EA039569B1 - Antibodies specific for hyperphosphorylated tau and methods of use thereof - Google Patents

Abstract

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

Description

Field of invention

The present invention relates to a new class of monoclonal antibodies that specifically bind to the phosphorylated serine residue 396 in the pathological hyperphosphorylated (PHF) tau protein (pS396), as well as methods of using these molecules and their tau-binding fragments in the treatment of Alzheimer's disease. and taupathy.

Link to sequence listing

This application contains one or more sequence listings in accordance with 37 C.F.R 1.821 ff, which are disclosed on machine-readable media (file name: 1049-WOPCT_FINAL_ST25_1.txt, created on June 13, 2017 and has a size of 65 kB), this file is included herein by reference in its entirety.

Background of the invention

Age-related neurodegenerative diseases such as Alzheimer's disease (AD) and dementia are currently one of the biggest social problems. The World Health Organization estimates that the cost of treating older people will continue to rise and the number of diagnosed cases of dementia 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 release modulators, such as acetylcholinesterase inhibitors, and NMDA receptor modulators. These therapeutics became available at the turn of the millennium and continue to form the cornerstone for alleviating the symptoms of memory deficits associated with dementia and AD. However, these drugs do not target the underlying causes of AD: the accumulation of β-amyloid (Ab) peptide and tau protein aggregates and the associated loss of neuronal synapses and ultimately neurons.

Long-term, community-wide studies involving older people (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) along with large genome-wide association studies (Lambert, J.C. et al. (2013) Nat. Genet. 45, 1452-1458) have shown that AD is is a heterogeneous combination of forms of dementia, with up to 10 percent of patients with advanced AD having no amyloid peptide pathology (Crary, J.F. et al. (2014) Acta Neuropathol. 128, 755-766). Additionally, basic pathological studies by Braak and Braak (Braak H. and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3-12) demonstrated a clear correlation between the degree of neurofibrillary tangle pathology and pre-mortem cognitive status. These observations have been supported by several investigators (Nelson, P.T. et al. (2012) J. Neuropathol. Exp. Neurol. 71, 362-381) and recent long-term biomarker studies that indicate that tau and hyperphosphorylated tau levels cerebrospinal fluid (CSF) proteins are elevated during the early and late stages of the disease (Jack, C.R., Jr. et al. (2013) Lancet Neurol. 12, 207-216).

As stated above, microtubule-associated tau protein and its hyperphosphorylated variant form the major component of intracellular neurofibrillary tangles, which are one of the main hallmarks of AD. Additionally, certain variants of the tau protein gene are associated with familial forms of frontotemporal dementia (FTD). The appearance of tau protein pathology in AD follows a well-defined spatial pattern, starting in the entorhinal cortex and then moving into the hippocampal and cortical regions (Braak, H. and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3 -12). The specific stage of tau pathology also correlates well with cognitive performance (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, these data form the basis of the hypothesis that tau plays a role in AD. It implies that intracellular accumulation of tau protein leads to the destruction of microtubules and the collapse of dendritic spines. As a result, communication between neurons is disrupted and cell death occurs. It has also recently been shown that tau itself can form endopathogenic molecules that can promote the spread of neurodegeneration from one cell to the next (Clavaguera, F. et al. (2009) Nat. Cell Biol. 11, 909-913).

I. Tay protein as an endopathogen

Clavaguera et al. have demonstrated that tau itself can act as an endopathogen (Clavaguera F. et al. (2009) Nat. Cell Biol. 11, 909-913). Brain extracts obtained at low speed centrifugation were isolated from tau-transgenic P301S mice (Allen, B. et al. (2002) J. Neurosci. 22, 9340-9351), diluted and injected into the hippocampus and cortical areas of young mice ALZ17. ALZ17 mice are a tau transgenic mouse strain that develops only a late form of the disease (Probst, A. et al. (2000) Acta Neuropathol. 99, 469-481). Injected ALZ17 mice rapidly developed dense filament pathology, and administration of tau immunodepleted brain extracts from P301S mice or extracts from wild-type mice did not induce tau pathology. Fractionation of brain extracts into soluble (S1) and sarcosyl insoluble (P3) tau protein (Sahara, N. et al. (2013) J.

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Alzheimer's. Dis. 33, 249-263) and their injection into ALZ17 mice demonstrated that the P3 fraction was the most capable of inducing pathology. It contains most of the intracellular hyperphosphorylated filamentous tau protein. Pathology could also be induced in most cases by injecting P301S extracts into the brains of wild-type mice, but no NFTs were generated. In subsequent studies, Clavaguera et al. showed that human tau protein extracted postmortem from brain tissue with other taupathies (argyrophilic granule disease (AGD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD)) can also induce tau protein pathology in the ALZ17 model (Clavaguera, F. et al. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 9535-9540). Several other models of priming action and tau clearance have been reported since the presentation of these data (Ahmed, Z. et al. (2014) Acta Neuropathol. 127, 667-683; Walker, L. C. et al. (2013) JAMA Neurol. 70 , 304-310). The main conclusion of these studies points to the mechanism by which the pathogenic tau protein in intracellular inclusions is secreted from the cell into the periplasmic space. The pathological material of the tau protein is then transported along the sheath of the synaptic vesicles in both anterograde and retrograde directions and is subsequently taken up by neighboring cells by bulk endocytosis. This mechanism explains why the diversity of pathology observed in human disease follows a well-defined anatomical pattern. Quite curiously, peripheral administration of pathological tau can accelerate the development of tau pathology in ALZ17 mice (Clavaguera, F. et al. (2014) Acta Neuropathol. 127, 299-301). This separation mechanism may explain the spread of disease 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 protein molecules

The discovery that tau can act as an endopathogen has prompted the search for pathogenic molecules that can be targeted as potential interventional therapies.

The microtubule-associated tau (MART) gene is located on chromosome 17 in the human genome and expresses six isoforms of tau in the adult brain. These isoforms result from alternative splicing of exons 2, 3, and 10 of the 16 exons in the MART gene. Exons 2 and 3 express a 29 amino acid repeat, and exon 10 expresses an additional microtubule-binding domain. As a result, tau protein isoforms will contain 0, 1, or 2 N-terminal repeats and 3 or 4 C-terminal microtubule-binding domains (tau protein 3R or 4R). Six isoforms of the tau protein are commonly 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 overhang of the tau protein (2N4R) consists of a 44 amino acid high glycine tail and residues 45-102 spanning two highly acidic regions (N1, N2 domains). Residues 151-243 contain two proline-rich regions (P1, P2 domains). The rest of the protein consists of four microtubule-binding domains (R1-R4), followed by a short C-terminal region.

The tau protein is soluble and highly labile for protein phosphorylation. Approximately 20% or 85 amino acid residues in the longest isoform of the tau protein are potential (Ser, Thr or Tyr) phosphorylation sites. It has been experimentally observed that about half of them 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) and that phosphorylation sites cluster around terminal residues of microtubule-binding domains. Tau protein undergoes dynamic phosphorylation and dephosphorylation during the cell cycle. It must dissociate from microtubules to allow mitosis to proceed. Its main role in postmitotic cells (differentiated neurons) is to act as a microtubule stabilizer, which ensures optimal axonal transport. It can only associate with microtubules in its mostly dephosphorylated form, so phosphorylation acts as a direct microtubule association/dissociation switch in the neuron. Under normal conditions, the cytosolic tau protein contains, on average, two phosphorylated sites. In paired helical filament 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). Paired helical filaments of hyperphosphorylated tau protein are a key feature of Alzheimer's disease (Kosik et. al. (1986) PNAS, 86, 40444048), a distinct shift in hyperphosphorylated tau mobility is observed in immunocytochemical analysis of human AD brain material.

Investigation of the tau protein using traditional structural techniques such as X-ray crystallography or NMR spectroscopy has been difficult, reflecting its metastable nature. Such studies have been carried out mainly on fragments of the non-phosphorylated tau protein domains. So far, the only structural study of the full-length tau protein (2N4R) using NMR spectroscopy revealed that the protein contains only rare

- 2 039569 fragments of a 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 high propensity to adopt a p-sheet structure. The first 200 residues of the backbone are significantly more ordered than the C-terminus, which spans microtubule-binding domains. The presence of a large number of specific long-range interactions in a protein in solution indicates that it exists in a highly disordered molten globule state (Ohgushi, M. and Wada, A. (1983) FEBS Lett. 164, 21-24).

In the material of coils, products of tau protein cleavage by proteases were identified, which are formed, in particular, under the action of caspase and calpain (Asp13, Glu391 and Asp421) (Gamblin T.C. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 10032 -10037). In particular, truncation at Asp421 has been studied in detail using an anti-tau C3 antibody that binds to the free end of Asp421. This truncation has been suggested to be an early event in the pathogenesis of AD associated with the induction of apoptosis (deCalignon A. et al. (2010) Nature 464, 1201-1204). N-terminal cleavage at Asp13 and C-terminal cleavage at Glu391 are considered late events in 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) has been identified in CSF from AD and PSP patients and has been hypothesized to be an early disease marker and particularly pathogenic (US14/092539; Bright, J. et al. ( 2014) Neurobiol Aging, 1-17). A similar fragment resulting from cleavage with calpain has been 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 ).

It has been proposed that, in addition to hyperphosphorylation and tau fragmentation, posttranslational acetylation (Cohen T.J. et al. (2011) Nat. Commun. 2, 252; Min, S.W. et al. (2010) Neuron 67, 953-966) and O-GlcNAc-ylation (Zhu Y. et al. (2014) J. Biol. Chem.) are pathology-determining processes in the formation of AD-associated tangle pathology.

III. Types of immunotherapy using tau protein

Types of immunotherapy are traditionally divided into passive and active approaches to vaccination. In an active approach to vaccination, a pathogenic agent, or an inactivated pathogenic form thereof, is injected into a patient and the immune system elicits an immune response. This triggers the maturation of B cells that produce high-affinity antibodies to the introduced antigen or a cellular response to it. In the passive approach to vaccination, triggering the immune system is avoided by infusion of an antibody specific to the antigen. The body's own purification system then removes the ligand bound to the antibody.

AC Immune provides a monoclonal mouse antibody to the tau protein phosphoserine-409. Antibodies to AD human brain tissue antigens and control brain tissue were profiled and selected based on their ability to recognize tangle pathology. Both humanized versions of the two antibodies hACI-36-2B6-Ab1 and hACI-36-3A8-Ab1 bind to a tau protein epitope within amino acids 401-418 (WO 2013/151762).

Roger Nitsch's group has isolated autoantibodies to tau protein from elderly healthy individuals without evidence of degenerative tauopathy. A number of antibodies were isolated using full length recombinant human tau protein (2N4R) to detect antibodies specific for tau protein. They were then screened for their ability to distinguish between tau protein isolates from diseased and healthy individuals. Three leader antibodies 4E4, 4A3 and 24B2 have been described in the patent literature (WO 2012049570; US 2012087861). Mapping of their epitopes indicates that they all recognize amino acids within and C-terminally from the microtubule-binding region at positions V339 to K369. These antibodies do not show any phosphospecificity.

C2N Diagnostics focuses primarily on the development of diagnostic tools for the early detection of neurodegenerative disease. Received antibodies to full-length human and mouse tau protein. Eight and five antibodies were identified, respectively recognizing human and mouse tau (Yanamandra, K. et al. (2013) Neuron 80, 402-414). Three antibodies with different binding kinetics were selected for in vivo evaluation. Namely, they were HJ9.3, HJ9.4 and HJ8.5, recognizing residues 306-320, 7-13 and 25-30 of the tau protein, respectively, the latter (HJ8.5) being specific to the human tau protein. Antibodies were also selected based on their ability to prevent the transmission of pathology in the original mechanized reporter analysis of the transcellular spread of tau pathology (Sanders, D. W. et al. (2014) Neuron 82, 1271-1288; Kfoury, N. et al. (2012) J Biol Chem 287, 19440-19451). Their assessment in studies with long-term i.c.v. injection into P301S transgenic mice demonstrated their ability to reduce levels of hyperphosphorylated tau protein as determined by immunohistochemistry of treated mice.

