg-ArcSwe), 4, 8, and 16 months old, were PET scanned with [(11)C]ABP688, which is highly specific to mGluR5, to investigate changes in mGluR5. Mouse brains were extracted postscan and mGluR5 and Aβ protofibril levels were assessed with immunoblotting and ELISA respectively. Receptor-dense brain regions (hippocampus, thalamus, and striatum) displayed higher [(11)C]ABP688 concentrations corresponding to mGluR5 expression pattern. Mice had similar uptake levels of [(11)C]ABP688 regardless of genotype or age. Immunoblotting revealed general decline in mGluR5 expression and elevated levels of mGluR5 in 16 months old tg-ArcSwe compared with wt mice. [(11)C]ABP688 could visualize mGluR5 in the mouse brain. In conclusion, mGluR5 levels were found to decrease with age and tended to be higher in tg-ArcSwe compared with wt mice, however these changes could not be quantified with PET.

  • 16.
    Fang, Xiaotian T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Meier, Silvio R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET-MRI Platform.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    High detection sensitivity with antibody-based PET radioligand for amyloid beta in brain2019In: NeuroImage, ISSN 1053-8119, E-ISSN 1095-9572, Vol. 184, p. 881-888Article in journal (Refereed)
    Abstract [en]

    PET imaging of amyloid-beta (A beta) deposits in brain has become an important aid in Alzheimer's disease diagnosis, and an inclusion criterion for patient enrolment into clinical trials of new anti-A beta treatments. Available PET radioligands visualizing A beta bind to insoluble fibrils, i.e. A beta plaques. Levels of prefibrillar A beta forms, e.g. soluble oligomers and protofibrils, correlate better than plaques with disease severity and these soluble species are the neurotoxic form of A beta leading to neurodegeneration. The goal was to create an antibody-based radioligand, recognizing not only fibrillary A beta , but also smaller and still soluble aggregates. We designed and expressed a small recombinant bispecific antibody construct, di-scFv 3D6-8D3, targeting the A beta N-terminus and the transferrin receptor (TfR). Natively expressed at the blood-brain barrier (BBB), TfR could thus be used as a brain-blood shuttle. Di-scFv 3D6-8D3 bound to A beta 1-40 with high affinity and to TfR with moderate affinity. Di-scFv [I-124] 3D6-8D3 was injected in two transgenic mouse models overexpressing human A beta and wild-type control mice and PET scanned at 14, 24 or 72 h after injection. Di-scFv [I-124] 3D6-8D3 was retained in brain of transgenic animals while it was cleared from wild-type lacking A beta . This difference was observed from 24 h onwards, and at 72 h, 18 months old transgenic animals, with high load of A beta pathology, displayed SUVR of 2.2-3.5 in brain while wildtype showed ratios close to unity. A subset of the mice were also scanned with [C-11] PIB. Again wt mice displayed ratios of unity while transgenes showed slightly, non-significantly, elevated SUVR of 1.2, indicating improved sensitivity with novel di-scFv [I-124] 3D6-8D3 compared with [C-11] PIB. Brain concentrations of di-scFv [I-124] 3D6-8D3 correlated with soluble A beta (p < 0.0001) but not with total A beta, i.e. plaque load (p = 0.34). We have successfully created a small bispecific antibody-based radioligand capable of crossing the BBB, subsequently binding to and visualizing intrabrain A beta in vivo. The radioligand displayed better sensitivity compared with [C-11] PIB, and brain concentrations correlated with soluble neurotoxic A beta aggregates.

  • 17.
    Fang, Xiaotian T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells2017In: Biological Procedures Online, E-ISSN 1480-9222, Vol. 19, article id 11Article in journal (Refereed)
    Abstract [en]

    Background: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.

    Methods: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost).

    Results: The protocol has been used to generate multiple variants of tetra-and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (A beta) aggregates in vivo.

    Conclusions: This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.

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  • 18.
    Faresjö, Rebecca
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Bonvicini, Gillian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Uppsala Univ, Dept Publ Healt & Caring Sci, Rudbeck Lab, Dag Hammarskjolds Vag 20, S-75185 Uppsala, Sweden.;BioArctic AB, Warfvinges Vag 35, S-11251 Stockholm, Sweden..
    Fang, Xiaotian T.
    Yale Univ, Yale PET Ctr, Dept Radiol & Biomed Imaging, 801 Howard Ave, New Haven, CT 06520 USA..
    Aguilar, Ximena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different size2021In: Fluids and Barriers of the CNS, E-ISSN 2045-8118, Vol. 18, no 1, article id 26Article in journal (Refereed)
    Abstract [en]

    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 I-125-iodinated ([I-125]) mAb3D6-scFv8D3 (n = 46) or [I-125]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 gamma-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 [I-125]mAb3D6-scFv8D3 (n = 12) or [I-125]di-scFv3D6-8D3 (n = 12), to estimate the relative levels of radiolabelled antibody in brain capillaries versus brain parenchyma. In vitro binding kinetics for [I-125]mAb3D6-scFv8D3 or [I-125]di-scFv3D6-8D3 to murine TfR were determined by LigandTracer. Results [I-125]di-scFv3D6-8D3 showed faster elimination from blood, lower brain C-max, and T-max, a larger parenchymal-to-capillary concentration ratio, and a net elimination from brain at an earlier time point after injection compared with the larger [I-125]mAb3D6-scFv8D3. However, the elimination rate from brain did not differ between the antibodies. The study also indicated that [I-125]di-scFv3D6-8D3 displayed lower avidity than [I-125]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.

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  • 19.
    Faresjö, Rebecca
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lindberg, Hanna
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, S-10691 Stockholm, Sweden..
    Stahl, Stefan
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, S-10691 Stockholm, Sweden..
    Lofblom, John
    KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, S-10691 Stockholm, Sweden..
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-A beta-Affibodies2022In: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 39, no 7, p. 1509-1521Article in journal (Refereed)
    Abstract [en]

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

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  • 20.
    Faresjö, Rebecca
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    O Sjöström, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Key2Brain AB, Nanna Svartz Väg 4, Solna, Sweden.
    Berglund, Magnus, M
    Key2Brain AB, Nanna Svartz Väg 4, Solna, Sweden.
    Eriksson, Jonas
    PET Centre, Uppsala University Hospital, Uppsala, Sweden; Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Single domain antibody conjugated to Aβ-binding scFv penetrates BBB via TfR to interact with AβManuscript (preprint) (Other academic)
    Abstract [en]

    Background: Camelid antibody fragments are interesting for use as radioligands for Positron Emission Tomography (PET), in central nervous system imaging, due to their fast clearance from blood. This study evaluated single variable domain of heavy chain (VHH) antibodies derived from llama, targeting the mouse and human transferrin receptor (TfR) for mediating increased brain uptake. In experiments, VHHs were combined with either a human Fc or with the single chain fragment of the amyloid beta (Aβ) antibody 3D6 (scFv3D6) to investigate intrabrain targeting.

