OA18779A - Antibodies Specific for Hyperphosphorylated Tau and Methods of Use Thereof. - Google Patents

Abstract

The present invention relates to a novel class of monoclonal antibody that specifically binds the phosphorylated serine 396 residue on pathological hyperphosphorylated (PHF) tau (pS396), as well as to methods of using these molecules and their tau binding fragments in the treatment of Alzheimer's disease and tauopathies.

Description

ANTIBODIES SPECIFIC FOR HYPERPHOSPHORYLATED TAU AND METHODS OF USE THEREOF

FIELD OF THE INVENTION

The présent invention relates to a novel class of monoclonal antibody that specifically binds the phosphorylated serine 396 residue on pathological hyperphosphorylated (PHF) tau (pS396), as well as to methods of using these molécules and their tau binding fragments in the treatment of Alzheimer's disease and tauopathies.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in computer-readable media (file name: 0995.txt, created on 23 June 2016, and having a size of 40 kB), which file is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Age-related neurodegenerative diseases such as Alzheimer's disease (AD) and dementia are one of the largest sociétal challenges today. The World Health Organization estimâtes that costs for care of the elderly will continue to increase and that the number of diagnosed dementia cases will triple by 2050 (World Health Organization and Alzheimer's Disease International - Status Report (2012) DEMENTIA: A public health priority, WHO). The first treatments for AD were neurotransmitter modulators such as acétylcholine esterase inhibitors and NMDA modulators. These thérapies became available at the turn of the millennium and still form the cornerstone for symptomatic relief of memory déficits related to dementia and AD. However, these drugs do not target the underlying causes of AD, accumulation of amyloid-β (Αβ) peptide and tau protein aggregates and associated loss of neuronal synapses and eventually neurons.

Longitudinal, community-wide studies of the elderly (Weiner, M.W. et al. (2014) ADNI online: http://www.adni-info.org/; Breteler, M.M. et al. (1992) Neuroepidemiology 11 Suppl 1, 23-28; Launer, L.J. (1992) Neuroepidemiology 11 Suppl 1, 2-13) together with large genome-wide association studies (Lambert, J.C. et al. (2013) Nat. Genet. 45, 1452-1458) hâve shown that AD is a heterogeneous mix of dementias where up to 10 percent of the advanced AD patients lack amyloid pathology (Crary, J.F. et al. (2014) Acta Neuropathol. 128, 755-766). Furthermore, séminal pathological studies by Braak & Braak (Braak, H. and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3-12) demonstrated a clear corrélation between the degree of neurofibrillary tangle pathology and cognitive state prior to autopsy. These observations hâve been reinforced by several investigators (Nelson, P.T. et al. (2012) J. Neuropathol.

Exp. Neurol. 71, 362-381), and in recent longitudinal biomarker studies, which indicate that cerebrospinal fluid (CSF) levels of tau and phospho-tau increase throughout early and late stages of the disease (Jack, C.R., Jr. et al. (2013) Lancet Neurol. 12, 207-216).

As indicated above, the microtubule-associated protein, tau, and its hyper-phosphorylated version, form the main constituent of intracellular neurofibrillary tangles, which are one of the main hallmarks of AD. Furthermore, spécifie genetic variants of tau are associated with familial forms of fronto-temporal dementia (FTD). Appearance of tau pathology in AD occurs in a distinct spatial pattern, starting in the entorhinal cortex, followed by hippocampal and cortical areas (Braak, H. and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3-12). The spécifie stage of tau pathology also correlates well with cognitive abilities (Nelson, P.T. et al. (2012) J. Neuropathol. Exp. Neurol. 71, 362-381; Braak, E. et al. (1999) Eur. Arch. Psychiatry Clin. Neurosci. 249 Suppl 3, 14-22). Taken together, this evidence forms the basis of a tau-based hypothesis for AD. It entails that the intracellular accumulation of tau leads to microtubule degeneration and spinal collapse. As a resuit, communication between neurons malfunctions and cell death follows. Recently, it has also been shown that tau itself may form an endo-pathogenic species that can transmit neurodegeneration from one cell to the next (Clavaguera, F. et al. (2009) Nat. Cell Biol. 11, 909-913).

I. Tau As An Endo-Pathogen

Clavaguera and colleagues hâve demonstrated that tau itself may act as an endo-pathogen (Clavaguera, F. et al. (2009) Nat. Cell Biol. 11, 909-913). Low spin brain extracts were isolated from P301S tau transgenic mice (Allen, B. et al. (2002) J. Neurosci. 22, 9340-9351), diluted and injected into the hippocampus and cortical areas of young ALZ17 mice. The ALZ17 mouse is a tau transgenic mouse line which only develops late pathology (Probst, A. et ai. (2000) Acta Neuropathol. 99, 469-481). The injected ALZ17 mice quickly developed solid filamentous pathology, and administration of immuno-depleted brain extracts from P301S mice or extracts from wild type mice did not induce tau pathology. Fractionation of the brain extracts in soluble (S1 ) and sarcosyl-insoluble tau (P3) (Sahara, N. et al. (2013) J. Alzheimer’s. Dis. 33, 249-263) and injection of these into ALZ17 mice demonstrated that the P3 fraction is most competent in inducing pathology. It contains most of the intracellular hyper-phosphorylated filamentous tau. The majority of pathology could also be induced when injecting P301S extracts into the brains of wild type mice, but no NFTs were formed. In subséquent studies, Clavaguera et al. hâve shown that human tau extracted from postmortem brain tissue of other tauopathies (Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP), and Corticobasal Degeneration (CBD)) may also induce tau pathology in the ALZ17 model (Clavaguera, F. et al. (2013) Proc. Natl. Acad. Sci. U.S.A.

110, 9535-9540). Since the présentation of these data, several other tau seeding and spreading models hâve been reported (Ahmed, Z. et al. (2014) Acta Neuropathol. 127, 667683; Walker, L.C. et al. (2013) JAMA Neurol. 70, 304-310). The main conclusion from these studies indicates a mechanism by which pathogenic tau in intracellular inclusions is secreted from the cell into the periplasmic space. The pathological tau material is then transported along the vesicular sheath in both anterograde and rétrogradé direction and subsequently taken up by neighboring cells by means of bulk endocytosis. This mechanism explains why the spread of pathology observed in human disease follows a distinct anatomical pattern. Intriguingly, peripheral administration of pathological tau may accelerate the formation of tau pathology in ALZ17 mice (Clavaguera, F. et al. (2014) Acta Neuropathol. 127, 299-301). This spreading mechanism may explain disease propagation in other proteinopathies (Goedert, M. et al. (2010) Trends Neurosci. 33, 317-325; Sigurdsson, E.M. et al. (2002) Trends Mol. Med. 8, 411-413).

II. Tau Species

The discovery that the tau protein may act as an endo-pathogen has spawned a search for “The Pathogenic Species that couid be targeted in potential interventive thérapies.

The microtubule-associated protein tau gene (MAPT) is located on chromosome 17 of the human genome and expresses six isoforms of the tau protein in adult human brain. These isoforms arise from the alternative splicing of exons 2, 3 and 10 of the 16 exons within the MAPT gene. Exons 2 and 3 express a 29 amino acid repeat and exon 10 expresses an additional microtubule binding domain. As a resuit, tau isoforms will contain 0, 1 or 2 Nterminal repeats and 3 or 4 C-terminal microtubule binding domains (3R or 4R tau). Commonly six isoforms of tau are expressed. The longest (2N4R) and shortest (0N3R) isoforms consist of 441 and 352 amino acids, respectively (Kolarova, M. et al. (2012) Int. J. Alzheimers. Dis. 2012, 731526). The N-terminal projection domain of tau (2N4R) consists of a 44 amino acid glycine-rich tail and residues 45-102 encompass two highly acidic régions (N1, N2-domains). Two proltne-rich régions are found at residues 151-243 (P1, P2 domains). The remainder of the protein is constituted by four microtubule binding domains (R1-R4), followed by a short C-terminal région.

Tau is very soluble and highly phosphorylation-labile protein. Approximately 20 percent or 85 of the amïno acid residues in the longest isoform of tau are potential (Ser, Thr or Tyr) phosphorylation sites. Roughly half of these hâve been observed experimentaliy (Hanger, D.P. et al. (2009) Trends Mol. Med. 15, 112-119; Hasegawa, M. et al. (1992) J. Biol. Chem. 267, 17047-17054), and are clustered around the terminal residues of the microtubule bînding domains. Tau is dynamically phosphorylated and de-phosphorylated during the cell cycle. It must dissociate from microtubules to allow for meiosis to occur. Its main rôle in post mitotic cells (the differentiated neuron) is to act as a microtubule stabilizer, allowing for optimal axonal transport. It can only associate with microtubules in its mostly dephosphorylated form, thus phosphorylation acts as a direct microtubule association/dissociation switch within the neuron. Under normal conditions, cytosolic tau contains on average two phosphorylated sites. In paired helical filamentous material, at least 7-8 sites are phosphorylated (Hanger, D.P. et al. (2009) Trends Mol. Med. 15, 112-119; Hasegawa, M. et al. (1992) J. Biol. Chem. 267, 17047-17054). Hyperphosphorylated, paired helical filamentous tau is a key hallmark of Alzheimer’s disease (Kosik et. al. (1986) PNAS, 86, 4044-4048), a distinct mobility shift of hyperphosphorylated tau is observed in immunecytochemical analysis of human AD brain material.

It has been difficult to study the tau protein with traditional structural techniques like x-ray crystallography or NMR spectroscopy, reflecting its meta-stable nature. Such studies hâve mainly been conducted on domain fragments of the un-phosphorylated protein. The only structural study to date on full-îength tau (2N4R), using NMR spectroscopy, reveals that the protein contains only sparse stretches of stable secondary structure (Mukrasch, M.D. et al. (2009) PLoS. Biol. 7, e34). This analysis indicates that the secondary structure of the peptide backbone has a large propensity for adapting a β-sheet structure. The backbone's first 200 residues are considerably more ordered than the C-terminus encompassing the microtubule binding domains. The presence of many spécifie long-range interactions within the protein in solution indicates that it exists in a largely disordered molten globular state (Ohgushi, M. and Wada, A. (1983) FEBS Lett. 164, 21-24).

Protease products of tau generated in particular by caspase and calpain (Asp13, Glu391 and Asp421) hâve been identified in tangle material (Gamblin, T.C. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 10032-10037). In particular, the truncation at Asp421 has been studied in detail using the tau C3 antibody, which binds to the free Asp421 terminus. This truncation has been postulated as an early event in AD pathogenesis associated with induction of apoptosis (deCalignon A. et al. (2010) Nature 464, 1201-1204). The N-terminal cleavage at Asp13 and the C-terminal cleavage at Glu391 are considered late events in the pathogenesis (deCalignon A. et al. (2010) Nature 464, 1201-1204; Delobel, P. et al. (2008) Am. J. Pathol. 172, 123-131). Recently, an additional N-terminal fragment (residues 1-224) was identified in CSF from AD and PSP patients, and has been hypothesized to be an early marker of disease and particularly pathogenic (US14/092539*; Bright, J. et al. (2014) Neurobiol. Ageing, 1-17). A similar calpain cleaved fragment was reported by other groups (Ferreira, A. and Bigio, E.H. (2011) Mol. Med. 17, 676-685; Reinecke, J.B. et al. (2011) PLoS. One. 6, e23865).

Apart from hyper-phosphorylation and tau fragmentation, post-translational acetylation(Cohen, T.J. et al. (2011) Nat. Commun. 2, 252; Min, S.W. et al. (2010) Neuron 67, 953-966) and O-GIcNAcylation (Zhu, Y. et al. (2014) J. Biol. Chem.) hâve been proposed to be pathology defining processes in the formation of tangle pathology associated with AD.

III. Tau Immunothérapies

Immunothérapies are traditionally separated into passive and active vaccine approaches. In an active vaccine approach, a pathogenic agent is injected into the patient and the innate immune system elicits an immune response. This triggers the maturation of B-cells generating high affinity antibodies against the administered antigen. In a passive vaccine approach, the triggering of the innate immune system is circumvented by infusing a spécifie antibody against the antigen. The inhérent clearance system then removes antibody bound ligand.

AC Immune is pursuing a mouse monoclonal antibody against phospho-serine 409 of tau. Antibodies were profiled against human AD and control brain tissue and were selected based on their ability to recognize tangle pathology. The humanized version of two antibodies, hACI-36-2B6-Ab1 and hACI-36-3A8-Ab1, both bind to a tau epitope within amino acids 401-418 (WO 2013/151762).

The group of Roger Nitsch hâve isolated tau auto-antibodies from elderly healthy individuals with no sign of degenerative tauopathy. A number of antibodies hâve been isolated using full length recombinant human tau (2N4R) to find tau spécifie antibodies. These were then screened for their ability to discriminate tau isolâtes from diseases and healthy individuals. Three lead antibodies, 4E4, 4A3 and 24B2, hâve been described in the patent literature (WO2012049570; US2012087861 ). Their epitope mapping indicates that ail recognize amino acids within and C-terminal to the microtubule binding région, from position V339 to K369. These antibodies do not exhibit any phospho-specificity.¹

C2N Diagnostics focus mainly on developing diagnostic tools for early détection of neurodegenerative disease. Antibodies were generated against full length human and mouse tau protein. Eight and five antibodies were identified, recognizing human and mouse tau, respectively (Yanamandra, K. et al. (2013) Neuron 80, 402-414). Three antibodies with different binding kinetics were selected for in vivo évaluation. Namely, HJ9.3, HJ9.4 and ¹ *US2014294831

HJ8.5, recognizing tau residues 306-320, 7-13 and 25-30, respectively, with the last one being spécifie for human tau. The antibodies were also selected based on their ability to prevent transfer of pathology in an ingenious mechanistic reporter assay of trans-cellular propagation of tau (Sanders, D.W. et al. (2014) Neuron 82, 1271-1288; Kfoury, N. et al. (2012) J. Biol. Chem. 287, 19440-19451). Their évaluation in chronic i.c.v. injection studies in P301S transgenic mice demonstrated their ability to reduce levels of hyperphosphorylated tau protein as determined by AT8 staining in immuno-histochemical analysis of the treated mice.

The antibodies of Peter Davies were developed originally as diagnostic tools that could differentiate between pathological and normal tau in AD and control brain material (Greenberg, S.G. and Davies, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5827-5831). Evaluation of the therapeutic utility of the PHF1 and MC1 antibodies was demonstrated in P301S and JPNL3 (P301L) (Boutajangout, A. et al. (2011) J. Neurochem. 118, 658-667; Chai, X. et al. (2011) J. Biol. Chem. 286, 34457-34467; D'Abramo, C. et al. (2013) PLoS. One. 8, e62402 mice). PHF1 recognizes a linear phospho-tau epitope (pS396, pS404) whereas MC1 is a conformation-dependent antibody that recognizes a structural tau epitope requiring two distinct parts of the linear sequence, an epitope within residues 46-202 and a C-termînal epitope between residues 312-342 (Jicha, G.A. et ai. (1997) J. Neurosci. Res. 48, 128-132). Injection of these two antibodies in chronic 12-13 week immunization studies resulted in substantial réduction of spinal cord and brainstem pathology among other brain régions, which translated to an atténuation of the motor déficit observed in these mice. (D’Abramo, C. et al. (2013) PLoS. One. 8, e62402).

ÎPerian / Bristol Meyers Squibb has developed tau antibodies against a postulated pathological tau species, composed of an N-terminal fragment of tau (etau: residues 1-224), which promoted hyperactivity in induced pluripotent stem cell based neuronal cultures. A portfolio of antibodies has been developed, but characterization has focused on antibodies IPN001 and IPN002 that recognize an N-terminal epitope within residues 9-18. Accordîngly, these antibodies detect elevated tau levels in CSF from staged AD and PSP patients that may be an early sign of disease. In vivo injections of the antibodies in JPNL3 (P301L) mice led to partial reversai of progressive motor déficits (US14/092539).

Einar Sigurdsson were the first program to demonstrate the efficacy of tau based immunotherapy. An active vaccine consisting of tau peptide 379-408[pS396, pS404] together with Adju-Phos adjuvant was used to immunize JPNL3 (P301L) mice. In this study a prominent réduction of tau pathology was observed in the vaccine treated mice when compared to control animais. An atténuation of tauopathy-related motor phenotype was detected as well. Its efficacy was confirmed in a different mouse model (htau/PS1) not driven by mutant tau (Boutajangout, A. et al. (2011) AAIC 2011 (7, issue 4, Supplément edn) p. s480-s431; Congdon, E.E. et al. (2013) J. Biol. Chem. 288, 35452-35465; Gu, J. et al. (2013) J. Biol. Chem. 288, 33081-33095).

Prothena has evaluated three tau antibodies in the K369I (K3) transgenic tau mouse and in a P301L mouse model. Antibodies with varying properties were selected for in-vivo évaluation. Two pS404 antibodies with different isotype (IgG 1/k and lgG2a/k) or a total (pan) anti-tau antibody (lgG1/k) were injected in a chronic paradîgm. K369I mice were treated with weekly injections for 21 weeks starting at 3 weeks of âge, and P301L mice were treated for 7 months with weekly injections starting at 4 months of âge. A réduction in tau positive neurofîbrillary inclusions was observed in the K3 mice with the lgG2a/k pS404 antibody. Both of the pS404 antibodies were able to reduce the levels of pS422 positive tau, whereas no réduction was observed in the pan-anti-tau antibody treated mice. These studies suggest that: 1) tau clearance may be isotype-dependent, and; 2) It may be important to target a tau species that is relevant to disease, as the total-anti-tau antibody was unable to reduce hyper-phosphorylated tau (PCT/US2014/025044).

The inventors of the présent invention hâve surprisingly found antibodies spécifie for the phosphorylated tau serine residue 396 (pS396); this is in contrast to the prier art antibodies which recognize primarily the tau proteins phosphorylated at both 396 and 404 residues, phosphorylated at the 404 residue only or at other residues on tau.

The inventors hâve developed antibodies which furthermore hâve a remarkable specificity and selectivity towards human pathological tau. There is a need for antibodies which are highly sélective and spécifie for pathogenic tau protein. The antibodies of the présent invention show a much higher degree of specificity and selectivity towards human pathological tau over non-pathologica! tau compared to the antibodies of WO2013/050567 (see Figure 1 of WO2013/050567). The antibodies of WO2012/045882 reported to hâve a spécifie binding, were elîcited from 6 to 9 residue amino acid sequences of Tau amino acids 393-401, 396-401, 394-400 and 393-400. This contrasts from the antibodies of the présent invention which were elîcited against pathogenic hyperphosphorylated tau comprising a longer amino acid sequence as described herein.

As shown in the Examples, comparison to five published tau antibodies: hACI-2B6 (described WO2013151762); IPN002 (described in WO 2014028777); HJ8.5 (described in WO 2014008404); the anti-Tau pS422 monoclonal antibody 2.10.3 (described in US8609097); PHF13 (a commerciaily available antibody (e.g. SigmaAldrich) recommended for détection of Tau phosphorylated at Ser 396 of mouse, rat and human origin and discussed by Sankaranarayanan (PLOSONE, DOI:10.1371/journal.pone.0125614 May 1, 2015 and Otvos (Biochemistry 1997, 36, 8114-8124); and the 4E4 antibody, (described as binding to V339, E342, D387, E391 and K395 in US8940272), showed that the antibodies, and epitope-binding fragments thereof, of the present invention exhibit a higher degree of specîficity and selectivity towards human pathological tau than any of the comparator antibodies.

Further, the antibodies, and epitope-binding fragments thereof, of the present invention show many advantageous features such as the ability to discriminate between pathological and non-pathological human tau protein, and in particular to bind tau associated with Alzheimer's (AD) pathology. In electrophysiological studies, the antibodies, and epitope-binding fragments thereof, of the invention were additionally able to reverse reduced paired puise facilitation and spontaneous miniature excitatory synaptic current (mEPSC).

SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies, and epitope-binding fragments thereof, capable of specifically binding to the phosphorylated residue serine 396 of human (2N4R îsoform) tau (SEQ ID NO:33) and to such antibodies that hâve been produced using a new method that allows such spécifie isolation and recovery. The antibodies are further characterized by their ability to discriminate between phosphorylated residues 396 and 404 such that they substantially do not bind the phosphorylated 404 residue.

Without being bound by a particular theory, evidence from the inventors demonstrates that the discrimination and selectivity of the antibodies of the present invention for human tau protein phosphorylated at residue 396 in the presence of tau protein phosphorylated at residue 404 but not at 396 is significant from a pathological and therapeutic perspective. The antibodies of the present invention are sélective for pathological tau in the presence of nonpathological - yet phosphorylated - tau. The antibodies of the present invention are able to deplete tau tangles of pathological tau in the presence of normal tau. Without being bound to a particular theory, it is believed that depleting tangles of tau comprising tau protein that has been phosphorylated at tau position 396 prevents seeding of pathological tau into tau tangles. Accordingly, one aspect of the invention relates to an antibody that is capable of selectively binding to 396-phosphorylated tau even when such molécules are in the presence of tau protein that has been phosphorylated at tau position 404. A related aspect of the invention relates to an antibody that is capable of selectively binding to 396phosphorylated tau even when such molécules are in the presence of non-pathogenic tau.

Further defined, the invention relates to an antibody sélective for pathological tau said pathological tau being hyperphosphorylated tau appearing as 64 kDa band (by Western Blot analysis) in transgenic mice overexpressing the human 2N4R isoform of tau.

One aspect of the invention is directed to an anti-tau antibody meeting the following test criteria: i) the antibody does not bind to non-phosphorylated tau; H) the antibody does not bind to tau phosphorylated at 404 and not phosphorylated at 396; iii) the antibody does bind to tau phosphorylated at 396; and iv) the antibody does bind to tau phosphorylated at both 396 and 404. The inventors hâve found that the binding under test criteria iii) and iv) are in the same order of magnitude and postulate that phosphorylation at position 404 does not interfère nor enhance the binding process. The inventors hâve further found that, contrarily to test criteria ii), binding to a tau protein which is not phosphorylated at 396 but is phosphorylated at 404, does not deplete tangles or clear pathological tau in test models.

One aspect of the invention is directed to an anti-tau antibody that, when used with immunedepleted rTg4510 extracts from transgenic mice, specifically reduces the hyperphosphorylated tau 64 and 70kDa bands by at least 90%, while reducing the 55 kDa tau band by not more than 10%. A further aspect of the invention is directed to an anti-tau antibody that specifically reduces the hyperphosphorylated tau 64 and 70kDa bands by at least 90%, while reducing the 55 kDa tau band by not more than 10%; or the capability, when used as described herein with extracts from human AD post-mortem brains, to specifically reduce the phosphorylated S396 hyperphosphorylated tau bands by at least 90%, while not reducing the non-hyperphosphorylated tau bands by more than 10%.

Another aspect of the invention is directed to a method of treating a patient with a taupathy, such as Alzheimer’s Disease, comprising depleting a tangle or attenuating the progression of said tangle, said tangle comprising hyperphosphorylated Tau, said method comprising contacting hyperphosphorylated Tau with an antibody of the invention such that the tangle is depleted, reduced in its content of hyperphosphorylated tau or progression of tangle formation is attenuated.

Alternative^ defined, the invention relates to a method of treating a patient with a taupathy, such as Alzheimer’s Disease, said method comprising contacting tangles with an antibody sélective for tau having residue 396 phosphorylated such that the tangle is depleted of hyperphosphorylated Tau.

More specifically the invention relates to any one of four monoclonal antibodies selected from the group comprising:

Antibody C5.2 wherein Antibody C5.2 comprises:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NQ:20;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22;

Antibody C8.3 wherein Antibody C8.3 comprises:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:25;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:26;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:27;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:28;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID ND:30;

Antibody C10-2 wherein Antibody C10-2 comprises:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14;

and

Antibody D1.2 wherein Antibody D1.2 comprises:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:1;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:2;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:3;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:4;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:6.

The amino acid sequences of the full light and heavy chains of an exemplary antibody C5.2, including the constant domains therein, are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively (as used in the Examples).

The amino acid sequences of the full light and heavy chains of an exemplary antibody C8.3, including the constant domains therein, are shown in SEQ ID NO:31 and SEQ ID NO:32, respectively (as used in the Examples).

The amino acid sequences of the full light and heavy chains of an interestîng antibody C102, including the constant domains therein, are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively (as used in the Examples). The amino acid sequence of the heavy Chain of humanized C10-2 antibody is shown in SEQ ID NO:35. The amino acid sequence of the light chain of humanized C10-2 antibody is shown in SEQ ID NO:36. One aspect of the invention relates to an antibody of the invention comprising SEQ ID NO:35 or SEQ ID NO:36, or both.

The amino acid sequences of the full light and heavy chains of an exemplary antibody D1.2, including the constant domains therein, are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively (as used in the Examples).

In an alternative embodiment, the antibody D1.2 comprises a light Chain having the amino acid sequence of SEQ ID NO:34, wherein the amino acid at position 3 is valine (whereas in the exemplary light Chain of SEQ ID NO:7, this amino acid is a méthionine). This light chain may be paired with a heavy chain as described above, i.e. having CDRs of SEQ ID NOs:4, 5 and 6. For example, the antibody may comprise a light chain having the amino acid sequence of SEQ ID NO:34 together with a heavy chain having the amino acid sequence of SEQ ID NO:8 (antibody “D1.2*”).

One aspect of the invention is directed to an antibody comprising:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10; and/or (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11.

A further aspect of the invention is directed to an antibody comprising, or additionally comprising:

(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13; and/or (c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

The antibodies, and epitope-binding fragments thereof, of the invention can be used in treating tauopathies such as Alzheimer's disease (AD), Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), TBI (traumatic brain injury, mild, acute or chronic), and chronic traumatic encephalopathy (CTE).

The antibodies, and epitope-binding fragments thereof, of the invention are furthermore intended for use in treating Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Binding to pathological material dot-blot

Figure 1 (Panels A-B) présents the results of a dot blot analysis displaying 500 ng S1 and P3 fractions (génération of the S1 and P3 fractions are disclosed in Example 3) derived from brains of AD patients (AD) and aged healthy individuals (con) or from 32 weeks old rTg4510 and non-transgenic (wt) littermates probed with 1 pg/ml D1.2 or C10-2 to assess détection of pathological tau (Example 3). The dot plot shows that D1.2 (Panel A) or C10-2 (Panel B) specifically reacts on disease material from AD patients or human (P301L) tau as expressed in transgenic mice (Tg4510).

Figure 2: Western blot analysis of D1.2 and C10-2 antibodies

Figure 2 (Panels A-B) présents the results of a Western blot analysis displaying 2 pg S1 and P3 fractions derived from brains of 32 week old rTg4510 and non-transgenic (wt) littermates or 20 pg S1 and P3 fractions derived from brains from AD patients (AD) and aged healthy individuals (con) probed with 1 pg/ml D1.2 (Panel A) or C10-2 (Panel B). S1 and P3 fractions were loaded at a ratio of 1:50 (based on tissue weight) which was derived from 0.01 mg tissue. In Western blot, normal P301L mutant human 4R0N tau is displayed at 55 kDa, while hyper-phosphorylated P301L mutant human 4R0N tau species is displayed at 64 and 70 kDa. In P3 fractions from AD hyper-phosphorylated tau is displayed at 54, 64, 69 and 74 kDa (Example 3). The figure illustrâtes that the antibodies specifically bind to hyperphosphorylated, mobility shifted tau protein.

Figure 3: Binding to pathological P3 material in MSD

Figure 3 (Panels A-D) présents the results of Meso Scale Discovery (MSD) ELISA binding of D1.2 (Panel A), C5-2 (Panel B), C10-2 (Panel C) and C8-3 (Panel D) to tau isolated from human AD and non-diseased control brains (Example 4). Similar to demonstrated in Figure

1, immobilisation of tau isolated from disease (AD) and healthy control brains on ELISA plates can be used to demonstrate that the antibodies in this invention specifically bind pathological tau species. Increasing concentrations of antibody lead to saturation binding. The quantity of bound antibody is detected with secondary anti-mouse antibody.

Figure 4: Peptide affinity and pS396 selectivity (peptide binding)

Figure 4 (Panels A-D) présents the résulte of an analysis of the spécifie binding of C10-2 (Panel A) and D1.2 (Panel B) to tau (386-409) peptides with ail combinations of phosphorylation at positions S396 and S404 (Example 5). Spécifie affinity towards human pathological material is difficult to assess, for this reason we use spécifie peptide binding to détermine the exact epitope affinity, using spécifie phosphorylated and un-phosphorylated peptides. Spécifie, dose response curves are shown for binding of antibodies C10-2 (Panel C) and D1.2 (Panel D) to the peptide: TDHGAEIVYK^(p)SPWSGDT^{p)SPRHL (SEQ ID NO:37) (pS396/pS404), phosphorylated at residues Ser396 and Ser404. Compétition binding was conducted with un-phosphorylated peptide (NP) and mono-phosphorylated peptides (pS396 and pS404). Additionally, a control peptide corresponding to phosphorylated serine 262 was included. The compétition binding demonstrates that ail binding is obtained through the phosphorylated 396 serine residue. Additionally, the data demonstrates that phosphorylation at residue 404 does not interfère with the binding of antibodies at phosphor-serine 396.