Antibodies by Peter Davies were originally developed as diagnostic tools that could discriminate between abnormal and normal tau in brain material from AD subjects and control brain material (Greenberg, S.G. and Davies, P. (1990)

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Proc. Natl. Acad. sci. U.S.A. 87, 5827-5831). Evaluation of the therapeutic utility of PHF1 and MC1 antibodies has been demonstrated in P301S and JPNL3 (P301L) mice (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). PHF1 recognizes a linear epitope of the phosphorylated tau protein (pS396, pS404), while MC1 is a conformation dependent antibody that recognizes a structural tau protein epitope that requires two distinct portions of the linear sequence, an epitope between residues 46-202 and C -terminal epitope within residues 312-342 (Jicha G.A. et al. (1997) J. Neurosci. Res. 48, 128-132). Injection of these two antibodies in long-term 12-13 week immunization studies resulted in a significant reduction in spinal cord and brainstem pathology among other brain regions, which translated into an alleviation of the motor deficits observed in these mice. (D'Abramo C. et al. (2013) PLoS. One. 8, e62402).

iPerian/Bristol Meyers Squibb developed anti-tau antibodies directed to putative pathological tau molecules consisting of an N-terminal tau fragment (eTau: residues 1224) that promoted hyperactivity in neuronal cultures from induced pluripotent stem cells. A portfolio of antibodies has been developed, but characterization has focused on the IPN001 and IPN002 antibodies, which recognize the N-terminal epitope within residues 9-18. Accordingly, these antibodies detect elevated levels of tau in CSF from patients with established AD and PSP, which may be an early sign of the disease. Antibody injections in JPNL3 (P301L) mice in vivo resulted in partial resolution of progressive motor deficits (US 14/092539).

Einar Sigurdsson announced the first program demonstrating the efficacy of tau-based immunotherapy. JPNL3 (P301L) mice were immunized with an active vaccine consisting of the 379-408 tau peptide [pS396, pS404] together with Adju-Phos adjuvant. In this study, a marked reduction in tau protein pathology was observed in vaccine-treated mice compared to control animals. A reduction in the motor phenotype associated with tauopathy was also detected. Its effectiveness was confirmed in another mouse model (htau/PS1) functioning without exposure to the mutant tau protein (Boutajangout A. et al. (2011) AAIC 2011 (7, issue 4, Supplement 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 evaluated three anti-tau antibodies in tau transgenic K369I (K3) mice and in a P301L mouse model. Antibodies with different properties were selected for in vivo evaluation. Two pS404-specific antibodies with different isotypes (IgG1/k and IgG2a/k) or a common (pan-specific) anti-tau antibody (IgG1/k) were injected as part of a long-term study paradigm. K369I mice were treated with weekly injections for 21 weeks starting at 3 weeks of age and P301L mice were treated for 7 months with weekly injections starting at 4 months of age. K3 mice treated with anti-pS404 IgG2a/k showed a decrease in tau-positive neurofibrillary inclusions. Both pS404-specific antibodies were able to reduce the level of pS422-positive tau protein, while no decrease was observed in mice treated with the anti-tau pan-specific antibody. These studies suggest that: 1) tau clearance may be dependent on antibody isotype and 2) targeting disease-relevant tau molecules may be important, as total tau antibody was unable to reduce levels hyperphosphorylated tau protein (PCT/US 2014/025044).

The present inventors surprisingly found that antibodies specific for the phosphorylated serine residue 396 of the tau protein (pS396) are effective in disease models; this is in contrast to prior art antibodies which recognize primarily tau proteins phosphorylated at both residue 396 and residue 404, phosphorylated only at residue 404 or other tau residues.

The authors of the present invention have developed antibodies, which are further characterized by significant specificity and selectivity for pathological human tau protein. The antibodies of the present invention show a much higher degree of specificity and selectivity for abnormal human tau compared to non-pathological tau than the antibodies of WO 2013/050567 (see Figure 1 of WO 2013/050567). The formation of antibodies from WO 2012/045882, which were reported to have specific binding, was elicited in response to amino acid sequences with 6-9 residues from amino acids 393-401, 396-401, 394-400 and 393-400 of the tau protein. This is in contrast to the antibodies of the present invention, which are generated in response to a pathogenic hyperphosphorylated tau protein containing the longer amino acid sequence described herein.

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

Brief description of the invention

The present invention relates to monoclonal antibodies and epitope-binding fragments thereof capable of specifically binding to the phosphorylated serine residue 396 of the human tau protein (isoform 2N4R) (SEQ ID NO: 1). The antibodies are further characterized by their ability to distinguish between phosphorylated residues 396 and 404 so that they hardly bind to the phosphorylated residue 404.

The antibodies of the present invention are selective for the abnormal tau protein in the presence of a non-pathological, yet phosphorylated, tau protein. The antibodies of the present invention are capable of causing selective depletion of tau tangles from abnormal tau in the presence of normal tau. Without wishing to be bound by any particular theory, it is believed that depletion of tau tangles containing tau protein that has been phosphorylated at position 396 of the tau protein prevents the priming action of abnormal tau protein to form tau tangles. Accordingly, one aspect of the present invention relates to an antibody capable of selectively binding to a tau protein phosphorylated at position 396 even when such molecules are in the presence of a tau protein that has been phosphorylated at position 404 of the tau protein. A related aspect of the present invention relates to an antibody capable of selectively binding to phosphorylated at position 396 tau protein even when such molecules are present in the presence of non-pathogenic tau protein. As further defined, the present invention relates to an antibody selective for abnormal tau protein, said abnormal tau protein being hyperphosphorylated tau protein appearing as a band corresponding to 64 kDa (in Western blot analysis) in transgenic mice overexpressing the 2N4R isoform of the human tau protein.

One aspect of the present invention is directed to an anti-tau antibody which, when used with immunodepleted extracts from rTg4510 transgenic mice, specifically reduces the 64 and 70 kDa hyperphosphorylated tau protein bands by at least 90% while reducing the 55 kDa tau band. kDa by no more than 10%. A further aspect of the present invention is directed to an anti-tau antibody that specifically reduces the 64 kD and 70 kD hyperphosphorylated tau bands by at least 90% while reducing the 55 kD tau band by no more than 10%; or which, when used as described herein with post-mortem human brain extracts from AD, has the ability to specifically reduce pS396 hyperphosphorylated tau bands by at least 90% without reducing non-hyperphosphorylated tau bands by more than 10% .

Another aspect of the present invention is directed to a method of treating a patient with tauopathy, such as Alzheimer's disease, comprising depleting tangles or attenuating the progression of formation of said tangles, said tangles containing hyperphosphorylated tau protein, said method comprising contacting hyperphosphorylated tau protein with antibody of the present invention in such a way that the tangles are depleted, their content of hyperphosphorylated tau protein is reduced, or the progression of tangle formation is attenuated.

As defined alternatively, the present invention relates to a method of treating a patient with tauopathy, such as Alzheimer's disease, which method comprises contacting coils with an antibody selective for a tau protein having a phosphorylated residue 396, such that in tangles are depleted of hyperphosphorylated tau protein.

One aspect of the present invention is directed to an anti-hyperphosphorylated human tau monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 40 and SEQ ID NO: 46;

(b) a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4; SEQ ID NO: 41 and SEQ ID NO: 47;

(c) a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 5; SEQ ID NO: 42 and SEQ ID NO: 48;

(d) a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 6; SEQ ID NO: 43; SEQ ID NO: 49; SEQ ID NO: 52 and SEQ ID NO: 55;

(e) a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 44; SEQ ID NO: 50; SEQ ID NO: 53 and SEQ ID NO: 56; and (f) a heavy chain CDR3 containing an amino acid sequence selected from the group,

- 5 039569 consisting of SEQ ID NO: 8, SEQ ID NO: 39; SEQ ID NO: 45; SEQ ID NO: 51; SEQ ID NO: 54 and SEQ ID

NO: 57.

One aspect of the present invention is directed to an anti-hyperphosphorylated human tau monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain selected from the group consisting of SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22 and SEQ ID NO: 23; and (b) a heavy chain selected from the group consisting of SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26 and SEQ ID NO: 27.

A further aspect of the present invention is directed to an anti-hyperphosphorylated human tau monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

An aspect of interest of the present invention is directed to an anti-hyperphosphorylated human tau monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

An aspect of interest of the present invention is directed to an anti-hyperphosphorylated human tau monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39;

and further comprising at least one of (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7.

The antibodies and epitope-binding fragments thereof of the present invention can be used in the treatment of taupathies such as Alzheimer's disease (AD), argyrophilic granule disease (AGD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), TBI (traumatic brain injury of the lung, acute or chronic forms) and chronic traumatic encephalopathy (CTE).

The antibodies and epitope-binding fragments thereof of the present invention are further intended for use in the treatment of psychosis, in particular AD-associated psychosis or psychosis in AD patients, and AD-associated apathy or apathy in AD patients.

Brief description of the figures

Fig. 1. Liquid phase P3 antigen uptake inhibition assay from AD subjects using humanized C10-2 and variants (C10-2_N32S and C10-2_N32S_A101T). As described in Example 3A, concentration-dependent inhibition of uptake of P3 antigens from material from subjects with AD P396-specific antibodies hC10-2 (squares), hC10-2_N32S (black circles) and hC10-2_N332_A101T (white circles) was examined. A P3 fraction from material from AD subjects was incubated for 60 minutes at room temperature (RT) with increasing antibody concentrations (0-1000 nM) before incubation with 200 ng/ml mouse C10-2 antibody immobilized in 96-well plates. Captured P3 antigens from material from AD subjects were detected with SULFO-TAG-labeled total tau (MSD) antibody.

The calculated IC ₅₀ values of hC10-2_N32S (black circles) and hC10-2_N332_A101T (white circles) antibodies were 44 nM and 14 nM, respectively. This is a marked improvement over hC10-2, as can be seen by comparing the curves in FIG. 1. Accordingly, in one aspect of the present invention, antibodies inhibit P3 from material from subjects with AD in the liquid phase inhibition assay described herein such that the signal is reduced by 50% at an antibody concentration of 100 nM or less, based on the liquid phase assay. inhibition of uptake of P3 antigens from material from subjects with AD.

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Fig. 2. Peptide inhibition assay illustrating the apparent affinity of hC10-2 and related variants. As described in Example 3B, concentration-dependent inhibition of antibody binding in liquid phase solution to Ptau 386-408 (pS396) peptide was examined with antibodies hd0-2 (squares), hC10-2_N32S (black circles) and hC10-2_N32_A101T (white circles) . Antibodies were preincubated at 1 ng/ml for 60 min. with increasing concentrations (0-10000 nM) of Ptau 386-408 (pS396) before incubation in wells coated with 100 ng/ml of Ptau 386-408 (pS396/pS404). Well-bound antibody was detected with SULFO-TAG labeled anti-human IgG (MSD).

As can be seen from FIG. 2, hC-10.2 antibody (IC ₅₀ = 24 nM), hC10.2_N32S antibody (IC ₅₀ = 50 nM), and hC10.2_N32S antibody, A101T (IC ₅₀ = 34 nM) have IC ₅₀ values of less than 100 nM and even less than 60 nM based on apparent affinity studies using a liquid phase solution with Ptau (P396) 386-408.

Fig. 3 (panels A-Z and AA-AG). Immunohistochemical detection of abnormal tau protein in brain samples obtained postmortem from donors with AD and in brains from rTg4510 mice. As described in Example 4, neurofibrillary tangles, neuropil filaments and dystrophic neurites were labeled in the prefrontal cortex from 3 different donors with AD hC10-2, hC102_N32S and hC10-2_N32S_A101T. The highest staining intensities were detected at the highest antibody concentrations. In the control sections of the brain, no immunoreactivity was observed. All 3 antibodies labeled phosphorylated tau protein in the brain from rTg4510 with an advanced pathology.

Staining increased in the direction from hC10-2 to hC10-2_N32S and to hC10-2_N32S_A101T. The highest staining intensities were detected in the case of hC10-2_N32S_A101T, then hC10-2_N32S and then hC10-2. Immunohistochemical detection of abnormal tau protein in brain samples from subjects with Alzheimer's disease was observed at concentrations of no more than 100 ng/ml hC102_N32S_A101T and hC10-2_N32S.

Fig. 4 (panels A-F). Staining of tau protein structures in rTg4510 mice treated with hC10-2. hC10.2 was administered i.v. (Panels A, C, E and F show rTg4510; panels B and D show tTA). Mice received a single injection of hC10-2 antibodies at a concentration of 80 mg/kg in a volume of 150 μl. Brain sections were obtained after 3 days according to the method described in Example 5. hC10-2 specifically labeled target structures in vivo in the hippocampus and cortex from rTg4510 brain samples but not from tTA control brain samples. Overlay images of AlexaFluor488 and Hoechst signals are shown in sections of the hippocampus.