    Methods: One novel and one previously disclosed species cross-reactive VHH towards murine TfR (mTfR) and human TfR (hTfR), as well as two VHHs with selective reactivity to mTfR and hTfR, respectively, were compared. The TfR binders were evaluated as recombinant fusion protein (FP) constructs fused with either a human Fc-fragment (FPFc) or with the Aβ-binding fragment scFv3D6 (FPscFv) at either C- or N-terminal positions of scFv3D6. The above FPs were radiolabeled with iodine-125 (125I) and biodistribution was studied ex vivo at 2 h, 6 h and 24 h after injection in wild-type (WT) mice and AD mouse model AppNL-G-F. Brain, blood, plasma and organ concentrations of the 125I-FPs were measured in a γ-counter. Autoradiography, nuclear track emulsion, and immunohistofluorescence imaging were used to study the brain distribution of the FPs. 

    Results: The constructs based on Fc fusions (FPFc) with binding affinity to mTfR displayed significantly higher brain uptake (around 1-3% ID/gbrain) in comparison with FPFc specific to only hTfR (control; 0.2% ID/gbrain). The VHHs reactive to mTfR fused to a scFv (FPscFv) showed an increased brain uptake 2 h after injection compared to control (FPscFv reactive to hTfR only). FPscFv with VHH linked to the N-terminus of scFv3D6 showed more efficient brain delivery than those fused with the C-terminal of scFv3D6. There was a 17-fold higher brain uptake in AppNL-G-F than WT mice for one of the cross-species  reactive FPscFv (FPscFv1B) at 24 h post-injection, and 2.5-fold higher at 6 h, in ex vivo studies. FPscFv1B  also showed consistently higher relative brain parenchymal localization compared to the other FPs, whether as Fc- or scFv fusion.  

    Conclusion: We showed that the novel cross-reactive VHHs tested herein displayed enhanced brain delivery in mice and that these could be successfully fused with an Aβ-binding scFv-fragment, maintaining high brain and preferential parenchymal delivery with increased retention to Aβ in brain. In summary, a FPscFv construct with affinity towards both  Aβ and mTfR showed differentiated and favorable distribution in AD-mice compared to WT already after 6 h (measured ex vivo); a relevant time point for clinical brain PET.

  • 21.
    Faresjö, Rebecca
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Age, dose, and binding to TfR on blood cells influence brain delivery of a TfR-transported antibody2023In: Fluids and Barriers of the CNS, E-ISSN 2045-8118, Vol. 20, no 1, article id 34Article in journal (Refereed)
    Abstract [en]

    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 125I-labeled mAb3D6-scFv8D3 or mAb3D6. Three different doses were used in the study, 0.05 mg/kg (low dose), 1 mg/kg (high dose), and 10 mg/kg (therapeutic dose), with equimolar doses for mAb3D6. The dose-corrected antibody concentrations in whole blood, blood cells, plasma, spleen, and brain were evaluated at 2 h post-administration. Furthermore, isolated brains were studied by autoradiography, nuclear track emulsion, and capillary depletion to investigate the intrabrain distribution of the antibodies, while binding to blood cells was studied in vitro using blood isolated from young and aged mice.

    Results

    The aged WT and tg-ArcSwe mice showed significantly lower brain concentrations of TfR-binding [125I]mAb3D6-scFv8D3 and higher concentrations in the blood cell fraction compared to young mice. For [125I]mAb3D6, no significant differences in blood or brain delivery were observed between young and aged mice or between genotypes. A low dose of [125I]mAb3D6-scFv8D3 was associated with increased relative parenchymal delivery, as well as increased blood cell distribution. Brain concentrations and relative parenchymal distribution of [125I]mAb3D6-scFv8D6 did not differ between tg-ArcSwe and WT mice at this early time point but were considerably increased compared to those observed for [125I]mAb3D6.

    Conclusion

    Age-dependent differences in blood and brain concentrations were observed for the bispecific antibody mAb3D6-scFv8D3 but not for the conventional Aβ antibody mAb3D6, indicating an age-related effect on TfR1-mediated brain delivery. The lowest dose of [125I]mAb3D6-scFv8D3 was associated with higher relative BBB penetration but, at the same time, a higher distribution to blood cells. Overall, 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.

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  • 22.
    Gustafsson, Gabriel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lööv, Camilla
    Massachusetts Gen Hosp, Dept Neurol, Charlestown, MA 02129 USA; Harvard Med Sch, Neurosci Program, Boston, MA 02115 USA.
    Persson, Emma
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lázaro, Diana F.
    Univ Med Ctr Gottingen, Dept Expt Neurodegenerat, Gottingen, Germany.
    Takeda, Shuko
    Massachusetts Gen Hosp, Dept Neurol, Charlestown, MA 02129 USA; Harvard Med Sch, Neurosci Program, Boston, MA 02115 USA.
    Bergström, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Balaj, Leonora
    Massachusetts Gen Hosp, Dept Neurol, Charlestown, MA 02129 USA; Massachusetts Gen Hosp, Dept Radiol, Charlestown, MA 02129 USA; Harvard Med Sch, Neurosci Program, Boston, MA 02115 USA.
    György, Bence
    Massachusetts Gen Hosp, Dept Neurol, Charlestown, MA 02129 USA; Massachusetts Gen Hosp, Dept Radiol, Charlestown, MA 02129 USA; Harvard Med Sch, Neurosci Program, Boston, MA 02115 USA.
    Hallbeck, Martin
    Linkoping Univ, Dept Clin & Expt Med, Dept Pathol, Linkoping, Sweden.
    Outeiro, Tiago F
    Univ Med Ctr Gottingen, Dept Expt Neurodegenerat, Gottingen, Germany; Max Planck Inst Expt Med, Gottingen, Germany; Newcastle Univ, Med Sch, Inst Neurosci, Framlington Pl, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England.
    Breakefield, Xandra O
    Massachusetts Gen Hosp, Dept Neurol, Charlestown, MA 02129 USA; Massachusetts Gen Hosp, Dept Radiol, Charlestown, MA 02129 USA; Harvard Med Sch, Neurosci Program, Boston, MA 02115 USA.
    Hyman, Bradley T
    Massachusetts Gen Hosp, Dept Neurol, Charlestown, MA 02129 USA; Harvard Med Sch, Neurosci Program, Boston, MA 02115 USA.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Massachusetts Gen Hosp, Dept Neurol, Charlestown, MA 02129 USA; Massachusetts Gen Hosp, Dept Radiol, Charlestown, MA 02129 USA; Harvard Med Sch, Neurosci Program, Boston, MA 02115 USA.
    Secretion and uptake of α-synuclein via extracellular vesicles in cultured cells2018In: Cellular and molecular neurobiology, ISSN 0272-4340, E-ISSN 1573-6830, Vol. 38, no 8, p. 1539-1550Article in journal (Refereed)
    Abstract [en]

    In Parkinson’s disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1–2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.

