Faculty Bibliography

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 (p < .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 (p < .0001). Likewise, htau WT expressing flies had a moderately short live span, which was prolonged by co-expressing the anti-tau scFv (p < .01). In addition, the htau expression impaired wing expansion after eclosion (p < .0001), and caused progressive abdomen expansion (p < .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 (p < .01-0.0001). Lastly, brain analyses revealed scFv-mediated tau clearance (p < .05-0.01), and its prevention of tau-mediated neurotoxicity (p < .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.

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.

Targeting tau with immunotherapies is currently the most common approach taken in clinical trials of patients with Alzheimer's disease. The most prominent pathological feature of tau is its hyperphosphorylation, which may cause the protein to aggregate into toxic assemblies that collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser³⁹⁶/Ser⁴⁰⁴ has received particular attention for therapeutic targeting because of its prominence and stability in diseased tissue. Herein, we present the antigen-binding fragment (Fab)/epitope complex structures of three different monoclonal antibodies (mAbs) that target the pSer⁴⁰⁴ tau epitope region. Most notably, these structures reveal an antigen conformation similar to a previously described pathogenic tau epitope, pSer⁴²², which was shown to have a β-strand structure that may be linked to the seeding core in tau oligomers. In addition, we have previously reported on the similarly ordered conformation observed in a pSer³⁹⁶ epitope, which is in tandem with pSer⁴⁰⁴. Our data are the first Fab structures of mAbs bound to this epitope region of the tau protein and support the existence of proteopathic tau conformations stabilized by specific phosphorylation events that are viable targets for immune modulation. The atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank under accession codes 6DC7 (8B2 apo), 6DC8 (8B2), 6DC9 (h4E6), and 6DCA (6B2).

Development of therapies for Alzheimer's disease has only resulted in a few approved drugs that provide some temporary symptomatic relief in certain patients. None of these compounds in clinical use halts or slows the progression of the disease. To date, several drugs targeting the amyloid-β peptide, and some against the tau protein, have failed in clinical trials. While there are various reasons for these failures, considering the following points may aid in improving the outcome of future trials. First, the tau protein should ideally be targeted intracellularly because most of tau pathology is within cells, neurons in particular. Second, an overriding emphasis in recent years has been on implementing drug-screening models that focus on prevention of seeding/spread of aggregates. Much less attention has been paid to identify compounds that inhibit neurotoxicity of these aggregates, which may not necessarily relate to their seeding/spread propensity. Ideally, all these markers should be readouts in the same assay or model. Third, diversity in conformers/strains of aggregates complicates drug development of small molecule aggregation inhibitors but is likely to be less of an issue for antibody-based treatments. Lastly, other more general targets associated with neurodegeneration should continue to be pursued but are in many ways more difficult to address than clearing amyloid-β and tau, the defining hallmarks of AD.

Programmed cell death protein 1 (PD-1) checkpoint blockade with an antibody has been shown to reduce amyloid-β plaques, associated pathologies and cognitive impairment in mouse models. More recently, this approach has shown effectiveness in a tauopathy mouse model to improve cognition and reduce tau lesions. Follow-up studies by other laboratories did not see similar benefits of this type of therapy in other amyloid-β plaque models. Here, we report a modest increase in locomotor activity but no effect on cognition or tau pathology, in a different more commonly used tauopathy model following a weekly treatment for 12 weeks with the same PD-1 antibody and isotype control as in the original Aβ- and tau-targeting studies. These findings indicate that further research is needed before clinical trials based on PD-1 checkpoint immune blockage are devised for tauopathies.

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.

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-β (Aβ) peptide and tau protein are the primary components of the plaques and tangles, respectively. In the decades since Aβ and tau were identified, development of therapies for AD has primarily focused on Aβ, but tau has received more attention in recent years, in part because of the failure of various Aβ-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 Aβ lesions, targeting of tau is expected to be more effective than Aβ clearance once the clinical symptoms are evident. With future improvements in diagnostics, these two hallmarks of the disease might be targeted prophylactically.

Tau antibodies have shown therapeutic potential for Alzheimer's disease and several are in clinical trials. As a microtubule-associated protein, tau relies on dynamic phosphorylation for its normal functions. In tauopathies, it becomes hyperphosphorylated and aggregates into toxic assemblies, which collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser396 has received particular attention because of its prominence and stability in tauopathies. Here we report the first structure of a monoclonal tau antibody in complex with the pathologically important phospho-Ser396 residue. Its binding region reveals tau residues Tyr394 to phospho-Ser396 stabilized in a β-strand conformation that is coordinated by a phospho-specific antigen binding site. These details highlight a molecular switch that defines this prominent conformation of tau and ways to target it. Overall, the structure of the antibody-antigen complex clarifies why certain phosphorylation sites in tau are more closely linked to neurodegeneration than others.