Fig. 5 (panels A-F). Staining of tau protein structures in rTg4510 mice treated with hC102_N32S. (Panels A, C and E show rTg4510; panels B, D and F show tTA). Mice received a single injection of hC10-2_N32S antibodies at a concentration of 80 mg/kg in a volume of 150 μl. Brain sections were obtained after 3 days according to the method described in Example 5. hC10-2_N32S specifically labeled target structures in vivo in the hippocampus and cortex from rTg4510 brain samples but not from tTA control brain samples. Overlay images of AlexaFluor488 and Hoechst signals are shown in sections of the hippocampus.

Fig. 6 (panels A-F). Staining of tau protein structures in rTg4510 mice after i.v. hC102_N32S_A101T injections. (Panels A, C and E show rTg4510; panels B, D and F show tTA). Mice received a single injection of antibodies hC10-2_N32S_A101T at a concentration of 80 mg/kg in a volume of 150 μl. Brain sections were obtained after 3 days according to the method described in Example 5. hC10-2_N32S_A101T specifically labeled target structures in vivo in the hippocampus and cortex from rTg4510 brain samples but not from tTA control brain samples. Overlay images of AlexaFluor488 and Hoechst signals are shown in sections of the hippocampus.

Comparison of FIG. 4-6 indicates that hC10.2, hC10-2_N32S and hC10-2_N32S_A101T cross the blood-brain barrier when injected intravenously. The figures further indicate that hC10-2_N32S and hC10-2_N32S_A101T labeled tau structures (immunoreactive for tau tangles) in the hippocampus and cerebral cortex with improved results compared to hC10-2.

Fig. 7 (panels A-D). Tau protein molecules recognized by pS396-specific antibodies in the brain from subjects with Alzheimer's disease (AD). As described in Example 6, in brain sections from AD subjects, tau tangles were either co-labeled with antibodies to E1 and p396 or positive for antibodies to pS396 alone (arrows). Sections were analyzed by fluorescence microscopy. A number of tau coils were labeled only with hC10-2 antibodies or hC102_N32S_A101T antibodies (arrows). Given that non-detectable coils are not stained with antibodies to the N-terminal tau fragment, tau molecules labeled with hC10-2 or hC102_N32S_A101T antibodies alone are likely to represent extracellular non-detectable coils.

Fig. 8A-8C. Detection of pathological tau protein using Western blotting. As described in Example 6, section Detection of Abnormal Tau by Western Blot, Abnormal Tau was detected by Western Blot using hC10.2, hC10-2_N32S, hC10

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2_N32S_A101T. Pooled forebrain samples from three rTg4510 mice and those from non-transgenic (non-Tg) control littermates euthanized at 32 weeks of age and pooled cortex samples from four AD donors and those from four healthy control (HC) donors were respectively fractionated into soluble (S1) fractions, TBS soluble precipitate (S1p) and sarcosyl insoluble (P3) fractions and analyzed by Western blotting against the phosphorylated tau pS396 epitope using 1 μg/ml hC10.2(A) , hC10-2_N32S (B), hC10-2_N32S_A101T (C). In the case of rTg4510, normal human tau protein 4R0N was displayed at 55 kDa, while hyperphosphorylated tau molecules were displayed at 64 and 70 kDa. In the case of AD, hyperphosphorylated tau molecules were displayed as four bands corresponding to 54, 64, 69, and 74 kDa, with varying amounts of the typical AD spot.

Each of hC10-2, hC10-2_N32S and hC10-2_N32S_A101T was selective for rTg4510 mouse tau over non-transgenic mice and for AD donor tau over healthy control donors. In addition, in soluble (S1) fractions, TBS-soluble precipitate (S1p) and sarcosyl-insoluble (P3) fractions, hC10-2, hC102_N32S, and hC10-2_N32S_A101T were each selective for the pathogenic 64 kDa tau protein from mice. rTg4510 compared to a normal 55 kDa tau protein from rTg4510 mice.

Fig. 9. Tau immunoprecipitation from brain samples from subjects with AD. As described in Example 6, section Pathological tau immunoprecipitation, tau immunoprecipitation using 10 µg hC10.2, hC10-2_N32S, hC10-2_N32S_A101T, using control human IgG1 (hIgG1) from 500 µg prepurified lysates of cerebral cortex homogenates brains pooled from four AD donors and healthy control (HC) donors were analyzed by Western blotting using polyclonal rabbit anti-tau antibody pS396 (tau protein pS396). In the case of AD, hyperphosphorylated tau molecules were displayed as four bands corresponding to 54, 64, 69, and 74 kDa, with varying amounts of the typical AD spot.

Fig. 10A-C. Quantification of tau protein aggregation by Cisbio assay. Wild-type (Wt) (WW) seed material showed no seeding effect and the background signal was subtracted from all seed samples. Homogenates from Tg4510 had an effective seeding effect and the seeding effect was not affected by treatment with B12, however, it was affected to varying degrees by treatment in seeding studies conducted using antibodies to the tau protein of the present invention (hC10-2_N32S_A101T > hC10- 2_N32S > hC10-2) at a concentration of 20 µg/ml. The graphical representations represent the results of four independent sets of experiments and are plotted as relative tau aggregation (signal fold relative to background normalized to total protein) and the relative content of insoluble p396 tau was quantified by western blot densitometry insoluble in Triton-X fractions (normalized signal fold to background level). All samples were normalized to the B12 isotype control antibody. In FIG. 10B-C show the quantification of tau protein aggregation using the Cisbio assay. HEK293 cells transfected with pcDNA for seeding showed no signal, confirming that incoming seeding material was not detected. Wt (wild-type) (WW) seed material did not seed, however, in contrast, homogenates from rTg4510 (CC) showed effective seeding compared to unseeded ones. This seeding effect was not affected by HEL treatment, but was partially abolished by anti-tau antibody treatment (C10-2 > D1.2 > hACI36-2B6-Ab1). The graphical representations represent three independent sets of samples and are plotted as relative tau aggregation (signal fold relative to background normalized to total protein). Example 6, Cellular Analysis and Aggregation Assay section, describes the following protocol.

Fig. 11. Quantification of Tau5 signal in Western blotting after immunodepletion of brain extracts from AD subjects using various amounts of hC10-2 and 2.10.3 antibodies. As described in Example 7, both antibodies removed a small fraction of the total tau protein from brain extracts from subjects with Alzheimer's disease.

Fig. 12. Quantification of P-S422 tau protein signal in Western blotting after immunodepletion of brain extracts from AD subjects using different amounts of hC10-2 antibodies (diamonds) and 2.10.3 (triangles). In FIG. results from Example 7 are shown. Serine-422 phosphorylated tau can be efficiently removed from brain extracts from AD subjects by immunodepletion using hC10-2 or 2.10.3. Both antibodies removed more than 90% of P-S422 tau, although more antibody 2.10.3 was required to achieve the same effect.

Fig. 13. Quantification of pS396 tau protein signal in Western blotting after immunodepletion of brain extracts from AD subjects using various amounts of hC10-2 and 2.10.3 antibodies. In FIG. the results from Example 7 are shown. Immuno-depletion with hC10-2 resulted in the removal of 88% of the tau protein phosphorylated at serine-396, while 2.10.3 resulted in the removal of only 55% of the tau protein pS396 from brain extracts from AD subjects .

Fig. 14. Quantification of P-S199/202 tau protein signal in Western blotting after immunodepletion of brain extracts from AD subjects using various amounts of hC10-2 and 2.10.3 antibodies. The figure shows the results from example 7. Immunodepletion with hC10-2 resulted in the purification of 69% of the tau protein phosphorylated at serine-199/202. Antibody 2.10.3 did not result in the same dose-dependent reduction.

Fig. 15. Western blot patterns of brain extracts from Alzheimer's subjects before and after immunodepletion. In FIG. the results from example 7 are shown. A 25 kDa fragment of tau protein phosphorylated at serine-396 is present. Immuno-depletion with hC10-2 resulted in a decrease in the 25 kDa tau band. The 2 other phosphospecific antibodies 2.10.3 and AT8 did not remove this 25 kDa molecule.

Fig. 16. Quantification of pS396 tau protein signal in Western blotting after immunodepletion of brain extracts from AD subjects using various amounts of N32S, N32Q, N32S_A101T, N32Q_A101T, N32Q_D55E, and N32S_D55E hC10-2 variants. As can be inferred from Example 8, the ability of the antibodies of the present invention to remove serine-396 phosphorylated tau protein from brain homogenates from AD subjects was significant. At less than 0.1 µg of antibody (75 ng assay point), variants resulted in a decrease in pS396 signal of at least 28% (except for N32Q, D55E, which was characterized by 16%), while C10.2 resulted in a decrease in pS396 signal by less than 6%.

Fig. 17 (panels A-D). Priming of tau tangles in the hippocampus induced by injection of brain extracts from subjects with AD. As set forth in Example 8a, at a dose of 15 mg/kg, mC10-2 treatment significantly reduced tangle pathology in the seeded hippocampus by 57% (P<0.05). There was a clear trend indicating that hC10-2 also reduced pathology. For comparison, 2.10.3 had no effect at the same dose.

Fig. 18. P-Ser396 and Tyr394 residues are located in the center of the antigen-binding site

The structure of Ile(392)-Val(393)-Tyr(394)-Lys(395)-pSer(396)-Pro(397)-Val(398) is shown. The main interaction with the antibody of the present invention involves the hydrophobic pocket, pSer396 and Y394 of the tau protein. There is a branched network of hydrogen bonds formed between the Y(394) side chain and the pSer396 phosphonate backbone and charge/polar interactions between them. The CDR1 of the HC antibodies of the present invention contains an 8-residue palindromic motif Polar amino acid - Hydrophobic amino acid - Polar amino acid - Charged amino acid - Charged amino acid - Polar amino acid - Hydrophobic amino acid - Polar amino acid His). The charged residues interact through an extensive network of hydrogen bonds, charge interactions, and charge/polar interactions between the antibody and the tau protein.

Fig. 19. Effectiveness of antibodies in the treatment paradigm

As described in Example 8b, HEK293 cells expressing hTau-P301L were primed with Tg4510 homogenates preincubated with the indicated antibodies, trypsinized and reprimed 24 hours later (antibodies were re-primed) and harvested 48 hours after seeding. . Total cell homogenates were examined for aggregated tau protein using the tau protein aggregation assay from Cisbio. Data are pooled data from 4 independent biological repeats +/S.E.M. normalized to CC+B12 (tg4510).

Cells expressing P301L-htau were primed with 40 μg brain homogenate from Tg4510 (total protein) pre-incubated overnight at 4°C with antibodies (20 μg/ml ~ 133 nM) in 6-well plates. Priming with CC+B12 resulted in a significant seeding response. hC10.2 had approximately 40% effect on aggregation. All other antibodies showed at least comparable effect to hC10.2. In particular, the hC10.2 N32S and N32S_A1O1T variants showed more pronounced effects in terms of reduced tau protein aggregation by 45% and 62%. The N32S_A101T variant showed a significantly more pronounced effect on aggregation compared to hC10.2. In FIG. humanized C10.2 has been shown to be effective in involving tau-induced seeding action, however, the addition of N32S, and in particular the double mutation of N32S and A101T, increases the neutralizing activity of the mAb.

Fig. 20. Studies on deamidation of variants under stress conditions

As described in Example 8c, deamidation of Asn residues at position 32 or 34 of the VL chain was monitored by analysis of the LC:T2 tryptic peptide [VTMTCQASQDTSIXLNWFQQKPGK; SEQ ID NO: 58] using LC-MS. X is Asn, Gln or Ser in the respective variants shown in FIG. 20. MS analysis makes it possible to calculate the relative content of deamidated peptides relative to non-deamidated ones. In the case of WT, variants A101T and D55E, there is significant deamidation in the LC:T2 peptide. It is also obvious that changing Asn32 to either Gln or

Ser completely prevents the deamidation of the peptide at other Asn34 residues. Also, no deamidation of Gln32 variants was detected. Similar results were observed for variants

Asn34.