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  • 23.
    Gustafsson, Sofia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gustavsson, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Roshanbin, Sahar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Blood-Brain Barrier Integrity in a Mouse Model of Alzheimer’s Disease With or Without Acute 3D6 Immunotherapy2018In: Neuropharmacology, ISSN 0028-3908, E-ISSN 1873-7064, Vol. 143, p. 1-9Article in journal (Refereed)
    Abstract [en]

    The blood-brain barrier (BBB) is suggested to be compromised in Alzheimer's disease (AD). The concomitant presence of vascular amyloid beta (AD) pathology, so called cerebral amyloid angiopathy (CAA), also predisposes impairment of vessel integrity. Additionally, immunotherapy against A beta may lead to further damage of the BBB. To what extent this affects the BBB passage of molecules is debated. The current study aimed to investigate BBB integrity to large molecules in transgenic mice displaying abundant A beta pathology and age matched wild type animals, with or without acute anti-A beta antibody treatment. Animals were administered a single i.v. injection of PBS or 3D6 (10 mg/kg), i.e. the murine version of the clinically investigated A beta antibody bapineuzumab, supplemented with [(125)]3D6. Three days post injections, a 4 kDa FITC and a 150 kDa Antonia Red dextran were administered i.v. to all animals. After termination, fluorescent detection in brain and serum was used for the calculation of dextran brain-to-blood concentration ratios. Further characterization of antibody fate and the presence of CAA were investigated using radioactivity measurements and Congo red staining. BBB passage of large molecules was equally low in wild type and transgenic mice, suggesting an intact BBB despite A beta pathology. Neither was the BBB integrity affected by acute 3D6 treatment. However, CAA was confirmed in the transgenes and local antibody accumulations were observed in the brain, indicating CAA-antibody interactions. The current study shows that independently of A beta pathology or acute 3D6 treatment, the BBB is intact, without extensive permeability to large molecules, including the 3D6 antibody.

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  • 24.
    Gustafsson, Sofia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lampa, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, UCR-Uppsala Clinical Research Center.
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Loryan, Irena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Heterogeneous drug tissue binding in brain regions of rats, Alzheimer’s patients and controls: impact on translational drug development2019In: Scientific Reports, E-ISSN 2045-2322, Vol. 9, article id 5308Article in journal (Refereed)
    Abstract [en]

    For preclinical and clinical assessment of therapeutically relevant unbound, free, brain concentrations, the pharmacokinetic parameter fraction of unbound drug in brain (fu,brain) is commonly used to compensate total drug concentrations for nonspecific brain tissue binding (BTB). As, homogenous BTB is assumed between species and in health and disease, rat BTB is routinely used. The impact of Alzheimer’s disease (AD) on drug BTB in brain regions of interest (ROI), i.e., fu,brain,ROI, is yet unclear. This study for the first time provides insight into regional drug BTB and the validity of employing rat fu,brain,ROI as a surrogate of human BTB, by investigating five marketed drugs in post-mortem tissue from AD patients (n = 6) and age-matched controls (n = 6). Heterogeneous drug BTB was observed in all within group comparisons independent of disease and species. The findings oppose the assumption of uniform BTB, highlighting the need of case-by-case evaluation of fu,brain,ROI in translational CNS research.

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  • 25.
    Gustavsson, Tobias
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Metzendorf, Nicole G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Wik, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Roshanbin, Sahar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Julku, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Chourlia, Aikaterini
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Nilsson, Per
    Karolinska Inst, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Stockholm, Sweden..
    Andersson, Ken G.
    BioArct AB, Stockholm, Sweden..
    Laudon, Hanna
    BioArct AB, Stockholm, Sweden..
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Long-term effects of immunotherapy with a brain penetrating Aβ antibody in a mouse model of Alzheimer's disease2023In: Alzheimer's Research & Therapy, E-ISSN 1758-9193, Vol. 15, no 1, article id 90Article in journal (Refereed)
    Abstract [en]

    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

    AppNL−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 [125I]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 [125I]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.

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  • 26.
    Gustavsson, Tobias
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    SPECT imaging of distribution and retention of a brain-penetrating bispecific amyloid-beta antibody in a mouse model of Alzheimer's disease2020In: Translational Neurodegeneration, ISSN 2047-9158, Vol. 9, no 1, article id 37Article in journal (Refereed)
    Abstract [en]

    Background Alzheimer's disease (AD) immunotherapy with antibodies targeting amyloid-beta (A beta) has been extensively explored in clinical trials. The aim of this study was to study the long-term brain distribution of two radiolabeled monoclonal A beta 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 (I-125)-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 A beta and CD31 immunostaining to investigate the intrabrain distribution of the antibodies and their interactions with A beta. Results Despite faster blood clearance, [I-125]RmAb158-scFv8D3 displayed higher brain exposure than [I-125]RmAb158 throughout the study. The brain distribution of [I-125]RmAb158-scFv8D3 was more uniform and coincided with parenchymal A beta pathology, while [I-125]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 A beta interactions for [I-125]RmAb158-scFv8D3, whereas [I-125]RmAb158 displayed retention and A beta 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.