Figure 5: Histological characterisation of pathology spécifie antibodies

Figure 5, Panel A shows that C10-2 (left column) and D1.2 (right column) antibodies bind to p-tau species in Tg4510 (top row) cell bodies and neuropii. No immunoreactivity is detected in non-Tg brain sections (bottom row). Figure 5, Panel B shows that C10-2 (left column) and D1.2 (right column) antibodies bind to p-tau species in cell bodies and neuropii threads in AD donor (AD) (top row). Control donor brains are devoid of immunoreactivity (bottom row) (Example 6).

Figure 6: Binding to pathological and non-pathological P3 for C10-2 and reference antibodies

Figure 6 (Panels A-E) présents results demonstrating the superiority of the antibody C10-2 (Panel C) of the present invention in recognizing pathological material compared to prior art antibodies 2-10-3 (Panel A), HACI-2B6 (Panel B), IPN 002 (Panel D), and HJ8.5 (Panel E). The Figure shows the spécifie binding of C10-2 to tau from healthy (as a control) and disease (AD) human brains, togetherwith the binding to tau from 10 month old Tg4510 mice expressing P301L mutant human tau. Increasing concentrations of antibody are added to P3 tau material immobilized on ELISA plates. Ratio of selectivity towards pathological tau is determined at full saturation with active species. The foîd selectivity for each of the prior art antibodies is shown in the figure (Example 7).

Figure 7: Prévention of seeding in HEK293 cells and in-vitro

Figure 7 (Panels A-C) présents a quantification of tau aggregation by Cisbio assay. Seeded pcDNA HEK293 cells showed no signal, confirming the absence of détection for input seeding material. Wt (wild type) seeding material (WW) showed no seeding, but in contrast rTg4510 homogenates (CC) seeded efficiently, compared to unseeded. This seeding effect was not affected by treatment with HEL, but was partially reversed by treatment with tau antibodies (C10-2>D1.2>hACI36-2B6-Ab1). Graphs (Panels A-C) represent three independent sets of samples and are plotted as relative tau aggregation (fold signal over background normalized to total protein) (Example 8).

Figure 8: Reversai of electrophysiological déficit

Figure 8 shows antibody reversai of paired puise facilitation (Panels B and D) and basal synaptic transmission (Panels A and C) déficits in CA1 evoked field potentials (C10-2, Panel A; D1.2, Panel B), illustrating the evoked filed potentials in CA1 subchronic treatment with C10-2 in Tg4510 mice with and tTa mice as a control. Animais were treated twiceweekly with a 15 mg/kg dose of antibody for two weeks (see Example 9). In Panel A (for C10-2) and Panel C (for D1.2), the Field potentials (fEPSP) slope is plotted against stimulation intensity. Panels A and C illustrate that in in vivo electrophysiological assessment of synaptic transmission and plasticity in the CA1 area of the hippocampus in

4.5 to 5.5 months old rTg4510 (iower 2 curves) and tTA (upper 2 curves) control mice i) basal synaptic transmission is significantly impaired in rTg4510 compared to tTA mice, and ii) paired-pulse facilitation is significantly reduced rTg4510 compared to tTA mice.

Paired-pulse facilitation, a short-term synaptic plasticity believed to rely on presynaptic mechanisms, was further measured in rTg4510 and tTA mice (Panel B for C10-2 and Panel D for D1.2). Briefly, a pair of stimuli with an inter-stimulus interval (ISI) varying from 25 to 1000 ms was applied to the Schaffer collateral, and the slope of the second fEPSP was compared to the slope of the first fEPSP. Facilitation was observed at ail ISIs, with a maximum facilitation at ISIs of 50 and 75 ms. Interestingly, a significantly lower PPF was observed in rTg4510 mice (second 2 bars) when compared tTA mice (first 2 bars).

Figure 9: OverView of screening as outlined in Figure 1-8

Antibodies were raised against the bi-phosphorylated peptide: TDHGAEIVYK^ip}SPWSGDT^<p>SPRHL (SEQ ID NO:37) covering residues 386-410 of 2N4R tau. Hybridomas are screened using dot-blot and MSD ELISA with immobilized human pathological and non-pathological tau (Example 4) to isolate clones that were highly spécifie towards the either of the phospho-epitopes S396 and/or S404 and at the same time specifically recognize hyper-phosphorylated tau from human Alzheimer’s disease brains. The ability to discriminate between pathological and non-pathological human tau protein in dot-blot and Western blot is used for sélection of hybridomas. 16 clones were selected of which four clones (D1.2, C10-2, C5.2 and C8.3) exhibit extraordinary capabilities for binding to human pathological material. Use of the spécifie immunization and screening protocol produces highly phospho-serine-396 (pS396) spécifie antibodies.

Figure 10: Residue pSer396 is bound at the center of the antigen binding site of mAb C5.2

The crystal structure of mAb C5.2 in a complex with phospho-peptide 386-410 at 1.9Â resolution. In this structure the électron density of residues 392-398 are resolved. Residue ^{PÎSer396 is bound at the center of the antigen binding site of mAb C5.2 In this structural study of anti-Tau mAbs, the epitope is bound across the heavy chain (bottom right) and light (bottom left) chains.

Figure 11: Antibody C5.2 interaction with phosphoserine tau (292-298) peptide

Figure 11 represents the interaction between antibody C5.2 with phosphoserine tau (292298) peptide. The structure of lle(392)-VAL(393)-Tyr(394)-Lys(395)-P-Ser(396)-Pro(397)Val(398) is shown. The main interaction involves the hydrophobie pocket formed by L3:H3, L3:F8*, H1:H13, H2:Y1, H2:Y3 and Y(394) of tau peptide. There is an extensive hydrogen bonding network formed between solvated ^<PlS(396) and L3;T4, H1:R10, H1:T11, H3:R1, H3:T3. In the employed nomenclature, the first letter (e.g., “L” of L3:H3) dénotés whether the involved CDR residue is a light chain CDR or a heavy chain CDR, the first number dénotés which CDR of such chain is involved (e.g., “L3 dénotés CDR3 of the light chain), the remaining terms (e.g., Ή3” of L3:H3) dénoté the name and position of the involved amino acid (e.g., Ή3 dénotés a histidine at the third residue position of the CDR); thus “L3:H3” dénotés the histidine residue at the third position of the of light chain CDR3. There are strong hydrogen bonding and charge/polar interactions between the Y(394) sidechain and the backbone with phosphonate of ^{P,S396 forms turn in peptide backbone. (*) L3:F8 is the C-terminal flanking framework residue of CDR L3.

The CDR sequences of C5.2 are:

CDRL1: QASQDTSINLN

CDR L2: GASNLED

CDR L3: LQHTYLP

CDRH1: KASGYTFTDRTIH

CDR H2: YIYPGDDSTKYNDNFKG (SEQ ID NO:17) (SEQID NO:18) (SEQID NO:19) (SEQ ID NO:2Û) (SEQ ID NO:21) (SEQID NO:22)

CDR H3: RGTMDY

Figure 12: Déplétion of Tau for seeding assay (HEK293)

Figure 12 (Panels A-B) shows immuno-depletion of rTg4510 brain homogenates using murine C10-2 (mC10-2) and humanized C10-2 (hC10-2). Western blots of depleted homogenates were detected with E1 (total tau; Panel A; Lower)) and C10-2 (pS396 tau; Panel A; Upper) and both mC10-2 and hC10-2 efficiently depleted hyperphosphorylated tau (upper bands on E1 blot and ail bands on C10-2 blot). Depleted homogenates were also analyzed for the déplétion of aggregated tau using the Cisbio assay. Panel B shows the change in aggregated Tau in samples. Déplétion studies with mC10-2 and hC10-2 removed tau aggregates by 99 and 99,5% respectively (Panel B).

Figure 13: Seeding assay (HEK293) with depleted material

Panels A-C shows depleted homogenates used to seed P301L-hTau in HEK293 cells. Homogenates from control animais (WW) did not seed, whereas rTg4510 homogenates (CC) seeded efficiently, as measured by the Cisbio aggregation assay on total cell lysâtes or by fractionation of HEK293 cells in 1% triton-X (quantification of insoluble hyperphosphorylated D1.2 and tau (Panel A, Upper and Lower)). Déplétion with HEL and hHEL antibodies did not affect seeding, whereas déplétion with mC10-2 and hC10-2 prevented tau aggregation 88% and 96% (Panel C) and insoluble tau 97% and 100% (Panel

B) respectively.

Figure 14: Immuno déplétion rTg4510 material (used for in vivo seeding studies)

Panels A-C demonstrate Western blot (Panel A; Upper, Lower) analysis of immunodepleted rTg4510 brain extracts. C10-2 and D1.2 specifically reduce the human hyperphosphorylated 64 kDa band by 90% and has no effect on the 55 kDa Tau Tau5, a commercial total Tau antibody does in contrast reduce normal 55 kDa Tau by 74% and no effect on the human 64 kDa Tau (Panels B-C).

Figure 15: Immuno déplétion AD material (used for în vivo seeding studies)

Figure 15 (Panels A-C) depicts Western blot (Panel A) analysis of immuno-depleted

Alzheimer brain extracts. Immuno-depletion using C10-2 and D1.2 does not reduce the total

Tau levais by more than 10%, but specifically lower hyperphosphorylated Tau (90% réduction) (Panels B-C).

Figure 16 (Panel A): Hippocampal Tau pathology in rTg4510 mice seeded with immuno depleted rTg4510 material

Figure 16 (Panel A) illustrâtes the quantification of Tau pathology in rTg4510 brains seeded with rTg4510 or AD brain homogenates. Prior to seeding the hyperphosphorylated Tau, but not normal Tau, had been reduced in the homogenates by 90-95% by using C10-2 or D1.2. By removing hyperphosphorylated tau from the homogenates, the homogenates do no longer induce seeding of Tau pathology.

Figure 16 (Panel B) Hippocampal tangle pathology in rTg4510 mice seeded with immuno depleted AD material

Figure 16 (Panel B) illustrâtes the quantification of Tau pathology in rTg4510 brains seeded with rTg4510 (A) or AD (B) brain homogenates. Prior to seeding the hyperphosphorylated Tau, but not normal Tau, had been reduced in the homogenates by 90-95% by using C10-2 or D1.2. By removing hyperphosphorylated tau from the homogenates, the homogenates do no longer induce seeding of Tau pathology.

Figure 17: Hippocampal tangle pathology in seeded rTg4510 mice treated with D1.2

Figure 17 depicts the quantification of tangle bearing neurons in hippocampus of seeded rTg4510 mice. The pathology increases with time (Ig G, 1 month; IgG 2 months; IgG 3 months). However, treating the mice with D1.2, the pathology is significantly lowered 1, 2 and 3 months after seeding. (D1.2 1 month; D1.2 2 months; D1.2 3 months).

Figure 18 Déplétion of Tau for seeding assay (HEK293)

Panel A shows immuno-depletion of rTg4510 brain homogenates using murine C10-2 (mC10-2) and humanized C10-2 (hC10-2). Western blots of depleted homogenates were detected with E1 (total tau) and C10-2 (pS396 tau) and both mC10-2 and hC10-2 efficiently depleted hyperphosphorylated tau (upper bands on E1 blot and ail bands on C10-2 blot). Depleted homogenates were analyzed for the déplétion of aggregated tau using the Cisbio assay. Panel B shows déplétion with mC10-2 and hC10-2 removed tau aggregates 99 and 99.5% respectively.

Figure 19 Seeding assay (HEK293) with depleted material

Panel A shows Tau fractionation (western on insoluble fraction. Panel B shows Western blow quantification. Panel C shows aggregated Tau in cell lysâtes. Depleted homogenates were used to seed P301L-hTau in HEK293 cells. Homogenates from control animais (WW) did not seed, whereas rTg4510 homogenates (CC) seeded efficiently, as measured by the Cisbio aggregation assay on total cell lysâtes or by fractionation of HEK293 cells in 1% triton-X (quantification of insoluble hyperphosphorylated D1.2+ tau). Déplétion with HEL and hHEL antibodies did not affect seeding, whereas déplétion with mC10-2 and hC10-2 (Panel

C) prevented tau aggregation by 88% and 96% and insoluble tau by 97% and 100% respectively (Panel B).

Figure 20 Immunoselectivity of C10-2 and D1.2 for hyperphopsphorylated tau over normal tau.

Immuno déplétion rTg4510 material used for in vivo seeding studies: Panel A show Western blot analysis of immuno-depleted rTg4510 brain extracts. Panel B shows that C10-2 and D1.2 specifically reduce the hyperphosphorylated 64 kDa band, phosphorylated at serine 396 over the Tau 55 kDa band, which does not comprise a significant amount of p396. In contrast, Tau5, a commercial total Tau antibody, does reduce normal 55 kDa Tau and is inefficient in binding to the 64 kDa Tau.

Figure 21 Immunoselectivity of C10-2 and D1.2 for hyperphopsphorylated tau over normal tau.

Immuno déplétion AD material (used for in vivo seeding studies: Panel A shows Western blot analysis of immuno-depleted Alzheimer brain extracts. Immuno-depletion using mC10-2 and D1.2 does not reduce the total Tau levels by more than 10% (Panel B), but specifically lower hyperphosphorylated Tau (90% réduction) (Panel C).

Figure 22 Hippocampal Tau pathology in rTg4510 mice

Panel A shows hippocampal Tau pathology in rTg4510 mice seeded with immuno depleted rTg4510 material. Panel B shows hippocampal tangle pathology in rTg4510 mice seeded with immuno depleted AD material. Quantification of Tau pathology in rTg4510 brains seeded with rTg4510 (A) or AD (B) brain homogenates. Prior to seeding the hyperphosphorylated Tau, but not normal Tau, had been reduced in the homogenates by 9095% by using antibodies C10-2 or D1.2. By removing hyperphosphorylated tau from the homogenates, the homogenates no longer induce seeding of Tau pathology.

Figure 23 Hippocampal tangle pathology in seeded rTg4510 mice treated with D1.2

Quantification of tangle bearing neurons in hippocampus of seeded rTg4510 mice. The Figure shows that pathology increases with time and by treating the mice with D1.2, the pathology is significantly lower 2 and 3 months after seeding.

Figure 24 Western blot analysis of immuno-depleted human AD extracts

The Figure illustrâtes that humanized version of C10-2 (hC10-2), as well as mC10-2 differ from the 2.10.3 (P-S422) antibody, in that although total tau remaining is not dramatically different (left hand panel) from 2.10.3, C10-2 (hC10-2), as well as mC10-2 remove more of the hyperphosphorylated Tau protein present in Alzheimer brain extracts by immuno déplétion methods. This is confirmed in Figure 25 by quantification.

Figure 25 Quantification of Aggregated Tau after immuno déplétion

The hC10-2 and mC10-2 antibodies differ from the 2.10.3 antibody in its ability to removes more of the aggregated Tau protein present in Alzheimer brain extracts by immuno déplétion methods.

Figure 26 Total tau remaining after immuno déplétion

Quantification of western blot signal after immuno depleting Alzheimer extracts using different amounts ofthe humanized C10-2 (A) and 2.10.3 antibody (♦). In Figure 26, quantification of total tau signal using Tau5 (ail tau isoforms were included in the analysis) is shown. Both antibodies remove a small fraction of tau from the Alzheimer brain préparation.

2.10.3, designed to hâve specificity for P-S422 tau removes up to 24% of the total tau amount, while C10-2 removes up to 15% of the total tau (see Figure 26).

Figure 27 Total tau remaining after immunodepletion of hyperphosphorylated tau

Figure 27 illustrâtes the quantification of the hyperphosphorylated tau, being phosphorylated at serine 422 (ail bands and the high molecular weight smear was included in the analysis). 2.10.3 (A) and C10-2 (♦) both remove more than 90% of the tau phosphorylated at Serine 422. However, the amount of antibody needed to remove 50% of the tau differ: for antibody

2.10.3, 0.42 pg antibody is needed whereas for C10-2, 0.27 pg is needed for the same effect.

Figure 28 Total tau remaining after immunodepletion of hyperphosphorylated tau

Quantification of the hyperphosphorylated tau, being phosphorylated at serine 396 (ail bands and the high molecular weight smear was included in the analysis). C10-2 (♦) efficiently 5 removes Tau being phosphorylated at serine 396 (Max effect: 88% and half of the effect is reached by using 0.30 pg antibody). 2.10.3 (A) removes a smaller fraction of tau being phosphorylated at the serine 396 (Max effect: 60% and half of that effect is reached when using 0.63 pg antibody). This indicates that ail Tau being phosphorylated at serine 422, also is phosphorylated at serine 396, but that there is a portion of hyperphosphorylated tau being 10 phosphorylated at serine 396 where the phosphorylated serine at positron 422 is not present.

Figure 29 Total tau remaining after immunodepletion of hyperphosphorylated tau

Quantification of the hyperphosphorylated tau, being phosphorylated at serine 199/202 (ail bands and the high molecular weight smear was included in the analysis). A large portion of the tau being removed by C10-2 (♦), is also phosphorylated at Serine 199/202, since 69% 15 of the tau having that phosphporylation is affected by the immunodepletion (50% of the effect when using 0,34 pg antibody). The 2.10.3 (A) immunodepletion does not give a sigmoidal dose response on the P-S199/202 tau although a drop in signal is seen with increasing amount of antibody (max 52% réduction when using the max amount of antibody (5 pg). This data indicates that the C10-2 antibody targeting the phosphorylated serine 396 20 binds a larger pool of the hyperphosporylated tau then the 2.10.3 antibody targeting the phosphorylated serine at the 422 position.

Figure 30 mD1.2 and mC10-2 inhibition of Tau antigen capture in mC10-2 coated plates

In fluid phase ELISA, where a mixture of rTg4510 P3 préparation and variable amounts of 25 C10-2 or D1.2 antibodies is added onto C10-2 coated plates. The more antibodies binding to

P3 tau in the solution, less available tau epitopes are able to bind to the plates. The amount of tau binding to the plates is determined by a sulfo-tagged human tau antibody. C10-2 (A) and D1.2 (□) hâve a different binding to the tau in solution, wherein C10-2 can compete out ail binding to the. plates (IC50 20 nM). D1.2, on the other hand, shows a very low level of 30 binding to the tau in the solution.

Figure 31 PHF13 and mC10-2 inhibition of Tau antigen capture in mC10-2 coated plates

In fluid phase ELISA, a mixture of AD P3 préparation and variable amounts of C10-2 and PHF13 antibodies were added onto C10-2 coated plates. The more antibodies binding to P3 tau in the solution, the less available tau epitopes are able to bind to the plates. The amount of tau binding to the plates is determined by a sulfo-tagged human tau antibody. C10-2 and PHF13 hâve a different binding to the tau in solution, wherein C10-2 can compete out ail binding to the plates (IC50 = 3 nM), whereas PHF13 does not.

Figure 32 Both mC10-2 and PFH-13 bind dose dependently to Ptau 386-408 (pS396/pS404)

Figure 32 shows mC10-2 and PHF-13 bind equally well in MSD plates coated with 100 ng/ml p-tau 386-408 (pS396/pS404. Increasing concentrations of antibodies (indicated on x-axis) was incubated in wells for 2 hrs followed by wash and détection of bound antibodies using sulfo tagged anti-human IgG antibodies. This indicates that the prepared PHF-13 used in subséquent examples is active.

Figure 33 Comparing mD1.2 and mC10-2 binding to AD-P3

Figure 33 shows mD1.2 and mC10-2 binds equally well in MSD plates coated with 1 pg/ml AD-P3. Increasing concentrations of antibodies (indicated on x-axis) incubated in the presence and absence of 10 uM p-tau 386-408 (pS396/pS404) peptide for 1 hour at room temp followed by incubation in wells for 2 hours followed prior to détection of bound antibodies using sulfo tagged anti-human IgG antibodies. IC50 values were 320 nM and 11 nM for capture of AD-P3 and AD-S1(p). In contrast mD1.2 showed significantly weaker inhibition of tau antigen capture with IC50 values of 589 and 503 nM - suggesting much lower affinity binding to soluble antigens.

Assay was performed in two steps A: 1 pg/ml AD-P3 and 20 ng/ml AD s1(p), respectively was incubated with increasing concentration of mD1.2 and mC10-2 and incubated 1 hour at room température to allow increasing antibody-antigen binding (occupancy). B: The samples were incubated on MSD plates coated with AD-P3 (1 pg/ml) for 2 hours followed by wash and détection of captured Tau antigens using sulfo tagged anti-total TauG antibodies.

Figure 34 mC10-2 but not PHF-13 binds efficiently to solid phase displayed AD-P3 antigens

Highly spécifie binding of mC10-2 but not PHF-13: Figure 34 shows that mC10-2 bind efficiently to AD-P3 antigens coated MSD plates (1 pg/ml). In comparison, the low binding activity of PHF-13 indicates lower affinity to physiological p-tau Antigens. Furthermore PHF13 demonstrated substantiel higher degree of non-specific binding in comparison to mC10-2 (see Table 6). Increasing concentrations of antibodies (indicated on x-axis) incubated for 2 hours followed prior to détection of bound antibodies using sulfo tagged anti-human IgG antibodies. Binding signal was corrected for non-specific binding activity (defined as signais measured in presence of 10 uM p-tau 386-408 (pS396/pS404) peptide. IC50 value were 3 nM for mC10-2 capture of AD-P3. In contrast PHF-13 showed virtually no inhibition.

Assay was performed in two steps. A: 1 pg/ml AD-P3 was incubated with increasing concentration of mC10-2 and PHF-13 and incubated 1 hour at room température to allow increasing antibody-antigen binding (occupancy).B: The samples were incubated on MSD plates coated with AD-P3 (1 pg/ml) for 2 hours followed by wash and détection of captured Tau antigens using sulfo tagged anti-total Tau antibodies.

SEQUENCES INCORPORATED BY REFERENCE

SEQ1D SEQID SEQID SEQID SEQID SEQID SEQID SEQ ID SEQID SEQ ID SEQ ID SEQID SEQID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID SEQ ID

NO:1

NO:2

NO:3

NO:4

NO:5

NO:6

NO:7

NO:8

NO:9

NO:10

NO:11

NO:12

NO:13

NO:14

NO:15

NO:16

NO:17

NO:18

NO:19

NO:20

NO:21

NO:22

NO:23

NO:24

NO:25

NO:26

NO:27

NO:28

NO:29

NO:30

NO:31

D1.2 Light Chain CDR1 D1.2 Light Chain CDR2 D1.2 Light Chain CDR3 D1.2 Heavy Chain CDR1 D1.2 Heavy Chain CDR2 D1.2 Heavy Chain CDR3 D1.2 Light Chain D1.2 Heavy Chain C10-2 Light Chain CDR1 C10-2 Light Chain CDR2 C10-2 Light Chain CDR3 C10-2 Heavy Chain CDR1 C10-2 Heavy Chain CDR2 C10-2 Heavy Chain CDR3 C10-2 Light Chain C10-2 Heavy Chain C5.2 Light Chain CDR1 C5.2 Light Chain CDR2 C5.2 Light Chain CDR3 C5.2 Heavy Chain CDR1 C5.2 Heavy Chain CDR2 C5.2 Heavy Chain CDR3 C5.2 Light Chain C5.2 Heavy Chain C8.3 Light Chain CDR1 C8.3 Light Chain CDR2 C8.3 Light Chain CDR3 C8.3 Heavy Chain CDR1 C8.3 Heavy Chain CDR2 C8.3 Heavy Chain CDR3 C8.3 Light Chain

SEQ ID NO:32 SEQ ID NO:33 SEQ ID NO:34 SEQ ID NO:35 SEQID NO:36

SEQ ID NO:37

C8.3 Heavy Chain

Human tau

D1.2* Light Chain humanized C10-2 Heavy Chain humanized C10-2 Light Chain tau residues 386-408 (pS396, pS404)

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “tau” is synonymous with “the tau protein” and refers to any of the tau protein isoforms (identified in, for example, UniProt as P10636, 1-9). The amino acid numbering of tau that is used herein is given with respect to isoform 2 (SEQ ID NO:33) as shown below, with méthionine (M) being amino acid residue 1:

SEQID NO:33:

MAEPRQEFEV PTEDGSEEPG TTAEEAGIGD IATPRGAAPP GSPGTPGSRS PDLKNVKSKI PGGGSVQIVY QSKIGSLDNI GDTSPRHLSN

MEDHAGTYGL SETSDAKSTP TPSLEDEAAG GQKGQANATR RTPSLPTPPT GSTENLKHQP KPVDLSKVTS

THVPGGGNKK VSSTGSIDMV

GDRKDQGGYT TAEDVTAPLV HVTQARMVSK IPAKTPPAPK REPKKVAVVR GGGKVQIINK KCGSLGNIHH IETHKLTFRE DSPQLATLAD

MHQDQEGDTD DEGAPGKQAA SKDGTGSDDK TPPSSGEPPK TPPKSPSSAK KLDLSNVQSK KPGGGQVEVK NAKAKTDHGA EVSASLAKQG

AGLKESPLQT AQPHTEIPEG KAKGADGKTK SGDRSGYSSP SRLQTAPVPM CGSKDNIKHV SEKLDFKDRV EIVYKSPVVS L

The présent invention relates to antibodies and epitope-binding fragments thereof that are capable of specifically binding to tau, and in particular to human tau, and in one embodiment exhibit the ability to specifically bind to the phosphorylated S396 residue (pS396) of human tau. The antibodies and epitope-binding fragments thereof of the invention, are further characterized by being incapable or substantially incapable of specifically binding to the phosphorylated 404 (pS404) residue on human tau, for example under antibody limited or non-saturating conditions. Furthermore, phosphorylation at pS404 does not interfère with the spécifie binding to pS396. As used herein, the notations “pS” and “^<PÎS” dénoté the amino acid residue phosphoserine. As used herein, an antibody is “substantially incapable of binding to an epitope if relative to another epitope such binding is less than 20%, less than 10%, less than 5%, less than 2%, and more preferably, less than 1% of the binding observed with such other epitope.

The term antibody (Ab) in the context of the présent invention refers to an immunoglobulin molécule or according to some embodiments of the invention, a fragment of an immunoglobulin molécule which has the ability to specifically bind to an epitope of a molécule (“antigen). Naturally occurring antibodies typically comprise a tetramer which is usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain, usually comprised of three domains (CH1, CH2 and CH3). Heavy chains can be of any isotype, including IgG (lgG1, lgG2, lgG3 and lgG4 subtypes). Each light chain is comprised of a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (CL). Light chains include kappa chains and lambda chains. The heavy and light chain variable domain is typically responsible for antigen récognition, while the heavy and light chain constant domain may médiate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune System (e.g., effector cells) and the first component (C1q) of the classical complément System. The VH and VL domains can be further subdivided into domains of hypervariability, termed “complementarity determining régions,” that are interspersed with domains of more conserved sequence, termed “framework régions (FR). Each VH and VL is composed of three CDR Domains and four FR Domains arranged from amino-terminus to carboxyterminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domains of the heavy and light chains contain a binding domain that interacts with an antigen. Of particular relevance are antibodies and their epitope-binding fragments that hâve been “isolated” so as to exist in a physical milieu distinct from that in which it may occur in nature or that hâve been modified so as to differ from a naturally occurring antibody in amino acid sequence.

The term epitope means an antigenic déterminant capable of spécifie binding to an antibody. Epitopes usually consist of surface groupings of molécules such as amino acids or sugar side chains and usually hâve spécifie three dimensional structural characteristics, as well as spécifie charge characteristics. Conformational and linear epitopes are distinguished in that the binding to the former, but not the latter, is always lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically epitope-binding peptide (in other words, the amino acid residue is within the footprint of the specifically epitopebinding peptide).

As used herein, the term “epitope-binding fragment of an antibody means a fragment, portion, région or domain of an antibody (regardless of how it is produced (e.g., via cleavage, recombinantly, synthetically, etc.)) that is capable of specifically binding to an epitope. An epitope-binding fragment may contain 1, 2, 3, 4, 5 or ail 6 of the CDR Domains of such antibody and, although capable of specifically binding to such epitope, may exhibit a specificity, affinity or selectivity toward such epitope that differs from that of such antibody. Preferably, however, an epitope-binding fragment will contain ail 6 of the CDR Domains of such antibody. An epitope-binding fragment of an antibody may be part of, or comprise, a single polypeptide chain (e.g., an scFv), or may be part of, or comprise, two or more polypeptide chains, each having an amino-temninus and a carboxyi terminus (e.g., a diabody, a Fab fragment, a Fab₂ fragment, etc.). Fragments of antibodies that exhibit epitope-binding ability can be obtained, for example, by protease cleavage of intact antibodies. More preferably, although the two domains of the Fv fragment, VL and VH, are naturally encoded by separate genes, polynucleotides that encode such gene sequences (e.g., their encoding cDNA) can be joined, using recombinant methods, by a flexible linker that enables them to be made as a single protein chain in which the VL and VH régions associate to form monovalent epitope-binding molécules (known as single-chain Fv (scFv); see e.g., Bird et al. , (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85:5879-5883). Alternatively, by employing a flexible linker that is too short (e.g., less than about 9 residues) to enable the VL and VH domains of a single polypeptide chain to associate together, one can form a bispecific antibody, diabody, or similar molécule (in which two such polypeptide chains associate together to form a bivalent epitope-binding molécule) (see for instance PNAS USA 90(14), 6444-8 (1993) for a description of diabodies). Examples of epitope-binding fragments encompassed within the présent invention include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in W02007059782; (ii) F(ab’)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge domain; (iii) an Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of a VL and VH domains, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;2i(ll) :484-90); (vi) camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan;5_(l): I II-24) and (vii) an isolated complementarity determining région (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molécules (known as single chain antibodies or sisngle chain Fv (scFv), see for instance Bird et al., Science 242, 423426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). These and other useful antibody fragments in the context of the présent invention are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (epitope-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.

