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| 2. |
- Syvänen, Stina, et al.
(författare)
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Fluorine-18-Labeled Antibody Ligands for PET Imaging of Amyloid-β in Brain
- 2020
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 11:24, s. 4460-4468
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are attractive as radioligands due to their outstanding specificity and high affinity, but their inability to cross the blood–brain barrier (BBB) limits their use for CNS targets. To enhance brain distribution, amyloid-β (Aβ) antibodies were fused to a transferrin receptor (TfR) antibody fragment, enabling receptor mediated transport across the BBB. The aim of this study was to label these bispecific antibodies with fluorine-18 and use them for Aβ PET imaging. Bispecific antibody ligands RmAb158-scFv8D3 and Tribody A2, both targeting Aβ and TfR, were functionalized with trans-cyclooctene (TCO) groups and conjugated with 18F-labeled tetrazines through an inverse electron demand Diels–Alder reaction performed at ambient temperature. 18F-labeling did not affect antibody binding in vitro, and initial brain uptake was high. Conjugates with the first tetrazine variant ([18F]T1) displayed high uptake in bone, indicating extensive defluorination, a problem that was resolved with the second and third tetrazine variants ([18F]T2 and [18F]T3). Although the antibody ligands’ half-life in blood was too long to optimally match the physical half-life of fluorine-18 (t1/2 = 110 min), [18F]T3-Tribody A2 PET seemed to discriminate transgenic mice (tg-ArcSwe) with Aβ deposits from wild-type mice 12 h after injection. This study demonstrates that 18F-labeling of bispecific, brain penetrating antibodies is feasible and, with further optimization, could be used for CNS PET imaging.
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| 3. |
- Abu Hamdeh, Sami, et al.
(författare)
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Brain tissue Aβ42 levels are linked to shunt response in idiopathic normal pressure hydrocephalus
- 2019
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Ingår i: Journal of Neurosurgery. - : Journal of Neurosurgery Publishing Group (JNSPG). - 0022-3085 .- 1933-0693. ; 130:1, s. 121-129
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Beretta, Chiara, et al.
(författare)
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Amyloid-β deposits in human astrocytes contain truncated and highly resistant proteoforms
- 2024
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Ingår i: Molecular and Cellular Neuroscience. - : Elsevier. - 1044-7431 .- 1095-9327. ; 128
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 5. |
- Beretta, Chiara, et al.
(författare)
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Astrocytes with Alzheimer’s disease pathology provoke lipid droplet mediated cell-to-cell propagation of MHC II complexes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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 brainConclusions. 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.
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| 6. |
- Beretta, Chiara, et al.
(författare)
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Extracellular vesicles from amyloid-beta exposed cell cultures induce severe dysfunction in cortical neurons
- 2020
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Ingår i: Scientific Reports. - : NATURE RESEARCH. - 2045-2322. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-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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| 7. |
- Bidesi, Natasha Shalina Radjani, et al.
(författare)
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Development of the First Tritiated Tetrazine : Facilitating Tritiation of Proteins
- 2022
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Ingår i: ChemBioChem (Print). - : John Wiley & Sons. - 1439-4227 .- 1439-7633. ; 23:23
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Tidskriftsartikel (refereegranskat)abstract
- Tetrazine (Tz)-trans-cyclooctene (TCO) ligation is an ultra-fast and highly selective reaction and it is particularly suited to label biomolecules under physiological conditions. As such, a 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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| 8. |
- Bonvicini, Gillian, et al.
(författare)
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Comparing in vitro affinity measurements of antibodies to TfR1 : Surface plasmon resonance versus on-cell affinity
- 2024
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Ingår i: Analytical Biochemistry. - : Elsevier. - 0003-2697 .- 1096-0309. ; 686
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Tidskriftsartikel (refereegranskat)abstract
- Despite years of utilizing the transferrin receptor 1 (TfR1) to transport large biomolecules into the brain, there is no consensus on how to optimally measure affinity to it. The aim of this study was to compare different methods for measuring the affinities of anti-TfR1 antibodies.Antibodies 15G11, OX26 and 8D3 are known to successfully carry large biologics across the blood-brain barrier in humans, rats, and mice, respectively. The affinity to their respective species of TfR1 was measured with different surface plasmon resonance setups in Biacore and an on-cell assay.When the antibody was captured and TfR1 was the analyte, the dissociation in Biacore was very slow. The dissociation was faster when the antibody was the analyte and TfR1 was the ligand. The Biacore setup with capture of N-terminal FLAG-tag TfR1 yielded the most similar apparent affinities as the cell assay.In conclusion, it is important to evaluate assay parameters including assay orientation, surface capture method, and antibody format when comparing binding kinetics for TfR1 antibodies. Although it seems possible to determine relative affinities of TfR1 antibodies using the methods described here, both the FLAG-tag TfR1 capture setup and cell assays likely yield apparent affinities that are most translatable in vivo.
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| 9. |
- Bonvicini, Gillian, et al.
(författare)
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Evaluation of valency effects on TfR-mediated brain delivery in vivo
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 10. |
- Bonvicini, Gillian
(författare)
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Harnessing the molecular Trojan horse : Evaluating properties of preclinical Aβ immunoPET radioligands for optimized brain delivery via the transferrin receptor
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With high specificity and selectivity to targets, antibodies are prime candidates for positron emission tomography (PET) radioligands. They do not passively cross the blood-brain barrier which has hindered their development for imaging intrabrain targets, like amyloid-β (Aβ) in Alzheimer’s disease. The molecular Trojan horse strategy with antibodies that bind to both the transferrin receptor (TfR) and an intrabrain target improves brain delivery of therapeutic antibodies. However, therapeutic antibodies are typically dosed substantially higher than antibody-based PET (immunoPET) radioligands.This thesis evaluated the effects of affinity, valency, and dose on the brain delivery of preclinical Aβ immunoPET radioligands via the TfR.Paper I investigated whether immunoPET with TfR-mediated brain delivery could image Aβ with similar sensitivity in rats as it has in mice. To our knowledge, this was the first time TfR-hijacking to deliver a radioligand to image Aβ was successfully demonstrated in rats; suggesting this strategy could eventually be translated to clinics.Affinity to TfR influences therapeutic delivery to the brain. In Paper II, we compared four Biacore setups and one on-cell assay for determining apparent affinities to the TfR. Absolute affinity determination was challenging since several assay conditions impacted the kinetic parameters. A directional TfR capture in Biacore may be optimal since it determined kinetic parameters while mimicking in vivo receptor conditions. Papers I and III investigated how antibody affinity affects brain delivery at tracer doses and indicated that stronger TfR affinity yielded higher brain delivery. The antibodies in Paper III lacked effector function. The resulting pharmacokinetic profiles in Aβ pathology-presenting mice indicated this may have improved target accumulation of the immunoPET radioligand.In Paper IV, we screened a novel library of monovalent and bivalent affinity variants of the anti-mouse TfR antibody, 8D3. A pair of monovalent and bivalent antibodies with an apparent affinity of 10 nM was identified and evaluated in vivo. Monovalent binding yielded higher brain uptake at a tracer dose but whether bivalent binding steered the antibody towards lysosomal degradation was unclear.In conclusion, monovalency, high affinity binding, and ablated effector function are likely beneficial properties for TfR-mediated brain delivery of an immunoPET radioligand at a tracer dose.
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