Fig. 21. Reduction of tau priming and aggregation in cortical neurons with anti-tau antibodies

Priming and tau aggregation in cultures of cortical neurons from rTg4510 embryonic mice were induced with 0.2 ng of pathological tau from P3 or S1 p fractions from 40 week old rTg4510 mice and measured using the Cisbio tau aggregation assay. On day 7 of culture (DIV), neurons were treated with a mixture of P3 or S1p and 10 μg of antibody or phosphate buffered saline (PBS). A complete change of environment was carried out on DIV11 with the removal of residual primers P3 and S1p and antibodies. Tau seeding was provided for an additional 4 days and neurons were lysed on DIV15 to measure seeding activity and tau aggregation. PBS and control human IgG antibody (counter, ab IgG) had no effect on priming and tau aggregation. Tau seeding and aggregation was partially reversed by treatment with anti-tau antibodies (hC10.2_A101T_N32S > hC10.2_N32S > hC10.2). The following reduction in priming action and tau protein aggregation was measured: 23% for hC10.2, 41-53% for hC10.2_N32S, and 48-60% for hC10.2_A101T_N32S. Bar graphs present data from two independent tau aggregation experiments normalized to neuronal protein as means ± SD. One-way ANOVA and Newman-Keuls test for multiple comparisons (PBS/control, ab IgG vs. hC10.2, hC10.2_N32S, hC10.2_A101T_N32S, ***p<0.001; hC10.2 and hC10.2_N32S, hC10 were used. 2_A101T_N32S, ###p<0.001).

Fig. 22. Dose-dependent staining of tau protein structures in rTg4510 mice after i.v. hC10-2_N32S injections.

hC10-2_N32S specifically methylated target structures in vivo in the hippocampus and cerebral cortex from rTg4510 brain samples. Images of the anterior cingulate cortex are shown. The strongest signals were observed at a dose of 20 and 80 mg/kg, weak signals - at a dose of 8 mg/kg, no visible signal - at a dose of 0.8 mg/kg.

Fig. 23. Seed-induced tau pathology in the hippocampus

The number of cells showing Gallyas coil staining in the seeded hippocampus was reduced by treatment with hC10-2 (50%), hC10-2_N32S (48%) and hC10-2_N32S_A101T (47%). Quantification was performed in every 6 sections covering the dorsal hippocampus, a total of 8 sections were used per animal. The number of cells reflects the sum of positive cells in all subregions of the hippocampus identified in 8 sections. For data analysis, one-way anova and Dunette's test for multiple comparisons were used.

Sequences included by reference

SEQ ID NO: 1 human tau protein (2N4R)

SEQ ID NO: 2 tau residues 386-408 (pS396, pS404)

SEQ ID NO: 3 CDR1 light chain C10-2

SEQ ID NO: 4 CDR2 C10-2 light chain

SEQ ID NO: 5 CDR3 C10-2 light chain

SEQ ID NO: 6 CDR1 heavy chain C10-2

SEQ ID NO: 7 CDR2 heavy chain C10-2

SEQ ID NO: 8 CDR3 heavy chain C10-2

SEQ ID NO: 9 mouse C10-2 light chain

SEQ ID NO: 10 mouse C10-2 heavy chain

SEQ ID NO: 11 humanized C10-2 heavy chain

SEQ ID NO: 12 humanized C10-2 light chain

SEQ ID NO: 13 humanized C10-2 heavy chain variant D55E

SEQ ID NO: 14 humanized C10-2 heavy chain variant D55Q

SEQ ID NO: 15 humanized C10-2 heavy chain variant D55S

SEQ ID NO: 16 N32S light chain variant of humanized C10-2

SEQ ID NO: 17 N32Q light chain variant of humanized C10-2

SEQ ID NO: 18 N34S light chain variant of humanized C10-2

SEQ ID NO: 19 N34Q light chain variant of humanized C10-2

SEQ ID NO: 20 light chain variant N32S, N34S of humanized C10-2

SEQ ID NO: 21 humanized C10-2 light chain variants N32Q, N34S

SEQ ID NO: 22 Humanized C10-2 light chain variant N32Q, N34Q

SEQ ID NO: 23 variant N32S, N34Q humanized C10-2 light chain

SEQ ID NO: 24 humanized C10-2 heavy chain variant A101T

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SEQ ID NO: 25 humanized C10-2 heavy chain variant D55E, A101T

SEQ ID NO: 26 humanized C10-2 heavy chain variant D55Q, A101T

SEQ ID NO: 27 humanized C10-2 heavy chain variant D55S, A101T

SEQ ID NO: 28 humanized C10-2 heavy chain CDR2 variant D55E

SEQ ID NO: 29 humanized C10-2 heavy chain variant D55Q

SEQ ID NO: 30 variant D55S heavy chain CDR2 of humanized C10-2

SEQ ID NO: 31 humanized C10-2 light chain N32S CDR1 variant

SEQ ID NO: 32 humanized C10-2 light chain variant N32Q CDR1

SEQ ID NO: 33 humanized C10-2 light chain N34S CDR1 variant

SEQ ID NO: 34 humanized C10-2 light chain variant N34Q CDR1

SEQ ID NO: 35 variant N32S, N34S light chain CDR1 of humanized C10-2

SEQ ID NO: 36 variant N32Q, N34S light chain CDR1 of humanized C10-2

SEQ ID NO: 37 variant N32Q, N34Q light chain CDR1 of humanized C10-2

SEQ ID NO: 38 variant N32S, N34Q humanized C10-2 light chain CDR1

SEQ ID NO: 39 humanized C10-2 heavy chain variant A101T CDR3

SEQ ID NO: 40 Humanized C10-2 light chain CDR1 according to IMGT numbering

SEQ ID NO: 41 Humanized C10-2 light chain CDR2 according to IMGT numbering

SEQ ID NO: 42 Humanized C10-2 light chain CDR3 according to IMGT numbering

SEQ ID NO: 43 Humanized C10-2 heavy chain CDR1 according to IMGT numbering

SEQ ID NO: 44 Humanized C10-2 heavy chain CDR2 according to IMGT numbering

SEQ ID NO: 45 Humanized C10-2 heavy chain CDR3 according to IMGT numbering

SEQ ID NO: 46 Humanized C10-2 light chain variant N32S CDR1 according to IMGT numbering SEQ ID NO: 47 Humanized C10-2 light chain variant N32S CDR2 according to IMGT numbering SEQ ID NO: 48 Humanized C10-2 light chain variant N32S CDR3 according to IMGT numbering IMGT SEQ ID NO: 49 variant A101T humanized C10-2 heavy chain CDR1 according to IMGT SEQ ID NO: 50 variant A101T humanized C10-2 heavy chain CDR2 according to IMGT SEQ ID NO: 51 variant A101T humanized C10-2 heavy chain CDR3 according to IMGT SEQ ID NO: 52 Humanized C10-2 heavy chain CDR1 according to Chotia numbering

SEQ ID NO: 53 Humanized C10-2 heavy chain CDR2 according to Chotia numbering

SEQ ID NO: 54 Humanized C10-2 heavy chain CDR3 according to Chotia numbering

SEQ ID NO: 55 humanized C10-2 heavy chain CDR1 variant A101T according to Chotia numbering

SEQ ID NO: 56 humanized C10-2 heavy chain variant A101T CDR2 according to Chotia numbering

SEQ ID NO: 57 humanized C10-2 heavy chain variant A101T CDR3 according to Chotia numbering

Detailed description of the invention

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

MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT

PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG

TTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK

IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSP

GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK SRLQTAPVPM

PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV

PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV

QSKIGSLDNI THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L

The present invention relates to antibodies and epitope-binding fragments thereof that are capable of specifically binding to tau protein, and in particular human tau protein, and in one embodiment exhibit the ability to specifically bind to the phosphorylated residue S396 (pS396) of human tau protein . The antibodies and epitope-binding fragments thereof of the present invention are further characterized in that they are unable or substantially unable to specifically bind to the phosphorylated serine residue 404 (pS404) in human tau protein, for example, under limited antibody or non-saturating conditions. Additionally, phosphorylation at pS404 does not interfere with specific binding to pS396-containing epitopes. As used herein, the designations pS and ^{p} S denote the phosphoserine amino acid residue, and subsequent numbers define the position of the residue relative to the sequence under SEQ ID NO: 1. As used herein, the antibody is substantially unable to bind 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 for that other epitope.

The term antibody (Ab) in the context of the present invention refers to an immunoglobulin molecule or, according to some embodiments of the present invention, a fragment of an immunoglobulin molecule that has the ability to specifically bind to an epitope of the molecule (antigen). Naturally occurring antibodies typically comprise a tetramer, which typically consists of at least two heavy chains (HC) and at least two light chains (LC). Each heavy chain is composed of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain, typically consisting of three domains (CH1, CH2 and CH3). Human heavy chains can be of any isotype, including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes). Each light chain is composed of a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (CL). Human light chains include kappa chains and lambda chains. The variable domain of the heavy and light chains is usually responsible for antigen recognition, while the constant domain of the heavy and light chains can mediate the binding of the immunoglobulin to tissues or host factors, including various cells of the immune system (for example, effector cells) and the first component (C1q) classical way of activation of the complement system. The VH and VL domains can be further subdivided into hypervariable domains called complementarity determining regions, which alternate with more conserved sequence domains called framework regions (FRs). Each VH and VL consists of three CDR domains and four FR domains, arranged from the amino end to the carboxyl end in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The heavy and light chain variable domains contain a binding domain that interacts with an antigen. Of particular relevance are antibodies and their epitope-binding fragments which have been isolated so that they exist in a physical environment different from that in which they may occur naturally, or which have been modified so that they differ in amino acid sequence from that found in the nature of the antibody or its epitobinding fragments.

The term epitope means an antigenic determinant capable of specifically binding to an antibody. Epitopes usually consist of surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structure characteristics as well as specific charge characteristics. Conformational and linear epitopes differ in that binding to the former, but not to the latter, is always lost in the presence of denaturing solvents. An epitope may contain amino acid residues directly involved in binding and other amino acid residues not directly involved in binding, such as amino acid residues effectively blocked by a peptide that specifically binds to the epitope (in other words, the amino acid residue is within the recognition region for the peptide, specifically binding to an epitope).

As used herein, the term epitope-binding fragment of an antibody means a fragment, portion, region, or domain of an antibody (whether obtained (e.g., by cleavage, recombinantly, synthetically, etc.)) that is capable of specifically binding to an epitope . An epitope-binding fragment may contain any of 1, 2, 3, 4, 5, or all 6 CDR domains of such an antibody and, while capable of specifically binding to such an epitope, may exhibit specificity, affinity, or selectivity for that epitope that different from the epitope for such an antibody. However, it is preferred that the epitope-binding fragment contains all 6 CDR domains of such an antibody. An epitope binding fragment of an antibody may be or comprise part of a single polypeptide chain (e.g., in scFv) or may be or contain part of two or more polypeptide chains each having an amino terminus and a carboxyl terminus (e.g., in a diatel, Fab fragment, Fab ₂ fragment, etc.). Antibody fragments that exhibit the ability to bind to an epitope can be obtained, for example, by digesting intact antibodies with proteases. More preferably, although the two domains of an Fv fragment, VL and VH, are naturally encoded by separate genes or polynucleotides that encode such gene sequences (e.g., their coding cDNA), can be recombinantly linked in a flexible manner. a linker that allows them to be produced as a single protein chain in which the VL and VH regions associate with each other to form monovalent epitope-binding molecules (known as single-stranded Fvs (scFvs), see e.g. Bird et al., ( 1988) Science 242:423-426 and Huston et al (1988) Proc Natl Acad Sci (USA) 85:5879-5883). Alternatively, by using a flexible linker that is too short (e.g., less than about 9 residues in length) to allow the VL and VH domains of the same polypeptide chain to associate with each other, a bispecific antibody, a diabody, can be formed. or a similar molecule (in which two such polypeptide chains associate with each other to form a bivalent epitope-binding molecule) (see, for example, PNAS USA 90(14), 6444-8 (1993) for a description of diabodies). Examples of epitope-binding fragments encompassed by the present invention include (i) a Fab' or Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO 2007059782; (ii) F(ab') ₂ fragments - bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge domain; (iii) an Fd fragment essentially consisting of VH and CH1 domains; (iv) an Fv fragment essentially consisting of VL and VH domains, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which essentially consists of a VH domain and also called a domain antibody (Holt et al; Trends Biotechnol. 2003 Nov; 2i(II): 484-90); (vi) camelid antibodies or nanobodies (Revets et al.; Expert Opin Biol Ther. 2005 Jan;5_(I): I II-24) and (vii) complementarity determining region (CDR). Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by recombinant methods with a synthetic linker, which allows them to be obtained as a single protein chain in which the VL and VH domains are in pair to form monovalent molecules (known as single chain antibodies or single chain Fvs (scFvs), see, for example, Bird et al., Science 242, 423-426 (1988), and Huston et al., PNAS USA 85, 5879- 5883 (1988)). These and other antibody fragments useful in the context of the present invention are further discussed herein. It is also to be understood that the term antibody, unless otherwise indicated, also includes antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and fragments of antibodies that retain the ability to specifically bind to an antigen (epitope-binding fragments), obtained using any known technique, such as as enzymatic digestion, peptide synthesis and recombinant techniques. The resulting antibody may be of any isotype. As used herein, an isotype refers to an immunoglobulin class (eg, IgG1, IgG2, IgG3, or IgG4) encoded by heavy chain constant domain genes. Such antibody fragments are prepared using conventional techniques known to those skilled in the art; however, suitable fragments capable of binding to the desired epitope can be readily screened for utility in the same manner as an intact antibody. In one embodiment, the Fc region of the antibodies of the present invention contains a mutation that modulates effector functions.

