Owing to their specificity and high-affinity binding, monoclonal antibodies have potential as positron emission tomography (PET) radioligands and are currently used to image various targets in ...peripheral organs. However, in the central nervous system, antibody uptake is limited by the blood-brain barrier (BBB). Here we present a PET ligand to be used for diagnosis and evaluation of treatment effects in Alzheimer's disease. The amyloid β (Aβ) antibody mAb158 is radiolabelled and conjugated to a transferrin receptor antibody to enable receptor-mediated transcytosis across the BBB. PET imaging of two different mouse models with Aβ pathology clearly visualize Aβ in the brain. The PET signal increases with age and correlates closely with brain Aβ levels. Thus, we demonstrate that antibody-based PET ligands can be successfully used for brain imaging.
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 Aβ 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
specificity. Thus, our innovative recombinant design markedly increases mAb158 brain uptake, which makes it a strong candidate for improved Aβ 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.
Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data ...suggests that spreading of amyloid-β (Aβ) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of Aβ
protofibrils by astrocytes results in the release of EVs containing neurotoxic Aβ. Here, we describe the cellular mechanisms behind EV-associated neurotoxicity in detail. EVs were isolated from untreated and Aβ
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 Aβ
protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM analysis demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathological lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from Aβ exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of Aβ-induced pathology.
Purpose
The triggering receptor expressed on myeloid cells 2 (TREM2) is expressed by brain microglia. Microglial activation, as observed in Alzheimer’s disease (AD) as well as in transgenic mice ...expressing human amyloid-beta, appears to increase soluble TREM2 (sTREM2) levels in CSF and brain. In this study, we used two different transgenic mouse models of AD pathology and investigated the potential of TREM2 to serve as an
in vivo
biomarker for microglial activation in AD.
Procedures
We designed and generated a bispecific antibody based on the TREM2-specific monoclonal antibody mAb1729, fused to a single-chain variable fragment of the transferrin receptor binding antibody 8D3. The 8D3-moiety enabled transcytosis of the whole bispecific antibody across the blood-brain barrier. The bispecific antibody was radiolabeled with I-125 (
ex vivo
) or I-124 (PET) and administered to transgenic AD and wild-type (WT) control mice. Radioligand retention in the brain of transgenic animals was compared to WT mice by isolation of brain tissue at 24 h or 72 h, or with
in vivo
PET at 24 h, 48 h, and 72 h. Intrabrain distribution of radiolabeled mAb1729-scFv8D3
CL
was further studied by autoradiography, while ELISA was used to determine TREM2 brain concentrations.
Results
Transgenic animals displayed higher total exposure, calculated as the AUC based on SUV determined at 24h, 48h, and 72h post injection, of PET radioligand
124
ImAb1729-scFv8D3
CL
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.
Alzheimer's disease (AD) immunotherapy with antibodies targeting amyloid-β (Aβ) has been extensively explored in clinical trials. The aim of this study was to study the long-term brain distribution ...of two radiolabeled monoclonal Aβ antibody variants - RmAb158, the recombinant murine version of BAN2401, which has recently demonstrated amyloid removal and reduced cognitive decline in AD patients, and the bispecific RmAb158-scFv8D3, which has been engineered for enhanced brain uptake via transferrin receptor-mediated transcytosis.
A single intravenous injection of iodine-125 (
I)-labeled RmAb158-scFv8D3 or RmAb158 was administered to AD transgenic mice (tg-ArcSwe). In vivo single-photon emission computed tomography was used to investigate brain retention and intrabrain distribution of the antibodies over a period of 4 weeks. Activity in blood and brain tissue was measured ex vivo and autoradiography was performed in combination with Aβ and CD31 immunostaining to investigate the intrabrain distribution of the antibodies and their interactions with Aβ.
Despite faster blood clearance,
IRmAb158-scFv8D3 displayed higher brain exposure than
IRmAb158 throughout the study. The brain distribution of
IRmAb158-scFv8D3 was more uniform and coincided with parenchymal Aβ pathology, while
IRmAb158 displayed a more scattered distribution pattern and accumulated in central parts of the brain at later times. Ex vivo autoradiography indicated greater vascular escape and parenchymal Aβ interactions for
IRmAb158-scFv8D3, whereas
IRmAb158 displayed retention and Aβ interactions in lateral ventricles.
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.
