Single-domain antibody-based noninvasive in vivo imaging of α-synuclein or tau pathology
Intracellular deposition of α-synuclein and tau are hallmarks of synucleinopathies and tauopathies, respectively. Recently, several dye-based imaging probes with selectivity for tau aggregates have been developed, but suitable imaging biomarkers for synucleinopathies are still unavailable. Detection of both of these aggregates early in the disease process may allow for prophylactic therapies before functional impairments have manifested, highlighting the importance of developing specific imaging probes for these lesions. In contrast to the β sheet dyes, single-domain antibodies, found in camelids and a few other species, are highly specific, and their small size allows better brain entry and distribution than whole antibodies. Here, we have developed such imaging ligands via phage display libraries derived from llamas immunized with α-synuclein and tau preparations, respectively. These probes allow noninvasive and specific in vivo imaging of α-synuclein versus tau pathology in mice, with the brain signal correlating strongly with lesion burden. These small antibody derivatives have great potential for in vivo diagnosis of these diseases.
Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity
BACKGROUND:Eleven tau immunoglobulin G (IgG) antibodies have entered clinical trials to treat tauopathies, including Alzheimer's disease, but it is unclear which IgG subclass/subtype has the ideal efficacy and safety profile. Only two subtypes, with or without effector function, have been examined in the clinic and not for the same tau antibody. The few preclinical studies on this topic have only compared two subtypes of one antibody each and have yielded conflicting results. METHODS:subclasses containing identical tau binding domains but differing Fc region. Unmodified sdAbs and their IgG subclasses were tested for efficacy in primary cultures and in vivo microdialysis using JNPL3 tauopathy mice. FINDINGS/RESULTS:subclasses varied greatly within and between sdAbs. For one of them, all its subtypes were non-toxic, only those with effector function cleared tau, and were more effective in vivo than unmodified sdAb. For the other sdAb, all its subtypes were toxic in tauopathy cultures but not in wild-type cells, suggesting that bivalent binding of its tau epitope stabilizes a toxic conformation of tau, with major implications for tau pathogenesis. Likewise, its subclasses were less effective than the unmodified sdAb in clearing tau in vivo. INTERPRETATION/CONCLUSIONS:These findings indicate that tau antibodies with effector function are safe and better at clearing pathological tau than effectorless antibodies, Furthermore, tau antibodies can provide a valuable insight into tau pathogenesis, and some may aggravate it. FUNDING/BACKGROUND:Funding for these studies was provided by the National Institute of Health (R01 AG032611, R01 NS077239, RF1 NS120488, R21 AG 069475, R21 AG 058282, T32AG052909), and the NYU Alzheimer's Disease Center Pilot Grant Program (via P30 AG008051).
Targeting tau only extracellularly is likely to be less efficacious than targeting it both intra- and extracellularly
Aggregation of the tau protein is thought to be responsible for the neurodegeneration and subsequent functional impairments in diseases that are collectively named tauopathies. Alzheimer's disease is the most common tauopathy, but the group consists of over 20 different diseases, many of which have tau pathology as their primary feature. The development of tau therapies has mainly focused on preventing the formation of and/or clearing these aggregates. Of these, immunotherapies that aim to either elicit endogenous tau antibodies or deliver exogenous ones are the most common approach in clinical trials. While their mechanism of action can involve several pathways, both extra- and intracellular, pharmaceutical companies have primarily focused on antibody-mediated clearance of extracellular tau. As we have pointed out over the years, this is rather surprising because it is well known that most of pathological tau protein is found intracellularly. It has been repeatedly shown by several groups over the past decades that antibodies can enter neurons and that their cellular uptake can be enhanced by various means, particularly by altering their charge. Here, we will briefly describe the potential extra- and intracellular mechanisms involved in antibody-mediated clearance of tau pathology, discuss these in the context of recent failures of some of the tau antibody trials, and finally provide a brief overview of how the intracellular efficacy of tau antibodies can potentially be further improved by certain modifications that aim to enhance tau clearance via specific intracellular degradation pathways.