The term bispecific antibody refers to an antibody containing two independent epitope-binding fragments, each of which targets independent targets. These targets may be epitopes present on different proteins or different epitopes present on the same target. Bispecific antibody molecules can be generated by compensatory amino acid changes in the HC constant domains of the original monospecific bivalent antibody molecules. The resulting heterodimeric antibody contains a single Fab that is contributed by two different parental monospecific antibodies. Amino acid changes in the Fc domain result in increased stability of the heterodimeric antibody with a 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 may also include molecules obtained by fusions of ScFv. The two monospecific scfvs are then independently linked to Fc domains that can form stable heterodimers to form a single bispecific molecule (Mabry et al., PEDS 23:3 115-127 (2010). Bispecific molecules have double binding capabilities.

The terms C10-2, human C10-2, hC10-2, HC10-2, hC10.2, Humanized C10-2, and humanized C10-2, as used herein and in the figures, are meant to be synonymous and are defined as antibody C10- 2. The term is intended to mean an antibody, or epitope-binding fragment thereof, containing an antibody light chain variable domain having:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5; and an antibody heavy chain variable domain having:

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having or consisting of the amino acid sequence of SEQ ID NO: 8.

The C10-2 antibody is a humanized antibody that can be defined as having the heavy chain of SEQ ID NO: 11, the light chain of SEQ ID NO: 12, or both. One embodiment of the present invention is directed to an antibody or epitope-binding fragment thereof comprising a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 12.

- 13 039569

The term mC10-2 as used herein and in the figures is meant to mean mouse C10-2 antibody and is defined under SEQ ID NOs: 9 and 10. Mouse C10.2 antibody is used as a control antibody and is not part of the present invention.

The terms hC10-2_N32S and C10-2_N32S, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody where the light chain has been mutated to at least contain a mutation of amino acid residue 32 in the N substitution type. to S, and are defined as the N32S antibody. The terms hC10-2_N32Q and C10-2_N32Q, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody wherein the light chain has been mutated to at least contain a mutation of amino acid residue 32 in the N substitution type. on Q, and are defined as the N32Q antibody.

The terms hC10-2_N34S and C10-2_N34S, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody wherein the light chain has been mutated to at least contain a mutation of amino acid residue 34 in the N substitution type. to S, and are defined as the N34S antibody. The terms hC10-2_N34Q and C10-2_N34Q, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody wherein the light chain has been mutated to at least contain an N mutation of amino acid residue 34. on Q, and are defined as the N34Q antibody.

The terms hC10-2_N32S_N34S and C10-2_N32S_N34S, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody wherein the light chain has been mutated to at least contain mutations of amino acid residues 32 and 34 of the N to S substitutions, and are defined as N32S, N34S antibodies. The terms hC102_N32Q_N34S and C10-2_N32Q_N34S, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody wherein the light chain has been mutated to at least contain mutations of amino acid residues 32 and 34 of the N to Q substitutions and N to S substitution types, respectively, and are defined as antibodies N32Q, N34S. The terms hC10-2_N32Q_N34Q and C10-2_N32Q_N34Q, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody wherein the light chain has been mutated to at least contain mutations of amino acid residues 32 and 34 of the replacing N with Q, and are defined as N32Q, N34Q antibodies. The terms hC10-2_N32S_N34Q and d0-2_N32S_N34Q, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody wherein the light chain has been mutated to at least contain mutations of amino acid residues 32 and 34 of the N to S substitutions and N to Q substitution types, respectively, and are defined as N32S, N34Q antibodies.

The terms hC10-2_D55E and C10-2_D55E, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody where the heavy chain has been mutated to at least contain a mutation of amino acid residue 55 to substitution type D to E, and are defined as the D55E antibody. The terms hC10-2_D55Q, C10-2_D55Q, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody, wherein the heavy chain has been mutated to at least contain a D mutation of amino acid residue 55 on Q, and are defined as the D55Q antibody. The terms hC10-2_D55S, C10-2_D55S, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody wherein the heavy chain has been mutated to at least contain a mutation of amino acid residue 55 of the substitutions D for S, and are defined as the D55S antibody.

The terms hC10-2_A101T and C10-2_A101T, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody where the heavy chain has been mutated to at least contain a mutation of amino acid residue 101 of the replacing A with T, and are defined as the A101T antibody.

Terms hC10-2_N32S_A101T, C10-2_N32S_A101T, hC10-2_A101T_N32S and

C10-2_A101T_N32S, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody wherein the heavy chain has been mutated to at least contain an A to T amino acid residue 101 mutation, and where the light chain has been mutated to at least contain an N to S amino acid residue 32 mutation, and are defined as the N32S, A101T antibody.

The terms hC10-2_N32Q_A101T, C10-2_N32Q_A101T, hC10-2_A101T_N32Q, and C102_A101T_N32Q, as used herein and in the figures, are intended to be synonymous and are variants of the C10-2 antibody where the heavy chain has been mutated to at least contain an A to T amino acid residue 101 mutation, and wherein the light chain has been mutated to at least contain an N to Q amino acid residue 32 mutation, and are defined as antibody N32Q, A101T.

The terms monoclonal antibody or monoclonal antibody composition as used herein refers to a preparation of antibody molecules of a single molecular composition. The traditional composition based on monoclonal antibodies is characterized by a single binding specificity and affinity for a particular epitope. In certain embodiments, the implementation of the monoclonal antibody may consist of more than one Fab-domain, thereby increasing the specificity for more than one target. The terms monoclonal antibody or composition based on monoclonal antibodies are not meant to be limited to any particular method of preparation (eg, recombinant, transgenic, hybridoma, etc.).

The antibodies of the present invention and their epitope-binding fragments are preferably humanized, in particular if they are used for therapeutic purposes. The term humanized refers to a molecule, usually produced by recombinant techniques, that has an epitope-binding site derived from an immunoglobulin from a non-human species and the remainder of the immunoglobulin structure based on the structure and/or sequence of the human immunoglobulin. The epitope-binding region may comprise either the entire non-human antibody variable domains fused to human constant domains, or only complementarity determining regions (CDRs), or portions thereof, of such variable domains grafted onto the appropriate human human variable domain framework regions. Framework residues of such humanized molecules may be wild-type (eg, fully human) or may be modified to contain one or more amino acid substitutions that do not occur in the human antibody whose sequence served as the basis for humanization. Humanization reduces or eliminates the likelihood that the constant domain of the molecule will act as an immunogen in humans, but the possibility of developing an immune response to a 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 obtaining constant domains of human origin, but also on modifying the variable domains in such a way as to reconstruct them as closely as possible to the human form. The variable domains of both the heavy and light chains are known to contain three complementarity determining regions (CDRs) that differ in response to the antigens in question and determine binding capacity, flanked by four framework regions (FRs) that are relatively conserved in this species and which presumably provide a backbone for the CDR. In cases where non-human antibodies are generated against a particular antigen, the variable domains can be reconstructed or humanized by grafting the CDRs derived from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this approach to various antibodies has been reported in 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 HIVNeutralizing 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, all CDR sequences are retained in humanized antibodies (eg, in a humanized mouse antibody that contains all six CDRs from mouse antibodies). In other embodiments, the humanized antibodies have one or more CDRs (one, two, three, four, five, six) altered from the parent antibody, also referred to as one or more CDRs derived from one or more CDRs of the parent antibody. The ability to humanize an antibody is well known (see, for example, US Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,859,205;

The term antibody XX is intended to mean an antibody or epitope-binding fragment thereof (e.g., C10-2 antibody) containing: (a) a light chain and a heavy chain, a heavy chain variable domain, or a heavy chain variable domain CDR1-3, as determined by their respective SEQ ID NO, or (b) a light chain variable domain and a heavy chain, a heavy chain variable domain, or a heavy chain variable domain CDR1-3 defined by their respective SEQ ID NOs, or (c) a light chain variable domain CDR1-3 defined by their corresponding to the corresponding SEQ ID NO, and a heavy chain, a heavy chain variable domain, or a heavy chain variable domain CDR1-3 as defined by or consisting of their respective SEQ ID NO. In certain embodiments, an antibody or epitope-binding fragment thereof is defined by its full heavy chain variable domain as defined by its SEQ ID NO and the light chain variable domain as defined by its SEQ ID NO.

Unless otherwise noted herein, the numbering of amino acid residues in the Fc region or constant domain of an antibody follows the EU numbering system, also referred to as 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.

In some antibodies, only a subset of the CDRs are needed to retain binding in a humanized antibody, namely the subset of CDR residues required for binding, referred to as SDRs. CDR residues not in contact with the corresponding epitope and not contained in the SDR can be identified based on previous studies. For example, Tyr, Ser, Gln, Lys, Phe, Gln residues corresponding to HC residues 60-65 under SEQ ID NO: 11 are often not necessary for isolation from Kabat CDR regions (they also occur in HC CDR2 (SEQ ID NO : 7) lying outside the 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 Hypervariable Regions 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 where one or more donor CDR residues are missing, or where the entire donor CDR is missing, the amino acid occupying the position may be an amino acid. slot that occupies the corresponding position (according to Kabat numbering) in the acceptor sequence of the antibody. The number of such CDR-acceptor-donor amino acid substitutions to be included reflects the balance of alternative opinions. Such substitutions are potentially advantageous in reducing the number of murine amino acids in a humanized antibody and thereby reducing potential immunogenicity. However, substitutions can also cause affinity changes, and significant reductions in affinity values are preferably avoided. Substitution positions within the CDR and amino acids to be replaced can also be empirically selected.

Antibodies are also characterized according to the IMGT numbering system, which is well defined in the art. Length (according to the number of amino acids, i.e., by the number of positions occupied) is an important and initial concept of IMGT-ONTOLOGY (http://www.imgt.org). CDR-IMGT lengths characterize IG and V-REGIONS of germline TR genes and V-DOMAINS of rearranged genes, cDNA, and proteins.

For example, antibodies can be characterized according to the Chothia numbering scheme http://www.bioinf.org.uk/abs/. The Chothia numbering scheme is identical to that of Kabat, however insertions occur in CDR-L1 and CDR-H1 at structurally defined positions. The numbering scheme of Chothia is based on the position of the structural regions of the loops. This means that the topologically equivalent remainders in these loops get the same label (in contrast to the Kabat scheme).

The fact that a single amino acid change in a CDR residue can result 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) enables the systematic identification of alternative functional CDR sequences. In one preferred method for producing such CDR variants, a CDR-encoding polynucleotide is mutated (e.g., by random mutagenesis or by a site-directed method (e.g., polymerase chain reaction-mediated amplification with primers that encode the mutant locus)) to obtain a CDR having a changed amino acid residue. By comparing the identity of the original (functional) CDR sequence at the corresponding residue with the identity of the substitution (non-functional) CDR sequence variant for this substitution, the substitution weight according to BLOSUM62.iij can be determined. The BLOSUM system provides an amino acid substitution matrix generated by analyzing a database of sequences for significant alignments (Eddy S.R. (2004) Where Did The BLOSUM62 Alignment Score Matrix Come 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 Altschul, S. F. (1991) Amino Acid Substitution Matrices From An Information Theoretic Perspective, J. Mol. Biol. 219, 555-565. Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij) Table 1 shows the weights of substitutions according to BLOSUM62.iij (the larger the weight, the more affect the function). If the epitope-binding fragment containing the resulting CDR cannot bind to the tau protein, for example, the substitution weight according to BLOSUM62.iij is considered to be insufficiently conserved, and a new candidate substitution having a higher substitution weight is selected and produced. So, for example, if the original residue was glutamate (E) and the non-functional replacement residue was histidine (H), then the substitution weight according to BLOSUM62.iiJ would be 0, and more conservative changes

- 16 039569 compounds (eg aspartate, asparagine, glutamine or lysine) are preferred.