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  • 27.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Fang, Xiaotian T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor2017In: Theranostics, E-ISSN 1838-7640, Vol. 7, no 2, p. 308-318Article in journal (Refereed)
    Abstract [en]

    The blood-brain barrier (BBB) is an obstacle for antibody passage into the brain, impeding the development of immunotherapy and antibody-based diagnostics for brain disorders. In the present study, we have developed a brain shuttle for active transport of antibodies across the BBB by receptor-mediated transcytosis. We have thus recombinantly fused two single-chain variable fragments (scFv) of the transferrin receptor (TfR) antibody 8D3 to the light chains of mAb158, an antibody selectively binding to A beta protofibrils, which are involved in the pathogenesis of Alzheimer's disease (AD). Despite the two TfR binders, a monovalent interaction with TfR was achieved due to the short linkers that sterically hinder bivalent binding to the TfR dimer. The design enabled efficient receptor-mediated brain uptake of the fusion protein. Two hours after administration, brain concentrations were 2-3% of the injected dose per gram brain, comparable to small molecular drugs and 80-fold higher than unmodified mAb158. After three days, fusion protein concentrations in AD transgenic mouse brains were 9-fold higher than in wild type mice, demonstrating high in vivo specificity. Thus, our innovative recombinant design markedly increases mAb158 brain uptake, which makes it a strong candidate for improved Aa immunotherapy and as a PET radioligand for early diagnosis and evaluation of treatment effect in AD. Moreover, this approach could be applied to any target within the brain.

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  • 28.
    Julku, Ulrika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Xiong, Mengfei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Wik, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Roshanbin, Sahar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Brain pharmacokinetics of mono- and bispecific amyloid-beta antibodies in wild-type and Alzheimer's disease mice measured by high cut-off microdialysis2022In: Fluids and Barriers of the CNS, E-ISSN 2045-8118, Vol. 19, article id 99Article in journal (Refereed)
    Abstract [en]

    Background: Treatment with amyloid-beta (A beta) targeting antibodies is a promising approach to remove A beta 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 A beta targeting antibodies have higher brain distribution, and can remove A beta 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 A beta-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 App(NL-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.

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  • 29.
    Kamali-Moghaddam, Masood
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Ekholm Pettersson, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Wu, Di
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Englund, Hillevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Darmanis, Spyros
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Lord, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Tavoosidana, Gholamreza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Gustafsdottir, Sigrun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Nilsson, Lars N. G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Sensitive detection of A beta protofibrils by proximity ligation: relevance for Alzheimer's disease2010In: BMC Neuroscience, E-ISSN 1471-2202, Vol. 11, p. 124-Article in journal (Refereed)
    Abstract [en]

    Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A beta 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 A beta 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 A beta protofibrils we have used a monoclonal antibody, mAb158, selective for A beta 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 Ab aggregates in solid- phase reactions, allowing detection of just 0.1 pg/ml A beta 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 Ab protofibril detection by up to 25- fold. The assay was used to measure soluble Ab aggregates in brain homogenates from mice transgenic for a human allele predisposing to A beta aggregation. Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of A beta aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.

  • 30.
    Kaya, Ibrahim
    et al.
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden..
    Brinet, Dimitri
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden.;Univ Gothenburg, Dept Chem & Mol Biol, S-41296 Gothenburg, Sweden..
    Michno, Wojciech
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden..
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Zetterberg, Henrik
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden.;Sahlgrens Univ Hosp, Clin Neurochem Lab, S-43180 Molndal, Sweden.;UCL, Inst Neurol, Dept Mol Neurosci, London WC1N 3BG, England..
    Blennow, Kaj
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden.;Sahlgrens Univ Hosp, Clin Neurochem Lab, S-43180 Molndal, Sweden..
    Hanrieder, Jorg
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden.;UCL, Inst Neurol, Dept Mol Neurosci, London WC1N 3BG, England.;Chalmers, Dept Chem & Chem Engn, S-41296 Gothenburg, Sweden..
    Delineating Amyloid Plaque Associated Neuronal Sphingolipids in Transgenic Alzheimer's Disease Mice (tgArcSwe) Using MALDI Imaging Mass Spectrometry2017In: ACS Chemical Neuroscience, E-ISSN 1948-7193, Vol. 8, no 2, p. 347-355Article in journal (Refereed)
    Abstract [en]

    The major pathological hallmarks of Alzheimer's disease (AD) are the progressive aggregation and accumulation of beta-amyloid (A beta) and hyperphosphorylated tau protein into neurotoxic deposits. A beta aggregation has been suggested as the critical early inducer, driving the disease progression. However, the factors that promote neurotoxic A beta aggregation remain elusive. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides, and proteins in biological tissue sections. In the present study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)-based imaging was used on transgenic Alzheimer's disease mouse (tgArcSwe) brain tissue to investigate the sphingolipid microenvironment of individual A beta plaques and elucidate plaque-associated sphingolipid alterations. Multivariate data analysis was used to interrogate the IMS data for identifying pathologically relevant, anatomical features based on their lipid chemical profile. This approach revealed sphingolipid species that distinctly located to cortical and hippocampal deposits, whose A beta identity was further verified using fluorescent amyloid staining and immunohistochemistry. Subsequent multivariate statistical analysis of the spectral data revealed significant localization of gangliosides and ceramides species to A beta positive plaques, which was accompanied by distinct local reduction of sulfatides. These plaque-associated changes in sphingolipid levels implicate a functional role of sphingolipid metabolism in A beta plaque pathology and AD pathogenesis. Taken together, the presented data highlight the potential of imaging mass spectrometry as a powerful approach for probing A beta plaque-associated lipid changes underlying AD pathology.

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  • 31.
    Lannfelt, Lars
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Moller, Christer
    Basun, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Osswald, Gunilla
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Satlin, Andrew
    Logovinsky, Veronika
    Gellerfors, Par
    Perspectives on future Alzheimer therapies: amyloid-beta protofibrils - a new target for immunotherapy with BAN2401 in Alzheimer's disease2014In: ALZHEIMERS RES THER, ISSN 1758-9193, Vol. 6, no 2, p. 16-Article, review/survey (Refereed)
    Abstract [en]

