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1.	Fang, Xiaotian, 1990-, et al. (författare) Fluorine-18 labeling of antibody-based radioligands for PET imaging of amyloid beta in brain Annan publikation (övrigt vetenskapligt/konstnärligt)
2.	Rokka, Johanna, et al. (författare) Journal of Labelled Compounds and Pharmaceuticals : Neurosciences session 2019 Ingår i: ISRS 2019. - : Wiley Online Library. ; , s. S78-S79 Konferensbidrag (refereegranskat)
3.	Syvänen, Stina, et al. (författare) Fluorine-18-Labeled Antibody Ligands for PET Imaging of Amyloid-β in Brain 2020 Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 11:24, s. 4460-4468 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.
4.	Abu Hamdeh, Sami, et al. (författare) Brain tissue Aβ42 levels are linked to shunt response in idiopathic normal pressure hydrocephalus 2019 Ingår i: Journal of Neurosurgery. - : Journal of Neurosurgery Publishing Group (JNSPG). - 0022-3085 .- 1933-0693. ; 130:1, s. 121-129 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.
5.	Allard, Christina, et al. (författare) Rasbiologiskt språkbruk i statens rättsprocess mot sameby : DN Debatt 2015-06-11 2015 Annan publikation (populärvet., debatt m.m.)abstract Statens hantering av forskningsresultat i rättsprocessen med Girjas sameby utgör ett hot mot Sverige som rättsstat och kunskapsnation. Åratal av svensk och internationell forskning underkänns och man använder ett språkbruk som skulle kunna vara hämtat från rasbiologins tid. Nu måste staten ta sitt ansvar och börja agera som en demokratisk rättsstat, skriver 59 forskare.
6.	Banka, Vinay, et al. (författare) Development of brain-penetrable antibody radioligands for in vivo PET imaging of amyloid-β and tau 2023 Ingår i: Frontiers in nuclear medicine. - : Frontiers Media S.A.. - 2673-8880. ; 3 Tidskriftsartikel (refereegranskat)abstract INTRODUCTION: Alzheimer's disease (AD) is characterized by the misfolding and aggregation of two major proteins: amyloid-beta (Aβ) and tau. Antibody-based PET radioligands are desirable due to their high specificity and affinity; however, antibody uptake in the brain is limited by the blood-brain barrier (BBB). Previously, we demonstrated that antibody transport across the BBB can be facilitated through interaction with the transferrin receptor (TfR), and the bispecific antibody-based PET ligands were capable of detecting Aβ aggregates via ex vivo imaging. Since tau accumulation in the brain is more closely correlated with neuronal death and cognition, we report here our strategies to prepare four F-18-labeled specifically engineered bispecific antibody probes for the selective detection of tau and Aβ aggregates to evaluate their feasibility and specificity, particularly for in vivo PET imaging.METHODS: We first created and evaluated (via both in vitro and ex vivo studies) four specifically engineered bispecific antibodies, by fusion of single-chain variable fragments (scFv) of a TfR antibody with either a full-size IgG antibody of Aβ or tau or with their respective scFv. Using [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.
7.	Beretta, Chiara, et al. (författare) Amyloid-β deposits in human astrocytes contain truncated and highly resistant proteoforms 2024 Ingår i: Molecular and Cellular Neuroscience. - : Elsevier. - 1044-7431 .- 1095-9327. ; 128 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.
