Altered amyloid-ß structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion.

Deposition of amyloid beta (Aß) into plaques is a major hallmark of Alzheimer's disease (AD). Different amyloid precursor protein (APP) mutations cause early-onset AD by altering the production or aggregation properties of Aß. We recently identified the Uppsala APP mutation (APPUpp), which causes Aß pathology by a triple mechanism: increased ß-secretase and altered α-secretase APP cleavage, leading to increased formation of a unique Aß conformer that rapidly aggregates and deposits in the brain. The aim of this study was to further explore the effects of APPUpp in a transgenic mouse model (tg-UppSwe), expressing human APP with the APPUpp mutation together with the APPSwe mutation. Aß pathology was studied in tg-UppSwe brains at different ages, using ELISA and immunohistochemistry. In vivo PET imaging with three different PET radioligands was conducted in aged tg-UppSwe mice and two other mouse models; tg-ArcSwe and tg-Swe. Finally, glial responses to Aß pathology were studied in cell culture models and mouse brain tissue, using ELISA and immunohistochemistry. Tg-UppSwe mice displayed increased ß-secretase cleavage and suppressed α-secretase cleavage, resulting in AßUpp42 dominated diffuse plaque pathology appearing from the age of 5-6 months. The γ-secretase cleavage was not affected. Contrary to tg-ArcSwe and tg-Swe mice, tg-UppSwe mice were [11C]PiB-PET negative. Antibody-based PET with the 3D6 ligand visualized Aß pathology in all models, whereas the Aß protofibril selective mAb158 ligand did not give any signals in tg-UppSwe mice. Moreover, unlike the other two models, tg-UppSwe mice displayed a very faint glial response to the Aß pathology. The tg-UppSwe mouse model thus recapitulates several pathological features of the Uppsala APP mutation carriers. The presumed unique structural features of AßUpp42 aggregates were found to affect their interaction with anti-Aß antibodies and profoundly modify the Aß-mediated glial response, which may be important aspects to consider for further development of AD therapies.

A Brain-Targeting Bispecific-Multivalent Antibody Clears Soluble Amyloid-Beta Aggregates in Alzheimer's Disease Mice.

Amyloid-ß (Aß) oligomers and protofibrils are suggested to be the most neurotoxic Aß species in Alzheimer's disease (AD). Hence, antibodies with strong and selective binding to these soluble Aß aggregates are of therapeutic potential. We have recently introduced HexaRmAb158, a multivalent antibody with additional Aß-binding sites in the form of single-chain fragment variables (scFv) on the N-terminal ends of Aß protofibril selective antibody (RmAb158). Due to the additional binding sites and the short distance between them, HexaRmAb158 displayed a slow dissociation from protofibrils and strong binding to oligomers in vitro. In the current study, we aimed at investigating the therapeutic potential of this antibody format in vivo using mouse models of AD. To enhance BBB delivery, the transferrin receptor (TfR) binding moiety (scFv8D3) was added, forming the bispecific-multivalent antibody (HexaRmAb158-scFv8D3). The new antibody displayed a weaker TfR binding compared to the previously developed RmAb158-scFv8D3 and was less efficiently transcytosed in a cell-based BBB model. HexaRmAb158 detected soluble Aß aggregates derived from brains of tg-ArcSwe and AppNL-G-F mice more efficiently compared to RmAb158. When intravenously injected, HexaRmAb158-scFv8D3 was actively transported over the BBB into the brain in vivo. Brain uptake was marginally lower than that of RmAb158-scFv8D3, but significantly higher than observed for conventional IgG antibodies. Both antibody formats displayed similar brain retention (72 h post injection) and equal capacity in clearing soluble Aß aggregates in tg-ArcSwe mice. In conclusion, we demonstrate a bispecific-multivalent antibody format capable of passing the BBB and targeting a wide-range of sizes of soluble Aß aggregates.

PET Imaging in Preclinical Anti-Aß Drug Development.