    The symptomatic drugs currently on the market for Alzheimer's disease (AD) have no effect on disease progression, and this creates a large unmet medical need. The type of drug that has developed most rapidly in the last decade is immunotherapy: vaccines and, especially, passive vaccination with monoclonal antibodies. Antibodies are attractive drugs as they can be made highly specific for their target and often with few side effects. Data from recent clinical AD trials indicate that a treatment effect by immunotherapy is possible, providing hope for a new generation of drugs. The first anti-amyloid-beta (anti-A beta) vaccine developed by Elan, AN1792, was halted in phase 2 because of aseptic meningoencephalitis. However, in a follow-up study, patients with antibody response to the vaccine demonstrated reduced cognitive decline, supporting the hypothesis that A beta immunotherapy may have clinically relevant effects. Bapineuzumab (Elan/Pfizer Inc./Johnson & Johnson), a monoclonal antibody targeting fibrillar A beta, was stopped because the desired clinical effect was not seen. Solanezumab (Eli Lilly and Company) was developed to target soluble, monomeric A beta. In two phase 3 studies, Solanezumab did not meet primary endpoints. When data from the two studies were pooled, a positive pattern emerged, revealing a significant slowing of cognitive decline in the subgroup of mild AD. The Arctic mutation has been shown to specifically increase the formation of soluble A beta protofibrils, an A beta species shown to be toxic to neurons and likely to be present in all cases of AD. A monoclonal antibody, mAb158, was developed to target A beta protofibrils with high selectivity. It has at least a 1,000-fold higher selectivity for protofibrils as compared with monomers of A beta, thus targeting the toxic species of the peptide. A humanized version of mAb158, BAN2401, has now entered a clinical phase 2b trial in a collaboration between BioArctic Neuroscience and Eisai without the safety concerns seen in previous phase 1 and 2a trials. Experiences from the field indicate the importance of initiating treatment early in the course of the disease and of enriching the trial population by improving the diagnostic accuracy. BAN2401 is a promising candidate for A beta immunotherapy in early AD. Other encouraging efforts in immunotherapy as well as in the small-molecule field offer hope for new innovative therapies for AD in the future.

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  • 32.
    Lord, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Gumucio, Astrid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Englund, Hillevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sundquist, Valentina Screpanti
    Söderberg, Linda
    Möller, Christer
    Gellerfors, Pär
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Pettersson, Frida Ekholm
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Nilsson, Lars N G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    An amyloid-beta protofibril-selective antibody prevents amyloid formation in a mouse model of Alzheimer's disease2009In: Neurobiology of Disease, ISSN 0969-9961, E-ISSN 1095-953X, Vol. 36, no 3, p. 425-434Article in journal (Refereed)
    Abstract [en]

    Human genetics link Alzheimer's disease pathogenesis to excessive accumulation of amyloid-beta (Abeta) in brain, but the symptoms do not correlate with senile plaque burden. Since soluble Abeta aggregates can cause synaptic dysfunctions and memory deficits, these species could contribute to neuronal dysfunction and dementia. Here we explored selective targeting of large soluble aggregates, Abeta protofibrils, as a new immunotherapeutic strategy. The highly protofibril-selective monoclonal antibody mAb158 inhibited in vitro fibril formation and protected cells from Abeta protofibril-induced toxicity. When the mAb158 antibody was administered for 4 months to plaque-bearing transgenic mice with both the Arctic and Swedish mutations (tg-ArcSwe), Abeta protofibril levels were lowered while measures of insoluble Abeta were unaffected. In contrast, when treatment began before the appearance of senile plaques, amyloid deposition was prevented and Abeta protofibril levels diminished. Therapeutic intervention with mAb158 was however not proven functionally beneficial, since place learning depended neither on treatment nor transgenicity. Our findings suggest that Abeta protofibrils can be selectively cleared with immunotherapy in an animal model that display highly insoluble Abeta deposits, similar to those of Alzheimer's disease brain.

  • 33.
    Magnusson, Kristina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Svedberg, Marie M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Philipson, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Soderberg, Linda
    Tegerstedt, Karin
    Holmquist, Mats
    Gellerfors, Pär
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hall, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Nilsson, Lars N. G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Specific Uptake of an Amyloid-beta Protofibril-Binding Antibody-Tracer in A beta PP Transgenic Mouse Brain2013In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 37, no 1, p. 29-40Article in journal (Refereed)
    Abstract [en]

    Evidence suggests that amyloid-beta (A beta) protofibrils/oligomers are pathogenic agents in Alzheimer's disease (AD). Unfortunately, techniques enabling quantitative estimates of these species in patients or patient samples are still rather limited. Here we describe the in vitro and ex vivo characteristics of a new antibody-based radioactive ligand, [I-125]mAb158, which binds to A beta protofibrils with high affinity. [I-125]mAb158 was specifically taken up in brain of transgenic mice expressing amyloid-beta protein precursor (A beta PP) as shown ex vivo. This was in contrast to [I-125]mAb-Ly128 which does not bind to A beta. The uptake of intraperitoneally-administered [I-125]mAb158 into the brain was age- and time-dependent, and saturable in A beta PP transgenic mice with modest A beta deposition. Brain uptake was also found in young A beta PP transgenic mice that were devoid of A beta deposits, suggesting that [I-125]mAb158 targets soluble A beta protofibrils. The radioligand was diffusely located in the parenchyma, sometimes around senile plaques and only occasionally colocalized with cerebral amyloid angiopathy. A refined iodine-124-labeled version of mAb158 with much improved blood-brain barrier passage and a shorter plasma half-life might be useful for PET imaging of A beta protofibrils.

  • 34.
    Meier, Silvio R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Rofo, Fadi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    metzendorf, Nicole
    Jamie, Morrison
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Therapeutic and diagnostic potential of a multivalent antibody against soluble amyloid-beta aggregatesManuscript (preprint) (Other academic)
  • 35.
    Meier, Silvio R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Pinpointing Brain TREM2 Levels in Two Mouse Models of Alzheimer's Disease2021In: Molecular Imaging and Biology, ISSN 1536-1632, E-ISSN 1860-2002, Vol. 23, p. 665-675Article in journal (Refereed)
    Abstract [en]

    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-scFv8D3CL 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 [124I]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.

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  • 36.
    Meier, Silvio R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Roshanbin, Sahar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lim Falk, Victoria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Saito, Takashi
    Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science, Wako, Japan; Department of Neurocognitive Science, Institute of Brain Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
    Saido, Takaomi C
    Laboratory for Proteolytic Neuroscience, RIKEN Center for Brain Science, Wako, Japan.
    Neumann, Ulf
    Neuroscience Research, Novartis Institutes for BioMedical Research, Basel, Switzerland.
    Rokka, Johanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Eriksson, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry. PET Centre, Uppsala University Hospital, Uppsala, Sweden.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    11C-PiB and 124I-antibody PET provide differing estimates of brain amyloid-β after therapeutic intervention2022In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 63, no 2, p. 302-309Article in journal (Refereed)
    Abstract [en]

    PET imaging of amyloid-β (Aβ) has become an important component of Alzheimer disease diagnosis. 11C-Pittsburgh compound B (11C-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 with 11C-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 with 11C-PiB to measure Aβ plaque load followed by a scan with the bispecific radioligand 124I-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 with 11C-PiB-PET and 125I-RmAb158-scFv8D3 SPECT. Brain tissue was isolated after scanning, and Aβ levels were assessed.

    Results: 124I-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. Reduced 125I-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 reduced 11C-PiB signal compared with untreated controls, and further, both NB-360 and control mice tended, although not reaching significance, to show higher 11C-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 with 11C-PiB PET, suggesting that these ligands detect different pools of Aβ.