8.	Beretta, Chiara, 1992- (författare) Astrocytes in Alzheimer’s disease : Exploring the impact of amyloid-β pathology on neurotoxicity, metabolism and inflammation. 2024 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Astrocytes play a central role in brain homeostasis, but are also tightly connected to the pathogenesis of Alzheimer’s disease (AD). Yet, their exact role in amyloid-beta (Aβ) pathology and chronic neuroinflammation is unclear. The aim of this thesis was to elucidate the impact of astrocytes in AD progression. For this purpose, astrocytes in different culture set-ups were exposed to soluble Aβ aggregates. The astrocytes engulf and process, but fail to fully degrade the Aβ aggregates, which are instead stored as large intracellular deposits. In Paper I, we show that extracellular vesicles (EVs), secreted from the Aβ-containing cells induce synaptic loss, axonal swelling and vacuolization of primary neurons, which consequently leads to apoptosis. Astrocytes play a central role in the brain’s energy metabolism and we were therefore interested in how Aβ pathology affects their metabolism. In Paper II, we report that Aβ accumulation in astrocytes disrupts mitochondrial fission/fusion homeostasis, resulting in decreased mitochondrial respiration and altered glycolysis. Interestingly, the astrocytes switch to fatty acid β oxidation with the aid of peroxisomes to maintain stable energy production. Another important task is to understand how astrocytes modify the ingested Aβ. In Paper III, we characterized the astrocytic Aβ inclusions by isolating them with magnetic beads. Our analysis showed that the astrocytes truncate and pack together the Aβ aggregates. Moreover, we found that astrocytes release specifically truncated forms of Aβ via different routes.Astrocytes’ involvement in lipid metabolism and inflammation has recently gained much interest, but many questions remain about the connection between these processes. In Paper IV, we show that Aβ pathology causes lipid droplet (LD) accumulation in astrocytes. Moreover, we could show that astrocytes frequently transfer LDs to neighboring cells, both through direct cell-to-cell contacts and via secretion. Astrocytes have previously been reported to express major histocompatibility complex II (MHCII) and have the capacity to perform as professional antigen presenting cells. Interestingly, our results demonstrate that LDs contain MHCII, identifying a link between LDs and inflammation in astrocytes.Taken together, this thesis contributes with important knowledge of the role of astrocytes in AD pathology.
9.	Beretta, Chiara, et al. (författare) Astrocytes with Alzheimer’s disease pathology provoke lipid droplet mediated cell-to-cell propagation of MHC II complexes 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.
10.	Beretta, Chiara, et al. (författare) Extracellular vesicles from amyloid-beta exposed cell cultures induce severe dysfunction in cortical neurons 2020 Ingår i: Scientific Reports. - : NATURE RESEARCH. - 2045-2322. ; 10 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.
11.	Bidesi, Natasha Shalina Radjani, et al. (författare) Development of the First Tritiated Tetrazine : Facilitating Tritiation of Proteins 2022 Ingår i: ChemBioChem. - : John Wiley & Sons. - 1439-4227 .- 1439-7633. ; 23:23 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.
12.	Bonvicini, Gillian, et al. (författare) Comparing in vitro affinity measurements of antibodies to TfR1 : Surface plasmon resonance versus on-cell affinity 2024 Ingår i: Analytical Biochemistry. - : Elsevier. - 0003-2697 .- 1096-0309. ; 686 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.
13.	Bonvicini, Gillian, et al. (författare) Evaluation of valency effects on TfR-mediated brain delivery in vivo 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.
14.	Bonvicini, Gillian (författare) Harnessing the molecular Trojan horse : Evaluating properties of preclinical Aβ immunoPET radioligands for optimized brain delivery via the transferrin receptor 2023 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.
15.	Bonvicini, Gillian, et al. (författare) ImmunoPET imaging of amyloid-beta in a rat model of Alzheimer's disease with a bispecific, brain-penetrating fusion protein 2022 Ingår i: Translational Neurodegeneration. - : BioMed Central (BMC). - 2047-9158. ; 11 Tidskriftsartikel (refereegranskat)abstract 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.
16.	Bonvicini, Gillian, et al. (författare) Stronger affinity to Transferrin receptor enhances detection of amyloid-β pathology with bispecific antibody radioligands at a tracer dose Annan publikation (övrigt vetenskapligt/konstnärligt)abstract 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.
17.	de la Vega, Maria Pagnon, et al. (författare) The Uppsala APP deletion causes early onset autosomal dominant Alzheimer's disease by altering APP processing and increasing amyloid beta fibril formation 2021 Ingår i: Science Translational Medicine. - : American Association for the Advancement of Science (AAAS). - 1946-6234 .- 1946-6242. ; 13:606 Tidskriftsartikel (refereegranskat)abstract 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.