As used herein, isotype refers to the immunoglobulin class (for instance lgG1, lgG2, lgG3 or lgG4) that is encoded by heavy Chain constant domain genes. Such antibody fragments are obtained using conventional techniques known to those of skill in the art; suitable fragments capable of binding to a desired epitope may be readily screened for utility in the same manner as an intact antibody.

The term “bispecific antibody” refers to an antibody containing two independent epitopebinding fragments that each target independent targets. These targets can be epitopes présent on different proteins or different epitopes présent on the same target. Bispecific antibody molécules can be made using compensatory amino acid changes in the constant domains of the HCs of the parent monospecific bivalent antibody molécules. The resulting heterodimeric antibody contains one Fabs contributed from two different parent monospecific antibodies. Amino acid changes in the Fc domain leads to increased stability of the heterodimeric antibody with bispecificity that is stable over time. (Ridgway et al., Protein Engineering 9, 617-621 (1996), Gunasekaran et al., JBC 285, 19637-1(2010), Moore et al., MAbs 3:6 546-557 (2011), Strop et al., JMB 420, 204-219 (2012), Metz et al., Protein Engineering 25:10 571-580 (2012), Labrijn et al., PNAS 110:113, 5145 -5150 (2013), Spreter Von Kreudenstein et al., MAbs 5:5 646-654 (2013)). Bispecific antibodies can also include molécules that are generated using ScFv fusions. Two monospecific scfv are then independently joined to Fc domains able to form stable heterodimers to generate a single bispecific molécule (Mabry et al., PEDS 23:3 115-127 (2010). Bispecific molécules hâve dual binding capabilities.

The term “antibody D1.2” is intended to dénoté an antibody or an epitope-binding fragment thereof, comprising, or consisting of, an antibody Light Chain Variable domain having:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:1;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:2; and (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:3;

and an antibody Heavy Chain Variable domain having:

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:4;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:6.

In one embodiment, the antibody D1.2 or epitope-binding fragment thereof may comprise or consist of the heavy chain of SEQ ID NO:8 and/or the light Chain of SEQ ID NO:7.

In a related embodiment, the antibody D1.2* or epitope-binding fragment thereof may comprise or consist of the heavy chain of SEQ ID NO:8 and/or the light chain of SEQ ID NO:34.

The term “antibody C10-2 is intended to dénoté an antibody or an epitope-binding fragment thereof comprising, or consisting of, an antibody Light Chain Variable domain having:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10; and (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11;

and an antibody Heavy Chain Variable domain having:

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

In one embodiment, the antibody C10-2 or epitope-binding fragment thereof may comprise or consist of the heavy chain of SEQ ID NO:16 and/or the light chain of SEQ ID NO:15.

In a further embodiment, the humanized the antibody C10-2 or epitope-binding fragment thereof may comprise or consist of the heavy chain of SEQ ID NO:35, the light chain of SEQ ID NO:36,, or both. One embodiment of the invention is directed to an antibody or epitopebinding fragment thereof comprising or consisting of the heavy chain of SEQ ID NO:35, the light chain of SEQ ID NO:36.

The term antibody C5.2” is intended to dénoté an antibody or epitope-binding fragment thereof comprising, or consisting of, an antibody Light Chain Variable domain having:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18; and (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19;

and an antibody Heavy Chain Variable domain having:

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22.

In one embodiment, the antibody C5.2 or epitope-binding fragment thereof may comprise or consist of the heavy chain of SEQ ID NO 24 and/or the light chain of SEQ ID NO:23.

The term “antibody C8.3” is intended to dénoté an antibody or an epitope-binding fragment thereof fragment thereof comprising, or consisting of, an antibody Light Chain Variable domain having:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:25;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:26; and (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:27;

and an antibody Heavy Chain Variable domain having:

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:28;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:30.

In one embodiment, the antibody C8.3 or epîtope-binding fragment thereof may comprise or consist of the heavy chain of SEQ ID NO 32 and/or the light chain of SEQ ID NO:31.

An anti-tau antibody is an antibody or an epîtope-binding fragment thereof, which binds specifically to tau or a tau fragment.

The terms monoclonal antibody or monoclonal antibody composition as used herein refer to a préparation of antibody molécules of single molecular composition. A conventional monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. In certain embodiments a monoclonal antibody can be composed of more than one Fab domain thereby increasing the specificity to more than one target. The terms monoclonal antibody or monoclonal antibody composition are not intended to be limited by any particular method of production (e.g., recombinant, transgenic, hybridoma, etc.).

The antibodies of the present invention and epîtope-binding fragments thereof will preferably be “humanized, particularly if employed for therapeutic purposes. The term “humanized” refer to a molécule, generally prepared using recombinant techniques, having an epitopebinding site derived from an immunoglobulin from a non-human species and a remaining immunoglobulin structure based upon the structure and /or sequence of a human immunoglobulin. The epîtope-binding site may comprise either complété non-human antibody variable domains fused to human constant domains, or only the compiementarity determining régions (CDRs) of such variable domains grafted to appropriate human framework régions of human variable domains. The framework residues of such humanized molécules may be wild type (e.g., fully human) or they may be modified to contain one or more amino acid substitutions not found in the human antibody whose sequence has served as the basis for humanization. Humanization lessens or éliminâtes the likelihood that a constant domain of the molécule will act as an immunogen in human individuals, but the possibility of an immune response to the foreign variable domain remains (LoBuglio, A.F. et al. (1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Another approach focuses not only on provîdîng human-derived constant domains, but modifying the variable domains as well so as to reshape them as closely as possible to human form. It is known that the variable domains of both heavy and light chains contain three complementarity- determining régions (CDRs) which vary in response to the antigens in question and détermine binding capability, flanked by four framework régions (FRs) which are relatively conserved in a given specîes and which putatîvely provide a scaffolding for the CDRs. When nonhuman antibodies are prepared with respect to a particular antigen, the variable domains can be “reshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs présent in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) “Reshaping Human Antibodies for Therapy, ” Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity, ” Science 239:1534-1536; Kettleborough, C. A. et al. (1991) “Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation, ” Protein Engineering 4:773-3783; Maeda, H. et al. (1991) “Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity, ” Human Antibodies Hybridoma 2:124-134; Gorman, S. D. et al. (1991) “Reshaping A Therapeutic CD4 Antibody, ” Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185; Tempest, P.R. et al. (1991) “Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo, ” Bio/Technology 9:266-271; Co, M. S. et al. (1991) “Humanized Antibodies For Antiviral Therapy, Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873; Carter, P. et al. (1992) “Humanization Of An Anti~p185her2 Antibody For Human Cancer Therapy, ” Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; and Co, M.S. et al. (1992) “Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen, J. ImmunoL 148:1149-1154. In some embodiments, humanized antibodies preserve ali CDR sequences (for example, a humanized mouse antibody which contains ali six CDRs from the mouse antibodies). In other embodiments, humanized antibodies hâve one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from one or more CDRs from the original antibody. The ability to humanize an antigen is well known (see, e.g., US Patents No. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).

The term “antibody “XX” is intended to dénoté an antibody or epitope-binding fragment thereof (for example antibody “C10-2”), comprising or consisting of the Light Chain, the Light Chain Variable domain, or the Light Chain Variable domain CDR1-3, as defined by its respective SEQ ID NO, and the Heavy Chain, Heavy Chain Variable Domain, or Heavy Chain Variable Domain CDR1-3 as defined by its respective SEQ ID NO. In certain embodiments the antibody or epitope-binding fragment thereof are defined by their entire Heavy Chain Variable Domain comprising as defined by their SEQ ID NO and their Light Chain Variable Domain as defined by their SEQ ID NO.

Unless otherwise specified herein, numbering of amino acid residues in the Fc région or constant domain of an antibody is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

As used herein, an antibody or an epitope-binding fragment thereof is said to “specifically” bind a région of another molécule (i.e., an epitope) if it reacts or associâtes more frequently, more rapidly, with greater duration and/or with greater affinity or avidity with that epitope relative to alternative epitopes. It is also understood by reading this définition that, for example, an antibody or epitope-binding fragment thereof that specifically binds to a first target may or may not specifically or preferentially bind to a second target. As used herein, the term binding în the context of the binding of an antibody to a predetermined antigen typically refers to binding with an affinity corresponding to a KD of about 10'⁷ M or less, such as about W⁸ M or less, such as about 10'⁹ M or less when determined by for instance surface plasmon résonance (SPR) technology in a BIAcore® 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dépendent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly spécifie), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold.

The term kd (sec -1 or 1 ls), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.

The term ka (M-1 x sec-1 or 1/Msec), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.

The term KD (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the kd by the ka.

The term KA (M-1 or 1/M), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the ka by the kd.

In one embodiment, the invention relates to an anti-tau antibody, or epitope-binding fragment thereof, which exhibits one or more of the following properties:

(i) a substantial inability to bind to non-phosphorylated tau;

(ii) a substantial inability to bind to tau that is phosphorylated at S404 and not phosphorylated at S396;

(iii) the ability to bind to tau phosphorylated at S396;

(iv) the ability to bind to tau phosphorylated at both S396 and at S404;

(v) the ability to selectively discriminate between phosphorylated tau residues S396 and S404 such that it is substantially unable to bind the phosphorylated 404 residue (pS404);

(vi) the ability to bind hyper-phosphorylated tau from human Alzheimer’s disease brains;

(vii) the ability to discriminate between pathological and non-pathological human tau protein; and/or (viii) the capability, when used as described herein with immune-depleted rTg4510 extracts from transgenic mice, to specifically reduce the hyperphosphorylated tau 64kDa and 70kDa bands by at least 90%, while not reducing the 55 kDa tau band by more than 10%; or the capability, when used as described herein with extracts from human AD post-mortem brains to specifically reduce the S396 phosphorylated hyperphosphorylated tau bands by at ieast 90%, while not reducing the non-hyperphosphorylated tau bands by more than 10%.

A further embodiment of the invention relates to an antibody generated by a method for generating high specificity, high affinity antibodies that are immunospecific for pathogenic hyperphosphorylated tau comprising residue a phosphorylated S396, wherein said method comprises the steps of:

(A) injecting an immunogen into a mammal, said immunogen comprising the bi-phosphorylated peptide comprising 18-40, such as at 18-30, such as 20-30 amino consecutive acid residues comprising TDHGAEIVYK^ip}SPWSGDT^(PÎSPRHL (SEQ ID NO:37) covering residues 386-410 of 2N4R tau., to thereby immunize said mammal;

(B) repeating said immunization of said mammal two or more times;

(C) screening a sérum sample from said repeatedly immunized mammal for the presence of high specificity, high affinity antibodies capable of binding pathogenic hyperphosphorylated tau comprising residue a phosphorylated S396, but substantially less capable of binding nonpathogenic tau; and (D) recovering said high specificity, high affinity antibodies.

As used herein, a “substantial inability to bind a tau molécule dénotés more than a 20% différence, more than a 40% différence, more than a 60% différence, more than an 80% différence, more than a 100% différence, more than a 150% différence, more than a 2-fold différence, more than a 4-fold différence, more than a 5-fold différence, or more than a 10fold différence in functionality, relative to the détectable binding mediated by a reference 10 antibody.

The term “sélective” and “immunoselective” when referring to the binding capabilities of an anti-tau antibody with respect to two epitopes is intended to dénoté that the observed binding under saturating conditions exhibits at least an 80% différence, at least a 95% différence, and most preferably a 100% différence (i.e., no détectable binding to one 15 epitope). The term “sélective and “immunoselective” when referring to a tau antibody is further intended to mean the antibody binds hyper-phosphorylated tau from human Alzheimer's disease brains and is able to discriminate between pathological and nonpathological human tau protein.

The terms TBS-extractable (S1 ), high salt/sarkosyl-extractable (S3), and sarkosyl-insoluble 20 (P3) fractions are fractions as obtained by the Tau biochemical fractionation described herein.

In some antibodies, only part of a CDR, namely the subset of CDR residues required for binding, termed the SDRs, are needed to retain binding in a humanized antibody. CDR 25 residues not contacting the relevant epitope and not in the SDRs can be identified based on previous studies (for example residues H60-H65 in CDR H2 are often not required), from régions of Kabat CDRs lying outside Chothia hypervariable loops (see, Kabat et al. (1992) Sequences of Proteins of Immunological Interest, National Institutes of Health Publication No. 91-3242; Chothia, C. et al. (1987) “Canonical Structures For The 30 Hypervariable Régions Of Immunoglobulins, J. Mol. Biol. 196:901-917), by molecular modeling and/or empirically, or as described in Gonzales, N.R. et al. (2004) “SDR Grafting Of A Murine Antibody Using Multiple Human Germline Templates To Minimize Its Immunogenicity, Mol. Immunol. 41:863-872. In such humanized antibodies at positions in which one or more donor CDR residues is absent or in which an entire donor CDR is omitted, the amino acid occupying the position can be an amino acid occupyîng the corresponding position (by Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids in the CDRs to include 5 reflects a balance of competing considérations. Such substitutions are potentially advantageous in decreasing the number of mouse amino acids in a humanized antibody and consequently decreasing potential immunogenicity. However, substitutions can also cause changes of affinity, and significant réductions in affinity are preferably avoided. Positions for substitution within CDRs and amino acids to substitute can also be selected empirïcally.

The fact that a single amino acid alteration of a CDR residue can resuit in loss of functional binding (Rudikoff, S. etc. (1982) “Single Amino Acid Substitution Altering Antigen-Binding Specificity, Proc. Natl. Acad. Sci. (USA) 79(6):1979-1983) provides a means for systematically identifying alternative functional CDR sequences. In one preferred method for obtaining such variant CDRs, a polynucleotide encoding the CDR is mutagenized (for 15 example via random mutagenesis or by a site-directed method (e.g., polymerase chainmediated amplification with primers that encode the mutated locus)) to produce a CDR having a substituted amino acid residue. By comparing the identity of the relevant residue in the original (functional) CDR sequence to the identity of the substituted (non-functional) variant CDR sequence, the BLOSUM62.iij substitution score for that substitution can be identified. The BLOSUM System provides a matrix of amino acid substitutions created by analyzing a database of sequences for trusted alignments (Eddy, S.R. (2004) “Where Did The BLOSUM62 Alignment Score Matrix Corne From? Nature Biotech. 22(8): 1035-1036; Henikoff, J.G. (1992) “Amino acid substitution matrices from protein blocks, ’’ Proc. Natl. Acad. Sci. (USA) 89:10915-10919; Karlin, S. et al. (1990) “Methods For Assessing The

Statistical Significance Of Molecular Sequence Features By Using General Scoring Schemes, Proc. Natl. Acad. Sci. (USA) 87:2264-2268; Altschul, S.F. (1991) “Amino Acid Substitution Matrices From An Information Theoretic Perspective, ” J. Mol. Biol. 219, 555565. Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table 1 présents the BLOSUM62.iij substitution scores (the higher the 30 score the more conservative the substitution and thus the more likely the substitution will not affect function). If an epitope-binding fragment comprising the résultant CDR fails to bind to tau, for example, then the BLOSUM62.iij substitution score is deemed to be insufficiently conservative, and a new candidate substitution is selected and produced having a higher substitution score. Thus, for example, if the original residue was glutamate (E), and the non35 functional substitute residue was histidine (H), then the BLOSUM62.iij substitution score will be 0, and more conservative changes (such as to aspartate, asparagine, glutamine, or lysine) are preferred.

Table 1
	A	R	N	D	C	Q	E	G	H	1	L	K	M	F	P	S	T	W	Y	V
A	+4	-1	-2	-2	0	-1	-1	0	-2	-1	-1	-1	-1	-2	-1	+1	0	-3	-2	0
R	-1	+5	0	-2	-3	+1	0	-2	0	-3	-2	+2	-1	-3	-2	-1	-1	-3	-2	-3
N	-2	0	+6	+1	-3	0	0	0	+1	-3	-3	0	-2	-3	-2	+1	0	-4	-2	-3
D	-2	-2	+ 1	+6	-3	0	+2	-1	-1	-3	-4	-1	-3	-3	-1	0	-1	-4	-3	-3
C	0	-3	-3	-3	+9	-3	-4	-3	-3	-1	-1	-3	-1	-2	-3	-1	-1	-2	-2	-1
Q	-1	+1	0	0	-3	+5	+2	-2	0	-3	-2	+1	0	-3	-1	0	-1	-2	-1	-2
E	-1	0	0	+2	-4	+2	+5	-2	0	-3	-3	+1	-2	-3	-1	0	-1	-3	-2	-2
G	0	-2	0	-1	-3	-2	-2	+6	-2	-4	-4	-2	-3	-3	-2	0	-2	-2	-3	-3
H	-2	0	+1	-1	-3	0	0	-2	+8	-3	-3	-1	-2	-1	-2	-1	-2	-2	+2	-3
I	-1	-3	-3	-3	-1	-3	-3	-4	-3	+4	+2	-3	+1	0	-3	-2	-1	-3	-1	+3
L	-1	-2	-3	-4	-1	-2	-3	-4	-3	+2	+4	-2	+2	0	-3	-2	-1	-2	-1	+ 1
K	-1	+2	0	-1	-3	+1	+1	-2	-1	-3	-2	+5	-1	-3	-1	0	-1	-3	-2	-2
M	-1	-1	-2	-3	-1	0	-2	-3	-2	+ 1	+2	-1	+5	0	-2	-1	-1	-1	-1	+ 1
F	-2	-3	-3	-3	-2	-3	-3	-3	-1	0	0	-3	0	+6	-4	-2	-2	+1	+3	-1
P	-1	-2	-2	-1	-3	-1	-1	-2	-2	-3	-3	-1	-2	-4	+7	-1	-1	-4	-3	-2
S	+1	-1	+ 1	0	-1	0	0	0	-1	-2	-2	0	-1	-2	-1	+4	+1	-3	-2	-2
T	0	-1	0	-1	-1	-1	-1	-2	-2	-1	-1	-1	-1	-2	-1	+1	+5	-2	-2	0
w	-3	-3	-4	-4	-2	-2	-3	-2	-2	-3	-2	-3	-1	+1	-4	-3	-2	+11	+2	-3
Y	-2	-2	-2	-3	-2	-1	-2	-3	+2	-1	-1	-2	-1	+3	-3	-2	-2	+2	+7	-1
V	0	-3	-3	-3	-1	-2	-2	-3	-3	+3	+ 1	-2	+ 1	-1	-2	-2	0	-3	-1	+4

The invention thus contemplâtes the use of random mutagenesis to identify improved CDRs.

In the context of the présent invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of Tables 2, 3, or 4:

Amino Acid Residue Classes For Conservative Substitutions:

Table 2
Acidic Residues	Asp (D) and Glu (E)
Basic Residues	Lys (K), Arg (R), and His (H)
Hydrophilic Uncharged Residues	Ser (S), Thr (T), Asn (N), and Gin (Q)
Aliphatic Uncharged Residues	Cly (G), Ala (A), Val (V), Leu (L), and Ile (I)
Non-polar Uncharged Residues	Cys (C), Met (M), and Pro (P)
Aromatic Residues	Phe (F), Tyr (Y), and Trp (W)__________________

Alternative Conservative Amino Acid Residue Substitution Classes:

Table 3
1	A	S	T
2	D	E
3	N	Q
4	R	K
5	I	L	M
6	F	Y	W

Alternative Physical and Functional Classifications of Amino Acid Residues:

Table 4
Alcohol Group-Containing Residues	S and T
Aliphatic Residues	I, L, Vand M
Cycloalkenyl-Associated Residues	F, H, W and Y
Hydrophobie Residues	A, C, F, G, H, I, L, M, R, T, V, W and Y
Negatively Charged Residues	D and E
Polar Residues	C, D, E, H, K, N, Q, R, S and T
Positively Charged Residues	H, K and R
Small Residues	A, C, D, G, N, P, S, T and V
Very Small Residues	A, G and S
Residues Involved In Turn Formation	A, C, D, E, G, H, K, N, Q, R, S, P and T
Flexible Residues	Q, T, K, S, G, P, D, E and R

More conservative substitutions groupings include: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using the principles described in, e.g., Creighton (1984) Proteins: Structure and Molecular Properties (2d Ed. 1993), W. H. Freeman and Company.

Phage display technology can alternatively be used to increase (or decrease) CDR affinity. This technology, referred to as affinity maturation, employs mutagenesis or “CDR walking and re-selection uses the target antigen or an antigenic epitope-binding fragment thereof to identify antibodies having CDRs that bind with higher (or lower) affinity to the antigen when compared with the initial or parental antibody (See, e.g. Glaser et al. (1992) J. Immunology 149:3903). Mutagenizing entire codons rather than single nucléotides results in a semirandomized répertoire of amino acid mutations. Libraries can be constructed consisting of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue. Mutants with increased (or decreased) binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased or decreased affinity to the antigen (e.g., ELISA) (See Wu et al. 1998, Proc. Natl. Acad. Sci. (U.S.A.) 95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDR walking which randomizes the Light Chain may be used possible (see, Schier et al., 1996, J. Mol. Bio. 263:551).

Methods for accomplishing such affinity maturation are described for example in: Krause, J.C. et al. (2011) “An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function Of A Human Antibody, MBio. 2(1) pii: e00345-10. doi: 10.1128/mBio.00345-10; Kuan, C.T. et al. (2010) “Affinity-Matured Anti-Glycoprotein NMB

Recombinant Immunotoxins Targeting Malignant Gliomas And Melanomas, ” Int. J. Cancer 10.1002/ijc.25645; Hackel, B. J. et al. (2010) “Stability And CDR Composition Biases Enrich Binder Functionality Landscapes, ” J. Mol. Biol. 401(1 ):84-96; Montgomery, D.L. étal. (2009) “Affinity Maturation And Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41, ” MAbs 1(5):462-474; Gustchina, E. et al. (2009) “Affinity Maturation By Targeted Diversification Of The CDR-H2 Loop OfA Monoclonal Fab Derived From A Synthetic Naïve Human Antibody Library And Directed Against The Internai Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With Improved HIV-1 Neutralization Potency And Breadth, Virology 393(1):112-119; Finlay, W.J. et al. (2009) “Affinity Maturation OfA Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The Complementarity-Determining Régions, ” J. Mol. Biol. 388(3):541-558; Bostrom, J. et al. (2009) “Improving Antibody Binding Affinity And Specificity For Therapeutic Development, ” Methods Mol. Biol. 525:353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR-Diversification, ” Mol. Immunol. 46(1):135-144; and Barderas, R. et al. (2008) “Affinity Maturation Of Antibodies Assisted By In Silico Modeling,” Proc. Natl. Acad. Sci. (USA) 105(26):9029-9034.

Thus, the sequence of CDR variants of encompassed antibodies or their epitope-binding fragments may differ from the sequence of the CDR of the parent antibody, D1.2, C10-2, C5.2 or C8.3, through substitutions; for instance substituted 4 amino acid residue, 3 amino acid residue, 2 amino acid residue or 1 of the amino acid residues. According to an embodiment of the invention it is furthermore envisaged that the amino acids in the CDR régions may be substituted with conservative substitutions, as defined in the 3 tables above. For example, the acidic residue Asp can be substituted with Glu without substantially affecting the binding characteristic of the antibody.

The term “normal tau refers to normal brain tau containing 2-3 moles of phosphate per mole of the protein.

The term “hyperphosphorylated tau refers to a poly-phosphorylated species of tau consistent with poly-anionic species induced mobility shift in Western Blot or to a tau species which has more than five, six or seven Serine, Threonine or Tyrosine sites phosphorylated.

The term “tau having residue 396 phosphorylated relates hyperphosphorylated tau wherein residue 396 is phosphorylated and residue 404 is or is not phosphorylated.

The term transgenic non-human animal refers to a non-human animal having a genome comprising one or more human heavy and/or light chain transgenes or trans-chromosomes (either integrated or non-integrated into the animais naturel genomic DNA) and which is capable of expressing fully humanized antibodies. For example, a transgenic mouse can hâve a humanized light chain transgene and either a humanized heavy chain transgene or humanized heavy chain trans-chromosome, such that the mouse produces humanized antitau antibody when immunized with tau antigen and/or cells expressing tau. The humanized heavy chain transgene may be integrated into the chromosomal DNA of the mouse, as is the case for transgenic mice, for instance HuMAb mice, such as HCo7 or HCol2 mice, or the humanized heavy chain transgene may be maintained extra-chromosomally, as is the case for trans-chromosomal KM mice as described in WO02/43478. Such transgenic and transchromosomal mice (collectiveîy referred to herein as transgenic mice) are capable of producing multiple isotypes of humanized monoclonal antibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-J recombination and isotype switching.

Transgenic, nonhuman animal can also be used for production of antibodies against a spécifie antigen by introducing genes encoding such spécifie antibody, for example by operatively linking the genes to a gene which is expressed in the milk of the animal.

The term “treatment” or “treating” as used herein means ameliorating, slowing, attenuating, or reversing the progress or severity of a disease or disorder, or ameliorating, slowing, attenuating, or reversing one or more symptoms or side effects of such disease or disorder. For purposes of this invention, “treatment” or “treating further means an approach for obtaining bénéficiai or desired clinical results, where “bénéficiai or desired clinical results include, without limitation, alleviation of a symptom, diminishment of the extent of a disorder or disease, stabilized (i.e., not worsening) disease or disorder state, delay or slowing of the progression a disease or disorder state, amelioration or palliation of a disease or disorder state, and remission of a disease or disorder, whether partial or total, détectable or undetectable.

An effective amount, when applied to an antibody or epitope-binding fragment thereof of the invention, refers to an amount sufficient, at dosages and for periods of time necessary, to achieve an intended biological effect or a desired therapeutic resuit including, without limitation, clinical results. The phrase “therapeutically effective amount, when applied to an antibody or an epitope-binding fragment thereof of the invention, is intended to dénoté an amount of the antibody, or epitope-binding fragment thereof, that is sufficient to ameliorate, palliate, stabilize, reverse, slow, attenuate or delay the progression of a disorder or disease state, or of a symptom of the disorder or disease. In an embodiment, the method of the présent invention provides for administration of the antibody, or epitope-binding fragment thereof, in combinations with other compounds. In such instances, the “effective amount” is the amount of the combination sufficient to cause the intended biological effect.

A therapeutically effective amount of an anti-tau antibody or epitope-binding fragment thereof of the invention may vary according to factors such as the disease state, âge, sex, and weight of the îndividual, and the ability of the anti-tau antibody, or epitope-binding fragment thereof, to elicit a desired response in the îndividual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically bénéficiai effects.

As indicated above, the present invention particularly relates to monoclonal antibodies, or epitope-binding fragments thereof, and to a completely new method for producing such molécules (and thus of such epitope-binding fragments thereof). This method is outlined in Figure 9. This ability of the new method to isolate monoclonal antibodies is exemplified herein by its use to isolate monoclonal antibodies that are capable of specifically binding to the phosphorylated residue serine 396 (^{piS396) of human tau (SEQ ID NO:33). These antibodies are further characterized by their ability to discriminate between phosphorylated residues serine 396 and serine 404 (pS404) such that they do not bind to tau protein with phosphorylated serine 404 unless the tau is also phosphorylated at residue 396.

The antibodies of the present invention, or epitope-binding fragment thereof, hâve been generated and isolated by use of a novel a method (Figure 9) which favors the sélection of ^{P}S396 spécifie antibodies (Figure 9). Furthermore, by applying this very strict antibody clone sélection procedure, antibodies hâve been obtained that are not only highly spécifie towards S396, but also highly sélective towards the phosphorylated ^ip,S396 epitope. These antibodies uniquely recognize tau from Alzheimer's disease brains. We also demonstrate that the screening procedure outlined in Figure 9 ensures the identification of antibodies which possess a functional and therapeutic utility.

Antibodies were raîsed against the bi-phosphorylated peptide: TDHGAEIVYK^(piSPWSGDT^{PlSPRHL (SEQ ID NO:37) covering residues 386-408 of 2N4R tau (Example 1). Mice were immunized with the phospho-peptide. Once sufficient antibody titres had been obtained, the mice were sacrificed and hybridomas were generated (Example 2). The hybridomas were screened using dot-blot (Example 3) and MSD ELISA with immobilized human pathological and non-pathological tau (Example 4). The ability to discriminate between pathological and non-pathological human tau protein in dot-blot and Western blot was used for the sélection of hybridomas. Sixteen clones were selected, of which four hybridoma clones were recovered that produced antibodies which exhibited extraordinary capabilities for binding to human pathological tau material.