Table 1

BUT R N D FROM Q E G n I L To M F R S T W Y V BUT +4 -one -2 -2 0 -one -one 0 -2 -one -one -one -one -2 -one +1 0 -3 -2 0 R -one +5 0 -2 -3 +1 0 -2 0 -3 -2 +2 -one -3 -2 -one -one -3 -2 -3 N -2 0 +6 +1 -3 0 0 0 +1 -3 -3 0 -2 -3 -2 +1 0 -four -2 -3 D -2 -2 +1 +6 -3 0 +2 -one -one -3 -four -one -3 -3 -one 0 -one -four -3 -3 FROM 0 -3 -3 -3 +9 -3 -four -3 -3 -one -one -3 -one -2 -3 -one -one -2 -2 -one Q -one +1 0 0 -3 +5 +2 -2 0 -3 -2 +1 0 -3 -one 0 -one -2 -one -2 E -one 0 0 +2 -four +2 +5 -2 0 -3 -3 +1 -2 -3 -one 0 -one -3 -2 -2 G 0 -2 0 -one -3 -2 -2 +6 -2 -four -four -2 -3 -3 -2 0 -2 -2 -3 -3 H -2 0 +1 -one -3 0 0 -2 +8 -3 -3 -one -2 -one -2 -one -2 -2 +2 -3 one -one -3 -3 -3 -one -3 -3 -four -3 +4 +2 -3 +1 0 -3 -2 -one -3 -one +3 L -one -2 -3 -four -one -2 -3 -four -3 +2 +4 -2 +2 0 -3 -2 -one -2 -one +1 To -one +2 0 -one -3 +1 +1 -2 -one -3 -2 +5 -one -3 -one 0 -one -3 -2 -2 m -one -one -2 -3 -one 0 -2 -3 -2 +1 +2 -one +5 0 -2 -one -one -one -one +1 F -2 -3 -3 -3 -2 -3 -3 -3 -one 0 0 -3 0 +6 -four -2 -2 +1 +3 -one R -one -2 -2 -one -3 -one -one -2 -2 -3 -3 -one -2 -four +7 -one -one -four -3 -2 S +1 -one +1 0 -one 0 0 0 -one -2 -2 0 -one -2 -one +4 +1 -3 -2 -2 t 0 -one 0 -one -one -one -one -2 -2 -one -one -one -one -2 -one +1 +5 -2 -2 0 W -3 -3 -four -four -2 -2 -3 -2 -2 -3 -2 -3 -one +1 -four -3 -2 +11 +2 -3 Y -2 -2 -2 -3 -2 -one -2 -3 +2 -one -one -2 -one +3 -3 -2 -2 +2 +7 -one V 0 -3 -3 -3 -one -2 -2 -3 -3 +3 +1 -2 +1 -one -2 -2 0 -3 -one +4

Thus, the present invention provides for the use of random mutagenesis to identify improved CDRs. In the context of the present invention, conservative substitutions can be defined as substitutions within the amino acid classes shown in one or more of the tables. 2, 3 or 4.

Classes of amino acid residues for conservative substitutions

table 2

acid residue Asp (D) and Glu (E) Basic remains Lys (K), Arg (R) and His (H) hydrophilic residues uncharged Ser (S), Thr (T), Asn (N) and Gin (Q) Aliphatic residues uncharged Gly (G), Ala (A), Vai (V), Leu (L) and He (I) Non-polar residues uncharged Cys (C), Met (M) and Pro (P) aromatic residues Phe (F), Tyr (Y) and Trp (W)

Alternative classes of amino acid residues for conservative substitutions

Table 3

- 17 039569

Alternative physical and functional classifications of amino acid residues

Table 4

Residues containing an alcohol group S&T Aliphatic residues 1, L, V and M Residues associated with cycloalkenyl F, H, WnY hydrophobic residues A, C, F, G, H, 1, L, M, R, T, V, WhY Negatively charged residues D and E polar remnants C, D, E, H, K, N, Q, R, S and T positively charged residues H, Ki R little leftovers A, C, D, G, N, P, S, ThV Very small remnants A, G and S Residues involved in the formation of a bend A, C, D, E, G, H, K, N, Q, R, S, R&T Flexible Remains Q, T, K, S, G, P, D, E and R

Groups of more conservative substitutions include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

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

Alternatively, phage display technology can be used to increase (or decrease) CDR affinity. This technique, called affinity maturation, uses mutagenesis or CDR walking and reselection using the target antigen or antigenic epitope binding fragment thereof to identify antibodies having CDRs that bind with higher (or lower) affinity to the antigen compared to original or parent antibody (see, for example, Glaser et al. (1992) J. Immunology 149:3903). Mutagenesis of whole codons rather than single nucleotides results in a semi-random set of amino acid mutations. Libraries can be constructed consisting of a pool of clone variants each differing in one amino acid change in one CDR and which contains variants representing each possible amino acid substitution for each CDR residue. Mutants with increased (or decreased) binding affinity for an antigen can be screened by contacting immobilized mutants with a labeled antigen. Any screening method known in the art (e.g., ELISA) can be used to identify mutant antibodies with increased or decreased affinity for the antigen (see Wu et al. 1998, Proc. Natl. Acad. Sci. (U.S.A.) 95:6037; Yelton et al., 1995, J. Immunology 155:1994). It is also possible to use CDR walking, during which the light chain undergoes random mutagenesis (see Schier et al., 1996, J. Mol. Bio. 263:551).

Methods for performing 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) AffinityMatured 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. et al. (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 Of A Monoclonal Fab Derived From A Synthetic Naive Human Antibody Library And Directed Against The Internal 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 Of A Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The Complementarity-Determining Regions, 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 the CDR variants of the covered antibodies or their epitope-binding fragments may differ from the sequence of the CDRs of the parent antibody, variants C10-2 and C10.2 due to substitutions; for example, 4 amino acid residues, 3 amino acid residues, 2 amino acid residues, or 1 amino acid residue have been replaced. According to an embodiment of the present invention, it is further contemplated that amino acids in the CDR regions may be substituted by conservative substitutions as defined in the 3 tables above. For example, the acidic residue Asp can be replaced by Glu without significantly affecting the binding characteristics of the antibody.

As used herein, an antibody or epitope-binding fragment thereof is said to be

- 18 039569 bind specifically to a region of another molecule (ie, an epitope) if they react with or associate with that epitope more frequently, more rapidly, with greater duration and/or with greater affinity or avidity compared to alternative epitopes. Also, when reading this definition, it should be understood that, for example, an antibody or epitope-binding fragment thereof that specifically binds to a first target may not bind to a second target, or may not specifically or preferentially do so. As used herein, the term binding in relation to 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 10 ^-8 M or less, such as , approximately 10 ^-9 M or less, when determined using, for example, Surface Plasmon Resonance (SPR) technology on a BIAcore® 3000 instrument using an antigen as a ligand and an antibody as an analyte, and binding to a predetermined antigen with affinity , corresponding to a KD at least ten times lower, such as at least 100 times lower, such as at least 1000 times lower, such as at least 10,000 times lower, for example, at least 100,000 times lower than its binding affinity for a non-specific antigen (eg, BSA, casein) other than from a predetermined antigen, or a closely related antigen. The degree by which the affinity is lower depends on the KD of the antibody, so if the KD of the antibody is very low (i.e. the antibody is highly specific), then the degree by which the affinity for the antigen is lower than the affinity for the non-specific antigen may be at least 10,000 times.

The term kd (c-1 or 1/c) as used herein refers to the dissociation rate constant for a particular antibody-antigen interaction. This value is also referred to as the koff value.

The term ka (M-1 x s-1 or 1/M- s) as used herein refers to the dissociation rate constant for a particular antibody-antigen interaction.

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

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

In one embodiment, the present invention provides an anti-tau antibody, or epitope-binding fragment thereof, that exhibits one or more of the following properties:

(i) virtually no ability to bind to non-phosphorylated tau protein;

(ii) virtually no ability to bind to a tau protein phosphorylated at S404 and not phosphorylated at S396;

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

(iv) the ability to bind to a tau protein phosphorylated in both S396 and S404;

(v) the ability to selectively discriminate between the phosphorylated residues S396 and S404 of the tau protein so that they are practically unable to bind to the phosphorylated residue 404 (pS404);

(vi) the ability to bind to hyperphosphorylated tau protein from the brains of people with Alzheimer's disease;

(vii) the ability to discriminate between pathological and non-pathological human tau protein and/or (viii) the ability to specifically reduce the 64 and 70 kDa hyperphosphorylated tau protein bands by at least 90% without reducing the tau protein band 55 kDa greater than 10% when used as described herein with immunodepleted extracts from rTg4510 transgenic mice or the ability to specifically reduce S396 phosphorylated hyperphosphorylated tau bands by at least 90% without reducing non-hyperphosphorylated tau bands by more than than 10% when used as described herein with brain extracts obtained post-mortem from people with AD.

An additional embodiment of the present invention relates to a method for producing highly specific, high affinity antibodies, wherein the method comprises the steps of:

(A) injecting an immunogen into a mammal so as to immunize said mammal;

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

(C) screening a serum sample from said booster mammal for the presence of the desired highly specific, high affinity antibodies, but much less capable of binding to another protein; and (D) recovering said highly specific, high affinity antibodies.

Thus, the present invention relates to a highly specific, high affinity antibody to pathogenic hyperphosphorylated tau protein containing a hyperphosphorylated S396 residue. Such antibodies can be obtained by adapting the above method for producing highly specific high affinity antibodies to the following:

(A) injecting an immunogen into a mammal, said immunogen comprising a diphosphorylated peptide containing 18-40, such as 18-30, such as

20-30 consecutive amino acid residues containing

TDHGAEIWK ^{p> SPWSGDT ^{p> SPRHL (SEQ ID NO: 2) encompassing residues 386-410 of the 2N4R tau protein so as to immunize said mammal;

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

(C) screening a blood serum sample from said multiply immunized mammal for the presence of highly specific, high affinity antibodies capable of binding to a pathogenic hyperphosphorylated tau protein containing a phosphorylated residue S396, but to a much lesser extent capable of binding to a non-pathogenic tau protein; and (D) recovering said highly specific, high affinity antibodies.

As used herein, virtually no ability to bind to a tau protein molecule means more than 20% difference, more than 40% difference, more than 60% difference, more than 80% difference, more than 100% difference, more than 150 % difference, greater than 2-fold difference, greater than 4-fold difference, greater than 5-fold difference, or greater than 10-fold difference in performance compared to detectable binding mediated by reference antibody.

The terms selective and immunoselective, when referring to the binding abilities that an anti-tau antibody has for two epitopes, are meant to mean that there is at least an 80% difference for observed binding under saturating conditions, at least 95% difference, and most preferably 100% difference (ie, no detectable binding to a single epitope). The terms selective and immunoselective, when referring to an anti-tau antibody, are further intended to mean that the antibody binds to hyperphosphorylated tau protein from the brains of individuals with Alzheimer's disease and is able to distinguish between pathological and non-pathological human tau.

The terms TBS extractable (S1), high saline/sarcosyl extractable (S3), and sarcosyl insoluble (P3) fractions refer to fractions obtained by the biochemical tau fractionation described herein.

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

The term hyperphosphorylated tau protein refers to polyphosphorylated tau protein molecules corresponding to a Western blot induced mobility shift by polyanionic molecules, or tau protein molecules having more than five, six, or seven phosphorylated serine, threonine, or tyrosine sites.

The term tau protein having a phosphorylated residue 396 refers to a hyperphosphorylated tau protein in which the serine residue 396 is phosphorylated.