■Synaptic density is decreased in a mouse model of alpha-synucleinopathy.■Ageing appears to be associated with decreased synaptic density in wild type mouse brain.■Model-independent AUC-based ...brain-to-blood ratio can be used to evaluate 11CUCB-J binding as an alternative to full pharmacokinetic modeling.
The positron emission tomography (PET) radioligand 11CUCB-J binds to synaptic vesicle protein 2A (SV2A) and is used to investigate synaptic density in the living brain. Clinical studies have indicated reduced 11CUCB-J binding in Alzheimer's disease (AD) and Parkinson's disease (PD) brains compared to healthy controls. Still, it is unknown whether 11CUCB-J PET can visualise synaptic loss in mouse models of these disorders. Such models are essential for understanding disease pathology and for evaluating the effects of novel disease-modifying drug candidates. In the present study, synaptic density in transgenic models of AD (ArcSwe) and PD (L61) was studied using 11CUCB-J PET. Data were acquired during 60 min after injection, and time-activity curves (TACs) in different brain regions and the left ventricle of the heart were generated based on the dynamic PET images. The 11CUCB-J brain concentrations were expressed as standardised uptake value (SUV) over time. The area under the SUV curve (AUC), the ratio of AUC in the brain to that in the heart (AUCbrain/blood), and the volume of distribution (VT) obtained by kinetic modelling using the heart TAC as input were compared between transgenic and age-matched wild type (WT) mice. The L61 mice displayed 11–13% lower AUCbrain/blood ratio and brain VT generated by kinetic modeling compared to the control WT mice. In general, also transgenic ArcSwe mice tended to show lower 11CUCB-J brain exposure than age-matched WT controls, but variation within the different animal groups was high. Older WT mice (18–20 months) showed lower 11CUCB-J brain exposure than younger WT mice (8–9 months). Together, these data imply that 11CUCB-J PET reflects synaptic density in mouse models of neurodegeneration and that inter-subject variation is large. In addition, the study suggested that model-independent AUCbrain/blood ratio can be used to evaluate 11CUCB-J binding as an alternative to full pharmacokinetic modelling.
Display omitted
Transferrin receptor 1 (TfR1) mediated brain delivery of antibodies could become important for increasing the efficacy of emerging immunotherapies in Alzheimer's disease (AD). However, age, dose, ...binding to TfR1 on blood cells, and pathology could influence the TfR1-mediated transcytosis of TfR1-binders across the blood-brain barrier (BBB). The aim of the study was, therefore, to investigate the impact of these factors on the brain delivery of a bispecific TfR1-transported Aβ-antibody, mAb3D6-scFv8D3, in comparison with the conventional antibody mAb3D6.
Young (3-5 months) and aged (17-20 months) WT and tg-ArcSwe mice (AD model) were injected with
I-labeled mAb3D6-scFv8D3 or mAb3D6. Three different doses were used in the study, 0.05 mg/kg (low dose), 1 mg/kg (high dose), and 10 mg/kg (therapeutic dose), with equimolar doses for mAb3D6. The dose-corrected antibody concentrations in whole blood, blood cells, plasma, spleen, and brain were evaluated at 2 h post-administration. Furthermore, isolated brains were studied by autoradiography, nuclear track emulsion, and capillary depletion to investigate the intrabrain distribution of the antibodies, while binding to blood cells was studied in vitro using blood isolated from young and aged mice.
The aged WT and tg-ArcSwe mice showed significantly lower brain concentrations of TfR-binding
ImAb3D6-scFv8D3 and higher concentrations in the blood cell fraction compared to young mice. For
ImAb3D6, no significant differences in blood or brain delivery were observed between young and aged mice or between genotypes. A low dose of
ImAb3D6-scFv8D3 was associated with increased relative parenchymal delivery, as well as increased blood cell distribution. Brain concentrations and relative parenchymal distribution of
ImAb3D6-scFv8D6 did not differ between tg-ArcSwe and WT mice at this early time point but were considerably increased compared to those observed for
ImAb3D6.
Age-dependent differences in blood and brain concentrations were observed for the bispecific antibody mAb3D6-scFv8D3 but not for the conventional Aβ antibody mAb3D6, indicating an age-related effect on TfR1-mediated brain delivery. The lowest dose of
ImAb3D6-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.