Spécifie binding to pathological and non-pathological tau was also determined by isolation of tau from diseased and non-diseased human AD brains and immobilization of this material on MSD ELISA plates (Example 4).

A further aspect of the invention relates to monoclonal antibody or an epitope-binding fragment thereof elicited against the bi-phosphorylated peptide comprising at least 18, such as at least 20 amino consecutive acid residues within TDHGAEIVYK^1P,SPWSGDT^<P>SRRHL (SEQ ID NO:37) covering residues 386-410 of 2N4R tau. In this aspect of the invention, the monoclonal antibody or an epitope-binding fragment thereof is typically elicited against the bi-phosphorylated peptide comprising 18-40, such as at 18-30, such as 20-30 amino consecutive acid residues comprising TDHGAEIVYK^{pîSPWSGDT^(p*SPRHL (SEQ ID NO:37) covering residues 386-410 of 2N4R tau.

A further aspect of the invention is directed to the monoclonal antibody or an epitope-binding fragment thereof of the invention, having a specificity for phosphoTau (pTau) from ADdiseased patients over age-matched healthy Controls, such that said monoclonal antibody or an epitope-binding fragment thereof has a specificity différence for phosphoTau (pTau) from AD-diseased patients over tau from age-healthy matched Controls of more than 50-fold, such as more than 100-fold increase in specificity for AD disease material compared to healthy control material in an ELISA based assay detect phosphoTau (pTau) in brain homogenates from AD and from healthy control subjects, using a phospho- and multimer-specific Setup 1 ELISA as described herein.

A related aspect of the invention is directed to the monoclonal antibody or an epitope-binding fragment thereof of the invention, having a specificity for AD-diseased Tau such that said monoclonal antibody or an epitope-binding fragment thereof has a specificity différence for AD over age-healthy matched Controls of more than 50-fold, such as more than 100-fold increase in specificity for AD disease material compared to healthy control material in an ELISA based assay detect phosphoTau (pTau) in brain homogenates from AD and from healthy control subjects, using a phospho- and multimer-specific Setup 1 ELISA.

Setup 1 ELISA method comprises the steps A) a capture of pathological human Tau antigens from AD brains using C10-2 coated plates; B) incubation of Tau antigens with increasing concentrations of pS396 spécifie antibodies; and C) détection of the Tau antigen capture and antibody mediated inhibition using sulfo-tagged anti human (total) Tau antibodies from MSD.

The invention further relates to an antibody generated by a method for generating high specificity, high affinity antibodies that are immunospecific for pathogenic hyperphosphorylated tau comprising residue a phosphorylated S396, wherein said method comprises the steps of:

(A) injecting an immunogen into a mammal, said immunogen comprising the biphosphorylated peptide comprising 18-40, such as at 18-30, such as 20-30 amino consecutive acid residues comprising TDHGAEIVYK^(plSPWSGDT^<p>SPRHL (SEQ ID NO:37) covering residues 386-410 of 2N4R tau, to thereby immunize said mammal;

(B) repeating said immunization of said mammal two or more times;

(C) screening a sérum sample from said repeatedly immunized mammal for the presence of high specificity, high affinity antibodies capable of binding pathogenic hyperphosphorylated tau comprising residue a phosphorylated S396, but substantially less capable of binding non-pathogenic tau; and (D) recovering said high specificity, high affinity antibodies.

More specifically, step A comprises: coating of MSD plates ( typically ovemight at 4 C) with C10-2 antibody, typically 0,5 pg/ml (capture antibody) in coating buffer, blocking (typically 1 hour at room température) and washing, typically 3 times. Step B comprises: mixing of samples of P3 lysate (typically 1:1000 = 2-4 pg/ml total protein) and/or S1(p) (typically 1:300=20-40 ng/ml total protein) from AD (pooled from 3 patients) with graded concentrations of pS396 peptide epitope spécifie antibody and incubating (typically 1 hour at room température). The reactions are subsequently incubated for 2 hours on plates prepared in step A. Step C comprises detecting C10-2 captured Tau was using sulfo-tagged human tau. Tau antibody (typically 1:50) from MSD following manufacturer instructions. Plates are analyzed on MSD SECTOR® S600. AD P3 and AD S1(p) are tested in a similar setup.

A further embodiment is directed to an antibody, or antigen-binding fragment thereof, capable of immunospecifically binding to the phosphorylated residue 396 of human tau (SEQ ID NO:33), which has been produced or manufactured in a cell line such as a human cell line, a mammal non-human cell line, an insect, yeast or bacteriai cell line.

The antibody, or antigen binding fragment thereof, capable of immunospecifically binding to the phosphorylated residue 396 of human tau (SEQ ID NO:33), produced in a CHO cell line,

HEK cell line, BHK-21 cell line, murine cell line (such as a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAP cell line and HuH-7 human cell line.

Spécifie affinîties and binding properties of D1.2 and C10-2 hâve been characterized using tau 386-410 (2R4N) peptides which are either phosphorylated or un-phosphorylated at position 396 or 404 (SEQ ID NOs:29-32). Using the spécifie immunization and screening protocol (Figure 9) outlined in this application will produce highly phosphor-serine-396 (pS396) spécifie antibodies as demonstrated in Figure 4.

In order to demonstrate that the antibodies are spécifie towards pathological tau, D1.2 and C10-2 antibodies hâve also been characterized by immune-histochemical analysis (Example 6). The antibodies exhibit highly spécifie binding to neurofibrillary tangles in Alzheimer’s disease brains and in sections from Tg4510 tau transgenic mice expressing human (P301L) mutant tau (Figure 5). No binding is observed to tissue from human control brains and from non-transgenic mouse brains, demonstrating that the antibodies specifically bind human tau and in particular tau associated with Alzheimer’s pathology.

The unique capability of these antibodies to recognize tau associated with disease pathology is demonstrated here in Example 7. We compare the binding of pathological vs. nonpathological tau in the assay described in Example 3. The comparison is to five published tau antibodies: hACI-2B6, IPN002, HJ8.5, 2.10.3, and 4E4. Figure 6 illustrâtes the binding of each of the reference antibodies towards tau from healthy and diseased brains, and binding to P301L human mutant tau isolated from 10 month old Tg4510 tau transgenic mice. This demonstrates that the isolated antibodies exhibit an exceptionally high degree of specificity and selectivity towards human pathological tau. This selectivity is superior to any of the comparator antibodies as shown in Table 5.

Table 5
mAb Tested	AD/ctrl	TG/wt
hACI-2B6	3	1
IPN002	3	37
HJ8.5	3	51
4E4	no binding	1
2.10.3	5	2
C5-2_C10-2	>100	118

At saturation binding antibodies D1.2 and C10-2 exhibit more than 100-fold selectivity towards P3 tau isolated from human AD brains.

To demonstrate that the selected antibodies hâve functional and therapeutic utility, antibodies were tested in in-vitro and in-cell tau-aggregation assays (Example 8). These assays are functional assays which demonstrate that the antibodies are able to interfère with the pathological aggregation process of tau. HEK293 cells are transiently transfected with human tau-P301 L-FLAG (4R0N). Subsequently the cells are exposed to tau extracts from human AD brains or from transgenic Tg4510 brains. This exposure to pathological tau promûtes tau uptake into cells and ïntracellular aggregation. Both immuno-depletion of taupreparations using antibodies D1.2 and C10-2, and direct treatment of cells with these antibodies is able to reduce the formation of tau aggregates dramatically (Figure 7).

Therapeutic utility of antibodies D1.2 and C10-2 has also been evaluated in the human tau/PS1 mouse (Example 9). This mouse model is a more AD disease relevant animal model which only generates AD pathology late in life (12-18 month of âge). However, the mice do exhibit tau hyper phosphorylation before the occurrence of solid tangle pathology. Mice were injected chronically for 13 weeks, twice weekly with 15 mg/kg dose. Antibody treated mice exhibit a dramatic réduction in phosphorylated tau as demonstrated in Figure 9, indicating that chronic treatment with antibodies D1.2 and C10-2 will reduce tangle pathology and thus subséquent neurodegeneration in vivo.

The antibodies of the invention specifically remove hyperphosphorylated Tau from rTg4510 mouse brain extracts by immunodepletion methods. Moreover, the antibodies of the invention do not remove the normal Tau from the homogenates, whereas the commercially available tau5 antibody does. In contrast to commercial antibodies which bind to tau proteins wherein phosphorylation at residue 404 or at both residues 404 and 396, the antibodies of the invention specifically remove the hyperphosphorylated tau by 95%, that is phosphorylated on serine 396. Experiments (Example 12) demonstrate that the antibody of the invention, despite only removing a very small fraction of the total tau in the brain homogenate (8%), the antibodies do however specifically remove the hyperphosphorylated tau (by 90%). Accordingly, one aspect of the invention is directed to a monoclonal antibody, or an epitope-binding fragment thereof, capable of immunospecifically binding to the pathogenic hyperphosphorylated tau. Furthemnore, in experiments wherein hyperphosphorylated Tau was removed using an antibody of the invention, the seeding activity is abolished. By removing hyperphosphorylated tau from the homogenates, the homogenates no longer induce seeding of Tau pathology. It has been proposed that réduction of seeding reduces the development of tangle formation and the progression of tauopathies, including Alzheimer’s disease. Accordingly, a further aspect of the invention is directed to an antibody of the invention for use in the réduction of the progression of AD or in the symptoms of AD.

More specifically, as detailed above, the invention relates to any one of four monoclonal antibodies selected from the group comprising:

Antibody D1.2 (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:1;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:2;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:3;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:4;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:5; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:6.

The antibody or epîtope-binding fragment thereof may comprise or consist of the heavy chain variable domain of SEQ ID NO:8 and/or the light chain variable domain of SEQ ID NO:7.

In a related embodiment, the antibody D1.2 or epîtope-binding fragment thereof may comprise or consist of the heavy chain of SEQ ID NO 8 and/or the light chain of SEQ ID NO:34.

Antibody C10-2 (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

The antibody or epîtope-binding fragment thereof may comprise or consist of the heavy chain variable domain of SEQ ID NO:15 and/or the light chain variable domain of SEQ ID NO:16.

The amino acid sequence of the heavy chain of humanized C10-2 antibody is shown in SEQ ID NO:35. The amino acid sequence of the light chain of humanized C10-2 antibody is shown in SEQ ID NO:36.

Altogether, the Examples show that the antibodies of the invention, including C10-2, bind efficiently to AD-P3 antigens coated MSD plates. In comparison, commercial antibodies such as PHF-13, hâve low binding activity. Furthermore PHF-13 demonstrated substantial higher degree of non-specific binding in comparison to the antibodies of the invention (see Table 6 A-Table 6D). Table 6 shows that mC10-2 fluid phase inhibition of Ptau antigen capture in C10-2 coated plate is effective (IC50= 10-20 nM) whereas mD1.2 is ineffective (IC50 = 5001000 nM). mC10-2 fluid phase inhibition of p-tau antigen capture in mC10-2 coated plate is effective with an of IC50= 10-20 nM) whereas PHF-13 is ineffective (IC50 = 500-1000 nM).

One aspect of the invention is directed to an antibody comprising (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11.

A further aspect of the invention is directed to an antibody comprising (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

A further aspect of the invention is directed to an antibody comprising (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO: 14 and one, two or three of (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10; and (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11.

Antibody C5,2 (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21 ; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22.

The antibody or epitope-binding fragment thereof may comprise or consist of the heavy chain variable domain of SEQ ID NO:23 and/or the light chain variable domain of SEQ ID NO:24.

As can be seen from the crystal structure of Figure 10, the epitope is bound across the heavy chain and light chains of C5.2. Accordingly, in a related embodiment, the antibody of the invention or epitope-binding fragment thereof comprises (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17; or (b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18; or (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19; and (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NQ:20; or (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21; or (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22.

Antibody C8.3 (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:25;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:26 (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:27;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:28;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:30.

The antibody or epitope-binding fragment thereof may comprise or consist of the heavy chain variable domain of SEQ ID NO:31 and/or the light chain variable domain of SEQ ID NO:32.

The antibody or epitope-binding fragment thereof is preferably a human or humanized antibody.

The antibodies and epitope-binding fragment thereof mentioned above may, according to one embodiment, further comprise a variant of such light and/or heavy chain CDR1, CDR2 or CDR3 (with no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.

As can be seen from Figure 11, HC CDR1, HC CDR2, HC CDR3 and LC CDR3 are, in at least one embodiment, important, for the binding to the 392-398 région of Tau. In one embodiment of the invention, the antibody of the invention, or epitope-binding fragment thereof comprises:

a) a Heavy Chain CDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, and SEQ ID NO:28;

b) a Heavy Chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:21, and SEQ ID NO:29; and

c) a Heavy Chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22, and SEQ ID NO:30; and

d) a Light Chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19, and SEQ ID NO:27.

The antibody of the invention, or epitope-binding fragment thereof may comprise

a) a Heavy Chain CDR1 comprising the amino acid sequence of SEQ ID NO:20;

b) a Heavy Chain CDR2 comprising the amino acid sequence of SEQ ID NO:21;

c) a Heavy Chain CDR3 comprising the amino acid sequence of SEQ ID NO:22; and

d) a Light Chain CDR3 comprising the amino acid sequence of SEQ ID NO:19.

In one aspect of the invention, the invention is directed to an antibody or epitope-binding fragments thereof, that forms a hydrophobie pocket formed by L3:H3, L3:F8*, H1:H13, H2:Y1, H2:Y3 with Y394 of the tau peptide. In an embodiment, the invention is directed to an antibody that competes with an antibody further described herein for fomning a hydrogen bonding network between solvated ^<P,S396 and L3:T4, H1:R10, H1:T11, H3:R1, H3:T3; (*) L3:F8 is the C-terminal flankîng framework residue of CDR L3 (see Figure 11).

As can be seen from the x-ray crystal structure, the antibody of the invention binds with two levels of selectivity. The first level of selectivity is selectivity for hyperphosphorylated pathological, tau and the second level of selectivity is selectivity for a phosphorylated serine residue wherein the phosphate of said phosphorylated serine is hydrogen bonded to the side chain of a tyrosine residue two residues removed from said phosphorylated serine. Accordingly, an interesting aspect of the invention is directed to an antibody or epitopebinding fragment thereof sélective for an amino acid motif of hyperphosphorylated tau comprising of a phosphorylated serine two residues removed from a tyrosine residue. Typically, the amino acid motif has the sequence:

Y - X - S(phosphorylated) - P wherein Y is tyrosine, X is a naturally occurring amino acid, P is proline and S(phosphorylated) is serine with a phosphorylated hydroxyl side chain .

Similarly, an interesting aspect of the invention is directed to an antibody or epitope-binding fragment thereof which binds to phosphorylated tau, preferably hyperphosphorylated tau, wherein said antibody or epitope-binding fragment thereof is sélective for the amino acid residue motif IA, wherein R is a side chain of a naturally occurring amino acid.

OR O

IA

Without being bound to a particular theory, it is believed that the antibody of the invention is sélective for amino acid motif IA when said motif is in a conformation adopted by pathological tau. Accordingly, the amino acid motif IA is typically the sequence selectively 10 recognized by the antibody of the invention. Accordingly, an interesting aspect of the invention is directed to an antibody or epitope-binding fragment thereof which binds to phosphorylated tau, preferably hyperphosphorylated tau, wherein said antibody or epitopebinding fragment thereof is sélective for the amino acid residue motif IB, wherein R is a side chain of a naturally occurring amino acid.

In a typical embodiment of this aspect of the invention, the invention is directed to an antibody or epitope-binding fragment thereof which binds to phosphorylated tau, preferably hyperphosphorylated tau, wherein said antibody or epitope-binding fragment thereof is sélective for the amino acid residue motif IB, wherein R is a side chain of a naturally occurring amino acid such as, but not limited to IC or ID.

4Θ

The present invention also provides a method of reducing tau tangle formation in a patient, comprising administering to the patient in need of such treatment, a therapeutically effective amount of an antibody of the invention, or epitope-binding fragments thereof.

One aspect of the invention is directed to a method of treating a taupathy using an antibody of the invention, or epitope-binding fragments thereof. Typically, the taupathy is selected from the group consisting of Alzheimer’s disease, Argyrophilic Grain Disease (AGD), Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD, psychiatrie symptoms of patients with Lewy body dementia, Progressive Supranuclear Palsy (PSP), Frontotemporal dementia (FTD or variants thereof), TBI (traumatic brain injury, acute or chronic), Corticobasal Degeneration (CBD), Picks Disease, Primary age-related tauopathy (PART), Neurofibrillary tangle-predominant senile dementia, Dementia pugilistica, Chronic traumatic encephalopathy, stroke, stroke recovery, neurodegeneration in relation to Parkinson’s disease, Parkinsonism linked to chromosome, Lytico-Bodig disease (Parkinsondementia complex of Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lipofuscinosis. More typically, the taupathy is selected from the group consisting of Alzheimer’s disease, Argyrophilic Grain Disease (AGD), Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD, psychiatrie symptoms of patients with Lewy body dementia, Progressive Supranuclear Palsy (PSP), Frontotemporal dementia (FTD or variants thereof), TBI (traumatic brain injury, acute or chronic), Corticobasal Degeneration (CBD), and Picks Disease. In particular, the tauopathies may be selected from Alzheimer’s disease, Argyrophilic Grain Disease (AGD), Psychosis due to AD or Psychosis in patients with AD, and psychiatrie symptoms of patients with Lewy body dementia.

Accordingly, a further aspect of the invention is directed to an antibody of the invention or epitope-binding fragments thereof, for use in the treatment of a taupathy. Typically, the taupathy is selected from the group consisting of Alzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD, psychiatrie symptoms of patients with Lewy body dementia, Progressive Supranuclear Palsy (PSP), Frontotemporal dementia (FTD or variants thereof), TBl (traumatic brain injury, acute or chronic), Corticobasal Degeneration (CBD), Picks Disease, Primary age-related tauopathy (PART), Neurofibrillary tangle-predominant senile dementia, Dementia pugilistica, Chronic traumatic encephalopathy, stroke, stroke recovery, neurodegeneration in relation to Parkinson’s disease, Parkinsonism linked to chromosome, Lytico-Bodig disease (Parkinsondementia complex of Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lipofuscinosis. More typically, the taupathy is selected from the group consisting of Alzheimer’s disease, Argyrophilic Grain Disease (AGD), Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD, psychiatrie symptoms of patients with Lewy body dementia, Progressive Supranuclear Palsy (PSP), Frontotemporal dementia (FTD or variants thereof), TBl (traumatic brain injury, acute or chronic), Corticobasal Degeneration (CSD), and Picks Disease. In particular, the tauopathies may be selected from Alzheimer’s disease, Argyrophilic Grain Disease (AGD), Psychosis due to AD or Psychosis in patients with AD, and psychiatrie symptoms of patients with Lewy body dementia.

A further aspect of the invention is directed to an antibody of the invention or epitope-binding fragments thereof, in a composition together with a pharmaceutically acceptable carrier, diluent, adjuvant and/or stabilizer. The antibodies of the invention, or epitope-binding fragments thereof, may be used in therapy for the treatment of a taupathy. Typically, the taupathy is selected from the group consisting of Alzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD, psychiatrie symptoms of patients with Lewy body dementia, Progressive Supranuclear Palsy (PSP), Frontotemporal dementia (FTD or variants thereof), TBl (traumatic brain injury, acute or chronic), Corticobasal Degeneration (CBD), Picks Disease, Primary age-related tauopathy (PART), Neurofibrillary tangle-predominant senile dementia, Dementia pugilistica, Chronic traumatic encephalopathy, stroke, stroke recovery, neurodegeneration in relation to Parkinson’s disease, Parkinsonism linked to chromosome, Lytico-Bodig disease (Parkinsondementia complex of Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis,

Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lîpofuscinosis. More typically, the taupathy is selected from the group consisting of Alzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD, psychiatrie symptoms of patients with Lewy body dementia, Progressive Supranuclear Palsy (PSP), Frontotemporal dementia (FTD or variants thereof), TBI (traumatic brain injury, acute or chronic), Corticobasal Degeneration (CBD), and Picks Disease. In particular, the tauopathies may be selected from Alzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis due to AD or Psychosis in patients with AD, and psychiatrie symptoms of patients with Lewy body dementia.

The treatment envisioned by the présent invention may be chronic and the patient may be treated at least 2 weeks, such as at least for 1 month, 6, months, 1 year or more.

The antibodies of the présent invention may, for example, be monoclonal antibodies produced by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or may be monoclonal antibodies produced by recombinant DNA or other methods, or more preferably may be produced by the novel method disclosed herein (Figure 9). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B lymphocyte cells obtained from mice immunized with an antigen of interest, for instance, in the form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest. Monoclonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or from nonhuman mammals such as rats, rabbits, dogs, sheep, goats, primates, etc.

In one embodiment, the antibody of the invention is a humanized antibody. Humanized monoclonal antibodies directed against tau may be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. Such transgenic and transchromosomîc mice include mice referred to herein as HuMAb (Humanized monoclonal antibody) mice and KM mice, respectively, and are collectively referred to herein as transgenic mice.

The HuMAb mouse contains a human immunoglobulin gene mini-locus that encodes unrearranged human heavy variable and constant (μ and Y) and light variable and constant (k) chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and K chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or K and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG, rç monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intem. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N., Ann. N. Y. Acad. Sci 764 536-546 (1995)). The préparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et a!., International Immunology 6, 579-591 (1994), Fishwild, D. étal., Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.

The HCo7, HCo12, HCo17 and HCo20 mice hâve a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 811-820 (1993)), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), and a KCo5 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)). Additionally, the HCo7 mice hâve a HCo7 human heavy chain transgene (as described in US 5,770,429), the HCo12 mice hâve a HCo12 human heavy chain transgene (as described in Example 2 of WO 01/14424), the HCo17 mice hâve a HCo17 human heavy Chain transgene (as described in Example 2 of WO 01/09187) and the HCo20 mice hâve a HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of the endogenous mouse heavy and kappa light chain loci.

In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 02/43478. HCo12-Balb/c, HCo17-Balb/c and HCo20-Balb/c mice can be generated by Crossing HCo12, HCo17 and HCo20 to KCo5[J/K](Balb) as described in WO 09/097006.

The rTg4510 mouse is a known tauopathy model providing temporal and spatial control over mutant tau transgene expression. In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain trans-chromosome composed of chromosome 14 epitope-binding fragment hCF (SC20) as described in WO 02/43478.

Splénocytes from these transgenic mice may be used to generate hybridomas that secrete humanized monoclonal antibodies according to well-known techniques. Humanized monoclonal or polyclonal antibodies of the présent invention, or antibodies, of the présent invention originating from other species may also be generated transgenically through the génération of another non-human mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. In connection with the transgenic production in mammals, antibodies may be produced in, and recovered from, the milk of goats, cows, or other mammals. See for instance US 5,827,690; US 5,756,687; US 5,750,172 and US 5,741,957.

The antibody of the invention may be of any isotype. The choice of isotype typically will be guided by the desired effector functions, such as ADCC induction. Exemplary isotypes are lgG1, lgG2, lgG3, and lgG4. Either of the human light chain constant domains, kappa or lambda, may be used. If desired, the class of an anti-tau antibody of the présent invention may be switched by known methods. For example, an antibody of the présent invention that was originally IgM may be class switched to an IgG antibody of the présent invention. Further, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGI to lgG2. Thus, the effector function of the antibodies of the présent invention may be changed by isotype switching to, e.g., an lgG1, lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In one embodiment an antibody of the présent invention is an IgG 1 antibody, for instance an lgG1, χ. An antibody is said to be of a particular isotype if its amino acid sequence is most homologous to that isotype, relative to other isotypes.

In one embodiment, the antibody of the invention is a full-length antibody, preferably an IgG antibody, in particular an lgG1, χ antibody. In another embodiment, the antibody of the invention is an antibody epitope-binding fragment or a single-chain antibody.

Antibodies and epitope-binding fragments thereof may e.g. be obtained by epitope-binding fragmentation using conventional techniques, and epitope-binding fragments screened for utility in the same manner as described herein for whole antibodies. For example, F(ab')₂ epitope-binding fragments may be generated by treating antibody with pepsin. The resulting 5 F(ab')₂ epitope-binding fragment may be treated to reduce disulfide bridges to produce Fab' epitope-binding fragments. Fab epitope-binding fragments may be obtained by treating an IgG antibody with papain; Fab' epitope-binding fragments may be obtained with pepsin digestion of IgG antibody. An F(ab') epitope-binding fragment may also be produced by binding Fab'-described below via a thioether bond or a disulfide bond. A Fab' epitope-binding I0 fragment is an antibody epitope-binding fragment obtained by cutting a disulfide bond of the hinge domain of the F(ab')₂. A Fab'- epitope-binding fragment may be obtained by treating an F(ab’)2 epitope-binding fragment with a reducing agent, such as dithiothreitol. Antibody epitope-binding fragment may also be generated by expression of nucleic acids encoding such epitope-binding fragments in recombinant cells (see for instance Evans et al., J. 15 Immunol. Meth. 184, 123-38(1995)). For example, a chimeric gene encoding a portion of an F(ab’)2 epitope-binding fragment couid include DNA sequences encoding the CH1 domain and hinge domain of the H Chain, followed by a translational stop codon to yield such a truncated antibody epitope-binding fragment molécule.

In one embodiment, the anti-tau antibody is a monovalent antibody, preferably a monovalent 20 antibody as described in W02007059782 (which is incorporated herein by reference in its entirety) having a délétion of the hinge région. Accordingly, in one embodiment, the antibody is a monovalent antibody, wherein said anti-tau antibody is constructed by a method comprising : i) providing a nucleic acid construct encoding the light chain of said monovalent antibody, said construct comprising a nucléotide sequence encoding the VL région of a 25 selected antigen spécifie anti-tau antibody and a nucléotide sequence encoding the constant CL région of an Ig, wherein said nucléotide sequence encoding the VL région of a selected antigen spécifie antibody and said nucléotide sequence encoding the CL région of an Ig are operably linked together, and wherein, in case of an lgG1 subtype, the nucléotide sequence encoding the CL région has been modified such that the CL région does not contain any 30 amino acids capable of forming disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the CL région in the presence of polyclonal human IgG or when administered to an animal or human being; ii) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucléotide sequence encoding the VH région of a selected antigen spécifie antibody and a 35 nucléotide sequence encoding a constant CH région of a human Ig, wherein the nucléotide sequence encoding the CH région has been modified such that the région corresponding to the hinge région and, as required by the Ig subtype, other régions of the CH région, such as the CH3 région, does not comprise any amino acid residues which participate in the formation of disuîphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the CH région of the human Ig in the presence of polyclonal human IgG or when administered to an animal human being, wherein said nucléotide sequence encoding the VH région of a selected antigen spécifie antibody and said nucléotide sequence encoding the CH région of said Ig are operably linked together; iii) providing a cell expression System for producing said monovalent antibody; iv) producing said monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ü) in cells of the cell expression System of (iii).

Similarly, in one embodiment, the anti-tau antibody of the invention is a monovalent antibody, which comprises:

(i) a variable domain of an antibody of the invention as described herein or an epitopebinding part ofthe said domain, and (Ü) a CH domain of an immunoglobulin or a domain thereof comprising the CH2 and CH3 domains, wherein the CH domain or domain thereof has been modified such that the domain corresponding to the hinge domain and, if the immunoglobulin is not an lgG4 subtype, other domains of the CH domain, such as the CH3 domain, do not comprise any amino acid residues, which are capable of forming disulfide bonds with an identical CH domain or other covalent or stable non-covalent inter-heavy chain bonds with an identical CH domain in the presence of polyclonal human IgG.

In a further embodiment, the heavy chain of the monovalent antibody of the invention has been modified such that the entire hinge région has been deleted.

In another further embodiment, the sequence ofthe monovalent antibody has been modified so that it does not comprise any acceptor sites for N-linked glycosylation.

The invention also includes “Bispecific Antibodies,” wherein an anti-tau binding région (e.g., a tau-binding région of an anti-tau monoclonal antibody) is part of a bivalent or polyvalent bispecific scaffold that targets more than one epitope, (for example a second epitope could comprise an epitope of an active transport receptor, such that the Bispecific Antibody would exhîbit improved transcytosis across a biological barrier, such as the Blood Brain Barder). Thus, in another further embodiment, the monovalent Fab of an anti-tau antibody may be joined to an additional Fab or sefv that targets a different protein to generate a bispecific antibody. A bispecific antibody can hâve a dual function, for example a therapeutic function imparted by an anti-tau binding domain and a transport function that can bind to a receptor molécule to enhance transfer cross a biological barrier, such as the blood brain barrier.