18.	Englund, Hillevi, 1980-, et al. (författare) Sensitive ELISA detection of amyloid-β protofibrils in biological samples 2007 Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 103:1, s. 334-345 Tidskriftsartikel (refereegranskat)abstract 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.
19.	Eriksson, Jonas, et al. (författare) [F-18]Tetrazine-trans-cyclooctene mediated labelling of antibodies for PET imaging of amyloid-beta 2018 Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer. - 1619-7070 .- 1619-7089. ; 45, s. S643-S643 Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
20.	Fang, Xiaotian, 1990-, et al. (författare) A small bispecific antibody-based construct based on bapineuzumab as a PET tracer for amyloid beta pathology in brain 2017 Ingår i: Meeting abstract. Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
21.	Fang, Xiaotian T., et al. (författare) Brain mGluR5 in mice with amyloid beta pathology studied with in vivo [(11)C]ABP688 PET imaging and ex vivo immunoblotting 2017 Ingår i: Neuropharmacology. - : Elsevier BV. - 0028-3908 .- 1873-7064. ; 113:Pt A, s. 293-300 Tidskriftsartikel (refereegranskat)abstract 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.
22.	Fang, Xiaotian T., et al. (författare) Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells 2017 Ingår i: Biological Procedures Online. - : Springer Science and Business Media LLC. - 1480-9222. ; 19 Tidskriftsartikel (refereegranskat)abstract 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.
23.	Fang, Xiaotian T., et al. (författare) High detection sensitivity with antibody-based PET radioligand for amyloid beta in brain 2019 Ingår i: NeuroImage. - : Elsevier BV. - 1053-8119 .- 1095-9572. ; 184, s. 881-888 Tidskriftsartikel (refereegranskat)abstract 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.
24.	Fang, Xiaotian T., 1990- (författare) Preclinical PET imaging of Alzheimer's disease progression 2017 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Amyloid PET imaging with [11C]PIB enabled detection of Aβ for the first time in vivo. However, [11C]PIB is a small molecule that binds only the insoluble Aβ plaque. Rather, the soluble Aβ aggregates are considered the cause of Alzheimer’s disease (AD). As such, a more sensitive and specific PET tracer is needed for tracking longitudinal AD pathology.Soluble Aβ aggregates likely interact with the metabotropic glutamate receptor 5 (mGluR5) to cause neurotoxic effects. However, with [11C]ABP688 PET we were unable to detect aberrant mGluR5 binding in AD mouse models, although we find elevated mGluR5 protein levels with immunoblotting.Antibodies are highly specific large molecules that can bind specifically to soluble Aβ aggregates, thus they can be a good marker for AD pathology. Unfortunately, due to their large size they cannot cross the blood-brain barrier (BBB). However, it is possible to shuttle antibodies into the brain by taking advantage of endogenous transporter systems on the BBB. By creating bispecific antibodies binding both to soluble Aβ aggregates and to the transferrin receptor (BBB target), we successfully transported the antibody into the brain and could visually detect soluble Aβ aggregates with PET.Recombinant expression further improved and optimized antibody design, creating smaller bispecific antibody-based constructs that had better pharmacokinetic properties allowing for earlier PET scanning (1 day instead of 3), and more sensitive signal.Lastly, using TCO-tetrazine click chemistry, we indirectly labeled our antibodies with fluorine-18, and could successfully perform PET already 11 h post-injection with a fluorine-18 labeled antibody.
