Tau is a member of the microtubule-associated family of proteins (MAPs) which are expressed predominantly in the brain (mainly founds in neurons but is also present at low levels in glial cells). This protein is encoded by a single gene, MAPT, located on human chromosome 17. In its native state, tau is present in a stable, unfolded monomeric conformation and it is a hydrophilic and highly soluble protein. Tau’s primary functions include the stabilization of microtubules and the coordinated movement of molecules along the microtubule and microtubule assembly. Tau is a phosophoprotein that a normal level of phosphorylation is required for its optimal function, whereas the hyperphosphorylated state makes tau to lose its biological activity. This protein has 85 phosphorylation sites in its human longest isoform. Normal brain tau contains 2-3 moles of phosphate per mole of the protein. In AD brain, tau is abnormally hyperphosphorylated at least three-fold greater than normal tau, this alteration leads to Neurofibrillary tangle (NFT) formation, a histopathological hallmark of the disease. The abnormal hyperphosphorylation of tau is also a hallmark of several other related neurodegenerative disorders, called Tauopathies. Tau phosphorylation at different sites has a different impact on its biological function and on its pathogenic role. An in vitro study demonstrated that phosphorylation of tau at many sites such as Ser262, Thr231, and Ser235 inhibits its binding to microtubules. Another study suggests that Ser199/Ser202/Thr205, Thr212, Thr231/Ser235, Ser262/Ser356, and Ser422 are among the critical phosphorylation sites that convert tau to an inhibitory molecule that sequesters normal microtubule-associated proteins from microtubule. Further phosphorylation at Thr231, Ser396, and Ser422 promotes self-aggregation of tau into filaments.

Recently, we have reported that phosphorylated tau at Thr231 is extremely neurotoxic and early driver of neurodegeneration upon tauopathies, such as AD. Our preclinical observations based on animal mouse models have clearly demonstrated that immunotherapy employing the pT231-tau mouse monoclonal antibody suppresses neurodegeneration in those TBI mouse models. Thus, we have generated a fully human pT231-tau antibody and examined the efficacy in suppressing neurodegeneration in transgenic AD mouse models and have shown our antibody efficiently neutralizes the pathogenic p-tau epitope.

The effect of antibody treatment (i.v.) on the ability of memory in the retention day. Color maps collapsed across all animals in each group (mean groups), showing the percent time in each location of the swimming pool during the no-platform probe test. The location of the platform is marked by a white box. Total time (s) that animals in each experimental group spent in the target zone were evaluated. Data are represented as mean ± SEM for 6 mice. One-way ANOVA followed by Tukey’s post hoc comparisons tests were performed.

To examine the anxiolytic properties of our mAb, we performed elevated-plus maze test in AD mice models. The healthy control animals explored significantly less time in the open arms than AD animals (p = 0.009 or p - 0.01) mice. Moreover, mAb-treated groups, similar to the control group, spent significantly less time in the open arms (p - 0.001) compared with AD groups. AD animals displayed more total open arm entries than the control and mAb-treated AD mice. As a result, all AD mice, exploring the two open arms, showed anxiety/risk-taking behavior by contrast, mAb-treated AD mice displayed minimal anxious behavior. Thus, our mAb not only eliminates pathogenic pT231-tau but also restores behavioral deficits triggered by AD.

Moreover, immunotherapy with our pT231-tau mAb improved ultrastructural consequences triggered by AD. It is clear that pathogenic pT231-tau is associated with neurodegenerative outcomes and microtubule and mitochondrial disruption. To understand the effectiveness of our mAb on neurodegenerative pathologies triggered by AD, we treated the animal models with anti pT231-tau mAb. We have shown that while AD pathogenesis result in brain ultrastructure disruption, our mAb treatment effectively eliminated tau pathology and structural defects in axonal microtubules (MTs; blue open arrows) and mitochondria (MI; red filled arrows).

Furthermore, we have clearly confirmed that while there is a profound tau pathology in AD animal models, the antibody efficiently suppresses tau hyperphosphorylation in the animal models (AT8 as early and PHF-1 as late tau pathology markers)

Also, we performed immunotherapy assessments on human AD neurons generated from fAD subjects iPSCs. Our preclinical observations have demonstrated that while AD neurons degenerated upon aging, pretreatment the culture with our pT231-tau human antibody suppresses neural cell death efficiently (left: control cells; middle: AD neurons; right panel: AD neurons treated with anti pT231-tau human antibody).

We have also co-immunostained human AD postmortem brain with our human antibody & mouse cis pT231-tau antibody and have found a prominent pT231-tau (but not cis p-tau ) epitope in those brains while there was not any positive signal in healthy control brains. There is a gradual increase in the pT231-tau epitope confirmed by immunostaining of human AD postmortem brains.

Moreover, we performed immunoblotting analysis on human AD and healthy brain extracts using our antibody and compared the results with anti cis pT231-tau mouse monoclonal antibody. Immunoblots of AD and healthy control human brain extracts stained with anti cis pT231-tau mouse monoclonal antibody (A) and our anti pT231-tau human monoclonal antibody (B). As shown in the blots, there is no significant difference between control and AD brain extracts when stained with cis p-tau antibody. Interestingly, our human pT231-tau recognizes six tau isoforms only in AD brain extract; demonstrating that the antibody specifically binds to pathogenic tau species.