Antibodies and epitope-binding fragments thereof of the invention, also include single chain antibodies. Single chain antibodies are peptides in which the heavy and light chain Fv domains are connected. In one embodiment, the présent invention provides a single-chain Fv (scFv) wherein the heavy and light chains in the Fv of an anti-tau antibody of the présent invention are joined with a flexible peptide linker (typically of about 10, 12, 15 or more amino acid residues) in a single peptide chain. Methods of producing such antibodies are described in for instance US 4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science 242, 423-426 (1988), Huston et al., PNAS USA 85, 5879-5883 (1988) and McCafferty et al., Nature 348, 552-554 (1990). The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used.

The antibodies and epitope-binding fragments thereof described herein may be modified by inclusion may be modified by inclusion of any suitable number of modified amino acids and/or associations with such conjugated substituents. Suitability in this context is generally determined by the ability to at least substantially retain the tau selectivity and/or the tau specificity associated with the non-derivatized parent anti-tau antibody. The inclusion of one or more modified amino acids may be advantageous in, for example, increasing polypeptide sérum half-life, reducing polypeptide antigenicity, or increasing polypeptide storage stability. Amino acid(s) are modified, for example, co-translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means. Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenylated (e.g., farnesylated, geranyl-geranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEGylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like. Référencés adéquate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols On CD-Rom, Humana Press, Totowa, NJ. The modified amino acid may, for instance, be selected from a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent.

The antibodies and epîtope-binding fragments thereof of the invention, may also be chemically modified by covalent conjugation to a polymer to for instance increase their circulating half-life. Exemplary polymers, and methods to attach them to peptides, are illustrated in for instance US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. Additional illustrative polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG with a molecular weight of between about 1,000 and about 40,000, such as between about 2,000 and about 20,000, e.g., about 3,000-12,000 g/mol).

The antibodies and epîtope-binding fragments thereof of the present invention may further be used in a diagnostic method or as a diagnostic imaging ligand.

In one embodiment, antibodies and epîtope-binding fragments thereof of the invention comprising one or more radiolabeled amino acids are provided. A radiolabeled anti-tau antibody may be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molécules is another possible feature). Non-limiting examples of such labels include, but are not limited to bismuth (²¹³Bi), carbon (¹¹C, ¹³C, ¹⁴C), chromium (⁵¹Cr), cobalt (⁵⁷Co, ⁶⁰Co), copper (⁶⁴Cu), dysprosium (¹⁶⁵Dy), erbium (^1B9Er), fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga), germanium (⁶⁸Ge), gold (¹⁹⁸Au), holmium (¹⁶⁶Ho), hydrogen (³H), indium (¹¹Ίη, ¹¹²ln, ¹¹³ln, ¹¹⁵ln), iodine (¹²¹l, ¹²³l, ¹²⁵l, ¹³¹l), iridium (¹⁹²lr), iron (⁵⁹Fe), krypton (^81mKr), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu), manganèse (⁵⁴Mn), molybdenum (Mo), nitrogen (¹³N, ¹⁵N), oxygen (¹⁵O), palladium (¹⁰³Pd), phosphorus (³²P), potassium (⁴²K), praseodymium (¹⁴²Pr), prométhium (¹⁴⁹Pm), rhénium (¹⁸⁶Re, ¹⁸⁸Re), rhodium (¹⁰⁵Rh), rubidium (⁸¹Rb, ⁸²Rb), ruthénium (⁸²Ru, ⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc), sélénium (⁷⁵Se), sodium (²⁴Na), strontium (⁸⁵Sr, ⁸⁹Sr, ^9ZSr), sulfur (³⁵S), technetium (⁹⁹Tc), thallium (^2O1T1), tin (¹¹³Sn, ¹¹⁷Sn), xénon (¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn) and zirconium (⁸⁹Zr). Zirconium (⁸⁹Zr) is particularly interesting. Methods for preparing radiolabeled amino acids and related peptide dérivatives are known in the art (see for instance Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2nd édition, Chaîner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581; US 4,735,210; US 5,101,827; US 5,102,990 (US RE35.500), US 5,648,471 and US 5,697,902. For example, a radioisotope may be conjugated by a chloramine T method (Lindegren, S. et al. (1998) “Chloramine-T In High-Specific-Activity Radioiodlnation Of Antibodies Using N-Succinimidyl3-(Trimethylstannyl)Benzoate As An Intermediate,” Nucl. Med. Biol. 25(7):659-665; Kurth, M. et al. (1993) “Site-Specific Conjugation Of A Radioiodinated Phenethylamine Dérivative To A Monoclonal Antibody Results In Increased Radioactivity Localization In Tumor,” J. Med. Chem. 36(9):1255-1261; Rea, D.W. et al. (1990) “Site-specifically radioiodinated antibody for targeting tumors, Cancer Res. 50(3 Suppl):857s-861s).

The invention also provides anti-tau antibodies and epitope-binding fragments thereof that are detectably labeled using a fluorescent label (such as a rare earth chelate (e.g., a europium chelate)), a fluorescein-type label (e.g., fluorescein, fluorescein isothiocyanate, 5carboxyfluorescein, 6-carboxy fluorescein, dichlorotriazinylamine fluorescein), a rhodaminetype label (e.g., ALEXA FLUOR® 568 (Invitrogen), TAMRA® or dansyl chloride), VIVOTAG 680 XL FLUOROCHROME™ (Perkin Elmer), phycoerythrin; umbelliferone, Lissamine; a cyanine; a phycoerythrin, Texas Red, BODIPY FL-SE® (Invitrogen) or an analogue thereof, ail of which are suitable for optical détection. Chemiluminescent labels may be employed (e.g., luminol, luciferase, luciferin, and aequorin). Such diagnosîs and détection can also be accomplished by coupling the diagnostic molécule of the présent invention to détectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, or to prosthetic group complexes such as, but not limited to, streptavidin/biotin and avïdin/biotin.

Chemiluminescent labels may be employed (e.g., luminol, luciferase, luciferin, and aequorin). Such diagnosis and détection can also be accomplished by coupling the diagnostic molécule of the présent invention to détectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, or to prosthetic group complexes such as, but not limited to, streptavidin/biotin and avidin/biotin. Paramagnetic labels can also be employed, and are preferably detected using Positron Emission Tomography (PET) or Single-Photon Emission Computed Tomography (SPECT). Such paramagnetic labels include, but are not limited to compounds containing paramagnetic ions of Aluminum (Al), Barium (Ba), Calcium (Ca), Cérium (Ce), Dysprosium (Dy), Erbium (Er), Europium (Eu), Gandolinium (Gd), Holmium (Ho), Iridium (lr), Lithium (Li), Magnésium (Mg), Manganèse (Mn), Molybdenum (M), Neodymium (Nd), Osmium (Os), Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), Ruthénium (Ru), Samarium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm), Tin (Sn), Titanium (Ti), Tungsten (W), and Zirconium (Zi), and particulariy, Co*², CR*², Cr*³, Cu*^z, Fe*², Fe*³, Ga*³, Mn*³, Ni*², Ti⁺³, V*³, and V*⁴, positron emitting metals using various positron émission tomographies, and nonradioactive paramagnetic métal ions.

Thus in one embodiment the anti-tau antibody or tau-binding fragment thereof of the invention may be labelled with a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotopic label or an enzyme label. The labelled antibody of fragment may be used in detecting or measuring the presence or amount of said tau in the brain of a subject.

al., Methods in Enzymol 153, 516-544 (1987), Mattanovich, D. et al. Methods Mol. Biol. 824, 329-358 (2012), Celik, E. étal. Biotechnol. Adv. 30(5), 1108-1118 (2012), Li, P. et al. Appl. Biochem. Biotechnol. 142(2), 105-124 (2007), Bôer, E. et al. Appl. Microbiol. Biotechnol. 77(3), 513-523 (2007), van der Vaart, J.M. Methods Mol. Biol. 178, 359-366 (2002), and

Holliger, P. Methods Mol. Biol. 178, 349-357 (2002)).

In an expression vector of the invention, anti-tau antibody-encoding nucleic acids may comprise or be associated with any suitable promoter, enhancer, and other expressionfacilitating éléments. Examples of such éléments include strong expression promoters (e.g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR 10 promoters), effective poly (A) termination sequences, an origin of réplication for plasmid product in E. coli, an antibiotic résistance gene as selectable marker, and/or a convenient cloning site (e.g., a polylïnker). Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE (the skilled artisan will recognize that such terms are actually descriptors of a degree of gene expression under certain conditions).

In an even further aspect, the invention relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody or epitope-binding fragment thereof of the invention as defined herein or a bispecific molécule of the invention as defined herein. Examples of host cells include yeast, bacteria, and mammalian cells, such as CHO or HEK cells. For example, in one embodiment, the présent invention provides a cell 20 comprising a nucleic acid stably integrated into the cellular genome that comprises a sequence coding for expression of an anti-tau antibody of the présent invention or an epitope-binding fragment thereof. In another embodiment, the présent invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of an anti-tau antibody or epitope-binding fragment thereof of the invention.

In a further aspect, the invention relates to a method for producing an anti-tau antibody of the invention, said method comprising the steps of a) culturing a hybridoma or a host cell of the invention as described herein above, and b) purifying the antibody of the invention from the culture media.

In one embodiment, the invention relates to a préparation that, as such term is used herein, comprises an anti-tau antibody as defined herein, and that is substantially free of naturallyarising antibodies that are either not capable of binding to tau or that do not materially alter the anti-tau functionality of the préparation. Thus, such a préparation does not encompass naturally-arising sérum, or a purified dérivative of such sérum, that comprises a mixture of an anti-tau antibody and another antibody that does not alter the functionality of the anti-tau antibody of the préparation, wherein such functionality is:

(i) a substantial inability to bind to non-phosphorylated tau;

(ii) a substantial inability to bind to tau that is phosphorylated at S404 and not phosphorylated at S396;

(iii) the ability to bind to tau phosphorylated at S396;

(iv) the ability to bind to tau phosphorylated at both S396 and at S404;

(v) the ability to selectively discriminate between phosphorylated tau residues S396 and S404 such that it is substantially unable to bind the phosphorylated 404 residue;

(vi) the ability to bind hyper-phosphorylated tau from human Alzheimer's disease brains;

(vii) the ability to discriminate between pathological and non-pathological human tau protein; and/or (viii) the capability, when used as described herein with immune-depleted rTg4510 extracts from transgenic mice, to specifically reduce the hyperphosphorylated tau 64kDa and 70kDa bands by at least 90%, while not reducing the 55 kDa tau band by more than 10% or the capability, when used as described herein with extracts from human AD post-mortem brains, to specifically reduce the S396 phosphorylated hyperphosphorylated tau bands by at least 90%, while not reducing the non-hyperphosphorylated tau bands by more than 10%.

The invention particularly relates to préparations of such an anti-tau antibody having a structural change in its amino acid sequence (in any of its CDRs, variable domains, framework residues and/or constant domains) relative to the structure of a naturallyoccurring anti-tau antibody, wherein said structural change causes the anti-tau antibody to exhibit a markedly altered functionality (i.e., more than a 20% différence, more than a 40% différence, more than a 60% différence, more than an 80% différence, more than a 100% différence, more than a 150% différence, more than a 2-fold différence, more than a 4-fold différence, more than a 5-fold différence, or more than a 10-fold différence in functionality) relative to the functionality exhibited by said naturally-occurring anti-tau antibody; wherein such functionality is:

(i) a substantial inability to bind to non-phosphorylated tau;

(ii) a substantial inability to bind to tau that is phosphorylated at S404 and not phosphorylated at S396;

(iii) the ability to bind to tau phosphorylated at S396;

(iv) the ability to bind to tau phosphorylated at both S396 and at S404;

(v) the ability to selectively discriminate between phosphorylated tau residues S396 and S404 such that it is substantially unable to bind the phosphorylated 404 residue;

(vi) the ability to bind hyper-phosphorylated tau from human Alzheimer's disease brains;

(vü) the ability to discriminate between pathological and non-pathological human tau protein; and/or (vîii) the capability, when used as described herein with immune-depleted rTg4510 extracts from transgenic mice, to specifically reduce the hyperphosphorylated tau 64kDa and 70kDa bands by at least 90%, while not reducing the 55 kDa tau band by more than 10%; or the capability, when used as described herein with extracts from human AD post-mortem brains to specifically reduce the S396 phosphorylated hyperphosphorylated tau bands by at least 90%, while not reducing the non-hyperphosphorylated tau bands by more than 10%.

The term “substantially free of naturally-arising antibodies refers to the complété absence of such naturally-arising antibodies in such préparations, or of the inclusion of a concentration of such naturally-arising antibodies in such préparations that does not materially affect the tau-binding properties of the préparations. An antibody is said to be “isolated” if it has no naturally-arising counterpart or has been separated or purified from components which naturally accompany it.

The term “naturally-arising antibodies,” as it relates to such préparations, refers to antibodies (including naturally-arising autoantibodies) elicited within living humans or other animais, as a natural conséquence to the functioning of their immune Systems.

Thus, the préparations of the présent invention do not exclude, and indeed explicitly encompass, such préparations that contain an anti-tau antibody and a deliberately added additional antibody capable of binding to an epitope that is not possessed by tau. Such préparations particularly include embodiments thereof wherein the préparation exhibits enhanced efficacy in treating Alzheimer’s disease (AD), Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP), and Corticobasal Degeneration (CBD). Furthermore, the présent invention is directed to préparations that contain an anti-tau antibody antibodies, or epitope-binding fragments thereof, intended for use in the treatment of Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD, and psychiatrie symptoms of patients with Lewy body dementia. Furthermore, the préparations of the présent invention contain an anti-tau antibody antibodies, or epitope-binding fragments thereof, that may be used in the treatment of stroke, stroke recovery, neurodegeneration in relation to Parkinson s disease.

In an even further aspect, the invention relates to a pharmaceutical composition comprising:

(i) a tau antibody, or epitope-binding fragment thereof, both as defined herein, or a préparation, as such term is defined herein, that comprises such an anti-tau antibody or epitope-binding fragment thereof; and (ii) a pharmaceutically-acceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2013.

The pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the chosen compound of the présent invention and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant négative impact on the desired biological properties of the chosen compound or pharmaceutical composition of the présent invention (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.)) on epitope binding.

A pharmaceutical composition of the présent invention may also include diluents, fillers, salts, buffers, détergents (e.g., a nonionic detergent, such as Tween-20 or Tween- 80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition. The diluent is selected to not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer’s solutions, dextrose solution, and Hank’s solution. In addition, the pharmaceutical composition or formulation may also include other carriers, or non-toxic, nontherapeutic, non-immunogenic stabilizers and the like. The compositions may also include large, slowly metabolized macromolecules, such as proteins, polysaccharides like chitosan, polylactic acids, polyglycoltc acids and copolymers (e.g., latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).

The actual dosage levels of the active ingrédients in the pharmaceutical compositions of the présent invention may be varied so as to obtain an amount of the active ingrédient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level will dépend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the présent invention employed, or the amide thereof, the route of administration, the time of administration, the rate of excrétion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the âge, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable route and mode, including: parentéral, topical, oral or intranasal means for prophylactic and/or therapeutic treatment. In one embodiment, a pharmaceutical composition of the présent invention is administered parenterally. The phrases parentéral administration and administered parenterally as used herein means modes of administration other than enterai and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, épidural and intrasternal injection and infusion.

Additional suitable routes of administering a compound of the présent invention in vivo and in vitro are well known in the art and may be selected by those of ordinary skill in the art.

In one embodiment that pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and ail suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a compound of the présent invention.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the présent invention include water, saline, phosphate buffered saline, éthanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloïdal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include stérile aqueous solutions or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the présent invention is contemplated.

Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the présent invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteïne hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;

(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) métal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the présent invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the présent invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds of the présent invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery Systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodégradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the préparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

ln one embodiment, the compounds of the présent invention may be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parentéral administration include stérile aqueous solutions or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the présent invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.

Pharmaceutical compositions for injection must typically be stérile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, micro-emulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be an aqueous or non-aqueous solvent or dispersion medium containing for instance water, éthanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required partîcle size in the case of dispersion and by the use of surfactants. In many cases, it will be préférable to include isotonie agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays antibody absorption, for example, monostearate salts and gelatin. Stérile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingrédients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a stérile vehicle that contains a basic dispersion medium and the required other ingrédients e.g. from those enumerated above. In the case of stérile powders for the préparation of stérile injectable solutions, examples of methods of préparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingrédient plus any additional desired ingrédient from a previousiy sterile-filtered solution thereof.

Stérile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingrédients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a stérile vehicle that contains a basic dispersion medium and the required other ingrédients from those enumerated above. In the case of stérile powders for the préparation of stérile injectable solutions, examples of methods of préparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingrédient plus any additional desired ingrédient from a previously sterile-filtered solution thereof.

Dosage regimens in the above methods of treatment and uses described herein are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parentéral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrète units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The spécification for the dosage unit forms of the present invention are dictated by and directly dépendent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inhérent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

The effective dosages and the dosage regimens for the antibodies or epitope-binding fragments thereof of the invention, dépend on the disease or condition to be treated and may be determined by persons skilled in the art. On any given day that a dosage is given, the dosage may range from about 0.0001 to about 100 mg/kg, and more usually from about 0.01 to about 5 mg/kg, of the host body weight. For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg body weight. Exemplary dosages thus include: from about 0.1 to about 10 mg/kg/body weight, from about 0.1 to about 5 mg/kg/body weight, from about 0.1 to about 2 mg/kg/body weight, from about 0.1 to about 1 mg/kg/body weight, for instance about 0.15 mg/kg/body weight, about 0.2 mg/kg/body weight, about 0.5 mg/kg/body weight, about 1 mg/kg/body weight, about 1.5 mg/kg/body weight, about 2 mg/kg/body weight, about 5 mg/kg/body weight, or about 10 mg/kg/body weight.

A physician having ordinary skill in the art may readily détermine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of an antibody or epitope-binding fragment thereof of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally dépend upon the factors described above. Administration may e.g. be intravenous, întramuscular, intraperitoneal, or subcutaneous. If desired, the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is préférable to administer the compound as a pharmaceutical composition as described above.

The labeled antibodies or epitope-binding fragments thereof of the invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases or disorders. The invention provides for the détection or diagnosis of a neurodegenerative or cognitive disease or disorder, including but not limited to Alzheimer’s disease, Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP), and Corticobasal Degeneration (CBD), comprising:

(a) assaying the existence of pyroglutamated Αβ fragments in cells or tissue samples of a subject using one or more antibodies that specifically bind to tau; and (b) comparing the level of the antigen with a control level, e.g. levels in normal tissue samples, whereby an increase in the assayed level of antigen compared to the control level of antigen is indicative of the disease or disorder, or indicative of the severîty of the disease or disorder.

The antibodies or epitope-binding fragments thereof of the invention can be used to assay tau or fragments of tau in a biological sample using immunohistochemical methods wellknown in the art. Other antibody-based methods useful for detecting protein include immunoassays such as the enzyme linked immunoassay (ELISA) and the radioimmunoassay assay (RIA) and mesoscale discovery platform based assays (MSD). Suitable antibody labels may be used in such kits and methods, and labels known in the art include enzyme labels, such as alkaline phosphatase and glucose oxidase; radioisotope labels, such as iodine (¹²⁵l, ¹³¹l), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹ln), and technetium (^99mTc); and luminescent labels, such as luminol and luciferase; and fluorescent labels, such as fluorescein and rhodamine.

The presence of labeled anti-tau antibodies or their tau-binding fragments may be detected in vivo for diagnostic purposes. In one embodiment, diagnosis comprises: a) administering to a subject an effective amount of such labeled molécule; b) waiting for a time interval following administration to allow the labeled molécule to concentrate at sites (if any) of Αβ déposition and to allow for unbound labeled molécule to be cleared to background level; c) determining a background level; and d) detecting the labeled molécule in the subject, such that détection of labeled molécule above the background level is indicative that the subject has the disease or disorder, or is indicative of the severity of the disease or disorder. In accordance with such embodinnent, the molécule is labeled with an imaging moiety suitable for détection using a particular imaging system known to those skilled in the art. Background levels may be determined by various methods known in the art, including comparing the amount of labeled antibody detected to a standard value previously determined for a particular imaging system. Methods and Systems that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as positron émission tomography (PET), magnetic résonance imaging (MRI), and sonography.

In a further aspect, the invention provides a monoclonal antibody, or an epitope-binding fragment thereof, as defined herein for use in therapy.

In a further aspect, the invention provides a monoclonal antibody, or an epitope-binding fragment thereof, as defined herein for use in treating, diagnosing or imaging of tauopathies.

In a further aspect, the invention provides a monoclonal antibody, or an epitope-binding fragment thereof, as defined herein for use in treating Alzheimer’s disease, Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP), and Corticobasal Degeneration (CBD).

In a further aspect, the invention provides a monoclonal antibody, or an epitope-binding fragment thereof, as defined herein for use in the manufacture of a médicament for treating, diagnosing or imaging tauopathies.

Preferably, the médicament is for treating Alzheimer’s disease (AD), Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP), and Corticobasal Degeneration (CBD) most preferably Alzheimer's disease (AD). The médicament is also preferably for the treatment of Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD, and psychiatrie symptoms of patients with Lewy body dementia.

In a further aspect, the invention provides a method of treating, diagnosing or imaging Alzheimer’s disease or other tauopathies in a subject, said method comprising administering the médicament monoclonal antibody or epitope-binding fragment thereof as defined herein, to said subject in an effective amount.

In a preferred embodiment, the treatment is chronic, preferably for at least 2 weeks, such as at least for 1 month, 6, months, 1 year or more.

In a further aspect, the invention provides a kit comprising the antibody, or fragment thereof, as defîned herein for use in therapy.

Embodiments

1. A monoclonal antibody, or an epitope-binding fragment thereof, capable of immunospecifîcally binding to the phosphorylated residue 396 of human tau, such as phosphorylated residue 396 of SEQ ID NO:33.

2. The antibody according to embodiment 1 consisting of an intact antibody.

3. The antibody or epitope-binding fragment thereof according to embodiment 1 or 2 comprising or consisting of an epitope-binding fragment selected from the group consisting of: an Fv fragment (e.g. single chain Fv and disulphide-bonded Fv); a Fablike fragment (e.g. Fab fragment, Fab' fragment and F(ab)2 fragment); a mini-body (Fv)2-CH3 domain, and a domain antibody (e.g. a single VH variable domain or VL variable domain).

4. The antibody or epitope-binding fragment thereof according to any preceding embodiment, wherein the antibody is selected from the group consisting of antibodies of subtype lgG1, lgG2, lgG3, or lgG4.

5. The monoclonal antibody or epitope-binding fragment thereof according to any of the previous embodiments which is human or humanized.

6. The monoclonal antibody, or epitope-binding fragment thereof, according to any one of the preceding embodiments wherein the antibody or epitope-binding fragment exhibits one or more of the following properties (a) selectivity and specificity for human pathological tau;

(b) a binding affinity (KD) for p-Tau 386-408 (pS396/pS404) (SEQ ID NO:33) between 0.5-10 nM, such as 1-5 nM or 1-2 nM

7. The monoclonal antibody, or epitope-binding fragment thereof, according to any one of the preceding embodiments, wherein said antibody does not substantially bind the phosphorylated 404 residue on tau (SEQ ID NO:33).

8. A monoclonal antibody, or an epitope-binding fragment thereof comprising:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.

9. The monoclonal antibody according to embodiment 8, comprising the heavy chain variable domain of SEQ ID NO:8 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence and/or the light chain variable domain of SEQ ID NO:7, having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.

10. A monoclonal antibody, or an epitope-binding fragment thereof, comprising:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.

11. The monoclonal antibody according to embodiment 10, comprising the heavy chain variable domain of SEQ ID NO:16 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence and or the light chain variable domain of SEQ ID NO:15 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.

12. A monoclonal antibody, wherein the epitope-binding fragment comprises:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.

13. The monoclonal antibody according to embodiment 12, comprising the heavy chain variable domain of SEQ ID NO:24 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence and or the light chain variable domain of SEQ ID NO:23 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.

14. A monoclonal antibody, or an epitope-binding fragment thereof comprising:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:26 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:28 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:30 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.

15. The monoclonal antibody according to embodiment 14, comprising the heavy chain variable domain of SEQ ID NO:32 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence and or the light chain variable domain of SEQ ID NO:31 or an amino acid sequence having no more than 4 amino acid différences, or no more than 3 amino acid différences, or no more than 2 amino acid différences, or no more than 1 amino acid différence.

16. The antibody or epitope-binding fragment thereof according to one of embodiments 1 to 7, wherein said antibody or fragment thereof competes with the antibody or epitope-binding fragment thereof defined in Embodiment 8-15 for binding to human tau.

17. The antibody or epitope-binding fragment thereof according to any preceding embodiment comprising an Fc domain.

18. The antibody or epitope-binding fragment thereof according to any preceding embodiment further comprising a moiety for increasing in vivo half-life.

19. The antibody or epitope-binding fragment thereof according to Embodiment 18, wherein the moiety for increasing the in vivo half-life is selected from the group consisting of polyethylene glycol (PEG), human sérum albumin, glycosylation groups, fatty acids and dextran.

20. The antibody or epitope-binding fragment thereof according to any preceding embodiment wherein the antibody further comprises a détectable moiety.

21. The antibody or epitope-binding fragment thereof according to Embodiment 20 wherein the détectable moiety is selected from the group consisting of: a fluorescent label; a chemiluminescent label; a paramagnetic label; a radio-isotopic label; or an enzyme label.

22. The antibody or epitope-binding fragment thereof according to Embodiments 20 or 21 wherein the détectable moiety comprises or consists of a radioisotope.

23. The antibody or epitope-binding fragment thereof according to Embodiment 22 wherein the radioisotope is selected from the group consisting of 99mTc, 1111n, 67Ga, 68Ga, 72As, 89Zr, 1231 and 201TI.

24. The antibody or epitope-binding fragment thereof according to Embodiment 21 wherein the détectable moiety comprises or consists of a paramagnetic isotope.

25. The antibody or epitope-binding fragment thereof according to Embodiment 24 wherein the paramagnetic isotope is selected from the group consisting of 157Gd, 55Mn, 162Dy, 52Cr and 56Fe.

26. The antibody or epitope-binding fragment thereof according to any of Embodiments 20 to 25 wherein the détectable moiety is détectable by an imaging technique such as SPECT, PET, MRI, optical or ultrasound imaging.

27. The antibody or epitope-binding fragment thereof according to any of Embodiments 20 to 26 wherein the détectable moiety is joîned to the antibody or epitope-binding fragment thereof indirectly, via a linking moiety.

28. The antibody or epitope-binding fragment thereof according to Embodiment 27 wherein the linking moiety is selected from the group consisting of dérivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), deferoxamine (DFO), dérivatives of dîethylenetriaminepentaacetic avid (DTPA), dérivatives of S-2(4-lsothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and dérivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA).

29. The monoclonal antibody, or epitope-binding fragment thereof wherein the Heavy Chain is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, and SEQ ID NO:35, and the Light Chain is selected from the group consisting ôf SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, and SEQ ID NO:36v

30. A monoclonal antibody, or epitope-binding fragment thereof comprising (a) a Heavy Chain CDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, and SEQ ID NO:28;

(b) a Heavy Chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:21, and SEQ ID NO:29; and (c) a Heavy Chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22, and SEQ ID NO:30; and (d) a Light Chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19, and SEQ ID NO:27.

31. The antibody of the invention, or epitope-binding fragment thereof according to any one of Embodiments 1 to 7 comprising

a) a Heavy Chain CDR1 comprising the amino acid sequence of SEQ ID NO:20;

(b) a Heavy Chain CDR2 comprising the amino acid sequence of SEQ ID NO:21;

(c) a Heavy Chain CDR3 comprising the amino acid sequence of SEQ ID NO:22; and (d) a Light Chain CDR3 comprising the amino acid sequence of SEQ ID NO:19.

32. An isolated nucleic acid molécule encoding an antibody or epitope-binding fragment thereof as defined in any of Embodiments 1 to 31.

33. A nucleic acid molécule according to Embodiment 32 wherein the molécule is a cDNA molécule.

34. A vector comprising a nucleic acid molécule as defined in Embodiment 32 or 33.

35. A recombinant host cell comprising a nucleic acid molécule as defined in any of Embodiments 32 to 34.

36. A method for producing an antibody or epitope-binding fragment as defined in any of Embodiments 1 to 31, the method comprising culturing a host cell as defined in Embodiment 35 under conditions which permit expression of the encoded antibody or epitope-binding fragment thereof.