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  • 37.
    Meier, Silvio R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Passive and receptor mediated brain delivery of an anti-GFAP nanobody2022In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 114-115, p. 128-134Article in journal (Refereed)
    Abstract [en]

    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 [125I]VHH-E9 and [125I]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, [125I]VHH-E9-scFv8D3 and [125I]scFv8D3 displayed 3-fold higher brain concentrations than [125I]VHH-E9. In vitro autoradiography showed distinct binding of both [125I]VHH-E9-scFv8D3 and [125I]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.

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  • 38.
    Meier, Silvio R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fang, Xiaotian T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Roshanbin, Sahar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. BioArctic AB, Stockholm, Sweden.
    Neumann, Ulf
    Novartis Inst BioMed Res, Neurosci Res, Basel, Switzerland.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Antibody-Based In Vivo PET Imaging Detects Amyloid-beta Reduction in Alzheimer Transgenic Mice After BACE-1 Inhibition2018In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 59, no 12, p. 1885-1891Article in journal (Refereed)
    Abstract [en]

    Visualization of amyloid-beta (A beta) pathology with PET has become an important tool for making a specific clinical diagnosis of Alzheimer disease (AD). However, the available amyloid PET radioligands, such as C-11-Pittsburgh compound B, reflect levels of insoluble A beta plaques but do not capture soluble and protofibrillar A beta forms. Furthermore, the plaque load appears to be fairly static during clinical stages of AD and may not be affected by A beta-reducing treatments. The aim of the present study was to investigate whether a novel PET radioligand based on an antibody directed toward soluble aggregates of A beta can be used to detect changes in A beta levels during disease progression and after treatment with a beta-secretase (BACE-1) inhibitor. Methods: One set of transgenic mice (tg-ArcSwe, a model of A beta pathology) aged between 7 and 16 mo underwent PET with the A beta protofibril-selective radioligand I-124-RmAb158-scFv8D3 (where RmAb is recombinant mouse monoclonal antibody and scFv is single-chain variable fragment) to follow progression of A beta pathology in the brain. A second set of tg-ArcSwe mice, aged 10 mo, were treated with the BACE-1 inhibitor NB-360 for 3 mo and compared with an untreated control group. A third set of tg-ArcSwe mice, also aged 10 mo, underwent PET as a baseline group. Brain tissue was isolated after PET to determine levels of A beta by ELISA and immunohistochemistry. Results: The concentration of I-124-RmAb158-scFv8D3, as measured in vivo with PET, increased with age and corresponded well with the ex vivo autoradiography and A beta immunohistochemistry results. Mice treated with NB-360 showed significantly lower in vivo PET signals than untreated animals and were similar to the baseline animals. The decreased I-124-RmAb158-scFv8D3 concentrations in NB-360-treated mice, as quantified with PET, corresponded well with the decreased A beta levels measured in postmortem brain. Conclusion: Several treatments for AD are in phase 2 and 3 clinical trials, but the possibility of studying treatment effects in vivo on the important, nonfibrillar, forms of A beta is limited. This study demonstrated the ability of the A beta protofibril-selective radioligand I-124-RmAb158-scFv8D3 to follow disease progression and detect treatment effects with PET imaging in tg-ArcSwe mice.

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  • 39.
    Michno, Wojciech
    et al.
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden.
    Nyström, Sofie
    Linkoping Univ, Dept Phys Chem & Biol, S-58183 Linkoping, Sweden.
    Wehrli, Patrick
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden.
    Lashley, Tammaryn
    UCL, Dept Neurodegenerat Dis, UCL Queen Sq Inst Neurol, London WC1N 3BG, England.
    Brinkmalm, Gunnar
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden.
    Guerard, Laurent
    Univ Gothenburg, Sahlgrenska Acad, Ctr Cellular Imaging, S-41390 Gothenburg, Sweden.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Kaya, Ibrahim
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden.
    Brinet, Dimitri
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden.
    Nilsson, K. Peter R.
    Linkoping Univ, Dept Phys Chem & Biol, S-58183 Linkoping, Sweden.
    Hammarström, Per
    Linkoping Univ, Dept Phys Chem & Biol, S-58183 Linkoping, Sweden.
    Blennow, Kaj
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden;Sahlgrens Univ Hosp, Clin Neurochem Lab, S-43180 Molndal, Sweden.
    Zetterberg, Henrik
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden;UCL, Dept Neurodegenerat Dis, UCL Queen Sq Inst Neurol, London WC1N 3BG, England;Sahlgrens Univ Hosp, Clin Neurochem Lab, S-43180 Molndal, Sweden;UCL, UK Dementia Res Inst, London WC1E 6BT, England.
    Hanrieder, Jörg
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, S-43180 Molndal, Sweden;UCL, Dept Neurodegenerat Dis, UCL Queen Sq Inst Neurol, London WC1N 3BG, England.
    Pyroglutamation of amyloid-βx-42 (Aβx-42) followed by Aβ1–40 deposition underlies plaque polymorphism in progressing Alzheimer's disease pathology2019In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, no 17, p. 6719-6732Article in journal (Refereed)
    Abstract [en]

    Amyloid- (A) 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 A species associated with amyloid polymorphism in brain tissue from individuals with sporadic AD (s-AD) and CU-AP. To this end, we interrogated A 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 A1-40 deposition. Using spatial in situ delineation with imaging MS (IMS), we show that A1-40 aggregates at the core structure of mature plaques, whereas A1-42 localizes to diffuse amyloid aggregates. Moreover, we observed that diffuse plaques have increased pyroglutamated Ax-42 levels in s-AD but not CU-AP, suggesting an AD pathology-related, hydrophobic functionalization of diffuse plaques facilitating A1-40 deposition. Experiments in tgAPP(Swe) mice verified that, similar to what has been observed in human brain pathology, diffuse deposits display higher levels of A1-42 and that A plaque maturation over time is associated with increases in A1-40. Finally, we found that A1-40 deposition is characteristic for cerebral amyloid angiopathy deposition and maturation in both humans and mice. These results indicate that N-terminal Ax-42 pyroglutamation and A1-40 deposition are critical events in priming and maturation of pathogenic A from diffuse into cored plaques, underlying neurotoxic plaque development in AD.