Increased neuronal activity in motor cortex reveals prominent calcium dyshomeostasis in tauopathy mice
Perturbed neuronal Ca2+ homeostasis is implicated in Alzheimer's disease, which has primarily been demonstrated in mice with amyloid-Î² deposits but to a lesser and more variable extent in tauopathy models. In this study, we injected AAV to express Ca2+ indicator in layer II/III motor cortex neurons and measured neuronal Ca2+ activity by two photon imaging in awake transgenic JNPL3 tauopathy and wild-type mice. Various biochemical measurements were conducted in postmortem mouse brains for mechanistic insight and a group of animals received two intravenous injections of a tau monoclonal antibody spaced by four days to test whether the Ca2+ dyshomeostasis was related to pathological tau protein. Under running conditions, we found abnormal neuronal Ca2+ activity in tauopathy mice compared to age-matched wild-type mice with higher frequency of Ca2+ transients, lower amplitude of peak Ca2+ transients and lower total Ca2+ activity in layer II/III motor cortex neurons. While at resting conditions, only Ca2+ frequency was increased. Brain levels of soluble pathological tau correlated better than insoluble tau levels with the degree of Ca2+ dysfunction in tauopathy mice. Furthermore, tau monoclonal antibody 4E6 partially rescued Ca2+ activity abnormalities in tauopathy mice after two intravenous injections and decreased soluble pathological tau protein within the brain. This correlation and antibody effects strongly suggest that the neuronal Ca2+ dyshomeostasis is causally linked to pathological tau protein. These findings also reveal more pronounced neuronal Ca2+ dysregulation in tauopathy mice than previously reported by two-photon imaging that can be partially corrected with an acute tau antibody treatment.
Tau antibody chimerization alters its charge and binding, thereby reducing its cellular uptake and efficacy
BACKGROUND:Bringing antibodies from pre-clinical studies to human trials requires humanization, but this process may alter properties that are crucial for efficacy. Since pathological tau protein is primarily intraneuronal in Alzheimer's disease, the most efficacious antibodies should work both intra- and extracellularly. Thus, changes which impact uptake or antibody binding will affect antibody efficacy. METHODS:Initially, we examined four tau mouse monoclonal antibodies with naturally differing charges. We quantified their neuronal uptake, and efficacy in preventing toxicity and pathological seeding induced by human-derived pathological tau. Later, we generated a human chimeric 4E6 (h4E6), an antibody with well documented efficacy in multiple tauopathy models. We compared the uptake and efficacy of unmodified and chimeric antibodies in neuronal and differentiated neuroblastoma cultures. Further, we analyzed tau binding using ELISA assays. FINDINGS/RESULTS:Neuronal uptake of tau antibodies and their efficacy strongly depends on antibody charge. Additionally, their ability to prevent tau toxicity and seeding of tau pathology does not necessarily go together. Particularly, chimerization of 4E6 increased its charge from 6.5 to 9.6, which blocked its uptake into human and mouse cells. Furthermore, h4E6 had altered binding characteristics despite intact binding sites, compared to the mouse antibody. Importantly, these changes in uptake and binding substantially decreased its efficacy in preventing tau toxicity, although under certain conditions it did prevent pathological seeding of tau. CONCLUSIONS:These results indicate that efficacy of chimeric/humanized tau antibodies should be thoroughly characterized prior to clinical trials, which may require further engineering to maintain or improve their therapeutic potential. FUND: National Institutes of Health (NS077239, AG032611, R24OD18340, R24OD018339 and RR027990, Alzheimer's Association (2016-NIRG-397228) and Blas Frangione Foundation.
Dynamic assessment of tau immunotherapies in the brains of live animals by two-photon imaging
Our original findings, showing the effectiveness of active and passive tau immunizations in mouse models, have now been confirmed and extended by many groups, with several clinical trials underway in Alzheimer's disease and progressive supranuclear palsy. Here, we report on a unique and sensitive two-photon imaging approach to concurrently study the dynamics of brain and neuronal uptake and clearance of tau antibodies as well as the acute removal of their pathological target in live animals. This in vivo technique is more sensitive to detect clearance of pathological tau protein than western blot tau analysis of brain tissue. In addition to providing an insight into the mechanisms involved, it allows for an efficient in vivo assessment of the therapeutic potential of tau antibodies, and may be applied to related protein misfolding diseases.