37. A préparation comprising the antibody or epitope-binding fragment thereof according to any one of the previous daims, wherein said préparation is substantially free of naturally-arising antibodies that are either not capable of binding to tau or that do not materially alter an anti-tau functionality of the préparation, wherein said functionality is selected from the group consisting of:

(i) a substantial inability to bind to non-phosphorylated tau;

(ii) a substantial inability to bind to tau that is phosphorylated at S404 and not phosphorylated at S396;

(iii) the ability to bind to tau phosphorylated at S396;

(iv) the ability to bind to tau phosphorylated at both S396 and at S404;

(v) the ability to selectively discriminate between phosphorylated tau residues S396 and S404 such that it is substantially unable to bind the phosphorylated 404 residue;

(vi) the ability to bind hyper-phosphorylated tau from human Alzheimer's disease brains;

(vii) the ability to discriminate between pathological and non-pathological human tau protein; and/or (viii) the capability, when used as described herein with immune-depleted rTg4510 extracts from transgenic mice, to specifically reduce the hyperphosphorylated tau 64kDa and 70kDa bands by at least 90%, while not reducing the 55 kDa tau band by more than 10% %; or the capability, when used as described herein with extracts from human AD post-mortem brains, to specifically reduce the S396 phosphorylated hyperphosphorylated tau bands by at least 90%, while not reducing the non-hyperphosphorylated tau bands by more than 10%.

38. A préparation comprising the antibody or epitope-binding fragment thereof according to any one of the previous claims, wherein said antibody or said epitope-binding fragment thereof possesses a structural change in its amino acid sequence, relative to the structure of a naturally-occurring anti-tau antibody, wherein said structural change causes said antibody or said fragment to exhibit an altered functionality relative to the functionality exhibited by said naturally-occurring anti-tau antibody, wherein said functionality is selected from the group consisting of:

(i) a substantiel inability to bind to non-phosphorylated tau;

(ii) a substantial inability to bind to tau that is phosphorylated at S404 and not phosphorylated at S396;

(iii) the ability to bind to tau phosphorylated at S396;

(iv) the ability to bind to tau phosphorylated at both S396 and at S404;

(v) the ability to selectively discriminate between phosphorylated tau residues S396 and S404 such that it is substantially unable to bind the phosphorylated 404 residue;

(vi) the ability to bind hyper-phosphorylated tau from human Alzheimer’s disease brains;

(vii) the ability to discriminate between pathological and non-pathological human tau protein; and/or (viii) the capability, when used as described herein with immune-depleted rTg4510 extracts from transgenic mice, to specifically reduce the hyperphosphorylated tau 64kDa and 70kDa bands by at least 90%, while not reducing the 55 kDa tau band by more than 10%; or the capability, when used as described herein with extracts from human AD post-mortem brains, to specifically reduce the S396 phosphorylated hyperphosphorylated tau bands by at least 90%, while not reducing the non-hyperphosphorylated tau bands by more than 10%.

39. A pharmaceutical composition comprising the monoclonal antibody or epitopebinding fragment thereof as defined in any of embodiments 1 to 31, or the préparation as defined in any of embodiments 37-38; and a pharmaceutical acceptable carrier.

40. The monoclonal antibody, or fragment thereof, of any of embodiments 1-31, the préparation of any of embodiments 37-38, or the pharmaceutical composition of embodiment 39, for use in medicine.

41. The monoclonal antibody, or fragment thereof, of any of embodiments 1-31, the préparation of any of embodiments 37-38, or the pharmaceutical composition of embodiment 39, for use in treating a tauopathy.

42. The monoclonal antibody, or fragment thereof, the préparation, or the pharmaceutical composition, according to embodiment 41 wherein the tauopathy is selected from the group consisting of Alzheimer's disease, Argyrophilic Grain Disease (AGD),

Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Psychosis, particularly Psychosis due to AD or Psychosis in patients with AD, and psychiatrie symptoms of patients with Lewy body dementia.

43. Use of the monoclonal antibody, or fragment thereof, of any of embodiments 1-31, the préparation of any of embodiments 37-38, or the pharmaceutical composition of embodiment 39 in the manufacturing of a médicament for treating a tauopathy.

44. The use of the monoclonal antibody, or fragment thereof, the préparation, or the pharmaceutical composition according to embodiment 43 wherein the tauopathy is selected from the group consisting of Alzheimer’s disease, Argyrophilic Grain

Disease (AGD), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD, Psychosis due to AD or Psychosis in patients with AD, and psychiatrie symptoms of patients with Lewy body dementia.

45. A method of treating Alzheimer’s disease or other tauopathies in a subject, said method comprising administering the monoclonal antibody, or fragment thereof, of any of embodiments 1-31, the préparation of any of embodiments 37-38, or the pharmaceutical composition of embodiment 39 to said subject in an effective amount

46. The method according to embodiment 45, wherein the treatment is chronic.

47. The method according to embodiment 46, wherein the chronic treatment is for at least 2 weeks, such as at least for 1 month, 6, months, 1 year or more.

48. The method according to any one of embodiments 45 to 47, wherein the subject is human.

49. A kit comprising the monoclonal antibody, or fragment thereof, of any of embodiments 1-31, the préparation of any of embodiments 37-38, or the pharmaceutical composition of embodiment 39 for use in medicine.

50. The monoclonal antibody, or fragment thereof, of any of embodiments 1-31, the préparation of any of embodiments 37-38, or the pharmaceutical composition of embodiment 39 for use in detecting or measuring the presence or amount of said tau in the brain of a subject.

51. The monoclonal antibody, or fragment thereof, the préparation or the pharmaceutical 30 composition of embodiment 50, wherein said détection or measurement comprises in vivo imaging of said anti-tau antibody bound to said tau.

52. The monoclonal antibody, or fragment thereof, the préparation or the pharmaceutical composition of embodiment 50, wherein said détection or measurement comprises ex vivo imaging of said anti-tau antibody or said fragment thereof, bound to said tau.

53. A monoclonal antibody, or an epitope-binding fragment thereof, capable of immunospecifically binding to the phosphorylated residue 396 of human tau (SEQ ID NO:33) in the presence of human tau phosphorylated at residue 404 but not phosphorylated at residue 396.

54. A monoclonal antibody or an epitope-binding fragment thereof that exhibits immunospecifically binding to human tau comprising a phosphorylated residue 396 according to the test criteria: i) the antibody does not substantially bind to nonphosphorylated tau; ii) the antibody does not substantially bind to tau phosphorylated at 404 when 396 is not phosphorylated; iii) the antibody does bind to tau phosphorylated at 396; and iv) the antibody does bind to tau when both 396 and 404 are phosphorylated,

55. A monoclonal antibody, raised against the bi-phosphorylated peptide: TDHGAEIVYK^ip}SPWSGDT^(p,SPRHL (SEQ ID NO:37) covering residues 386-410 of 2N4R tau, or an epitope-binding fragment thereof, capable of immunospecifically binding to the phosphorylated residue 396 of human tau (SEQ ID NO:33).

56. The monoclonal antibody according to embodiment 55, wherein hybridomas are screened with human pathological and non-pathological tau to isolate clones that both i) were immunospecific towards the either of the phospho-epitopes S396 and ii) specifically recognize hyper-phosphorylated tau from human Alzheimer’s disease brains, wherein said antibodies are able to discriminate between pathological and non-pathological human tau protein.

57. A method of removing hyperphosphorylated Tau from a tangle said tangle comprising hyperphosphorylated Tau said method comprising contacting hyperphosphorylated Tau with an antibody, said antibody sélective for Tau having residue 396 phosphorylated, so as to resuit the tangle being depleted of 90% of hyperphosphorylated tau.

58. A method of delaying the progression of Alzheimer’s Disease in a patient said method comprising reducing or attenuating the accumulation of pathological tau protein in said patient, said method comprising administering an antibody which removes a tau protein with a phosphorylated 396 residue.

59. A method of delaying the progression of Alzheimer’s Disease in a patient said method comprising removing the tau proteins that seed for pathological tau proteins, wherein tau proteins having residue 396 phosphorylated are removed.

60. A method of treating a patient with Alzheimer’s Disease comprising removing hyperphosphorylated Tau from a tangle said tangle comprising hyperphosphorylated Tau and normal Tau by contacting hyperphosphorylated Tau with an antibody sélective for Tau having residue 396 phosphorylated.

61. A method according to any of embodiments 57 to 59 comprising the use of an antibody as defined in any one of embodiments 1 to 31, 40-42 or 50 to 56.

62. An isolated monoclonal antibody, or an isolated epitope-binding fragment thereof, capable of immunospecifically binding to the phosphorylated residue 396 of human tau (SEQ ID NO:33).

63. A recombinant human or recombinant humanized monoclonal antibody, or an isolated epitope-binding fragment thereof, capable of immunospecifically binding to the phosphorylated residue 396 of human tau (SEQ ID NO:33).

64. A recombinant monoclonal antibody, or an epitope-binding fragment thereof, raised against the bi-phosphorylated peptide: TDHGAEIVYK{p}SPWSGDT{p}SPRHL (SEQ ID NO:37) covering residues 386-410 of 2N4R tau, wherein said recombinant monoclonal antibody, or an epitope-binding fragment thereof, is capable of immunospecifically binding to the phosphorylated residue 396 of human tau (SEQ ID NO:33).

65. A pharmaceutical composition comprising an isolated monoclonal antibody, or an isolated epitope-binding fragment thereof, wherein said isolated monoclonal antibody, or an isolated epitope-binding fragment thereof is as defined in any one of the above embodiments.

66. A chimeric monoclonal antibody or an isolated epitope-binding fragment thereof, capable of immunospecifically binding to the phosphorylated residue 396 of human tau (SEQ ID NO:33).

67. An antibody, or antigen-binding fragment thereof, as defined in any of embodiments 1-31 and 51-56 which has been produced or manufactured in a cell line such as a human cell line, a mammal non-human cell line, an insect, yeast or bacterial cell line.

68. The antibody, or antigen binding fragment thereof, according to embodiment 67 produced in a CHO cell line, HEK cell line, BHK-21 cell line, murine cell line (such as a myeloma cell line), fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAP cell line and HuH-7 human cell line.

EXAMPLES

Example 1: Immunisation of mice with phospho-peptides 396/404

C56/BL6 and FVB mice were immunised with 10 pg P30 conjugated phosphorylated tau 386-408 (pS396/pS404) (SEQ ID NO:37) formulated in TiterMax adjuvant.

Mice (C56/BL6 and FVB strains, female and male. 2-to 3-month-old mice were immunized with peptide epitope P30 conjugated phosphorylated tau 386-408.

Immunogenic P30 conjugated phosphorylated tau 386-408 (pS396/pS404) peptide was formulated in TiterMax (400 pg/ml peptide mixed 1:1 vokvol) following the TiterMax/vendor protocol and mice were injected subcutaneously with 20 pg peptide (100 μΙ) of antigen. Control mice were injected with adjuvant only. Ail peptide-immunised mice were boosted with 0.5 pg peptide/Titermax (10 pg/ml peptide formulated as described above and injected) at monthly intervals. The mice were finally boosted with P30 conjugated phosphorylated tau 386-408 (pS396/pS404) without Titermax 3 days prior to fusion of splénocytes with SP-2 cells. Hybridomas were selected for re-cloning cycles after exhibiting positive binding to ELISA plates that had been coated with 1 pg/ml phosphorylated tau 386-408 (pS396/pS404), and exhibiting preferential binding activity to S1 and P3 antigens from AD and TG4510 brain lysate (described below in Example 3). Such binding was compared with the binding activity of such antibodies to brain lysate from Controls, using dot blots and brain lysate coated ELISA or MSD plates.

Example 2: Hybridoma génération

The mice were boosted with P30 conjugated phosphorylated tau 386-408 (pS396/pS404) without Titermax 3 days prior to fusion of splénocytes with SP-2 cells. Hybridomas were selected for re-cloning cycles after positive binding in ELISA plates coated with 1 pg/ml phosphorylated tau 386-408 (pS396/pS404), and preferential binding activity to S1 and P3 antigens from AD and TG4510 brain lysate in comparison to brain lysate from Controls using dot blots and brain lysate coated ELISA or MSD plates.

Example 3 Western blot and dot-blot analysis of spécifie antibodies

Tau biochemical fractionation

Brain tissues from humans or rTg4510 mice overexpressing the human tau mutation P301L were homogenized in 10 volumes of Tris-buffered saline containing protease and phosphatase inhibitors as follows: 50 mM Tris/HCI (pH 7.4); 274 mM NaCI; 5 mM KCI; 1% protease inhibitor mixture (Roche); 1% phosphatase inhibitor cocktail I & Il (Sigma); and 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma). The homogenates were centrifuged at 27,000 x g for 20 min at 4°C to obtain supematant (S1) and pellet fractions. Pellets were rehomogenized in 5 volumes of high salt/sucrose buffer (0.8 M NaCI, 10% sucrose, 10 mM Tris/HCI, [pH 7.4], 1 mM EGTA, 1 mM PMSF) and centrifuged as above. The supernatants were collected and încubated with sarkosyl (1% final concentration; Sigma) for one hour at 37°C, followed by centrifugation at 150,000 x g for one hour at 4°C to obtain sarkosylinsoluble pellets, referred to as P3 fraction. The P3 pellet was resuspended in TE buffer (10 mM Tris/HCI [pH 8.0], 1 mM EDTA) to a volume équivalent to half of the original volume used for the brain homogenates.

Western and dot blots

Fractionated tissue extracts S1 and P3 were dissolved in SDS-sample buffer containing 0.1 M DTT. The heat-treated samples (95°C for 10 min) were separated by gel electrophoresis on 4-12% Bis-Tris SDS-PAGE gels (Invitrogen) and transferred onto PVDF membranes (BioRad Laboratories, Hercules, CA). Dot blot samples were spotted directly onto nitrocellulose membranes (Amersham, Pittsburgh, PA) at known concentrations across samples. Both Western and dot blot membranes were blocked in 5% non-fat dry milk in TBS-Tween (0.5%) pH 7.4, followed by incubation in 1 pg/ml D1.2 or C10-2 overnight at 4 °C. Membranes were washed and incubated with peroxidase-conjugated anti-mouse IgG (1:5000; Jackson ImmunoResearch, West Grove, PA). Bound antibodies were detected using an enhanced chemiluminescence system (ECL PLUS kit; PerkinElmer). Quantitation and Visual analysis of Western and dot blot immunoreactivity was performed with a computer-linked LAS-4000 Biolmaging Analyzer System (Fujifilm, Tokyo, Japan) and Multi Gauge v3.1 software (Fujifilm). Protein loading was adjusted by the volume of original fractions and can be converted to original tissue wet weight. Results are shown in Figure 1 and Figure 2.

Exampie 4 Screening and sélection of 396/404 antibodies using immobilized human pathological material

Hybridoma supernatants were screened for antibody binding in nunc plates coated with 1 pg/ml peptide phosphorylated tau 386-408 (pS396/pS404) using 0.1 M carbonate buffer pH 9.

Positive supernatants were subsequently diluted 1:50 -1:800 in PBS, 0.1 % BSA and 0.1 NP40 for binding in ELISA or MSD plates coated with brain (P3 pellet, see Example 3) lysate antigens from AD and healthy Controls (HC), respectively. Brain lysate antigens were diluted 1500 fold in 0.1 M Carbonate buffer pH9 prior to incubation/coating of ELISA or MSD plates. Wells were subsequently blocked 2 hrs at room température (PBS, 3 mg/ml BSA, 0.1% NP-40) and antibody binding activity detected with HRP (DAKO) and sulfotag (MSD, product #) conjugated anti-mouse IgG following vendor protocol. Sélections of antibodies (D1-2, C5-2, C8-3 and C10-2) diluted in PBS with 0.1% BSA were characterised by dose response and showed sub-nanomolar - nanomolar binding activity to AD-P3 antigen coated plates were furthermore characterised for binding-activity to sélection of spécifie and control peptides. Results are shown in Figure 3.

Example 5: Peptide specificity and binding affinity

Antibodies positive for binding to pathological tau were further characterised for apparent affinity (IC50) and selectivity/specificity to a range of phospho-peptide (p) epitopes. MSD plates were coated with 100 ng/ml phosphorylated tau 386-408 (pS396/pS404) as described above. Antibodies against phosphorylated tau were analysed in dose response assays to identify antibody concentrations providing appropriate analytical signal level (typical 5,00020,000 RU in MSD corresponding to 0.5-2% of maximal instrumental signal or OD signais of 1.0 - 1.5 at 450 nm in ELISA. A sélection of antibodies was incubated with graded concentrations (0-1000 nM) of phosphorylated tau 386-408 (pS396/404) for 2 hrs/room température. The reactions were subsequently applied to peptide coated MSD plates coated with 100 ng/ml peptide phosphorylated tau 386-408 (pS396/pS404) as described above and binding activity measured. IC50 values from the inhibition assays correspond to apparent affînities (KD) between 10 -100 nM.

Specificity and phospho-selectivity: An appropriate concentration of monoclonal antibody was incubated with 100 nM double phosphorylated (pS396/pS404) non-phosphorylated or monophosphorylated (pS396 or pS404) phosphorylated tau 386-408 and analysed for binding activity (inhibition assays). Control phosphorylated tau peptides (phosphorylated tau 260-270 (pS262) or phosphorylated tau 240-270 (pS262) and recombinant non phosphorylated tau protein was analysed for comparison. Ail AD-P3 antigen positive antibodies showed strong preference for phosphorylated peptide tau 386-408 (pS396/pS404) and monophosphorylated peptide phosphorylated tau 386-408 (pS396) and no binding activity for mono-phosphorylated peptide phosphorylated tau 386-408 (pS404) and non-phosphorylated peptide tau 386-408. Control phosphor-peptides tau 240-270 and phosphorylated tau. Results are shown in Figure 4 and Figure 32.

Example 6: Histological characterrzation of antibodies by

Immunohistochemistry

Mouse brain tissues were collected from 8 months old rTg4510 mice (overexpressing human P301L-tau under the CamKII promoter) and non-transgenic littermate (non-Tg), fixed in 4% paraformaldéhyde and embedded in paraffin. Paraffin-embedded human brain samples of frontal cortex were acquired from Tissue Solutions (Glasgow, UK). Tissue from donors with diagnosed end stage Alzheimer’s disease was compared to age-matched non-demented control donors. Four um thick sections were deparaffinized and subjected to antigen retrieval by microwaving the sections in 10 mM Citrate buffer, pH 6, for 10 minutes. Endogenous peroxidases were blocked with 1% hydrogen peroxidase followed by 5% normal swine sérum in PBS/1%BSA/0.3%Triton X-100 (PBS-BT). Sections were incubated overnight at 4 °C with D1.2 and C10-2 antibodies diluted in PBS-BT at a range of concentrations. The sections were washed in PBS, 0.25% BSA, 0.1% Triton X-100, before being incubated with a biotinylated secondary swine anti-mouse antibody (E0464; DAKO, Glostrup, Denmark) at 1:500 for 1 hour. Following additional washing, StreptAvidin-Biotin Complex kit (Vector Laboratories, Burlingame, CA) was applied and immunoreactivity was visualized with diaminobenzidine. Sections were counterstained with hematoxylin. Results are shown in Figure 5.

Example 7: Selectivity of antibodies towards pathological tau

MSD plates were coated with solubilized P3 antigens from AD brain (diluted 1:1500) or TG4510 brain (diluted 1:3000). Results are shown in Figure 6.

Détection is performed as described in Example 4 above.

Example 8: HEK cell seeding assay

HEK293 cells were transiently transfected with human tau-P301L-FLAG in 6-well plates 24 h after plating, followed 24 h later by incubation with brain homogenate for 24 h, followed by splitting and replating cells and harvesting after an additional 24 h. Cells were lysed and sonicated in PBS, supplemented with 1% triton X, Phos-stop and complété phosphatase and protease inhibitors (Roche) buffer and ultracentrifugated at 100,000 x g for 30 min. The pellet was resuspended in SDS, sonicated and ultracentrifugated for 30 min at 100,000 x g. Supernatants were analyzed by western blotting. Cells expressing human tau-P301L showed insoluble (SDS fraction, E1/FLAG détection), hyperphosphorylated (D1.2}pS396 détection) tau upon seeding with total brain homogenates from rTg4510 tau transgenic mice. Cells treated with control brain homogenate from mice showed an absence of aggregated hyperphosphorylated human tau. Additionally, total cell lysâtes of HEK293 cells were analyzed using the tau aggregation assay from Cisbio. This assay is based on time-resolved fluorescence using the same antibody for both donor (Tb3+ conjugated) and accepter (d2 conjugated) antibody in FRET. A 10 μΙ sample was mixed with 10 μΙ antibody mix and incubated for 20h. The plate was read on the Pherastar plate reader to assess time-resolved fluorescence (FRET signal measured/integrated after switching of the excitation light). The assay measures aggregated tau both in human autopsy material, rTg4510 mice and in seeded HEK cells with high specificity and sensitivity. Results are shown in Figure 7 and show that the seeding effect was not affected by treatment with HEL, but was partially reversed by treatment with tau antibodies (C10-2>D1.2>hACI36-2B6-Ab1).

Example 9: Reversai of functional (electrophysiology (elphys)) response invivo for D1.2 and C10-2

In vivo electrophysiological assessment of synaptic transmission and plasticity in the CA1 area of the hippocampus in 4.5 to 5.5 months old rTg4510 and tTA control mice showed that

i) basal synaptic transmission is significantly impaired in rTg4510 compared to tTA mice, and ii) paîred-pulse facilitation is significantly reduced rTg4510 compared to tTA mice.

AH experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) for the care and use of laboratory animais and the Danish législation regulating animal experiments.

rTg4510 and tTA male mice (Taconic Europe A/S) aged 5 to 5.5 months were used in the présent study at the time of the recordings. Mice were grouped-housed in controlled température (22 ± 1.5°C) and humidity conditions (55-65%) and kept in a 12:12 hour light/dark cycle (lights on at 06:00h). Food and water were available ad libitum.

Animais were anesthetized with an intraperitoneal (i.p.) injection of urethane (1.2 g/kg). Mice were then mounted in a stereotaxic frame, their température adjusted to 37.5°C via a heating pad, and the skull was exposed. A platinum wire was placed in the frontal bone to act as a reference, and an additional hole was drilled for insertion of the recording and stimulating électrodes in the hippocampus, at the following coordinates according to the atlas of Paxinos and Franklin (Paxinos and Franklin, 2001): recording, 1.5-1.7 mm posterior to Bregma, 1.ΟΙ .2 mm latéral to the midline, 1.4-1.7 mm below the surface of the brain; stimulation, 1.8-2.0 mm posterior to Bregma, 1.5-1.7 mm latéral to the midline, 1.3-1.7 mm below the surface of the brain. Animais were left in the stereotaxic frame throughout the whole duration of the recordings and their level of anesthésia was regularly checked.

Field potentials (fEPSP) were evoked in the CA1 by electrical stimulation of the Schaffer collateral every 30 s, and the depth of the recording electrode was adjusted until a négative fEPSP was recorded in response to a unipolar square puise. The slope of the evoked fEPSP was typically measured between 30 and 70% of the maximum amplitude of the fEPSP.

Once an optimal fEPSP was induced, basal synaptic transmission was assessed by the relationship between stimulation intensity and slope of the evoked fEPSP (input-output relationship). The different intensities of stimulation were 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 μΑ, and were applied successively in increasing order, with 2 to 3 repeats at each intensity. Basal synaptic transmission was found to be significantly impaired in rTg4510 compared to tTA mice.

Paired-pulse facilitation, a short-term synaptic plasticity believed to rely on presynaptic mechanisms, was further measured in rTg4510 and tTA mice. Briefly, a pair of stimuli with an inter-stimulus interval (ISI) varying from 25 to 1000 ms was applied to the Schaffer collateral, and the slope of the second fEPSP was compared to the slope of the first fEPSP. Facilitation was observed at ail ISIs, with a maximum facilitation at [Sis of 50 and 75 ms. Interestingly, a significantly lower PPF was observed in rTg4510 mice when compared tTA mice.

The identified impairments in basal synaptic transmission and paired-pulse facilitation in rTg4510 mice were further used as readout to test antibody efficacy.

Recordings were performed in ail experiments 2 to 4 days following administration of 4 doses of antibody twice per week for 2 weeks, i.p.). Basal synaptic transmission and pairedpulse facilitation were recorded in both hippocampi in each animal when possible, and further used as individual experiments. Results are shown in Figure 8 and show antibody reversai of paired puise facilitation and basal synaptic transmission déficits in CA1 evoked field potentials.

Example 10: Immunodepletion of Tau from rTg4510 brain extracts pg mouse and humanized C10-2 antibody was immobilized to 300 pl of Magnetic dynabead suspension (Immunoprécipitation Kit Dynabeads Protein G Novex, Cat no 10007D). After thorough washing the beads were mixed with 60 pl rTg4510 brain extract and incubated at room température for 10 minutes. The magnetic beads were separated from the extract and the extracts were analysed by western blot. Déplétion with mC10-2 and hC10-2 removed tau aggregates 99 and 99,5% respectively. Results are shown in Figure 12.

Example 11: HEK cell seeding assay using immunodepleted extracts

HEK293 cells were transiently transfected with human tau-P301L-FLAG in 6-well plates. 24 h later cells were incubated with brain homogenate that had been immunodepleted using humanized or mouse C10-2. After 24 h cells were re-plated and harvested after an additional 24 h. Cells were lysed and sonicated in TBS, supplemented with 1% triton X, phosphatase and protease inhibitors (Roche) and ultracentrifugated at 100,000 x g for 30 min. The pellet was resuspended in 1% SDS, sonicated and ultracentrifugated for 30 min at 100,000 x g. Supernatants were analyzed by western blotting. Cells expressing human tau-P301L showed insoluble (SDS fraction, E1/FLAG détection), hyperphosphorylated tau (D1.2/pS396 Tau, running at a higher molecular weight) upon seeding with total brain homogenates from rTg4510 tau transgenic mice. Cells treated with control brain homogenate from tTA mice showed an absence of aggregated hyperphosphorylated human tau. Additionally, total cell lysâtes of HEK293 cells were analyzed using the tau aggregation assay from Cisbio. Déplétion with HEL and hHEL antibodies did not affect seeding, whereas déplétion with mC10-2 and hC10-2 prevented tau aggregation 88 and 96% and insoluble tau 97 and 100% respectively. Results are shown in Figure 13.

Example 11: Immunodepletion of Tau from rTg4510 brain extracts

100 pg mouse C10-2, D1.2 and Tau5 (Invitrogen) antibody was immobilized to 500 μΙ of Magnetic dynabead suspension (Immunoprécipitation Kit Dynabeads Protein G Novex, Cat no 10007D). After thorough washing the beads were mixed with 100 pl rTg4510 brain extract and incubated at room température for 10 minutes. The magnetic beads were separated from the extract and the extracts were analysed by western blot. C10-2 and D1.2 do not remove the normal Tau from the homogenates, as the commercially available Tau5 antibody does. In contrast, two antibodies of the invention specifically remove the hyperphosphorylated tau (64kDa) by 95%, that is tau phosphorylated on serine 396. Results are shown in Figure 14.

Example 12: Immunodepletion of Tau from Alzheimer’s brain extracts

100 pg mouse C10-2 and D1 antibody was îmmobilized to 500 μΙ of Magnetic dynabead suspension (Immunoprécipitation Kit Dynabeads Protein G Novex, Cat no 10007D). After thorough washing the beads were mixed with 100 μΙ Alzheimer brain extract and incubated at room température for 10 minutes. The magnetic beads were separated from the extract and the extracts were analysed by western blot. D1.2 and C10-2 does only remove a very small fraction of the total tau in the brain homogenate (8%). The antibodies do however specifically remove the hyperphosphorylated tau (90%), spécifie for AD patients. Results are shown in Figure 15.

Example 13: Seeding in rTg4510 mice using immunodepleted extracts

Transgenic mice expressing human mutated Tau (P301L 0N4R) under a tet-off responstve element in CamK2 positive neurons (rTg4510) was used. This model normally starts developing Tau pathology at 3 months of âge, but by feeding the mothers with doxycycline during pregnancy and for the first 3 weeks of the pup's life, the pathology develops at a later stage (starting after 6 months of âge). The doxycycline pre-treated mice used in the studies were 2,5 months old at the time-point of injection. Mice were anesthetized by isoflouran inhalation fixed in a stereotactic frame. The seuil was exposed and adjusted until bregma and lambda was in level. A hole was drilled in the seuil 2 mm latéral (right) and 2,4 mm posterior of the bregma. A 10 μΙ syringe beveled tip (SGE) was used to inject the seeding material 1,4 mm ventral to the brain surface at the at the above mentioned co-ordinates. 2 μΙ of the immunodepleted extracts, described in Examples 11 and 12, was slowly infused at the site (1 μΙ/minute) and the syringe was left for 5 minutes before removing it. The wound was closed by stiches and mice were heated while waking up. The mice were housed for 3 months and then sacrificed and perfusion fixed with 4% PFA.

Immunohistochemistry: Fixed brains were eut into 35 pm coronal sections at NSA and every 6⁰¹ section was stained for Tau tangles (Gallyas silver stain) and for hyperphosphorylated Tau (AT8). Positively stained neurons (soma) were counted in ipsi- and contralatéral sides of hippocampi of ail brains. Ail sub-regions of hippocampus were included. Eight sections were counted per brain. Results reflect the sum of positive neurons from the 8 sections. The background signal was determined in 2 non-injected mice. By removing hyperphosphorylated tau from the homogenates, the homogenates do no longer induce seeding of Tau pathology. Results are shown in Figure 16. Quantification of Tau pathology in rTg4510 brains seeded with rTg4510 (A) or AD (B) brain homogenates. Prior to seeding the hyperphosphorylated Tau, but not normal Tau, had been reduced in the homogenates by 90-95% by using C10-2 or D1.2. By removing hyperphosphorylated tau from the homogenates, the homogenates no longer induce seeding of Tau pathology.