25.	Faresjö Melander, Rebecca, 1990- (författare) Factors influencing transferrin receptor-mediated brain delivery : Evaluating preclinical antibody-based proteins for PET imaging in Alzheimer’s disease 2023 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Antibody-based proteins targeting amyloid-beta (Aβ) could be used as radioligands in positron emission tomography (PET) to study Alzheimer’s disease (AD) pathology in the living brain. The prospective advantages of antibody-based PET are to detect pathology earlier, with higher sensitivity, and to evaluate treatment effects of emerging immunotherapies against Aβ. However, antibodies and other proteins are too large to cross the blood-brain barrier (BBB). This can be circumvented by fusing antibodies with transferrin-receptor (TfR) binders that penetrate the BBB via receptor-mediated transcytosis. In this thesis, I evaluated different bispecific antibody-based proteins that bind both TfR and Aβ. The overall aim was to determine which factors are important for TfR-mediated brain delivery of these proteins and their use as PET radioligands. In paper I, we studied a large, high TfR-avidity antibody compared with a smaller antibody fragment fusion with lower TfR avidity. The small antibody had fast elimination from blood and was cleared from the brain earlier than the large antibody, thus providing better signal-to-noise ratio for brainPET. In paper II, antibody-like proteins (affibodies), even smaller than the previously studied antibody, had enhanced TfR-mediated brain delivery but had an imbalance in binding to TfR and Aβ. This resulted in poor pathology-related retention of 125I-radiolabeled affibodies. In paper III, we observed that aged mice had poorer brain delivery of the bispecific antibody, mAb3D6-scFv8D3, compared with young mice. Age was also related to increased blood cell binding of the bispecific antibody, and a lower dose resulted in higher relative delivery to the brain parenchyma. In paper IV, we evaluated single domain llama-based antibodies, VHHs, which bound both mouse and human TfR, and were characterized by rapid elimination from blood and brain. The VHHs were fused to an Aβ binding antibody fragment, scFv3D6, which enabled increased brain retention of the 125I-radiobeled antibodies in an AD mouse model, and, thus, provided high contrast to healthy controls.In conclusion, antibody format, size, mouse age, dose, and TfR binding were important factors influencing brain delivery and retention.
26.	Faresjö, Rebecca, 1990-, et al. (författare) Age, dose, and binding to TfR on blood cells influence brain delivery of a TfR-transported antibody 2023 Ingår i: Fluids and Barriers of the CNS. - : BioMed Central (BMC). - 2045-8118. ; 20:1 Tidskriftsartikel (refereegranskat)abstract BackgroundTransferrin 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.MethodsYoung (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.ResultsThe 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.ConclusionAge-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.
27.	Faresjö, Rebecca, et al. (författare) Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different size 2021 Ingår i: Fluids and Barriers of the CNS. - : BioMed Central (BMC). - 2045-8118. ; 18:1 Tidskriftsartikel (refereegranskat)abstract Background Transferrin receptor (TfR1) mediated enhanced brain delivery of antibodies have been studied extensively in preclinical settings. However, the brain pharmacokinetics, i.e. brain entry, distribution and elimination are still not fully understood for this class of antibodies. The overall aim of the study was to compare the brain pharmacokinetics of two BBB-penetrating bispecific antibodies of different size (210 vs 58 kDa). Specifically, we wanted to investigate if the faster systemic clearance of the smaller non-IgG antibody di-scFv3D6-8D3, in comparison with the IgG-based bispecific antibody mAb3D6-scFv8D3, was also reflected in the brain. Methods Wild-type (C57/Bl6) mice were injected with 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.
28.	Faresjö, Rebecca, et al. (författare) Single domain antibody conjugated to Aβ-binding scFv penetrates BBB via TfR to interact with Aβ Annan publikation (övrigt vetenskapligt/konstnärligt)abstract 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.
29.	Faresjö, Rebecca, et al. (författare) Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-Aβ-Affibodies 2022 Ingår i: Pharmaceutical research. - : Springer Nature. - 0724-8741 .- 1573-904X. ; 39:7, s. 1509-1521 Tidskriftsartikel (refereegranskat)abstract Affibodies targeting amyloid-beta (Aβ) could potentially be used as therapeutic and diagnostic agents in Alzheimer’s disease (AD). Affibodies display suitable characteristics for imaging applications such as high stability and a short biological half-life. The aim of this study was to explore brain delivery and retention of Aβ protofibril-targeted affibodies in wild-type (WT) and AD transgenic mice and to evaluate their potential as imaging agents. Two affibodies, Z5 and Z1, were fused with the blood–brain barrier (BBB) shuttle single-chain variable fragment scFv8D3. In vitro binding of 125I-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, [125I]I-Z5 and [125I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 showed increased brain concentrations of 0.53 ± 0.16% and 1.20 ± 0.35%ID/g brain. At 24 h post injection, brain retention of [125I]I-Z1 and [125I]I-Z5 was low, while [125I]I-scFv8D3-Z1 and [125I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [125I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around Aβ deposits. Affibody-scFv8D3 fusions displayed increased brain and parenchymal delivery compared with the non-fused affibodies. However, fast brain washout and a suboptimal balance between Aβ and mTfR1 affinity resulted in low intrabrain retention around Aβ deposits.