Neuronally expressed anti-tau scFv prevents tauopathy-induced phenotypes in Drosophila models
We have derived single-chain variable fragments (scFv) from tau antibody hybridomas and previously shown their promise as imaging diagnostic agents. Here, we examined the therapeutic potential of anti-tau scFv in transgenic Drosophila models that express in neurons wild-type (WT) human tau (htau) or the human tauopathy mutation R406W. scFv expressing flies were crossed with the tauopathy flies and analyzed. Overall, the survival curves differed significantly (pâ€¯<â€¯.0001). Control flies not expressing htau survived the longest, whereas R406W expressing flies had the shortest live span, which was greatly prolonged by co-expressing the anti-tau scFv (pâ€¯<â€¯.0001). Likewise, htau WT expressing flies had a moderately short live span, which was prolonged by co-expressing the anti-tau scFv (pâ€¯<â€¯.01). In addition, the htau expression impaired wing expansion after eclosion (pâ€¯<â€¯.0001), and caused progressive abdomen expansion (pâ€¯<â€¯.0001). These features were more severe in htau R406W flies than in htau WT flies. Importantly, both phenotypes were prevented by co-expression of the anti-tau scFv (pâ€¯<â€¯.01-0.0001). Lastly, brain analyses revealed scFv-mediated tau clearance (pâ€¯<â€¯.05-0.01), and its prevention of tau-mediated neurotoxicity (pâ€¯<â€¯.05-0.001). In summary, these findings support the therapeutic potential of an anti-tau scFv, including as gene therapies, and the use of Drosophila models for such screening.
Tau-targeting therapies for Alzheimer disease: current status and future directions
Alzheimer disease (AD) is the most common cause of dementia in older individuals. AD is characterized pathologically by amyloid-β (Aβ) plaques and tau neurofibrillary tangles in the brain, with associated loss of synapses and neurons, which eventually results in dementia. Many of the early attempts to develop treatments for AD focused on Aβ, but a lack of efficacy of these treatments in terms of slowing disease progression led to a change of strategy towards targeting of tau pathology. Given that tau shows a stronger correlation with symptom severity than does Aβ, targeting of tau is more likely to be efficacious once cognitive decline begins. Anti-tau therapies initially focused on post-translational modifications, inhibition of tau aggregation and stabilization of microtubules. However, trials of many potential drugs were discontinued because of toxicity and/or lack of efficacy. Currently, the majority of tau-targeting agents in clinical trials are immunotherapies. In this Review, we provide an update on the results from the initial immunotherapy trials and an overview of new therapeutic candidates that are in clinical development, as well as considering future directions for tau-targeting therapies.
Tau-targeting therapies for Alzheimer disease
Alzheimer disease (AD) is the most common form of dementia. Pathologically, AD is characterized by amyloid plaques and neurofibrillary tangles in the brain, with associated loss of synapses and neurons, resulting in cognitive deficits and eventually dementia. Amyloid-Î² (AÎ²) peptide and tau protein are the primary components of the plaques and tangles, respectively. In the decades since AÎ² and tau were identified, development of therapies for AD has primarily focused on AÎ², but tau has received more attention in recent years, in part because of the failure of various AÎ²-targeting treatments in clinical trials. In this article, we review the current status of tau-targeting therapies for AD. Initially, potential anti-tau therapies were based mainly on inhibition of kinases or tau aggregation, or on stabilization of microtubules, but most of these approaches have been discontinued because of toxicity and/or lack of efficacy. Currently, the majority of tau-targeting therapies in clinical trials are immunotherapies, which have shown promise in numerous preclinical studies. Given that tau pathology correlates better with cognitive impairments than do AÎ² lesions, targeting of tau is expected to be more effective than AÎ² clearance once the clinical symptoms are evident. With future improvements in diagnostics, these two hallmarks of the disease might be targeted prophylactically.
Amyloid-β targeting immunisation in aged non-human primate (Microcebus murinus)
Non-human primates have an important translational value given their close phylogenetic relationship to humans. Studies in these animals remain essential for evaluating efficacy and safety of new therapeutic approaches, particularly in aging primates that display Alzheimer's disease (AD) -like pathology. With the objective to improve amyloid-β (Aβ) targeting immunotherapy, we investigated the safety and efficacy of an active immunisation with an Aβ derivative, K6Aβ1-30-NH2, in old non-human primates. Thirty-two aged (4-10 year-old) mouse lemurs were enrolled in the study, and received up to four subcutaneous injections of the vaccine in alum adjuvant or adjuvant alone. Even though antibody titres to Aβ were not high, pathological examination of the mouse lemur brains showed a significant reduction in intraneuronal Aβ that was associated with reduced microgliosis, and the vaccination did not lead to microhemorrhages. Moreover, a subtle cognitive improvement was observed in the vaccinated primates, which was probably linked to Aβ clearance. This Aβ derivative vaccine appeared to be safe as a prophylactic measure based on the brain analyses and because it did not appear to have detrimental effects on the general health of these old animals.