Positron emission tomography (PET), a medical imaging technique allowing for studies of the living human brain, has gained an important role in clinical trials of novel drugs against Alzheimer's disease (AD). For example, PET data contributed to the conditional approval in 2021 of aducanumab, an antibody directed towards amyloid-beta (Aß) aggregates, by showing a dose-dependent reduction in brain amyloid after treatment. In parallel to clinical studies, preclinical studies in animal models of Aß pathology may also benefit from PET as a tool to detect target engagement and treatment effects of anti-Aß drug candidates. PET is associated with a high level of translatability between species as similar, non-invasive protocols allow for longitudinal rather than cross-sectional studies and can be used both in a preclinical and clinical setting. This review focuses on the use of preclinical PET imaging in genetically modified animals that express human Aß, and its present and potential future role in the development of drugs aimed at reducing brain Aß levels as a therapeutic strategy to halt disease progression in AD.

Passive and receptor mediated brain delivery of an anti-GFAP nanobody.

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 (Aß) 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 Aß 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.

11C-PiB and 124I-Antibody PET Provide Differing Estimates of Brain Amyloid-ß After Therapeutic Intervention.

PET imaging of amyloid-ß (Aß) has become an important component of Alzheimer disease diagnosis. 11C-Pittsburgh compound B (11C-PiB) and analogs bind to fibrillar Aß. However, levels of nonfibrillar, soluble, aggregates of Aß appear more dynamic during disease progression and more affected by Aß-reducing treatments. The aim of this study was to compare an antibody-based PET ligand targeting nonfibrillar Aß with 11C-PiB after ß-secretase (BACE-1) inhibition in 2 Alzheimer disease mouse models at an advanced stage of Aß 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 Aß plaque load followed by a scan with the bispecific radioligand 124I-RmAb158-scFv8D3 to investigate nonfibrillar aggregates of Aß. 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 Aß 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 Aß 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 Aß levels after anti-Aß therapy in ArcSwe and AppNL-G-F mice with pronounced Aß pathology. In contrast, the decreased Aß levels could not be quantified with 11C-PiB PET, suggesting that these ligands detect different pools of Aß.

Pinpointing Brain TREM2 Levels in Two Mouse Models of Alzheimer's Disease.

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.

Chemical imaging of evolving amyloid plaque pathology and associated Aß peptide aggregation in a transgenic mouse model of Alzheimer's disease.

One of the major hallmarks of Alzheimer's disease (AD) pathology is the formation of extracellular amyloid ß (Aß) plaques. While Aß has been suggested to be critical in inducing and, potentially, driving the disease, the molecular basis of AD pathogenesis is still under debate. Extracellular Aß plaque pathology manifests itself upon aggregation of distinct Aß 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 Aß aggregation processes provides a promising target for early interventions. However, the chain of events of when, where and what Aß 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 Aß 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 Aß (Aß1-42). Plaque maturation was found to be characterized by a relative increase in deposition of Aß1-40, which was associated with the appearance of a cored morphology for those plaques. Finally, other C-terminally truncated Aß species (Aß1-38 and Aß1-39) exhibited a similar aggregation pattern as Aß1-40, suggesting that these species have similar aggregation characteristics. These results suggest that initial plaque formation is seeded by Aß1-42; a process that is followed by plaque maturation upon deposition of Aß1-40 as well as deposition of other C-terminally modified Aß species.

High detection sensitivity with antibody-based PET radioligand for amyloid beta in brain.