30.	Gustafsson, Gabriel, et al. (författare) Secretion and uptake of α-synuclein via extracellular vesicles in cultured cells 2018 Ingår i: Cellular and molecular neurobiology. - : Springer Science and Business Media LLC. - 0272-4340 .- 1573-6830. ; 38:8, s. 1539-1550 Tidskriftsartikel (refereegranskat)abstract 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.
31.	Gustafsson, Sofia, et al. (författare) Blood-Brain Barrier Integrity in a Mouse Model of Alzheimer’s Disease With or Without Acute 3D6 Immunotherapy 2018 Ingår i: Neuropharmacology. - : Elsevier BV. - 0028-3908 .- 1873-7064. ; 143, s. 1-9 Tidskriftsartikel (refereegranskat)abstract 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.
32.	Gustafsson, Sofia, et al. (författare) Heterogeneous drug tissue binding in brain regions of rats, Alzheimer’s patients and controls : impact on translational drug development 2019 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9 Tidskriftsartikel (refereegranskat)abstract 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.
33.	Gustavsson, Tobias, et al. (författare) Long-term effects of immunotherapy with a brain penetrating Aβ antibody in a mouse model of Alzheimer's disease 2023 Ingår i: Alzheimer's Research & Therapy. - : BioMed Central (BMC). - 1758-9193. ; 15:1 Tidskriftsartikel (refereegranskat)abstract BackgroundBrain-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.MethodsAppNL−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.ResultsNeither 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.ConclusionsBoth 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.
34.	Gustavsson, Tobias, et al. (författare) SPECT imaging of distribution and retention of a brain-penetrating bispecific amyloid-beta antibody in a mouse model of Alzheimer's disease 2020 Ingår i: Translational Neurodegeneration. - : BMC. - 2047-9158. ; 9:1 Tidskriftsartikel (refereegranskat)abstract 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.
35.	Hultqvist, Greta, et al. (författare) Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor 2017 Ingår i: Theranostics. - : IVYSPRING INT PUBL. - 1838-7640. ; 7:2, s. 308-318 Tidskriftsartikel (refereegranskat)abstract 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.
36.	Julku, Ulrika, et al. (författare) Brain pharmacokinetics of mono- and bispecific amyloid-beta antibodies in wild-type and Alzheimer's disease mice measured by high cut-off microdialysis 2022 Ingår i: Fluids and Barriers of the CNS. - : Springer Nature. - 2045-8118. ; 19 Tidskriftsartikel (refereegranskat)abstract 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.
37.	Kamali-Moghaddam, Masood, et al. (författare) Sensitive detection of A beta protofibrils by proximity ligation : relevance for Alzheimer's disease 2010 Ingår i: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 11, s. 124- Tidskriftsartikel (refereegranskat)abstract 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.
38.	Kaya, Ibrahim, et al. (författare) Delineating Amyloid Plaque Associated Neuronal Sphingolipids in Transgenic Alzheimer's Disease Mice (tgArcSwe) Using MALDI Imaging Mass Spectrometry 2017 Ingår i: ACS Chemical Neuroscience. - : AMER CHEMICAL SOC. - 1948-7193. ; 8:2, s. 347-355 Tidskriftsartikel (refereegranskat)abstract 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.