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 .sup.125I-iodinated (.sup.125I) mAb3D6-scFv8D3 (n = 46) or .sup.125Idi-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 .sup.125ImAb3D6-scFv8D3 (n = 12) or .sup.125Idi-scFv3D6-8D3 (n = 12), to estimate the relative levels of radiolabelled antibody in brain capillaries versus brain parenchyma. In vitro binding kinetics for .sup.125ImAb3D6-scFv8D3 or .sup.125Idi-scFv3D6-8D3 to murine TfR were determined by LigandTracer. Results .sup.125Idi-scFv3D6-8D3 showed faster elimination from blood, lower brain C.sub.max, and T.sub.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 .sup.125ImAb3D6-scFv8D3. However, the elimination rate from brain did not differ between the antibodies. The study also indicated that .sup.125Idi-scFv3D6-8D3 displayed lower avidity than .sup.125ImAb3D6-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. Keywords: Bispecific antibody, Brain pharmacokinetics, Transferrin receptor, BBB
Currently, several amyloid beta (Aβ) antibodies, including the protofibril selective antibody BAN2401, are in clinical trials. The murine version of BAN2401, mAb158, has previously been shown to ...lower the levels of pathogenic Aβ and prevent Aβ deposition in animal models of Alzheimer's disease (AD). However, the cellular mechanisms of the antibody's action remain unknown. We have recently shown that astrocytes effectively engulf Aβ
protofibrils, but store rather than degrade the ingested Aβ aggregates. In a co-culture set-up, the incomplete degradation of Aβ
protofibrils by astrocytes results in increased neuronal cell death, due to the release of extracellular vesicles, containing N-truncated, neurotoxic Aβ.
The aim of the present study was to investigate if the accumulation of Aβ in astrocytes can be affected by the Aβ protofibril selective antibody mAb158. Co-cultures of astrocytes, neurons, and oligodendrocytes, derived from embryonic mouse cortex, were exposed to Aβ
protofibrils in the presence or absence of mAb158.
Our results demonstrate that the presence of mAb158 almost abolished Aβ accumulation in astrocytes. Consequently, mAb158 treatment rescued neurons from Aβ-induced cell death.
Based on these findings, we conclude that astrocytes may play a central mechanistic role in anti-Aβ immunotherapy.
Antibodies are highly specific for their target molecules, but their poor brain penetrance has restricted their use as PET ligands for imaging of targets within the CNS. The aim of this study was to ...develop an antibody-based radioligand, using the TribodyTM format, for PET imaging of soluble amyloid-beta (Aβ) protofibrils, which are suggested to cause neurodegeneration in Alzheimer's disease. Antibodies, even when expressed in smaller engineered formats, are large molecules that do not enter the brain in sufficient amounts for imaging purposes. Hence, their transport across the blood-brain barrier (BBB) needs to be facilitated, for example through interaction with the transferrin receptor (TfR). Thus, a Fab fragment of the TfR antibody 8D3 was fused with two single chain variable fragments (scFv) of the Aβ protofibril selective antibody mAb158. Five TribodyTM proteins (A1-A5) were generated with different linkers between the Fab-8D3 and scFv-158. All proteins bound to TfR and Aβ protofibrils in vitro. Three of the proteins (A1-A3) were radiolabeled with iodine-125 and studied ex vivo in wild-type (wt) and transgenic mice overexpressing human Aβ. The systemic pharmacokinetics were similar with half-lives in blood of around 9h for all three ligands. Brain concentrations at 2h were around 1% of the injected dose per gram brain tissue, which is similar to what is observed for small molecular radioligands and at least 10-fold higher than antibodies in general. At 72h, transgenic mice showed higher concentrations of radioactivity in the brain than wt mice (12, 15- and 16-fold for A1, A2 and A3 respectively), except in the cerebellum, an area largely devoid of Aβ pathology. A3 was then labelled with iodine-124 for in vivo positron emission tomography (PET) imaging. Brain concentrations were quantified in six different regions showing a clear distinction both quantitatively and visually between wt and transgenic mice and a good correlation with Aβ pathology. We have thus produced a recombinant, bispecific protein, actively transported into the brain, for PET imaging within the CNS. In a longer perspective, this technique may enable imaging of other proteins involved in neurodegenerative diseases for which imaging agents are completely lacking today.
Display omitted
•Receptor mediated transcytosis enabled use of an antibody-based ligand for brain PET.•Amyloid-beta protofibrils could be visualized with the antibody-based radioligand.•PET quantification of protofibril levels correlated with levels in brain homogenates.•Our study demonstrated that antibodies can be applied for imaging of brain targets.
PDF