Homogenates from rTg4510 or Alzheimer brains can seed Tau pathology in rTg4510 mice at a stage when endogenous Tau pathology has not developed. By removing the hyperphosphorylated Tau from the homogenates by using D1.2 or C10-2, as described in Examples 11 and 12. the seeding activity is abolished.

Example 14: Antibody treatment in seeded rTg4510 mice

Doxycyclin treated rTg4510 mice (as described in Example 13) were chronically treated with mouse D1.2 or control antibody, 15 mg/kg/week startîng at 2 months of âge. At 2,5 months rTg4510 brain extract was infused into the hippocampus (as described in Example 13). Mice were sacrificed 1,2 and 3 months after the brain infusion and immunohistochemistry and the following analysis was performed as described in Example 13. D1.2 treatment significantly reduced Tau pathology 2 and 3 months after seeding had been initiated. Results are shown in Figure 17.

Quantification of tangle bearing neurons in hippocampus of seeded rTg4510 mice. The pathology increase with time and by treating the mice with D1.2 the pathology is significantly lower 2 and 3 months after seeding. Quantification of tangle bearing neurons in hippocampus of seeded rTg4510 mice. The pathology increase with time and by treating the mice with D1.2 the pathology is significantly lower 2 and 3 months after seeding.

Homogenates from rTg4510 or Alzheimer brains can seed Tau pathology in rTg4510 mice at a stage when endogenous Tau pathology has not developed. By systemically treating the mice with D1.2 the development of tau pathology can be significantly reduced.

Example 15: Immunodepletion of Tau from Alzheimer’s brain extracts using humanized Tau antibodies

100 pg mouse and humanized C10-2 as well as prior art antibodies 2.10.3 and HJ8.5 antibody (source) was immobilized to 500 μΙ of Magnetic dynabead suspension (Immunoprécipitation Kit Dynabeads Protein G Novex, Cat no 10007D). After thorough washing the beads were mixed with 100 μΙ Alzheimer brain extract and incubated at room température for 10 minutes. The magnetic beads were separated from the extract and the extracts were analysed by western blot and the CisBio assay. The mouse and humanized C10-2 efficiently removed 93 and 97 % of the pS396 phosphorylated Tau, but only 22 and 17% of the total Tau compared to the hHel control antibody. The 2.10.3 antibody removed 91% of Serine 396 phosphorylated tau and 10% of total tau. It seems like the 2.10.3 is less efficient in removing one of the hyper phosphorylated bands in comparison to the C10-2 antibodies (the middle 64kDa band). The HJ8.5 antibody has a completely different profile then both the C10-2 and 2.10.3 antibodies. by removing the majority of tau, 89% of total tau and 88% of pS396 Tau. Results are shown in Figures 23-24.

Example 16: Immunodepletion of aggregated Tau from Alzheimer’s brain extracts using humanized Tau antibodies

The immunodepleted AD extracts described in Example 13 was also analysed by using the Tau aggregation assay described in example 10. The C10-2 and HJ8.5 antibodies are more efficiently removing aggregated Tau from the AD material then the 2.10.3 antibody. In order of efficiency: HJ8.5 (99%), hC10-2 (98%), mC10-2 (96%) and 2.10.3 (90%) ail in comparison to hHel antibody. Results are shown in Figure 25.

Example 17: Immunodepletion of Tau pg antibody (humanized C10-2 or 2.10.3) was immobilized to 125 pl of Magnetic dynabead suspension (Immunoprécipitation Kit Dynabeads Proteïn G Novex, Cat no 10007D). After thorough washing the coated beads were mixed with variable amounts of non-coated. washed beads. Starting from 100% Ab coated beads, corresponding to 5 pg antibody, down to 100% non-coated beads. The total amount of beads was the same in ail samples. The beads were mixed with 20 pl AD extract and incubated at room température for 10 minutes. The magnetic beads were separated from the extract and the extracts were aliquoted, snap frozen and kept at -80 C until use.

Analysis of déplétion using western blot

Samples were boiled in 1x LDS loading buffer and 100 mM DTT. A volume corresponding to 3 pl of extracts were loaded on a 4-12% Bis-Tris NuPAGE Gel (LifeTech Novex). After electrophoresis, the proteins were blotted over to a Immobilon-FL PVDF membrane (0,45pm, IPFL10100, Millipore). The membrane was blocked with SEA blocking buffer (Prod#37527, Thermo). Tau and P-tau levels were assessed in the samples using Tau5 (Abcam ab80579, 1:2000) mouse C10-2 (1 pg/ml), P-S199/202 (Invitrogen 44768 G, 1:1000), P-S422 (Abcam ab79415. 1:750), human IPN (1pg/ml). Gapdh and actin were used as a loading Controls (Abcam ab9484, 1:2000, Sigma A5441, 1:20000). Secondary fluorophore conjugated IgG antibodies was used (IRDye 800CW Goat anti-Human, IRDye 800CW, Goat anti-rabbit, IRDye 680 Goat anti-mouse, LI-COR biosciences) and the signal was quantified using Odyssey CLx and Image studio software (LI-COR biosciences).

Quantification of individual bands as well as signal in whole lanes was done and from this sigmoidal dose-response curves were plotted and when possible max effect and EC50 values were estimated.

Resuits

Both antibodies remove a small fraction of tau from the Alzheimer brain préparation. 2.10.3, designed to hâve specificity for P-S422 tau removes up to 24% of the total tau amount, while C10-2 removes up to 15% of the total tau (see Figure 26).

2.10.3 and C10-2 both remove more than 90% of the tau phosphorylated at Serine 422 although the amount of antibody needed to remove 50% of the P-S422 tau differ, for 2.10.3, 0,42 pg antibody is needed and for C10-2, 0.27 pg is needed for the same effect (see Figure 27).

C10-2 efficiently remove Tau being phosphorylated at serine 396 (Max effect: 88% and half of the effect is reached by using 0,30 pg antibody). 2.10.3 removes a smaller fraction of tau being phosphorylated at the serine 396 (Max effect: 60% and half of that effect is reached when using 0,63 pg antibody)(see Figure 28). This indicates that ail Tau being phosphorylated at serine 422, also is phosphorylated at serine 396, but that there is a portion of hyperphosphorylated tau being phosphorylated at serine 396 where the phosphorylated serine at position 422 is not présent.

A large portion of the tau, being removed by C10-2, is also phosphorylated at Serine 199/202, since 69% of the tau having that phosphporylation is affected by the immunodepletion (50% of the effect when using 0,34 pg antibody)(see figure 29). The 2.10.3 immunodepletion does not give a sigmoidal dose response on the P-S199/202 tau although a drop in signal is seen with increasing amount of antibody (max 52% réduction when using the max amount of antibody (5 pg)(see Figure 29).

This data indicates that the C10-2 antibody targeting the phosphorylated serine 396 binds a larger pool of the hyperphosporylated tau then the 2.10.3 antibody targeting the phosphorylated serine at the 422 position.

Example 18: Antibody mediated inhibition of mC10-2 spécifie capture of pathological Tau antigens in AD brain lysâtes

Materials and methods

Material

Coating buffer: Carbonate buffer pH 8.5, 150 mM NaCL. Blocking buffer: 3% BSA (fraction V), 0.1% NP40 in PBS pH7.4. Washing buffer: 0.1 % BSA (fraction V), 0.1 % NP40 in PBS, pH 7,4. Sulfotag goat total humanized Tau antibody (MSD D221LA-1,50 pg/ml)

Method aim to measure capture of pathological human Tau antigens from AD brains using C10-2 coated plates (step A) after incubation of Tau antigens with increasing concentrations of pS396 spécifie antibodies (step B). The Tau antigen capture and antibody mediated inhibition was detected using sulfo-tagged anti human (total) Tau antibodies from MSD

A: MSD plates were coated (o/n at 4C) with 0.5 pg/ml mC10-2 (capture antibody) in coating buffer and subsequently blocked for 1 hour at room termperature) and washed 3 times. (Figure 30)

B: Samples P3 lysate (1:1000 = 2-4 pg/ml total protein) and/or S1(p) (1:300=20-40 ng/ml total protein) from AD (pool from 3 patient) were mixed with graded concentrations of pS396 peptide epitope spécifie antibody and incubated for 1 hour at room température. The réactions were subsequently incubated 2 hours on plates prepared in step A. (Figure 31)

C: C10-2 captured Tau was detected using sulfo-tagged human tau. Tau antibody (1:50) from MSD following manufacture instruction. Plates were analyzed on MSD SECTOR® S 600. AD P3 and AD S1 (p) was tested in similar setup. (Figure 33/34)

Table 6A mouse C10-2, + tau peptide, 10μΜ

	mean Signal	signal	signai
PBS/0,1%BSA	388	403	373
C10-2 3ng/ml	366	384	348
C10-2 10ng/ml	383	398	367
C10-2 30ng/ml	345	384	306
C10-2 100ng/ml	357	401	313
C10-2 300ng/ml	407	434	379
C10-2 1000ng/ml	451	462	439
C10-2 10000ng/ml	870	920	820

Table 6B mouse C10-2,

+ PBS/0,1% BSA	mean Signal signal signal
PBS/0,1%BSA + PBS	303 293 312

C10-2 3ng/ml+ PBS	1881	1890	1871
C10-2 10ng/ml+ PBS	5721	5863	5579
010-2 30ng/ml+ PBS	11922	12044	11799
C10-2 100ng/ml+ PBS	21833	21925	21741
C10-2 300ng/ml+ PBS	30410	30311	30508
C10-2 1000ng/m!+ PBS	38524	38233	38814
C10-2 10000ng/ml+ PBS	51171	51253 51089

Table 6C mouse clone PHF 13, + tau peptide, 10μΜ mean

	Signal	signal	signal
PBS/0,1%BSA	287	286	287
PHF 13 1000000	280	284	276
PHF 13 300000	299	305	292
PHF 13 100000	355	370	340
PHF 13 30000	481	472	490
PHF 13 10000M	953	1019	886
PHF 13 3000	2182	2279	2084
PHF 13 1000	6896	7542	6249
Table 6D
mouse clone PHF 13, ± PBS/0,1% BSA	mean
	Signal	signal	signal
PBS/0,1%BSA + PBS	281	282	280
PHF 13 1000000+ PBS	335	358	312
PHF 13 300000+ PBS	560	568	551
PHF 13 100000+ PBS	852	856	847
PHF 13 30000+ PBS	1579	1661	1496
PHF 13 10000+ PBS	2882	2899	2864
PHF 13 3000+ PBS	5792	6126	5458
PHF 13 1000+ PBS	12639	13654	11624

Table 6A-6D:Tau antigen capture inhibition

SEQUENCE LISTING <110> H. Lundbeck A/S <120> Tau 396 antibodies <130> 0995-WO-PCT <160> 37 <170> Patentln version 3.5 <210> 1 <211> 16 <212> PRT <213> Artificial <220>

<223> D1.2 CDR 1 Light Chain <400> 1

Arg Ser Ser Gin Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

10 15 <210>

<211>

<212>

<213>

PRT

Artificial <220>

<223> D1.2 CDR 2 Light Chain <400>2

Lys Val Ser Asn Arg Phe Ser 15

<210>	3
<211>	7
<212>	PRT
<213> '	Artificial

<220>

<223> D1.2 CDR 3 Light Chain <400>3

Ser Gin Ser Thr His Val Pro <210> 4 <211> 13 <212> PRT <213> Artificial <220>

<223> D1.2 CDR 1 Heavy Chaiin <400> 4

Lys Ala Ser Gly Asn Thr Phe Thr Asp Tyr Glu Ile His

10 <210> 5 <211> 17 <212> PRT <213> Artificial <220>

<223> D1.2 CDR 2 Heavy Chain <400> 5

Ala Ile Asp 1

Pro Glu Thr

Gly Asn Thr Ala Tyr Asn Gin Lys Phe Lys

15

Gly <210> <211> <212> <213>

<220>

PRT

Artificial <223> D1.2 CDR 3 Heavy Chain <400> 6

Ser Arg Gly Phe Asp Tyr 1 5 <210>

<211>

<212>

<213>

219

PRT

Artificial

<220> <223>	D1.2 Light Chain
<400>	7

Asp 1

Val

Met

Met

Thr 5

Gin

Thr

Pro

Leu

Ser 10

Leu

Pro

Val

Ser

Leu 15

Gly

Asp

Gin

Ala

Ser 20

Ile

Ser

cys

Arg

Ser 25

Ser

Gin

Ser

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Val 30

His

Ser

Asn

Gly

Asn 35

Thr

Tyr

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His

Trp 40

His

Leu

Gin

Lys

Pro 45

Gly

Gin

Ser

Pro

Lys 50

Phe

Leu

Ile

Tyr

Lys 55

Val

Ser

Asn

Arg

Phe 60

Ser

Gly

Val

Pro

Asp 65

Arg

Phe

Ser

Gly

Ser 70

Gly

Ser

Gly

Thr

Asp 75

Phe

Thr

Leu

Lys

Ile 80

Ser Arg Val Glu Ala Glu Asp

Thr His Val Pro Phe Thr Phe

100

Arg Ala Asp Ala Ala Pro Thr

115

Gin Leu Thr Ser Gly Gly Ala

130 135

Tyr Pro Lys Asp Ile Asn Val

145 150

Gin Asn Gly Val Leu Asn Ser

165

Thr Tyr Ser Met Ser Ser Thr

180

Arg His Asn Ser Tyr Thr Cys

195

Leu Gly Val Tyr Phe Cys Ser Gin Ser

95

Gly Ser Gly Thr Lys Leu Glu Ile Lys

105 110

Val Ser Ile Phe Pro Pro Ser Ser Glu

120 125

Ser Val Val Cys Phe Leu Asn Asn Phe

140

Lys Trp Lys Ile Asp Gly Ser Glu Arg

155 160

Trp Thr Asp Gin Asp Ser Lys Asp Ser

170 175

Leu Thr Leu Thr Lys Asp Glu Tyr Glu

185 190

Glu Ala Thr His Lys Thr Ser Thr Ser

200 205

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215 <210> 8 <211> 451 <212> PRT <213> Artificial <220>

<223> D1.2 Heavy Chain <400> 8

Gin 1

Val

Gin

Leu

Gin 5

Gin

Ser

Gly

Ala

Glu 10

Leu

Val

Arg

Pro

Gly 15

Ala

Ser

Val

Thr

Leu 20

Ser

Cys

Lys

Ala

Ser 25

Gly

Asn

Thr

Phe

Thr 30

Asp

Tyr

Glu

Ile

His 35

Trp

Val

Lys

Gin

Thr 40

Pro

Val

His

Gly

Leu 45

Glu

Trp

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Ala 50

Ile

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Pro

Glu

Thr 55

Gly

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Tyr 60

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Lys 65

Gly

Lys

Ala

Arg

Leu 70

Thr

Ala

Asp

Lys

Ser 75

Ser

Ser

Thr

Ala

Tyr 80

Met

Glu

Leu

Arg

Ser 85

Leu

Thr

Ser

Glu

Asp 90

Ser

Ala

Val

Tyr

Tyr 95

Cys

Thr

Arg

Ser

Arg 100

Gly

Phe

Asp

Tyr

Trp 105

Gly

Gin

Gly

Thr

Thr 110

Leu

Thr

Val

Ser

Ser 115

Ala

Lys

Thr

Thr

Pro 120

Pro

Ser

Val

Tyr

Pro 125

Leu

Ala

Pro

Gly

Cys 130

Gly

Asp

Thr

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Gly 135

Ser

Ser

Val

Thr

Leu 140

Gly

Cys

Leu

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Lys 145

Gly

Tyr

Phe

Pro

Glu 150

Ser

Val

Thr

Val

Thr 155

Trp

Asn

Ser

Gly

Ser 160

Leu

Ser

Ser

Ser

Val 165

His

Thr

Phe

Pro

Ala 170

Leu

Leu

Gin

Ser

Gly 175

Leu

Tyr

Thr

Met

Ser 180

Ser

Ser

Val

Thr

Val 185

Pro

Ser

Ser

Thr

Trp 190

Pro

Gin

Thr

Val 195

Thr

Cys

Ser

Val

Ala 200

His

Pro

Ala

Ser

Ser 205

Thr

Thr

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Lys 210

Lys

Leu

Glu

Pro

Ser 215

Gly

Pro

Ile

Ser

Thr 220

Ile

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Pro

Pro 225

Pro

Cys

Lys

Glu

Cys 230

His

Lys

Cys

Pro

Ala 235

Pro

Asn

Leu

Glu

Gly

Pro

Ser

Val

Phe 245

Ile

Phe

Pro

Pro

Asn 250

Ile

Lys

Asp

Val

Leu 255

Ile

Ser

Leu

Thr 260

Pro

Lys

Val

Thr

Cys 265

Val

Val

Val

Asp

Val 270

Ser

Asp

Asp

Pro 275

Asp

Val

Arg

Ile

Ser 280

Trp

Phe

Val

Asn

Asn 285

Val

Glu

His

Thr 290

Ala

Gin

Thr

Gin

Thr 295

His

Arg

Glu

Asp

Tyr 300

Asn

Ser

Thr

Arg 305

Val

Val

Ser

Ala

Leu 310

Pro

Ile

Gin

His

Gin 315

Asp

Trp

Met

Ser

Lys

Glu

Phe

Lys

Cys 325

Lys

Val

Asn

Asn

Lys 330

Asp

Leu

Pro

Ser

Pro 335

Glu

Arg

Thr

Ile 340

Ser

Lys

Ile

Lys

Gly 345

Leu

Val

Arg

Ala

Pro 350

Gin

Tyr

Ile

Leu 355

Pro

Pro

Pro

Ala

Glu 360

Gin

Leu

Ser

Arg

Lys 365

Asp

Val

Leu

Thr 370

Cys

Leu

Val

Val

Gly 375

Phe

Asn

Pro

Gly

Asp 380

Ile

Ser

Val

Trp 385

Thr

Ser

Asn

Gly

His 390

Thr

Glu

Glu

Asn

Tyr 395

Lys

Asp

Thr

Ala

Ser

Val

Cys

Gly

240

Met

Glu

Val

Ile

Gly

320

Ile

Val

Ser

Glu

Pro

400

Val

Leu

Asp

Ser

Asp 405

Gly

Ser

Tyr

Phe

Ile 410

Tyr

Ser

Lys

Leu

Asp 415

Ile

Lys

Thr

Ser

Lys 420

Trp

Glu

Lys

Thr

Asp 425

Ser

Phe

Ser

Cys

Asn 430

Val

Arg

His

Glu

Gly 435

Leu

Lys

Asn

Tyr

Tyr 440

Leu

Lys

Lys

Thr

Ile 445

Ser

Arg

Ser

Pro Gly Lys

450 <210>

<211>

<212>

<213>

P RT

Arti ficial <220>

<223> CIO.2 CDR 1 Light Chain <400> 9

Gin Ala Ser Gin Gly Thr Ser Ile Asn Leu Asn 1 510

<210>	10
<211>	7
<212>	PRT
<213>	Arti ficial
<220>
<223>	CIO.2 CDR 2 Light Chain
<400>	10

Gly Ala Ser Asn Leu Glu Asp 15

<210>	11
<211>	7
<212>	PRT
<213>	Artificial

<220>

<223> CIO.2 CDR 3 Light Chain <400>11

100

Leu Gin His Thr Tyr Leu Pro 1 5 <210>

<211>

<212>

<213>

P RT

Artificial <220>

<223> CIO.2 CDR 1 Heavy Chain <400> 12

Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg Thr Ile His 1 5 10 <210>

<211>

<212>

<213>

P RT

Arti ficial <220>

<223> CIO.2 CDR 2 Heavy Chain <400> 13

Tyr Ile Tyr Pro Gly Asp Gly Ser 1 5

Thr Lys Tyr Asn Glu Asn Phe Lys

15

Gly <210>

<211>

<212>

<213>

P RT

Arti ficial <220>

<223> CIO.2 CDR 3 Heary Chain <400> 14

Arg Gly Ala Met Asp Tyr 1 5 <210>

<211>

<212>

<213>

214

P RT

Artificial

101 <220>

<223> CIO.2 Light Chain <400> 15

Asp 1

Val

Gin

Met

Ile 5

Gin

Ser

Pro

Ser

Ser 10

Leu

Ser

Ala

Ser

Leu 15

Gly

10

Asp

Ile

Val

Thr

Met

Thr

Cys

Gin

Ala

Ser

Gin

Gly

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20

25

30

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15

35

40

45

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Ser

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Val

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50

55

60

20

Ser

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Tyr

Gly

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Thr

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Ser

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65

70

75

80

25

Glu

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Met

Ala

Thr

Tyr

Phe

Cys

Leu

Gin

His

Thr

Tyr

Leu

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Phe

85

90

95

30

Thr

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Ser

Gly

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Lys

Arg

Ala

Asp

Ala

Ala

100

105

110

Pro

Thr

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Ser

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Phe

Pro

Pro

Ser

Ser

Glu

Gin

Leu

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Ser

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35

115

120

125

Gly

Ala

Ser

Val

Val

Cys

Phe

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Asn

Asn

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Tyr

Pro

Lys

Asp

I le

130

135

140

40

Asn

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Lys

Trp

Lys

Ile

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Gly

Ser

Glu

Arg

Gin

Asn

Gly

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Leu

145

150

155

160

45

Asn

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Trp

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Ser

Thr

Tyr

Ser

Met

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165

170

175

50

Ser

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Leu

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Leu

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Tyr

Glu

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His

Asn

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Tyr

180

185

190

Thr

Cys

Glu

Ala

Thr

His

Lys

Thr

Ser

Thr

Ser

Pro

Ile

Val

Lys

Ser

55

195

200

205

102

Phe Asn Arg Asn Glu Cys

210

<210>	16
<211>	439
<212>	P RT
<213>	Artificial
<220>
<223>	CIO.2 Heavy Chain
<400>	16

Gin 1

Val

Gin

Leu

Gin 5

Gin

Ser

Asp

Ala

Glu 10

Leu

Val

Lys

Pro

Gly 15

Ala

Ser

Val

Lys

Ile 20

Ser

Cys

Lys

Ala

Ser 25

Gly

Tyr

Thr

Phe

Thr 30

Asp

Arg

Thr

Ile

His 35

Trp

Val

Lys

Gin

Arg 40

Pro

Glu

Gin

Gly

Leu 45

Glu

Trp

Ile

Gly

Tyr 50

Ile

Tyr

Pro

Gly

Asp 55

Gly

Ser

Thr

Lys

Tyr 60

Asn

Glu

Asn

Phe

Lys 65

Gly

Lys

Ala

Thr

Leu 70

Thr

Ala

Asp

Lys

Ser 75

Ser

Ser

Thr

Ala

Tyr 80

Met

Gin

Leu

Asn

Ser 85

Leu

Thr

Ser

Glu

Asp 90

Ser

Ala

Val

Tyr

Phe 95

Cys

Ala

Arg

Arg

Gly 100

Ala

Met

Asp

Tyr

Trp 105

Gly

Gin

Gly

Thr

Ser 110

Val

Thr

Val

Ser

Ser 115

Ala

Lys

Thr

Thr

Pro 120

Pro

Ser

Val

Tyr

Pro 125

Leu

Ala

Pro

Gly

Ser 130

Ala

Ala

Gin

Thr

Asn 135

Ser

Met

Val

Thr

Leu 140

Gly

Cys

Leu

Val

Lys 145

Gly

Tyr

Phe

Pro

Glu 150

Pro

Val

Thr

Val

Thr 155

Trp

Asn

Ser

Gly

Ser 160

103

Leu Ser Ser Gly

Val His Thr Phe Pro 165

Ala Val Leu Gin Ser 170

Asp Leu

175

Tyr

Thr

Leu

Ser 180

Ser

Ser

Val

Thr

Val 185

Pro

Ser

Ser

Thr

Trp 190

Pro

Ser

Glu

Thr

Val 195

Thr

Cys

Asn

Val

Ala 200

His

Pro

Ala

Ser

Ser 205

Thr

Lys

Val

Asp

Lys 210

Lys

Ile

Val

Pro

Arg 215

Asp

Cys

Gly

Cys

Lys 220

Pro

Cys

Ile

Cys

Thr Val 225

Asp Val

Asp Ile

Asp Val

Asn Ser

290

Trp Leu

305

Pro Ala

Ala Pro

Asp Lys

Ile Thr

370

Pro Glu

Val Ser

230

Ser Val

Phe Ile

Leu Thr

Ser Lys

260

Glu Val

275

Thr Phe

Asn Gly

Pro Ile

Gin Val

340

Val Ser

355

Val Glu

Ile Thr

245

Asp Asp

His Thr

Arg Ser

Lys Glu

310

Glu Lys

325

Tyr Thr

Leu Thr

Trp Gin

Leu Thr

Pro Glu

Ala Gin

280

Val Ser

295

Phe Lys

Thr Ile

Ile Pro

Cys Met

360

Trp Asn

375

Pro Lys

250

Val Gin

265

Thr Gin

Glu Leu

Cys Arg

Ser Lys

330

Pro Pro

345

Ile Thr

Gly Gin

Phe Pro 235

Val Thr

Phe Ser

Pro Arg

Pro Ile

300

Val Asn 315

Thr Lys

Lys Glu

Asp Phe

Pro Ala

380

Pro Lys

Pro Lys

240

Cys Val

Val Val

255

Trp Phe

270

Glu Glu

285

Met His

Ser Ala

Gly Arg

Gin Met

350

Phe Pro

365

Glu Asn

Val Asp

Gin Phe

Gin Asp

Ala Phe

320

Pro Lys

335

Ala Lys

Glu Asp

Tyr Lys

104

Asn 385

Thr

Gin

Pro

Ile

Met 390

Asp

Thr

Asp

Gly

Ser 395

Tyr

Phe

Val

Tyr

Ser 400

Lys

Leu

Asn

Val

Gin

Lys

Ser

Asn

Trp

Glu

Ala

Gly

Asn

Thr

Phe

Thr

405

410

415

Cys

Ser

Val

Leu

His

Glu

Gly

Leu

His

Asn

His

His

Thr

Glu

Lys

Ser

420

425

430

Leu

Ser

His

Ser

Pro

Gly

Lys

435 <210> 17 <211> 11 <212> PRT <213> Artificial <220>

<223> C5.2 CDR 1 Light Chain <400> 17

Gin Ala Ser Gin Asp Thr Ser Ile Asn Leu Asn

5 10

<210>	18
<211>	7
<212>	PRT
<213>	Artificial
<220> <223>	C5.2 CDR 2 Light Chain
<400>	18

Gly Ala Ser Asn Leu Glu Asp 1 5

<210>	19
<211>	7
<212>	PRT
<213>	Artificial
<220>
<223>	C5.2 CDR 3 Light Chain
<400>	19

105

Leu Gin His Thr Tyr Leu Pro 1 5 <210> 20 <211> 13 <212> PRT <213> Artificial <220>