PET imaging of amyloid-beta (Aß) 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-Aß treatments. Available PET radioligands visualizing Aß bind to insoluble fibrils, i.e. Aß plaques. Levels of prefibrillar Aß forms, e.g. soluble oligomers and protofibrils, correlate better than plaques with disease severity and these soluble species are the neurotoxic form of Aß leading to neurodegeneration. The goal was to create an antibody-based radioligand, recognizing not only fibrillary Aß, but also smaller and still soluble aggregates. We designed and expressed a small recombinant bispecific antibody construct, di-scFv 3D6-8D3, targeting the Aß 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 Aß1-40 with high affinity and to TfR with moderate affinity. Di-scFv [124I]3D6-8D3 was injected in two transgenic mouse models overexpressing human Aß and wild-type control mice and PET scanned at 14, 24 or 72 h after injection. Di-scFv [124I]3D6-8D3 was retained in brain of transgenic animals while it was cleared from wild-type lacking Aß. This difference was observed from 24 h onwards, and at 72 h, 18 months old transgenic animals, with high load of Aß pathology, displayed SUVR of 2.2-3.5 in brain while wild-type showed ratios close to unity. A subset of the mice were also scanned with [11C]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 [124I]3D6-8D3 compared with [11C]PIB. Brain concentrations of di-scFv [124I]3D6-8D3 correlated with soluble Aß (p < 0.0001) but not with total Aß, 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 Aß in vivo. The radioligand displayed better sensitivity compared with [11C]PIB, and brain concentrations correlated with soluble neurotoxic Aß aggregates.

Antibody-Based In Vivo PET Imaging Detects Amyloid-ß Reduction in Alzheimer Transgenic Mice After BACE-1 Inhibition.

Visualization of amyloid-ß (Aß) 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 11C-Pittsburgh compound B, reflect levels of insoluble Aß plaques but do not capture soluble and protofibrillar Aß forms. Furthermore, the plaque load appears to be fairly static during clinical stages of AD and may not be affected by Aß-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 Aß can be used to detect changes in Aß levels during disease progression and after treatment with a ß-secretase (BACE-1) inhibitor. Methods: One set of transgenic mice (tg-ArcSwe, a model of Aß pathology) aged between 7 and 16 mo underwent PET with the Aß protofibril-selective radioligand 124I-RmAb158-scFv8D3 (where RmAb is recombinant mouse monoclonal antibody and scFv is single-chain variable fragment) to follow progression of Aß 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 Aß by ELISA and immunohistochemistry. Results: The concentration of 124I-RmAb158-scFv8D3, as measured in vivo with PET, increased with age and corresponded well with the ex vivo autoradiography and Aß 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 124I-RmAb158-scFv8D3 concentrations in NB-360-treated mice, as quantified with PET, corresponded well with the decreased Aß 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 Aß is limited. This study demonstrated the ability of the Aß protofibril-selective radioligand 124I-RmAb158-scFv8D3 to follow disease progression and detect treatment effects with PET imaging in tg-ArcSwe mice.

Pharmacokinetics, biodistribution and brain retention of a bispecific antibody-based PET radioligand for imaging of amyloid-ß.

Monoclonal antibodies (mAbs) have not been used as positron emission tomography (PET) ligands for in vivo imaging of the brain because of their limited passage across the blood-brain barrier (BBB). However, due to their high affinity and specificity, mAbs may be an attractive option for brain PET if their brain distribution can be facilitated. In the present study, a F(ab')2 fragment of the amyloid-beta (Aß) protofibril selective mAb158 was chemically conjugated to the transferrin receptor (TfR) antibody 8D3 to enable TfR mediated transcytosis across the BBB. The generated bispecific protein, 8D3-F(ab')2-h158, was subsequently radiolabeled and used for microPET imaging of Aß pathology in two mouse models of AD. [124I]8D3-F(ab')2-h158 was distributed across the BBB several fold more than unmodified mAbs in general and its accumulation in the brain reflected disease progression, while its concentration in blood and other organs remained stable across all age groups studied. Cerebellum was largely devoid of 8D3-F(ab')2-h158 in young and middle aged mice, while mice older than 18 months also showed some accumulation in cerebellum. In a longer perspective, the use of bispecific antibodies as PET ligands may enable in vivo 'immunohistochemistry' also of other proteins in the brain for which PET radioligands are lacking.

A bispecific Tribody PET radioligand for visualization of amyloid-beta protofibrils - a new concept for neuroimaging.

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 (Aß) 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 Aß 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 Aß 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 Aß. 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 Aß 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 Aß 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.