AD is the leading cause of senile dementia. Its prevalence is predicted to increase at least threefold between 2000 and 2050, rendering AD an increasing worldwide public health problem . The current most effective treatment approach for AD—cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists—is purely symptomatic and provides benefit for only up to 12 months . Therefore, the aim of the currently proposed therapeutic approaches and strategies for AD is to counteract the fundamental underlying pathological processes leading to the development and progression of the disease.
The main neuropathological hallmarks of AD are NFTs composed of tau protein and senile plaques consisting of amyloid-β. It has been shown that diseased modified tau proteins play an essential role in the clinical manifestation of AD [7, 9, 56]. An increased understanding of the molecular mechanisms underlying the pathological transformations of tau has opened up the possibility of specifically targeting pathologically modified tau protein for therapeutic purposes. As a result, a number of therapeutic approaches that directly or indirectly target the tau misfolding cascade have emerged [2, 57, 58]. One of these promising therapeutic approaches is immunotherapy targeting various tau species. Several independent studies have shown that either active or passive immunotherapy could prevent tau aggregation or clear tau aggregates and reduce tau hyperphosphorylation [22–24, 26, 59–62]. However, it has been discussed that phosphorylated tau antigens, used predominantly in the abovementioned studies, display some potential risks, as these phosphorylation sites are associated mainly with matured NFTs and not with early stages of tangle formation . Moreover, phosphorylation is the main physiological mechanism regulating tau structure and function, and, therefore, the major concern caused by active immunisation with phospho-tau peptides is an immune response towards the physiological tau species .
In this report, we present a preclinical immunoproteomic platform for the identification of structural determinants on tau protein essential for its pathological interaction. Our strategy was based on our findings that truncation of tau protein significantly changes the structure and function of the molecule. Thus, mis-disordered truncated tau is a substrate for pathological tau–tau interactions [27, 30, 31]. To identify the critical structural determinants on tau responsible for pathological tau–tau interaction, we developed molecular imprinting technology based on the unique properties of mAbs recognizing structural changes of naturally disordered proteins. Using hypothesis-driven research approaches, we proposed a targeted vaccine development strategy consisting of the following steps: (1) identification of a mAb that is able to prevent tau from its pathological tau–tau interaction, (2) in vivo validation of the therapeutic activity of the antibody with special emphasis on reduction of neurofibrillary pathology and sarcosyl-insoluble tau, (3) identification of the epitopes on tau recognised by therapeutic antibody, (4) identification of the three-dimensional structure of these binding sites and (5) characterisation of the antibody’s ability to discriminate between disease-modified and physiological tau protein. The chosen algorithm allows us to identify structural determinants essential for pathological tau–tau interaction and validate it as a novel therapeutic target for tau immunotherapy in AD.
In order to identify an antibody that can protect tau from pathological tau assembly, we have developed an in vitro assay for screening of antibody potency to inhibit pathological tau–tau interaction. Previously, it has been shown that the key regions responsible for tau–tau interactions are located in the microtubule-binding domain of tau [37, 64, 65]; however, their pathologic gain of function is regulated by the truncation of distant parts of the tau protein molecule . The phenomenon of regulation of protein function by truncation is particularly prominent in the intrinsically disordered proteins involved in neurodegenerative diseases . It has been shown that tau truncation is one of the key pathognomonic features of neurodegenerative processes in AD . Furthermore, removal of tau protein termini triggers a tau misfolding cascade and the development of tau neurofibrillary pathology in animal models [30, 31]. In our present study, we found truncated tau protein 151-391/4R to be particularly suitable for screening for antibodies inhibiting pathological tau–tau interaction. It allowed us to identify DC8E8 antibody as extremely efficient in inhibiting tau–tau interaction. Proteomic mapping revealed that the antibody was capable of binding to four independent and yet highly homologous binding regions, each of which is a separate epitope, one in each of the MTBR regions. DC8E8 represents the first specific antibody recognizing four previously unidentified functional regions of tau (structural determinants, epitopes) regulating pathological tau–tau interaction.
In line with this, DC8E8 inhibits the formation of dimers, trimers and oligomers by mis-disordered tau. Therefore, identified DC8E8 epitopes are involved in tau conformational changes leading to tau oligomerisation as the first step of tau fibrillisation. As DC8E8 is capable of discriminating between pathological and physiological tau proteins, at least one of these four epitopes is conformational.
It is probable that binding of DC8E8 to physiological tau at one or more of these epitopes impedes in the same manner certain conformational changes in the tau that are needed for the oligomerisation of tau. In other words, targeting of these epitopes by antibody influences the structure of adjacent regions with β-structural propensities (for example, 274–281, 306–311
). Thus, binding of DC8E8 to one of these four epitopes within normal tau is capable of preventing one of the earliest pathological changes in tau, connected with the formation of β-sheets within tau. All of the four epitopes targeted by DC8E8 are present in the sequence of the AD paired helical filament (PHF) core
. Inhibition of these motifs by DC8E8 and subsequent prevention of PHF core formation is a good explanation for the mechanism underlying how DC8E8 is capable of interfering with the multiple tau-mediated activities contributing to AD pathology, including (1) transition from physiological tau to pathological tau; (2) formation of tau dimers, trimers and other tau oligomers; and (3) formation of insoluble tau aggregates.
We have determined the X-ray structure of a DC8E8 binding site revealing a relatively flexible, deep binding pocket. The pronounced flexibility of CDR L1 and CDR H3 (Figure
6A and B) can facilitate recognition of four independent homologous epitopes on tau. The topology of the DC8E8 binding pocket indicates a protruding shape adopted by the HXPGGG epitope in the tau–DC8E8 complex, suggesting a 180° turn on the tau chain. As we have shown that DC8E8 targets the regulatory motif of tau–tau interaction, it is likely that formation of such a protruding turn by the HXPGGG tau sequence and binding of this turn by DC8E8 efficiently blocks interaction of downstream aggregation-prone tau motifs
[37, 64, 65].
We have proven the antibody’s therapeutic efficacy in an in vivo model. To date, tau-targeted immunisation has been explored exclusively in different mutant tau mouse models because these mice develop NFTs in their brains
. However, no tau mutations have thus far been observed in AD. Tau protein mutations on chromosome 17 correlate with cognitive and motor impairment in frontotemporal dementia linked to chromosome 17
[67, 68]. It is important to note, however, that posttranslational modifications of tau protein, such as truncation
[37, 69, 70], abnormal hyperphosphorylation
[73, 74], ubiquitination
 and nitration
, play key roles in tangle development. They can significantly influence conformational characteristics of tau and impose novel toxic properties onto mis-disordered tau. Thus, in order to test the biological function of the inhibitory antibody DC8E8, we used an animal model of AD based on an AD-relevant disease modification of tau—truncation. Our transgenic mice of the strain R3m/4 express truncated tau derived from human AD brains and display early onset of AD tau pathology, which renders this model an ideal test system for immunotherapy targeting tau neurofibrillary lesions. We treated transgenic animals with DC8E8 for 4 months starting at their second month of life. The antibody was able to significantly reduce early and late tau pathology, showing that DC8E8 targets and disables disease-modified mis-disordered tau. Importantly, antibody DC8E8 recognises all forms of tau lesions, including pretangles and intracellular and extracellular NFTs, in both preclinical and fully developed human AD. Therefore, it is reasonable to expect that the antibody will exert the same pattern of therapeutic activity in AD patients.