<223> C5.2 CDR 1 Heavy Chain <400> 20

Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg Thr Ile His

5 10 <210> 21 <211> 17 <212> PRT <213> Artificial <220>

<223> C5.2 CDR 2 Heavy Chain <400> 21

Tyr Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Asn Asp Asn Phe Lys

10 15

Gly <210>

<211>

<212>

<213>

PRT

Artificial <220>

<223> C5.2 CDR 3 Heavy Chain <400> 22

Arg Gly Thr Met Asp Tyr 1 5 <210>

<211>

<212>

<213>

214

PRT

Artificial

106 <220>

<223> C5.2 Light Chain <400> 23

Asp 1

Val

Gin

Met

Ile 5

Gin

Ser

Pro

Ser

Ser 10

Leu

Ser

Ala

Ser

Leu 15

Gly

Asp

Ile

Val

Thr 20

Met

Thr

Cys

Gin

Ala 25

Ser

Gin

Asp

Thr

Ser 30

Ile

Asn

Leu

Asn

Trp 35

Phe

Gin

Gin

Lys

Pro 40

Gly

Lys

Ala

Pro

Lys 45

Leu

Leu

Ile

Tyr

Gly 50

Ala

Ser

Asn

Leu

Glu 55

Asp

Gly

Val

Pro

Ser 60

Arg

Phe

Ser

Gly

Ser 65

Arg

Tyr

Gly

Thr

Asp 70

Phe

Thr

Leu

Thr

Ile 75

Ser

Ser

Leu

Glu

Asp 80

Glu

Asp

Met

Ala

Thr 85

Tyr

Phe

Cys

Leu

Gin 90

His

Thr

Tyr

Leu

Pro 95

Phe

Thr

Phe

Gly

Ser 100

Gly

Thr

Lys

Leu

Glu 105

Ile

Lys

Arg

Ala

Asp 110

Ala

Ala

Pro

Thr

Val 115

Ser

Ile

Phe

Pro

Pro 120

Ser

Ser

Glu

Gin

Leu 125

Thr

Ser

Gly

Gly

Ala 130

Ser

Val

Val

Cys

Phe 135

Leu

Asn

Asn

Phe

Tyr 140

Pro

Lys

Asp

Ile

Asn 145

Val

Lys

Trp

Lys

Ile 150

Asp

Gly

Ser

Glu

Arg 155

Gin

Asn

Gly

Val

Leu 160

Asn

Ser

Trp

Thr

Asp 165

Gin

Asp

Ser

Lys

Asp 170

Ser

Thr

Tyr

Ser

Met 175

Ser

Ser

Thr

Leu

Thr 180

Leu

Thr

Lys

Asp

Glu 185

Tyr

Glu

Arg

His

Asn 190

Ser

Tyr

Thr

Cys

Glu

Ala

Thr

His

Lys

Thr

Ser

Thr

Ser

Pro

I le

Val

Lys

Ser

107

195

200

205

Phe Asn Arg Asn Glu Cys

210 <210>

<211>

<212>

<213>

<220>

439

PRT

Artificial <223> C5.2 Heavy Chain <400> 24

Gin Val 1

Ser Val

Thr Ile

Gly Tyr

Lys Ala 65

Met Gin

Ala Arg

Val Ser

Gly Ser

130

Lys Gly

145

Gin Leu

Lys Ile

His Trp 35

Ile Tyr

Lys Ala

Leu Asn

Arg Gly

100

Ser Ala

115

Ala Ala

Tyr Phe

Gin Gin

Ser Cys

Val Lys

Pro Gly

Thr Leu

Ser Leu 85

Thr Met

Lys Thr

Gin Thr

Pro Glu

150

Ser Asp

Lys Ala

Gin Arg

Asp Asp

Thr Ala

Thr Ser

Asp Tyr

Thr Pro

120

Asn Ser

135

Pro Val

Ala Glu

Ser Gly

Pro Glu

Ser Thr

Asp Lys

Asp Asp

Trp Gly 105

Pro Ser

Met Val

Thr Val

Leu Val

Tyr Thr

Gin Gly

Lys Tyr 60

Ser Ser

Ser Ala

Gin Gly

Val Tyr

Thr Leu

140

Thr Trp

155

Lys Pro

Phe Thr

Leu Glu 45

Asn Asp

Asn Thr

Val Tyr

Thr Ser

110

Pro Leu

125

Gly Cys

Asn Ser

Gly Ala 15

Asp Arg

Trp Ile

Met Phe

Ala Tyr

Phe Cys 95

Val Thr

Ala Pro

Leu Val

Gly Ser

160

108

Leu Ser

Ser Gly

Tyr Thr

Glu Thr

Asp Lys

210

Thr Val

225

Asp Val

Asp Ile

Asp Val

Asn Ser

290

Trp Leu

305

Pro Ala

Ala Pro

Asp Lys

Ile Thr

Leu Ser

180

Val Thr

195

Lys Ile

Pro Glu

Leu Thr

Ser Lys

260

Glu Val

275

Thr Phe

Asn Gly

Pro Ile

Gin Val

340

Val Ser

355

Val Glu

Val His 165

Ser Ser

Cys Asn

Val Pro

Val Ser

230

Ile Thr

245

Asp Asp

His Thr

Arg Ser

Lys Glu

310

Glu Lys

325

Tyr Thr

Leu Thr

Trp Gin

Thr Phe

Val Thr

Val Ala

200

Arg Asp

215

Ser Val

Leu Thr

Pro Glu

Ala Gin

280

Val Ser

295

Phe Lys

Thr Ile

Ile Pro

Cys Met

360

Trp Asn

Pro Ala

170

Val Pro

185

His Pro

Cys Gly

Phe Ile

Pro Lys

250

Val Gin

265

Thr Gin

Glu Leu

Cys Arg

Ser Lys

330

Pro Pro

345

Ile Thr

Gly Gin

Val Leu

Ser Ser

Ala Ser

Cys Lys

220

Phe Pro

235

Val Thr

Phe Ser

Pro Arg

Pro Ile

300

Val Asn

315

Thr Lys

Lys Glu

Asp Phe

Pro Ala

Gin Ser

Thr Trp

190

Ser Thr

205

Pro Cys

Pro Lys

Cys Val

Trp Phe

270

Glu Glu

285

Met His

Ser Ala

Gly Arg

Gin Met

350

Phe Pro

365

Glu Asn

Asp Leu 175

Pro Ser

Lys Val

Ile Cys

Pro Lys

240

Val Val

255

Val Asp

Gin Phe

Gin Asp

Ala Phe

320

Pro Lys

335

Ala Lys

Glu Asp

Tyr Lys

109

370

375

380

Asn 385

Thr

Gin

Pro

Ile

Met 390

Asp

Thr

Asp

Gly

Ser 395

Tyr

Phe

Val

Tyr

Ser 400

Lys

Leu

Asn

Val

Gin 405

Lys

Ser

Asn

Trp

Glu 410

Ala

Gly

Asn

Thr

Phe 415

Thr

Cys

Ser Val

Leu His Glu Gly Leu His Asn His

420 425

His Thr Glu Lys Ser

430

Leu Ser His Ser Pro Gly Lys

435

<210> <211> <212> <213>	25 11 PRT Artificial
<220>
<223>	C8.3 CDR 1 Light Chain
<400>	25
Gin Ala Ser Gin Gly Thr Ser Ile Asn Leu Asn
1	5 10
<210>	26
<211>	7
<212>	PRT
<213>	Arti f icial
<220>
<223>	C8.3 CDR 2 Light Chain
<40Û>	26
Gly Ser Ser Asn Leu Glu Asp
1	5
<210>	27
<211>	7
<212>	PRT
<213>	Artificial
<220>
<223>	C8.3 CDR 3 Light Chain

110 <400> 27

Leu Gin His Ser Tyr Leu Pro

5 <210>

<211>

<212>

<213>

P RT Artificial <220>

<223> C8.3 CDR 1 Heavy Chain <400> 28

Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg Thr Ile His

10 <210> 29 <211> 17 <212> PRT <213> Artificial <220>

<223> C8.3 CDR 2 Heavy Chain <400> 29

Tyr Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Asn Phe Lys

10 15

Gly

<210>	30
<211>	6
<212>	PRT
<213>	Artificial

<220>

<223> C8.3 CDR 3 Heavy Chain <400> 30

Arg Gly Ala Met Asp Tyr <210> <211> <212>

214

PRT

111

<213>	Artificial
<220>
<223>	C8.3 Light Chain
<400>	31

Asp 1

Val

Gin

Met

Ile 5

Gin

Ser

Pro

Ser

Ser 10

Leu

Ser

Ala

Ser

Leu 15

Gly

Asp

Ile

Val

Thr 20

Met

Thr

Cys

Gin

Ala 25

Ser

Gin

Gly

Thr

Ser 30

Ile

Asn

Leu

Asn

Trp 35

Phe

Gin

Gin

Lys

Pro 40

Gly

Lys

Ala

Pro

Lys 45

Leu

Leu

Ile

Tyr

Gly 50

Ser

Ser

Asn

Leu

Glu 55

Asp

Gly

Val

Pro

Ser 60

Arg

Phe

Ser

Gly

Ser 65

Arg

Tyr

Gly

Thr

Asp 70

Phe

Thr

Leu

Thr

Ile 75

Ser

Ser

Leu

Glu

Asp 80

Glu

Asp

Met

Ala

Thr 85

Tyr

Phe

Cys

Leu

Gin 90

His

Ser

Tyr

Leu

Pro 95

Phe

Thr

Phe

Gly

Ser 100

Gly

Thr

Lys

Leu

Glu 105

Ile

Lys

Arg

Ala

Asp 110

Ala

Ala

Pro

Thr

Val 115

Ser

Ile

Phe

Pro

Pro 120

Ser

Ser

Glu

Gin

Leu 125

Thr

Ser

Gly

Gly

Ala 130

Ser

Val

Val

Cys

Phe 135

Leu

Asn

Asn

Phe

Tyr 140

Pro

Lys

Asp

Ile

Asn 145

Val

Lys

Trp

Lys

Ile 150

Asp

Gly

Ser

Glu

Arg 155

Gin

Asn

Gly

Val

Leu 160

Asn

Ser

Trp

Thr

Asp 165

Gin

Asp

Ser

Lys

Asp 170

Ser

Thr

Tyr

Ser

Met 175

Ser

Ser

Thr

Leu

Thr 180

Leu

Thr

Lys

Asp

Glu 185

Tyr

Glu

Arg

His

Asn 190

Ser

Tyr

112

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200

Pro Ile Val

205

Lys Ser

Phe Asn Arg Asn Glu Cys

210

10	<210> <211> <212> <213>	32 439 PRT Artificial
15	<220> <223>	C8.3 Heavy Chain
	<400>	32

20

Gin 1

Val

Gin

Leu

Gin 5

Gin

Ser

Asp

Ala

Glu 10

Leu

Val

Asn

Pro

Gly 15

Ala

25

Ser

Val

Lys

Ile 20

Ser

Cys

Lys

Ala

Ser 25

Gly

Tyr

Thr

Phe

Thr 30

Asp

Arg

Thr

Ile

His 35

Trp

Val

Lys

Gin

Arg 40

Pro

Glu

Gin

Gly

Leu 45

Glu

Trp

Ile

30

Gly

Tyr 50

Ile

Tyr

Pro

Gly

Asp 55

Gly

Ser

Thr

Lys

Tyr 60

Asn

Glu

Asn

Phe

35

Lys 65

Gly

Lys

Ala

Thr

Leu 70

Thr

Ala

Asp

Lys

Ser 75

Ser

Ser

Thr

Ala

Tyr 80

40

Met

Gin

Leu

Asn

Ser 85

Leu

Ala

Ser

Glu

Asp 90

Ser

Ala

Val

Tyr

Phe 95

Cys

45

Ala

Arg

Arg

Gly 100

Ala

Met

Asp

Tyr

Trp 105

Gly

Gin

Gly

Thr

Ser 110

Val

Thr

Val

Ser

Ser 115

Ala

Lys

Thr

Thr

Pro 120

Pro

Ser

Val

Tyr

Pro 125

Leu

Ala

Pro

50

Gly

Ser 130

Ala

Ala

Gin

Thr

Asn 135

Ser

Met

Val

Thr

Leu 140

Gly

Cys

Leu

Val

55

Lys

Gly

Tyr

Phe

Pro

Glu

Pro

Val

Thr

Val

Thr

Trp

Asn

Ser

Gly

Ser

113

145

150

155

160

Leu

Ser

Ser

Gly

Val 165

His

Thr

Phe

Pro

Ala 170

Val

Leu

Gin

Ser

Asp 175

Leu

Tyr

Thr

Leu

Ser 180

Ser

Ser

Val

Thr

Val 185

Pro

Ser

Ser

Thr

Trp 190

Pro

Ser

Glu

Thr

Val 195

Thr

Cys

Asn

Val

Ala 200

His

Pro

Ala

Ser

Ser 205

Thr

Lys

Val

Asp

Lys 210

Lys

Ile

Val

Pro

Arg 215

Asp

Cys

Gly

Cys

Lys 220

Pro

Cys

Ile

Cys

Thr 225

Val

Pro

Glu

Val

Ser 230

Ser

Val

Phe

Ile

Phe 235

Pro

Pro

Lys

Pro

Lys 240

Asp

Val

Leu

Thr

Ile 245

Thr

Leu

Thr

Pro

Lys 250

Val

Thr

Cys

Val

Val 255

Val

Asp

Ile

Ser

Lys 260

Asp

Asp

Pro

Glu

Val 265

Gin

Phe

Ser

Trp

Phe 270

Val

Asp

Asp

Val

Glu 275

Val

His

Thr

Ala

Gin 280

Thr

Gin

Pro

Arg

Glu 285

Glu

Gin

Phe

Asn

Ser 290

Thr

Phe

Arg

Ser

Val 295

Ser

Glu

Leu

Pro

Ile 300

Met

His

Gin

Asp

Trp 305

Leu

Asn

Gly

Lys

Glu 310

Phe

Lys

Cys

Arg

Val 315

Asn

Ser

Ala

Ala

Phe 320

Pro

Ala

Pro

I le

Glu 325

Lys

Thr

Ile

Ser

Lys 330

Thr

Lys

Gly

Arg

Pro 335

Lys

Ala

Pro

Gin

Val 340

Tyr

Thr

Ile

Pro

Pro 345

Pro

Lys

Glu

Gin

Met 350

Ala

Lys

Asp

Lys

Val 355

Ser

Leu

Thr

Cys

Met 360

I le

Thr

Asp

Phe

Phe 365

Pro

Glu

Asp

114

Ile

Thr 370

Val

Glu

Trp

Gin

Trp 375

Asn

Gly

Gin

Pro

Ala 380

Glu

Asn

Tyr

Lys

5

Asn 385

Thr

Gin

Pro

Ile

Met 390

Asp

Thr

Asp

Gly

Ser 395

Tyr

Phe

Val

Tyr

Ser 400

10

Lys

Leu

Asn

Val

Gin 405

Lys

Ser

Asn

Trp

Glu 410

Ala

Gly

Asn

Thr

Phe 415

Thr

Cys

Ser

Val

Leu 420

His

Glu

Gly

Leu

His 425

Asn

His

His

Thr

Glu 430

Lys

Ser

15

Leu

Ser

His 435

Ser

Pro

Gly

Lys

20

<210> <211> <212> <213> .

33 441 PRT Artificial

25

<220> <223> <400>

No 32 33

J Human Ί

Pau

30

Met 1

Ala

Glu

Pro

Arg 5

Gin

Glu

Phe

Glu

Val 10

Met

Glu

Asp

His

Ala 15

Gly

35

Thr

Tyr

Gly

Leu 20

Gly

Asp

Arg

Lys

Asp 25

Gin

Gly

Gly

Tyr

Thr 30

Met

His

40

Gin

Asp

Gin 35

Glu

Gly

Asp

Thr

Asp 40

Ala

Gly

Leu

Lys

Glu 45

Ser

Pro

Leu

Gin

Thr 50

Pro

Thr

Glu

Asp

Gly 55

Ser

Glu

Glu

Pro

Gly 60

Ser

Glu

Thr

Ser

45

Asp 65

Ala

Lys

Ser

Thr

Pro 70

Thr

Ala

Glu

Asp

Val 75

Thr

Ala

Pro

Leu

Val 80

50

Asp

Glu

Gly

Ala

Pro 85

Gly

Lys

Gin

Ala

Ala 90

Ala

Gin

Pro

His

Thr 95

Glu

55

Ile

Pro

Glu

Gly

Thr

Thr

Ala

Glu

Glu

Ala

Gly

Ile

Gly

Asp

Thr

Pro

115

100

105

110

Ser

Leu

Glu 115

Asp

Glu

Ala

Ala

Gly 120

His

Val

Thr

Gin

Ala 125

Arg

Met

Val

Ser

Lys 130

Ser

Lys

Asp

Gly

Thr 135

Gly

Ser

Asp

Asp

Lys 140

Lys

Ala

Lys

Gly

Ala Asp 145

Gly Gin

Pro Ala

Asp Arg

Arg Ser . 210

Lys Val

225

Ser Arg

Lys Ser

Gly Lys

Ser Lys

290

Ser Val

305

Gly Lys

Lys Gly

Pro Lys

180

Ser Gly 195

Arg Thr

Ala Val

Leu Gin

Lys Ile

260

Val Gin

275

Cys Gly

Gin Ile

Thr Lys

150

Gin Ala

165

Thr Pro

Tyr Ser

Pro Ser

Val Arg

230

Thr Ala 245

Gly Ser

Ile Ile

Ser Lys

Val Tyr

310

Ile Ala

Asn Ala

Pro Ser

Ser Pro

200

Leu Pro

215

Thr Pro

Pro Val

Thr Glu

Asn Lys

280

Asp Asn

295

Lys Pro

Thr Pro

Thr Arg

170

Ser Gly

185

Gly Ser

Thr Pro

Pro Lys

Pro Met

250

Asn Leu

265

Lys Leu

Ile Lys

Val Asp

Arg Gly 155

Ile Pro

Glu Pro

Pro Gly

Ala Ala

Ala Lys

Pro Lys

190

Thr Pro

205

Pro Thr

220

Ser Pro

235

Pro Asp

Lys His

Asp Leu

His Val

300

Leu Ser

315

Arg Glu

Ser Ser

Leu Lys

Gin Pro

270

Ser Asn

285

Pro Gly

Lys Val

Pro Pro

160

Thr Pro

175

Ser Gly

Gly Ser

Pro Lys

Ala Lys

240

Asn Val

255

Gly Gly

Val Gin

Gly Gly

Thr Ser

320

116

Lys Cys Gly Ser Leu Gly Asn Ile

325

His His Lys Pro Gly Gly Gly Gin

330 335

Val Glu Val Lys Ser Glu Lys Leu

340

Asp Phe Lys Asp Arg Val Gin Ser

345 350

Lys Ile Gly Ser Leu Asp Asn Ile

355 360

Thr His Val Pro Gly Gly Gly Asn

365

Lys Lys Ile Glu Thr His Lys Leu

370 375

Thr Phe Arg Glu Asn Ala Lys Ala

380

Lys Thr Asp His Gly Ala Glu Ile

385 390

Val Tyr Lys Ser Pro Val Val Ser

395 400

Gly Asp Thr Ser Pro Arg His Leu

405

Ser Asn Val Ser Ser Thr Gly Ser

410 415

Ile Asp Met Val Asp Ser Pro Gin

420

Leu Ala Thr Leu Ala Asp Glu Val

425 430

Ser Ala Ser Leu Ala Lys Gin Gly

435 440

Leu <210> 34 <211> 219 <212> PRT <213> Artificial <220>

<223> D1.2* Light Chain <400> 34

Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1015

Asp Gin Ala Ser Ile Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser

2530

Asn Gly Asn Thr Tyr Leu His Trp His Leu Gin Lys

3540

Pro Gly Gin Ser

Pro Lys Phe Leu Ile Tyr Lys Val

Ser Asn Arg Phe Ser Gly Val Pro

117

Asp 65

Arg

Phe

Ser

Gly

Ser 70

Gly

Ser

Gly

Thr

Asp 75

Phe

Thr

Leu

Lys

Ile 80

Ser

Arg

Val

Glu

Ala 85

Glu

Asp

Leu

Gly

Val 90

Tyr

Phe

Cys

Ser

Gin 95

Ser

Thr

His

Val

Pro 100

Phe

Thr

Phe

Gly

Ser 105

Gly

Thr

Lys

Leu

Glu 110

Ile

Lys

Arg

Ala

Asp 115

Ala

Ala

Pro

Thr

Val 120

Ser

Ile

Phe

Pro

Pro 125

Ser

Ser

Glu

Gin

Leu 130

Thr

Ser

Gly

Gly

Ala 135

Ser

Val

Val

Cys

Phe 140

Leu

Asn

Asn

Phe

Tyr 145

Pro

Lys

Asp

Ile

Asn 150

Val

Lys

Trp

Lys

Ile 155

Asp

Gly

Ser

Glu

Arg 160

Gin

Asn

Gly

Val

Leu 165

Asn

Ser

Trp

Thr

Asp 170

Gin

Asp

Ser

Lys

Asp 175

Ser

Thr

Tyr

Ser

Met 180

Ser

Ser

Thr

Leu

Thr 185

Leu

Thr

Lys

Asp

Glu 190

Tyr

Glu

Arg

His

Asn 195

Ser

Tyr

Thr

Cys

Glu 200

Ala

Thr

His

Lys

Thr 205

Ser

Thr

Ser

Pro

I le 210

Val

Lys

Ser

Phe

Asn 215

Arg

Asn

Glu

Cys

<210>

<211>

<212>

<213>

444

PRT

Artificial <220>

<223> hC10.2 Heavy Chain <400> 35

Gin Val Gin Leu Val Gin Ser Gly Ala 1 5

Glu Val Val Lys Pro Gly Ala

15

118

Ser Val·

Lys Ile

Ser Cys

Lys Ala

Thr Ile

Gly Tyr

Gin Gly 65

Met Glu

Ala Arg

Val Ser

Ser Ser

130

Lys Asp

145

Leu Thr

Leu Tyr

Thr Gin

Val Asp

210

Pro Pro

His Trp 35

Val Arg

Gin Ala

Ile Tyr

Pro Gly

Asp Gly 55

Arg Ala

Thr Leu

Thr Ala

Ser Gly 25

Pro Gly

Ser Thr

Asp Thr

Tyr Thr

Gin Gly

Lys Tyr

Ser Ala 75

Leu Ser

Ser Leu 85

Arg Ser

Glu Asp

Thr Ala

Phe Thr

Leu Glu 45

Ser Gin

Ser Thr

Val Tyr

Asp Arg

Trp Ile

Lys Phe

Ala Tyr

Tyr Cys 95

Arg Gly

100

Ala Met

Asp Tyr

Trp Gly

105

Gin Gly

Thr Ser

110

Val Thr

Ser Ala

115

Ser Thr

Lys Gly

120

Pro Ser

Val Phe

Pro Leu

125

Ala Pro

Lys Ser

Tyr Phe

Ser Gly

Ser Leu

180

Thr Tyr

195

Lys Arg

Cys Pro

Thr Ser

Pro Glu

150

Val His

165

Ser Ser

Ile Cys

Val Glu

Ala Pro

Gly Gly

135

Thr Ala

Ala Leu

140

Gly Cys

Leu Val

Pro Val

Thr Val

Ser Trp

155

Asn Ser

Gly Ala

160

Thr Phe

Val Val

Asn Val

200

Pro Lys

215

Glu Leu

Pro Ala

170

Thr Val

185

Asn His

Ser Cys

Leu Gly

Val Leu

Gin Ser

Ser Gly

175

Pro Ser

Lys Pro

Asp Lys

220

Gly Pro

Ser Ser

190

Leu Gly

Ser Asn

205

Thr His

Ser Val

Thr Lys

Thr Cys

Phe Leu

119

225

230

235

Phe

Pro

Pro

Lys

Pro 245

Lys

Asp

Thr

Leu

Met 250

I le

Ser

Arg

Thr

Pro 255

Val

Thr

Cys

Val 260

Val

Val

Asp

Val

Ser 265

His

Glu

Asp

Pro

Glu 270

Val

Phe

Asn

Trp 275

Tyr

Val

Asp

Gly

Val 280

Glu

Val

His

Asn

Ala 285

Lys

Thr

Pro

Arg 290

Glu

Glu

Gin

Tyr

Asn 295

Ser

Thr

Tyr

Arg

Val 300

Val

Ser

Val

Thr 305

Val

Leu

His

Gin

Asp 310

Trp

Leu

Asn

Gly

Lys 315

Glu

Tyr

Lys

Cys

Val

Ser

Asn

Lys

Ala 325

Leu

Pro

Ala

Pro

I le 330

Glu

Lys

Thr

Ile

Ser 335

Ala

Lys

Gly

Gin 340

Pro

Arg

Glu

Pro

Gin 345

Val

Tyr

Thr

Leu

Pro 350

Pro

Arg

Glu

Glu 355

Met

Thr

Lys

Asn

Gin 360

Val

Ser

Leu

Thr

Cys 365

Leu

Val

Gly

Phe 370

Tyr

Pro

Ser

Asp

Ile 375

Ala

Val

Glu

Trp

Glu 380

Ser

Asn

Gly

Pro 385

Glu

Asn

Asn

Tyr

Lys 390

Thr

Thr

Pro

Pro

Val 395

Leu

Asp

Ser

Asp

Ser

Phe

Phe

Leu

Tyr 405

Ser

Lys

Leu

Thr

Val 410

Asp

Lys

Ser

Arg

Trp 415

Gin

Gly

Asn

Val 420

Phe

Ser

Cys

Ser

Val 425

Met

His

Glu

Ala

Leu 430

His

His

Tyr

Thr 435

Gin

Lys

Ser

Leu

Ser 440

Leu

Ser

Pro

Gly

240

Glu

Lys

Lys

Leu

Lys

320

Lys

Ser

Lys

Gin

Gly

400

Gin

Asn

120 <210> 36 <211> 214 <212> PRT <213> Artificial 5 <220>

<223> hC10.2 LC <400> 36

Asp 1

Val

Gin

Met

Thr 5

Gin

Ser

Pro

Ser

Ser 10

Leu

Ser

Ala

Ser

Val 15

Gly

15

Asp

Arg

Val

Thr 20

Met

Thr

Cys

Gin

Ala 25

Ser

Gin

Asp

Thr

Ser 30

Ile

Asn

20

Leu

Asn

Trp 35

Phe

Gin

Gin

Lys

Pro 40

Gly

Lys

Ala

Pro

Lys 45

Leu

Leu

Ile

25

Tyr

Gly 50

Ala

Ser

Asn

Leu

Glu 55

Thr

Gly

Val

Pro

Ser 60

Arg

Phe

Ser

Gly

Ser 65

Arg

Ser

Gly

Thr

Asp 70

Phe

Thr

Leu

Thr

Ile 75

Ser

Ser

Leu

Gin

Pro 80

30

Glu

Asp

Met

Ala

Thr 85

Tyr

Tyr

Cys

Leu

Gin 90

His

Thr

Tyr

Leu

Pro 95

Phe

35

Thr

Phe

Gly

Ser 100

Gly

Thr

Lys

Leu

Glu 105

Ile

Lys

Arg

Thr

Val 110

Ala

Ala

40

Pro

Ser

Val 115

Phe

Ile

Phe

Pro

Pro 120

Ser

Asp

Glu

Gin

Leu 125

Lys

Ser

Gly

45

Thr

Ala 130

Ser

Val

Val

Cys

Leu 135

Leu

Asn

Asn

Phe

Tyr 140

Pro

Arg

Glu

Ala

Lys 145

Val

Gin

Trp

Lys

Val 150

Asp

Asn

Ala

Leu

Gin 155

Ser

Gly

Asn

Ser

Gin 160

50

Glu

Ser

Val

Thr

Glu 165

Gin

Asp

Ser

Lys

Asp 170

Ser

Thr

Tyr

Ser

Leu 175

Ser

55

Ser

Thr

Leu

Thr

Leu

Ser

Lys

Ala

Asp

Tyr

Glu

Lys

His

Lys

Val

Tyr

121

Claims (12)

1. A monodonal antibody, or an epitope-binding fragment thereof, capable of immunospecifically binding to the phosphorylated residue 396 of human tau (SEQ ID NO:33) and specifically recognize hyper-phosphorylated tau from human Alzheimer’s disease brains.

2. The monoclonal antibody according to claim 1 consisting of an intact antibody.

3. The monoclonal antibody or epitope-binding fragment thereof according to claim 1 comprising or consisting of an epitope-binding fragment selected from the group consisting of: an Fv fragment (; a Fab fragment, Fab' fragment and F(ab)₂ fragment; and a single V_H variable domain or V_L variable domain.

4. The monoclonal antibody or epitope-binding fragment thereof according to any preceding daims, wherein the antibody is selected from the group consisting of antibodies of subtype lgG1, lgG2, lgG3 or lgG4.

5. The monoclonal antibody or epitope-binding fragment thereof according to any of the previous daims which is human or humanized.

6. A monodonal antibody, or epitope-binding fragment thereof, according to any one of daims 1 to 5, comprising:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11 ;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13; and (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

7. The monoclonal antibody according to daim 6, comprising the heavy chain of SEQ ID NO:16 and the light chain of SEQ ID NO:15.

8. A pharmaceutical composition comprising the monoclonal antibody or epitope-binding fragment thereof according to any of daims 1 to 7 and a pharmaceutical acceptable carrier.

9. A nucleic acid encoding a monoclonal antibody or epitope-binding fragment thereof according to any one of daims 1 to 7, or encoding a constituent chain thereof.

10 The monoclonal antibody, or epitope-binding fragment thereof, according to any one of daims 1 to 7 or the pharmaceutical composition of claim 8, for use in therapy.

11. The monoclonal antibody, or epitope-binding fragment thereof, according to any one of daims 1 to 7 or the pharmaceutical composition of claim 8, for use in treating, diagnosing or imaging a tauopathy.

12. The monoclonal antibody, or epitope-binding fragment thereof according to any one of daims 1 to 7 or pharmaceutical composition according to daim 8 for use in treating a tauopathy selected from the group consisting of of Alzheimer’s disease, Argyrophilic Grain Disease (AGD), Psychosis, including Psychosis due to AD or Psychosis in patients with AD, psychiatrie symptoms of patients with Lewy body dementia, Progressive Supranuclear Palsy (PSP), Frontotemporal dementia (FTD or variants thereof), TBI (traumatic brain injury, acute or chronic), Corticobasal Degeneration (CBD), Picks Disease, Primary age-related tauopathy (PART), Neurofibrillary tanglepredominant senile dementia, Dementia pugilistica, Chronic traumatic encephalopathy, stroke, stroke recovery, neurodegeneration in relation to Parkinson's disease, Parkinsonism linked to chromosome, Lytico-Bodig disease (Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lipofuscinosis.