39.	Lannfelt, Lars, et al. (författare) Perspectives on future Alzheimer therapies : amyloid-beta protofibrils - a new target for immunotherapy with BAN2401 in Alzheimer's disease 2014 Ingår i: ALZHEIMERS RES THER. - : Springer Science and Business Media LLC. - 1758-9193. ; 6:2, s. 16- Forskningsöversikt (refereegranskat)abstract 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.
40.	Lord, Anna, et al. (författare) An amyloid-beta protofibril-selective antibody prevents amyloid formation in a mouse model of Alzheimer's disease 2009 Ingår i: Neurobiology of Disease. - : Elsevier BV. - 0969-9961 .- 1095-953X. ; 36:3, s. 425-434 Tidskriftsartikel (refereegranskat)abstract 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.
41.	Lord, Anna, 1979-, et al. (författare) An antibody against Amyloid-β protofibrils mediates their clearance and prevents amyloid formation in a mouse model of Alzheimer’s disease Annan publikation (populärvet., debatt m.m.)
42.	Magnusson, Kristina, et al. (författare) Specific Uptake of an Amyloid-beta Protofibril-Binding Antibody-Tracer in A beta PP Transgenic Mouse Brain 2013 Ingår i: Journal of Alzheimer's Disease. - 1387-2877 .- 1875-8908. ; 37:1, s. 29-40 Tidskriftsartikel (refereegranskat)abstract 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.
43.	Meier, Silvio R., et al. (författare) 11C-PiB and 124I-antibody PET provide differing estimates of brain amyloid-β after therapeutic intervention 2022 Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 63:2, s. 302-309 Tidskriftsartikel (refereegranskat)abstract 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.
44.	Meier, Silvio R., et al. (författare) Antibody-Based In Vivo PET Imaging Detects Amyloid-beta Reduction in Alzheimer Transgenic Mice After BACE-1 Inhibition 2018 Ingår i: Journal of Nuclear Medicine. - : SOC NUCLEAR MEDICINE INC. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 59:12, s. 1885-1891 Tidskriftsartikel (refereegranskat)abstract 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.
45.	Meier, Silvio R., et al. (författare) Passive and receptor mediated brain delivery of an anti-GFAP nanobody 2022 Ingår i: Nuclear Medicine and Biology. - : Elsevier. - 0969-8051 .- 1872-9614. ; 114-115, s. 128-134 Tidskriftsartikel (refereegranskat)abstract Purpose: Antibody-based constructs, engineered to enter the brain using transferrin receptor (TfR) mediated transcytosis, have been successfully used as PET radioligands for imaging of amyloid-beta (Aβ) in preclinical studies. However, these radioligands have been large and associated with long circulation times, i.e. non-optimal properties for neuroPET radioligands. The aim of this study was to investigate the in vivo brain delivery of the radiolabeled nanobody VHH-E9 that binds to glial fibrillary acidic protein (GFAP) expressed by reactive astrocytes, without and with fusion to a TfR binding moiety, as potential tools to detect neuroinflammation.Methods: Three protein constructs were recombinantly expressed: 1) The GFAP specific nanobody VHH-E9, 2) VHH-E9 fused to a single chain variable fragment of the TfR binding antibody 8D3 (scFv8D3) and 3) scFv8D3 alone. Brain delivery of the constructs was investigated at 2 h post injection. Binding to GFAP was studied with autoradiography while in vivo brain retention of [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.