The term non-human transgenic animal refers to a non-human animal having a genome containing one or more human heavy and/or light chain-encoding transgenes or transchromosomes (whether or not integrated into the animal's natural genomic DNA) that is capable of expressing fully human antibodies. For example, a transgenic mouse may have a transgene encoding a human light chain and either a transgene encoding a human heavy chain or a transchromosome encoding a human heavy chain, whereby the mouse produces a human anti-tau antibody when immunized with antigenic tau and/or cells expressing the tau protein. The transgene encoding the human heavy chain may be integrated into the chromosomal DNA of the mouse, as is the case with transgenic mice, e.g. transchromosomal KM mice as described in WO 02/43478. Such transgenic and transchromosomal mice (collectively referred to herein as transgenic mice) are capable of producing multiple isotypes of human monoclonal antibodies to a given antigen (such as IgG, IgA, IgM, IgD, and/or IgE) by undergoing V-D-J recombination and isotype switching.

A non-human transgenic animal can also be used to generate antibodies to a specific antigen by introducing genes encoding that specific antibody, for example by operably linking the genes to a gene expressed in the animal's milk.

The term treatment or treatment as used herein means alleviating, slowing, ameliorating, or eliminating the progression or severity of a disease or disorder, or alleviating, slowing, alleviating, or eliminating one or more symptoms or side effects of such a disease or disorder. For the purposes of the present invention, treating or administering treatment further means an approach to obtain useful or desired clinical results, wherein useful or desired clinical results include, but are not limited to, alleviation of a symptom, reduction in a disorder or disease, stabilized

- 20 039569 (ie, not worsening) disease or disorder condition, delaying or slowing the progression of the disease or disorder condition, alleviating or mitigating the disease or disorder condition, and remission of the disease or disorder, either partially or completely.

An effective amount applied to an antibody or epitope-binding fragment thereof of the present invention refers to an amount that, when administered at the required doses and for the required periods of time, is sufficient to achieve the intended biological effect or desired therapeutic result, including without limitation clinical results. . The phrase "therapeutically effective amount" when used with respect to an antibody or epitope-binding fragment thereof of the present invention is meant to mean an amount of the antibody or epitope-binding fragment thereof which is sufficient to alleviate, mitigate, stabilize, eliminate, retard, attenuate or delay the progression of a disorder or disease state, or symptom of a disorder or disease. In an embodiment, the method of the present invention provides for the administration of an antibody, or an epitope-binding fragment thereof, in combination with other compounds. In such a case, an effective amount is the number of combinations sufficient to produce the desired biological effect.

A therapeutically effective amount of an anti-tau antibody or epitope-binding fragment thereof of the present invention may vary depending on factors such as disease state, age, sex and weight of the individual, as well as the ability of the anti-tau antibody or epitope-binding fragment thereof to elicit the desired response. at the individual. A therapeutically effective amount is also the amount at which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody or antibody portion.

As stated above, the present invention specifically relates to monoclonal antibodies or epitope-binding fragments thereof, and to an entirely new method for producing such molecules (and therefore such epitope-binding fragments thereof). This ability to isolate monoclonal antibodies using the new method is presented herein using an example of its use for the isolation of monoclonal antibodies capable of specifically binding to the phosphorylated serine residue 396 (P-S396, pS396, ^{p} S396) of the human tau protein (SEQ ID NO : one). These antibodies are further characterized by their ability to discriminate between phosphorylated serine-396 and serine-404 residues (P-S404, pS404) such that they do not bind to tau protein with phosphorylated serine-404 unless the tau protein also phosphorylated at residue 396.

The antibodies of the present invention, or their epitope-binding fragment, have been generated and isolated using a novel method that facilitates the selection of pS396-specific antibodies. Additionally, by applying this very stringent antibody clone selection procedure, antibodies have been generated that are not only highly specific for S396, but also highly selective for the phosphorylated epitope of pS396. These antibodies uniquely recognize tau from the Alzheimer's brain. The screening procedure provides for the identification of antibodies with functional and therapeutic utility.

Antibodies were induced against the diphosphorylated TDHGAEIVYK{p}SPWSGDT{p}SPRHL peptide (SEQ ID NO: 2) spanning residues 386-408 of the 2N4R tau protein. Mice were immunized with the phosphorylated peptide. After obtaining sufficient antibody titers, mice were sacrificed and hybridomas were obtained. Hybridomas were screened by dot blot analysis and MSD-ELISA with immobilized pathological and non-pathological human tau protein. The ability to discriminate between pathological and non-pathological human tau protein in dot blot and western blot analysis was used to select hybridomas. Sixteen clones were selected, from which four hybridoma clones were isolated, which produced antibodies characterized by extremely high ability to bind to the pathological material of the human tau protein.

Specific binding to pathological and non-pathological tau protein was also determined by isolating tau protein from diseased and non-diseased AD human brains and immobilizing this material on MSD-ELISA plates (Example 4).

A further aspect of the present invention relates to a monoclonal antibody or epitope-binding fragment thereof, the formation of which occurs in response to a diphosphorylated peptide containing at least 18, such as at least 20, consecutive amino acid residues within TDHGAEIWK ^{P} SPVVSGDT ^{P> SPRHL (SEQ ID NO: 2) encompassing residues 386410 of the 2N4R tau protein. In this aspect of the present invention, the production of a monoclinal antibody or epitope-binding fragment thereof is typically elicited in response to a diphosphorylated peptide containing 18-40, such as 18-30, such as 20-30, consecutive amino acid residues containing tdhgaeiwk<p>spwsgdt<p>sprhl (SEQ id no: 2) encompassing residues 386-410 of the 2N4R tau protein.

A further aspect of the present invention is directed to a monoclonal antibody or

- 21 039569 epitope-binding fragment of the present invention, having specificity for phosphorylated tau protein (pTau) from patients affected by AD, compared with healthy controls in the corresponding age group, so that the specified monoclonal antibody or its epitope-binding fragment is characterized by a difference in specificity in relation to phosphorylated tau protein (pTau) from patients affected by AD compared to tau protein from healthy controls in the corresponding age group, representing more than 50-fold, such as more than 100-fold increase in specificity in against material affected by AD compared to material from healthy controls in an ELISA-based assay for the detection of phosphorylated tau protein (pTau) in brain homogenates from subjects with AD and from healthy controls using Specific Design 1 ELISA or MSD for phosphorylated and multimeric th molecules described in this document.

A related aspect of the present invention is directed to a monoclonal antibody or epitope-binding fragment thereof of the present invention having specificity for a tau protein from AD-affected material, such that said monoclonal antibody or epitope-binding fragment thereof has a difference in specificity for AD-affected material compared to material from healthy controls in the appropriate age group, representing a greater than 50-fold, such as greater than 100-fold, increase in specificity for AD-affected material compared to material from healthy controls in an ELISA or MSD based analysis to detect phosphorylated tau protein (pTau) in brain homogenates from subjects with AD and from healthy controls using a specific ELISA scheme 1 for phosphorylated and multimeric molecules.

The method for performing ELISA or MSD Scheme 1 comprises the steps of A) ensuring capture of pathological antigenic human tau proteins from brain samples from AD subjects on C10-2 coated plates; B) incubating antigenic tau proteins with increasing concentrations of pS396 specific antibodies; and C) detecting residual free antigenic tau captured by immobilized C10.2 with SULFO-TAG labeled anti-human (total) tau antibodies from MSD.

More specifically, step A comprises coating MSD plates (typically overnight at 4°C) with C10-2 antibody, typically at 0.5 μg/ml (capture antibody) in coating buffer, blocking (typically for 1 hour at room temperature) and washing, typically 3 times. Step B involves mixing lysate samples P3 (typically diluted at 1:1000 = 2-4 µg/mL total protein) and/or S1(p) (typically diluted at 1:300 = 20-40 ng/mL total protein) protein) from material from subjects with AD (pooled from 3 patients) with an antibody specific for the pS396 peptide epitope at stepped concentrations and incubation (typically for 1 hour at room temperature). The reactions are then incubated for 2 hours in the plates prepared in step A. Step C involves detection of C10-2 captured tau protein with SULFO-TAG labeled anti-human tau antibody (typically at 1:50) from MSD according to the manufacturer's instructions. The plates are analyzed on a SECTOR® S600 from MSD. P3 from material from AD subjects and S1(p) from material from AD subjects are tested according to a similar scheme.

A further embodiment is directed to an antibody or antigen-binding fragment thereof capable of specifically binding to the phosphorylated serine residue 396 of the human tau protein (SEQ ID NO: 1) that has been made or produced in a cell line such as a human cell line, a mammalian cell line, non-human, insect, yeast, or bacterial cell line.

An antibody or an antigen-binding fragment thereof capable of specifically binding to the phosphorylated residue 396 of human tau protein (SEQ ID NO: 1) is produced in a CHO cell line, a HEK cell line, a BHK-21 cell line, a mouse cell line (such as a myeloma cell line). ), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines, and human HuH-7 cell lines.

The specific affinity and binding properties of variants C10-2 and C10.2 were characterized using tau protein (2R4N) peptides 386-410 phosphorylated or not at position 396 or 404. Using the specific immunization and screening protocol summarized in this application, get highly specific for phosphoserine-396 (pS396) antibodies.

To demonstrate that the antibodies are specific for the pathological tau protein, C10-2 antibodies were also characterized by immunohistochemistry. The antibodies exhibit highly specific binding to neurofibrillary tangles in brain samples from Alzheimer's subjects (tau tangles) and in sections from Tg4510 tau transgenic mice expressing the human (P301L) tau mutant. Binding to tissue from control human brains and from non-transgenic mouse brains was not observed, demonstrating that the antibodies specifically bind to human

- 22 039569 tau protein, and in particular, tau protein associated with Alzheimer's pathology.

Antibody C10-2

One aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

A further aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

Alternatively defined using the IMGT definition, one aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 40;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 41;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 42;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 43;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45.

Alternatively defined using the Chotia definition, one aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 52;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 53; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 54.

The unique ability of the antibodies of the present invention to recognize the tau protein associated with the pathology characteristic of the disease has been shown. The binding of pathological and non-pathological tau protein was compared. The comparison is made against five published anti-tau antibodies: hACI-2B6, IPN002, HJ8.5, 2.10.3 and 4E4. Antibody 2.10.3 is a commercially available recombinant monoclonal antibody that specifically binds to the serine-422 phosphorylated tau protein (pS422). HJ8.5 is a commercially available monoclonal antibody that only recognizes the N-terminal region of the human tau protein (epitope corresponding to amino acid residues 25-30). The antibody belongs to the IgG2b isotype. Therapeutic anti-tau NI-1O5-4E4 antibody is a commercially available antibody. The table shows that the isolated antibodies exhibit an exceptionally high degree of specificity and selectivity for human pathological tau protein. This selectivity is superior to that of any of the compared antibodies shown in Table 1. 5.

Table 5 mAb tested AD/control TG/wt hACI-2B6 3 one IPN002 3 37 HJ8.5 3 51 4E4 absence binding one 2.10.3 5 2 Antibody C10-2 >100 118

When saturated, the C10-2 antibody exhibits more than 100-fold selectivity for the P3 tau protein isolated from human AD brain samples.

To demonstrate that the selected antibodies have functional and therapeutic utility, the antibodies were tested in in vitro and in-cell tau aggregation assays (Example 8). These assays are functional assays that demonstrate that antibodies are able to interfere with the pathological tau aggregation process. HEK293 cells are transiently transfected with P301L-FLAG (4R0N) human tau protein. Cells are then exposed to tau-containing extracts from human AD brain samples or from Tg4510 transgenic brain samples. This exposure to pathological tau promotes tau uptake by cells and its intracellular aggregation. Both immunodepletion of tau preparations with the C10-2 antibody and direct treatment of cells with these antibodies can provide a significant reduction in the formation of tau protein aggregates.

The therapeutic utility of the C10-2 antibody was also evaluated in mice expressing human tau/PS1. This mouse model is a more appropriate animal model of AD disease, in which AD pathology develops only at a later age (12-18 months of age). However, mice exhibit tau hyperphosphorylation prior to the appearance of dense tangle pathology. Mice were injected with a dose of 15 mg/kg twice a week for an extended period of 13 weeks. Mice treated with the antibody show a significant decrease in the level of phosphorylated tau protein, indicating that prolonged treatment with C10-2 will reduce tangle pathology and thus subsequent neurodegeneration in vivo.

