Abstract Body

Tau has become an unavoidable therapeutic target in Alzheimer's disease and progressive supranuclear palsy. Decreasing the expression of tau is the most commonly used strategy. In addition to anti-sense oligonucleotides, anti-tau immunotherapy is one of the most advanced therapeutic approaches. However, targeting tau remains a challenge. Tau is primarily an intracellular protein. In such paradigm, immunological tools must reach the cytoplasm of the cells to be targeted. However, tau protein is also secreted, and its pro-aggregative species are believed to participate in tau pathology spreading by a prion-like mechanism. In this hypothesis, immunological tools should block extracellular pathological species. From a technological point of view, immunological tools have largely evolved and there is now a very wide panel: monoclonal antibody (immunoglobulin, IgG), Fab fragment, single-chain variable fragment (Fv), VHH single domain antibody (or nanobody), ... Each of them has advantage.

In the laboratory, we work with IgGs that recognise different tau regions or pathological post-translational modifications (phosphorylation or acetylation). We have also developed numerous anti-tau VHHs that can be used as recombinant protein or by viral vectorisation. We use many experimental models of tauopathy (FRET seeding assay, murine models (seeding/spreading)...) for preclinical studies.

In this work, we will discuss the advantages and disadvantages of using a total anti-tau immunoglobulin versus pathological anti-tau. We will also see the opportunity to use VHH-based gene therapy for certain tauopathies considered as orphan disease (FTLD-MAPT).

Altogether, we show that Tau is a promising therapeutic target, but targeted epitope is crucial to avoid side-effects.

Aims

The mechanisms leading to Tau aggregation are still ill-defined, different molecular features have been identified as involved in the aggregation process. The peptide PHF6 (Paired Helical Filament-6, residues 306-VQIVYK-311) is described as a nucleus of Tau aggregation. Different strategies have been proposed as therapeutic approaches in tauopathies, including immunotherapy. However, it remains important to better define the best region(s) of Tau to target , as well as the Tau species (non/phosphorylated, non/soluble or aggregated) and their localization (intra/extra-cellular). In view of this challenge, we have used VHHs (Variable domain of the Heavy- chain of the Heavy-chain-only-antibodies) for their interesting properties and relative ease of generation.

Methods

In partnership with Hybrigenics Company, a synthetic phage display library of VHHs was screened against full-length recTau protein. The epitopes recognized by the selected VHHs, were defined using Nuclear Magnetic Resonance spectroscopy. A VHH targeting an epitope in the microtubule binding domain of tau, corresponding to the PHF6, was selected. Further optimizations of this VHH, using yeast two-hybrid were performed, increasing its intracellular binding capacity and its binding affinity, resulting in a family of VHHs targeting PHF6. These VHHs were next screened for their inhibitory effect towards tau aggregation in vitro, in a cellular model (FRET analysis) as well as in tau transgenic mice.

Results

VHH seem to have the capacity of blocking tau seeding in all cases.

Conclusions

The promising preliminary results of the in vivo open the way for new studies using these VHHs as molecular tools to decipher the best target in Tau immunothérapies.