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  • 40.
    Michno, Wojciech
    et al.
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, Mölndal, Sweden.
    Wehrli, Patrick
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, Mölndal, Sweden.
    Meier, Silvio R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Zetterberg, Henrik
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, Molndal, Sweden;Sahlgrens Univ Hosp, Clin Neurochem Lab, Mölndal, Sweden;UCL, UK Dementia Res Inst, London, England;UCL, Queen Sq Inst Neurol, Dept Neurodegenerat Dis, London, England.
    Blennow, Kaj
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, Mölndal, Sweden;Sahlgrens Univ Hosp, Clin Neurochem Lab, Molndal, Sweden.
    Hanrieder, Jörg
    Univ Gothenburg, Sahlgrenska Acad, Dept Psychiat & Neurochem, Mölndal, Sweden;UCL, Queen Sq Inst Neurol, Dept Neurodegenerat Dis, London, England.
    Chemical imaging of evolving amyloid plaque pathology and associated A β peptide aggregation in a transgenic mouse model of Alzheimer's disease2020In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 152, no 5, p. 602-616Article in journal (Refereed)
    Abstract [en]

    One of the major hallmarks of Alzheimer's disease (AD) pathology is the formation of extracellular amyloid β (A β) plaques. While A β has been suggested to be critical in inducing and, potentially, driving the disease, the molecular basis of AD pathogenesis is still under debate. Extracellular A β plaque pathology manifests itself upon aggregation of distinct A β 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 A β aggregation processes provides a promising target for early interventions. However, the chain of events of when, where and what A β 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 A β 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 A β (A β 1-42). Plaque maturation was found to be characterized by a relative increase in deposition of A β 1-40, which was associated with the appearance of a cored morphology for those plaques. Finally, other C-terminally truncated A β species (A β 1-38 and A β 1-39) exhibited a similar aggregation pattern as A β 1-40, suggesting that these species have similar aggregation characteristics. These results suggest that initial plaque formation is seeded by A β 1-42; a process that is followed by plaque maturation upon deposition of A β 1-40 as well as deposition of other C-terminally modified A β species.

  • 41.
    Nikitidou, Elisabeth
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Rudbeck Lab, Mol Geriatr, Uppsala, Sweden..
    Söllvander, Sofia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Rudbeck Lab, Mol Geriatr, Uppsala, Sweden..
    Zyśk, Marlena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences. Rudbeck Lab, Mol Geriatr, Uppsala, Sweden..
    Söderberg, L.
    BioArctic Neurosci, Stockholm, Sweden..
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Rudbeck Lab, Mol Geriatr, Uppsala, Sweden..
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Rudbeck Lab, Mol Geriatr, Uppsala, Sweden..
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Rudbeck Lab, Mol Geriatr, Uppsala, Sweden..
    The A beta protofibril selective antibody mAb158 prevents accumulation of A beta in astrocytes and rescues neurons from A beta induced apoptosis2017In: Glia, ISSN 0894-1491, E-ISSN 1098-1136, Vol. 65, no S1, p. E170-E170Article in journal (Other academic)
  • 42.
    O'Callaghan, Paul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Noborn, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Li, Jin-ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zhang, Xiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Apolipoprotein E increases cell association of amyloid-β 40 through heparan sulfate and LRP1 dependent pathways2014In: Amyloid: Journal of Protein Folding Disorders, ISSN 1350-6129, E-ISSN 1744-2818, Vol. 21, no 2, p. 76-87Article in journal (Refereed)
  • 43.
    Olsen, Malin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Aguilar, Ximena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Fang, Xiaotian T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET-MRI Platform.
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Astroglial Responses to Amyloid-Beta Progression in a Mouse Model of Alzheimer's Disease2018In: Molecular Imaging and Biology, ISSN 1536-1632, E-ISSN 1860-2002, Vol. 20, no 4, p. 605-614Article in journal (Refereed)
    Abstract [en]

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by amyloid-beta (A beta) deposition, hyperphosphorylation of tau, and neuroinflammation. Astrocytes, the most abundant glial cell type in the nervous system, respond to neurodegenerative disorders through astrogliosis, i.e., converting to a reactive inflammatory state. The aim of this study was to investigate how in vivo quantification of astrogliosis using positron emission tomography (PET) radioligand deuterium-l-[C-11]deprenyl ([C-11]DED), binding to enzyme monoamine oxidase-B (MAO-B) which is overexpressed in reactive astrocytes during AD, corresponds to expression of glial fibrillary acidic protein (GFAP) and vimentin, i.e., two well-established markers of astrogliosis, during A beta pathology progression. APP(ArcSwe) mice (n = 37) and wild-type (WT) control mice (n = 23), 2-16-month old, were used to investigate biomarkers of astrogliosis. The radioligand, [C-11]DED, was used as an in vivo marker while GFAP, vimentin, and MAO-B were used to investigate astrogliosis and macrophage-associated lectin (Mac-2) to investigate microglia/macrophage activation by immunohistochemistry of the mouse brain. A beta and GFAP levels were also measured with ELISA in brain homogenates. The intrabrain levels of aggregated A beta and reactive astrocytes were found to be elevated in APP(ArcSwe) compared with WT mice. GFAP and vimentin expression increased with age, i.e., with A beta pathology, in the APP(ArcSwe) mice. This was not the case for in vivo marker [C-11]DED that showed elevated binding of the same magnitude in APP(ArcSwe) mice compared with WT mice at both 8 and 16 months. Further, immunohistochemistry indicated that there was limited co-expression of MAO-B and GFAP. MAO-B levels are increased early in A beta pathology progression, while GFAP and vimentin appear to increase later, most likely as a consequence of abundant A beta plaque formation. Thus, [C-11]DED is a useful PET radioligand for the detection of changes in MAO-B at an early stage of AD progression but does not measure the total extent of astrogliosis at advanced stages of A beta pathology.