46.	Meier, Silvio R., et al. (författare) Pinpointing Brain TREM2 Levels in Two Mouse Models of Alzheimer's Disease 2021 Ingår i: Molecular Imaging and Biology. - : Springer. - 1536-1632 .- 1860-2002. ; 23, s. 665-675 Tidskriftsartikel (refereegranskat)abstract PURPOSE: The triggering receptor expressed on myeloid cells 2 (TREM2) is expressed by brain microglia. Microglial activation, as observed in Alzheimer's disease (AD) as well as in transgenic mice expressing human amyloid-beta, appears to increase soluble TREM2 (sTREM2) levels in CSF and brain. In this study, we used two different transgenic mouse models of AD pathology and investigated the potential of TREM2 to serve as an in vivo biomarker for microglial activation in AD.PROCEDURES: We designed and generated a bispecific antibody based on the TREM2-specific monoclonal antibody mAb1729, fused to a single-chain variable fragment of the transferrin receptor binding antibody 8D3. The 8D3-moiety enabled transcytosis of the whole bispecific antibody across the blood-brain barrier. The bispecific antibody was radiolabeled with I-125 (ex vivo) or I-124 (PET) and administered to transgenic AD and wild-type (WT) control mice. Radioligand retention in the brain of transgenic animals was compared to WT mice by isolation of brain tissue at 24 h or 72 h, or with in vivo PET at 24 h, 48 h, and 72 h. Intrabrain distribution of radiolabeled mAb1729-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.
47.	Meier, Silvio R., 1990- (författare) Preclinical PET imaging and therapy of Alzheimer's disease 2021 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract The main histopathological hallmarks of Alzheimer’s disease are extracellular amyloid-beta (Aβ) plaques and intracellular neurofibrillary tangles, containing tau protein. Because of misfolded and aggregated proteins, activated microglia and astroglia react with a neuroinflammatory response, which may contribute to disease progression and severity. To date, there is no treatment available that stops the underlying mechanisms of the disease, but several new drug candidates entered clinical trials every year during the last decade. New treatments, aiming to clear Aβ from the brain parenchyma or to reduce Aβ production, are dependent on diagnostic tools to follow changes in brain Aβ pathology in vivo. The presence of brain amyloid, verified with positron emission tomography (PET), is a regularly used criterion for enrolling patients in clinical trials. However, current amyloid radioligands such as [11C]Pittsburgh Compound B ([11C]PiB) have some disadvantages, e.g. early saturation during disease progression and reduced binding to diffuse Aβ pathology. Currently available radioligands for imaging of neuroinflammation are also suboptimal. In this thesis, we investigated the potential of a brain-penetrating, bispecific Aβ antibody as a PET ligand to detect effects of treatment. In paper I and II, we demonstrated that this ligand can follow Aβ disease progression and that Aβ reduction due to treatment with a BACE-1 inhibitor can be quantified in a mouse model of AD. In paper II we also compared antibody-PET with [11C]PiB-PET and showed that the two ligands provided differing read-outs.In paper III we created and investigated an antibody-based radioligand against the triggering receptor expressed on myeloid cells 2. Compared to wild type mice, transgenic animals displayed higher total in vivo exposure, calculated as the area under the concentration curve based on PET at 24 h, 48 h and 72 h post injection. However, differences were not evident in single time point PET images.In paper IV we investigated brain delivery of a nanobody against GFAP with and without active transcytosis over the blood-brain barrier in vivo. Brain uptake with active transcytosis was two times higher. However, brain retention after 8 h, 24 h or 48 h did not differ between transgenic and wild type mice. In paper V we studied the potential of a hexavalent and bispecific antibody construct against soluble Aβ aggregates for PET or immunotherapy in vivo. Its brain retention increased with age when applied at tracer doses in genetically modified mice. However, when applied at therapeutic dose, it had no or very low impact on Aβ levels measured in brain homogenates.
48.	Meier, Silvio R., et al. (författare) Therapeutic and diagnostic potential of a multivalent antibody against soluble amyloid-beta aggregates Annan publikation (övrigt vetenskapligt/konstnärligt)
49.	Michno, Wojciech, 1992, et al. (författare) Chemical imaging of evolving amyloid plaque pathology and associated A β peptide aggregation in a transgenic mouse model of Alzheimer's disease 2020 Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 152:5, s. 602-616 Tidskriftsartikel (refereegranskat)abstract 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.
50.	Michno, Wojciech, 1992, et al. (författare) Pyroglutamation of amyloid-βx-42 (Aβx-42) followed by Aβ1–40 deposition underlies plaque polymorphism in progressing Alzheimer's disease pathology 2019 Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 294:17, s. 6719-6732 Tidskriftsartikel (refereegranskat)abstract 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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