Faculty Bibliography

The majority of clinical trials targeting the tau protein in Alzheimer's disease and other tauopathies are tau immunotherapies. Because tau pathology correlates better with the degree of dementia than amyloid-β lesions, targeting tau is likely to be more effective in improving cognition than clearing amyloid-β in Alzheimer's disease. However, the development of tau therapies is in many ways more complex than for amyloid-β therapies as briefly outlined in this review. Most of the trials are on humanized antibodies, which may have very different properties than the original mouse antibodies. The impact of these differences are to a large extent unknown, can be difficult to decipher, and may not always be properly considered. Furthermore, the ideal antibody properties for efficacy are not well established and can depend on several factors. However, considering the varied approaches in clinical trials, there is a general optimism that at least some of these trials may provide functional benefits to patients suffering of various tauopathies.

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.

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.

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.

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).

Introduction/UNASSIGNED:The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies. Methods/UNASSIGNED:Highly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays. Results/UNASSIGNED:of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau. Discussion/UNASSIGNED:Targeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.