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  • 44.
    Pagnon de la Vega, María
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Ge, Junyue
    Department of Psychiatry and Neurochemistry, University of Gothenburg, Gothenburg, Sweden .
    Giedraitis, Vilmantas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hanrieder, Jörg
    Department of Psychiatry and Neurochemistry, University of Gothenburg, Gothenburg, Sweden .
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    The Uppsala APP mutation promotes wildtype Aβ aggregation and deposition in vivoManuscript (preprint) (Other academic)
  • 45.
    Pagnon de la Vega, María
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Giedraitis, Vilmantas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Michno, Wojciech
    Department of Psychiatry and Neurochemistry, University of Gothenburg, Gothenburg, Sweden .
    Kilander, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Güner, Gökhan
    German Center for Neurodegenerative Diseases (DZNE) and Neuroproteomics, School of Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany .
    Zielinski, Mara
    Institute of Biological Information Processing, Structural Biochemistry (IBI-7) and JuStruct, Jülich Center for Structural Biology, Forschungszentrum Jülich, 52425 Jülich, Germany.
    Löwenmark, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Brundin, RoseMarie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Danfors, Torsten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Söderberg, Linda
    BioArctic AB, Stockholm, Sweden.
    Alafuzoff, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Lars
    Department of Pharmacology, University of Oslo and Oslo University Hospital, Oslo, Norway.
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Willbold, Dieter
    Institute of Biological Information Processing, Structural Biochemistry (IBI-7) and JuStruct, Jülich Center for Structural Biology, Forschungszentrum Jülich, 52425 Jülich, Germany .
    Müller, Stephan A.
    German Center for Neurodegenerative Diseases (DZNE) and Neuroproteomics, School of Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany .
    Schöder, Gunnar F.
    Institute of Biological Information Processing, Structural Biochemistry (IBI-7) and JuStruct, Jülich Center for Structural Biology, Forschungszentrum Jülich, 52425 Jülich, Germany .
    Hanrieder, Jörg
    Department of Psychiatry and Neurochemistry, University of Gothenburg, Gothenburg, Sweden .
    Lichtenthaler, Stefan F.
    German Center for Neurodegenerative Diseases (DZNE) and Neuroproteomics, School of Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany .
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    The Uppsala APP deletion causes early onset autosomal dominant Alzheimer’s disease by altering APP processing and increasing amyloid-β fibril formationIn: Science Translational Medicine, ISSN 1946-6234, E-ISSN 1946-6242, Science Translational MedicineArticle in journal (Other academic)
  • 46.
    Pagnon de la Vega, María
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Giedraitis, Vilmantas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Clinical geriatrics.
    Hooley, Monique
    Univ Edinburgh, UK Dementia Res Inst, Edinburgh Med Sch, Edinburgh, Scotland.;Univ Edinburgh, Ctr Discovery Brain Sci, Edinburgh, Scotland..
    Konstantinidis, Evangelos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Meier, Silvio R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Rokka, Johanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Eriksson, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry. Uppsala Univ Hosp, PET Ctr, Uppsala, Sweden.
    Aguilar, Ximena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Spires-Jones, Tara L.
    Univ Edinburgh, UK Dementia Res Inst, Edinburgh Med Sch, Edinburgh, Scotland.;Univ Edinburgh, Ctr Discovery Brain Sci, Edinburgh, Scotland..
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics. BioArctic AB, Stockholm, Sweden.
    Nilsson, Lars N. G.
    Oslo Univ Hosp, Dept Pharmacol, Oslo, Norway.;Oslo Univ Hosp, Oslo, Norway..
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics. Univ Hlth Network, Krembil Brain Inst, Toronto, ON, Canada; Univ Toronto, Tanz Ctr Res Neurodegenerat Dis, Dept Med & Lab Med, Toronto, ON, Canada; Univ Toronto, Tanz Ctr Res Neurodegenerat Dis, Dept Pathobiol, Toronto, ON, Canada.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Molecular Geriatrics.
    Altered amyloid-β structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion2024In: Acta neuropathologica communications, E-ISSN 2051-5960, Vol. 12, no 1, article id 22Article in journal (Refereed)
    Abstract [en]

    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 [11C]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.

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  • 47.
    Pagnon de la Vega, María
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hooley, Monique
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. UK Dementia Research Institute, Edinburgh Medical School, University of Edinburgh, UK.
    Giedraitis, Vilmantas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Kostantinidis, Evangelos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Meier, Silvio R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Rokka, Johanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Eriksson, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET-MRI Platform. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Aguilar, Ximena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Spires-Jones, Tara
    UK Dementia Research Institute, Edinburgh Medical School, University of Edinburgh, UK.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Nilsson, Lars
    Department of Pharmacology, University of Oslo and Oslo University Hospital, Oslo, Norway.
    Lannfelt, Lars
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Transgenic mice harboring the Uppsala APP mutation display altered APP processing and accelerated Aβ42 pathology with distinct structural featuresManuscript (preprint) (Other academic)
  • 48.
    Rofo, Fadi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Buijs, Jos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Ridgeview Instruments, S-75237 Uppsala, Sweden..
    Falk, Ronny
    BioArctic AB, S-11251 Stockholm, Sweden..
    Honek, Ken
    BioArctic AB, S-11251 Stockholm, Sweden..
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences. BioArctic AB, S-11251 Stockholm, Sweden..
    Lilja, Anna M.
    BioArctic AB, S-11251 Stockholm, Sweden..
    Metzendorf, Nicole G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Gustavsson, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Soderberg, Linda
    BioArctic AB, S-11251 Stockholm, Sweden..
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Novel multivalent design of a monoclonal antibody improves binding strength to soluble aggregates of amyloid beta2021In: Translational Neurodegeneration, ISSN 2047-9158, Vol. 10, no 1, article id 38Article in journal (Refereed)
    Abstract [en]

    Background: Amyloid-beta (A beta) 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 A beta, 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 A beta 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 A beta protofibrils. Different ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to A beta aggregates of different sizes. Finally, the ability of the antibodies to protect cells from A beta-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 A beta aggregates. Conclusion: We provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of A beta aggregates. This approach should be general and work for any aggregated protein or repetitive target.

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  • 49.
    Rofo, Fadi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Meier, Silvio R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Metzendorf, Nicole G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Morrison, Jamie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Petrovic, Alex
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    A Brain-Targeting Bispecific-Multivalent Antibody Clears Soluble Amyloid-Beta Aggregates in Alzheimer's Disease Mice2022In: NEUROTHERAPEUTICS, ISSN 1933-7213, Vol. 19, no 5, p. 1588-1602Article in journal (Refereed)
    Abstract [en]

    Amyloid-beta (A beta) oligomers and protofibrils are suggested to be the most neurotoxic A beta species in Alzheimer's disease (AD). Hence, antibodies with strong and selective binding to these soluble A beta aggregates are of therapeutic potential. We have recently introduced HexaRmAb158, a multivalent antibody with additional A beta-binding sites in the form of single-chain fragment variables (scFv) on the N-terminal ends of A beta 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 A beta 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 A beta 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 A beta aggregates.

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  • 50.
    Rofo, Fadi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Metzendorf, Nicole
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Saubi, Cristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Suominen, Laura
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Godec, Ana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Blood-brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer’s disease2022In: Alzheimer's Research & Therapy, E-ISSN 1758-9193, Vol. 14, no 1, article id 180Article in journal (Refereed)
    Abstract [en]

    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.

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