The antibodies of the present invention specifically remove hyperphosphorylated tau protein from rTg4510 mouse brain extracts using immunodepletion methods. In addition, the antibodies of the present invention do not remove normal tau protein from homogenates, while the commercially available Tau5 antibody does. Unlike commercial antibodies that bind to tau proteins, which are phosphorylated at residue 404 or at both residue 404 and residue 396, the antibodies of the present invention specifically remove 95% of the hyperphosphorylated tau protein, which is phosphorylated at serine-396 . Experiments (Example 12) demonstrate that although the antibody of the present invention removes only a very small fraction of the total tau protein in the brain homogenate (8%), the antibodies nevertheless specifically remove hyperphosphorylated tau protein (by 90 %). Accordingly, one aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, capable of specifically binding to pathogenic hyperphosphorylated tau protein. Additionally, in experiments in which hyperphosphorylated tau protein was removed with an antibody of the present invention, seeding activity was eliminated. By removing the hyperphosphorylated tau protein from the homogenates, the homogenates no longer induce a seeding action to form tau pathology. It has been suggested that with a decrease in the initial action, the development of tangle formation and the progression of taupathies, including Alzheimer's disease, decrease. Accordingly, a further aspect of the present invention is directed to an antibody of the present invention for use in reducing the progression of AD or the intensity of symptoms of AD.

Antibody D55E

One aspect of the present invention relates to a variant of the C10-2 antibody, the D55E antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) A light chain CDR3 having the amino acid sequence of SEQ ID NO: 5.

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

An additional embodiment of the D55E antibody aspect of the present invention relates to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 13.

Alternatively, a certain additional embodiment of the D55E antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5; and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Antibody D55Q

Another aspect of the present invention relates to a variant of the C10-2 antibody, the D55Q antibody. This aspect of the present invention is directed to a monoclonal antibody or epitope-binding antibody thereof.

- 24 039569 fragment containing:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(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: 8.

An additional embodiment of the D55Q antibody aspect of the present invention relates to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 14.

Alternatively, a specific additional embodiment of the D55Q antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(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: 8.

Antibody D55S

Another aspect of the present invention relates to a variant of the C10-2 antibody, the D55S antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 30; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

An additional embodiment of the D55S antibody aspect of the present invention relates to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 15.

Alternatively, a specific additional embodiment of the D55S antibody aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 30; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Studies using D55S antibody, D55Q antibody, D55E antibody indicate that the mutation of this residue leads to the formation of an antibody with unchanged binding properties compared to these antibodies before and after treatment at low pH for a long period of time at room temperature, which indicates that there is no isomerization at low pH, or that any isomerized protein has unchanged binding properties compared to pretreatment.

Antibody N32S

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N32S antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and

- 25 039569 (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

An additional embodiment of the N32S antibody aspect of the present invention relates to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 16; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

Alternatively, a specific further embodiment of the N32S antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

An alternative definition of an N32S antibody, using the IMGT definition, is a monoclonal antibody or epitope-binding fragment thereof containing:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 46;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 47;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 48;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 43;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45.

A further alternative definition of an N32S antibody, using the Chotia definition, is a monoclonal antibody or epitope-binding fragment thereof containing:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 52;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 53; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 54.

As can be seen from FIG. 1, the IC ₅₀ of the N32S antibody (black circles) is reduced compared to the C10-2 antibody: the IC ₅₀ of N32S is calculated to be 44 nM. This marked improvement over C10-2 was not expected. As confirmed by FIG. 1, one aspect of the present invention is directed to an antibody that inhibits P3 from AD subjects in the liquid phase inhibition assay described herein such that the signal is reduced by 50% at an antibody concentration of 100 nM or less. In one embodiment, an antibody of the present invention has an IC ₅₀ of 0.1 nM to 100 nM, such as a concentration of 50 nM or less, such as 0.1 nM to 50 nM, based on a liquid phase inhibition assay uptake of P3 antigens from material from subjects with AD.

The data obtained using the N32S antibody indicate that a mutation at position 32 of the C10-2 antibody light chain or C10-2 antibody CDR1 LC in a batch substitution pattern results in increased apparent affinity (IC ₅₀ ) in peptide inhibition assays. In addition, a mutation at position 32 with a serine or glutamine substitution (antibody N32S and antibody N32Q) abolished deamidation at both positions 32 and 34, as shown in FIG. 20. The specific activity determined from in vitro seeding and aggregation studies is retained in both the N32S and N32Q variants.

The possibility of potential deamidation was identified at C10-2 at a position that could lead to heterogeneity of protein batches. C10-2 was characterized by heterogeneous binding to P3 antigens from material from AD subjects, with a small proportion with high apparent affinity (2-5 nM) and predominant binding with low apparent affinity (200-1000 nM) (Fig. 1). Heterogeneous binding may reflect different subpopulations of deamidated and non-deamidated C10-2. The variant was obtained with a substitution (N32S) that prevented deamidation. Shows improved binding activity, as indicated by the overall reduced IC ₅₀ (higher apparent affinity) compared to C10-2. An additional A101T substitution was introduced resulting in uniform binding in a manner characterized by high apparent affinity. The improved binding activity at both N32S and N32S-A1O1T indicates that more stable and homogeneous antibodies are obtained with the above mutations.

Antibody N32Q

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N32Q antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

- 26 039569 (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

An additional embodiment of the N32Q antibody aspect of the present invention relates to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 17; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

Alternatively, a specific further embodiment of the N32Q antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

As indicated, the data obtained using the N32S antibody indicate that a mutation at position 32 of the light chain of the C10-2 antibody or CDR1 LC of the C10-2 antibody, in a serine substitution type, results in increased apparent affinity (IC ₅₀ ), with the mutation at type of substitution for glutamine (N32Q antibody) results in unchanged binding activity. However, a mutation at position 32 with a serine or glutamine substitution (antibody N32S and antibody N32Q) abolished deamidation at both positions 32 and 34, as shown in FIG. 20. Accordingly, both the N32S antibody and the N32Q antibody are advantageous.

The specific activity determined from in vitro seeding and aggregation studies is retained in both the N32S and N32Q variant.

Antibody N34S

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N34S antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 33;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

An additional embodiment of the N34S antibody aspect of the present invention relates to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 18; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

Alternatively, a specific further embodiment of the N34S antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 33;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Antibody N34Q

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N34Q antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

- 27 039569 (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

An additional embodiment of the N34Q antibody aspect of the present invention relates to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 19; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

Alternatively, a specific further embodiment of the N34Q antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Antibody N32S,N34S

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N32S, N34S antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 35;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Alternatively, a specific further embodiment of the N32S, N34S antibody aspect of the present invention refers to a monoclinal antibody or epitope-binding fragment thereof comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 20; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

Alternatively, a specific further embodiment of the N32S, N34S antibody aspect of the present invention refers to a monoclinal antibody or epitope-binding fragment thereof comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 35;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Antibody N32Q, N34S

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N32Q, N34S antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 36;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Alternatively, a specific further embodiment of the N32Q, N34S antibody aspect of the present invention refers to a monoclinal antibody or epitope-binding fragment thereof comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 21;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

Alternatively, a specific further embodiment of the N32Q, N34S antibody aspect of the present invention refers to a monoclinal antibody or epitope-binding fragment thereof comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 36;

- 28 039569 (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Antibody N32Q, N34Q

Another aspect of the present invention relates to a variant of the C10-2 antibody, N32Q, N34Q antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 37;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Alternatively, a specific further embodiment of the N32Q, N34Q antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 22; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

Alternatively, a specific further embodiment of the N32Q, N34Q antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 37;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Antibody N32S, N34Q

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N32S, N34Q antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Alternatively, a specific further embodiment of the N32S, N34Q antibody aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 23; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

Alternatively, a specific further embodiment of the N32S, N34Q antibody aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Antibody A101T

Another aspect of the present invention relates to a variant of the C10-2 antibody, the A101T antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

- 29 039569 (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Alternatively, a specific further embodiment of the A101T antibody aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

Alternatively, a specific further embodiment of the A101T antibody aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Data obtained using the A101T antibody indicate that mutation of the heavy chain of the C10-2 antibody or CR3 of the heavy chain of the C10-2 antibody results in a two-fold increase in peptide binding and a 10-20-fold increase in binding to the P3 material. Additionally, variants containing the A101T mutation are characterized by increased specific activity in in vitro seeding assays.

Antibody N32S, A101T

Another aspect of the present invention relates to a variant of the C10-2 antibody, N32S antibody, A101T. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Alternatively, a specific further embodiment of the N32S antibody, A101T aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 16;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

Alternatively, a specific further embodiment of the N32S antibody, A101T aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39;

and further comprising at least one of (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7.

Using the IMGT definition, an N32S, A101T antibody is a monoclonal antibody or epitope-binding fragment thereof containing:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 46;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 47;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 48;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 49;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 50; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 51.

Using the Chotia definition, an N32S, A101T antibody is a monoclonal antibody or epitope-binding fragment thereof containing:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

- 30 039569 (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 55;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 56; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 57.

As can be seen from FIG. 1, the IC ₅₀ of the N32S, A101T antibody (open circles) is significantly reduced compared to the C10-2 antibody: the IC ₅₀ of N32S, A101T is calculated to be 14 nM. This marked improvement over C10-2 was not expected. Based on FIG. 1, one aspect of the present invention is directed to an antibody that inhibits P3 from material from subjects with AD in the liquid phase inhibition assay described herein such that the signal is reduced by 50% at a concentration of 100 nM antibody or less as, eg 10 to 100 nM antibody, such as at a concentration of 50 nM or less, such as 10 to 50 nM antibody. As shown in FIG. 20, the N32S antibody, A101 is very stable with respect to deamidation. As shown in FIG. 19, the N32S antibody, A101T was characterized by a more pronounced decrease in aggregation.

Antibody N32Q, A101T

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N32Q antibody, A101T. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Alternatively, a specific further embodiment of the N32Q, A101T antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 17; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

Alternatively, a specific further embodiment of the N32Q, A101T antibody aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39;

and further comprising at least one of (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7.

Antibody N32S, D55E

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N32S, D55E antibody. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Alternatively, a specific further embodiment of the N32S, D55E antibody aspect of the present invention refers to a monoclinal antibody or epitope-binding fragment thereof comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 16;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 13.

Alternatively, a specific further embodiment of the N32S, D55E antibody aspect of the present invention refers to a monoclinal antibody or epitope-binding fragment thereof comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28;

and further comprising at least one of (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and

- 31 039569 (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Antibody N32Q, D55E

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N32Q antibody, D55E. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Alternatively, a specific further embodiment of the N32Q, D55E antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 17;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 13.

Alternatively, a specific further embodiment of the N32Q, D55E antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32; and

a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and further comprising at least one of (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

Antibody N34S, A101T

Another aspect of the present invention relates to a variant of the C10-2 antibody, N34S antibody, A101T. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 33;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Alternatively, a specific further embodiment of the N34S antibody, A101T aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 18; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

Alternatively, a specific further embodiment of the N34S antibody, A101T aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 33; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39;

and further comprising at least one of (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7.

Antibody N34Q, A101T

Another aspect of the present invention relates to a variant of the C10-2 antibody, the N34Q antibody, A101T. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

- 32 039569 (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Alternatively, a specific further embodiment of the N34Q, A101T antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 19; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

Alternatively, a specific further embodiment of the N34Q, A101T antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39;

and further comprising at least one of (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7.

Antibody D55E, A101T

Another aspect of the present invention relates to a variant of the C10-2 antibody, the D55E antibody, A101T. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Alternatively, a specific further embodiment of the D55E, A101T antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 25.

Alternatively, a certain additional embodiment of the D55E, A101T antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5; and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Antibody D55Q, A101T

Another aspect of the present invention relates to a variant of the C10-2 antibody, the D55Q antibody, A101T. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(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: 39.

Alternatively, a specific further embodiment of the D55Q, A101T antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 26.

Alternatively, a specific additional embodiment of the D55Q, A101T antibody aspect of the present invention refers to a monoclinal antibody, or epitope binding fragment thereof, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(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: 39.

Antibody D55S, A101T

Another aspect of the present invention relates to a variant of the C10-2 antibody, the D55S antibody, A101T. This aspect of the present invention is directed to a monoclonal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 30; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Alternatively, a certain additional embodiment of the D55S, A101T antibody aspect of the present invention refers to a monoclinal antibody, or epitope-binding fragment thereof, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 27.

Alternatively, a certain additional embodiment of the D55S, A101T antibody aspect of the present invention refers to a monoclonal antibody, or epitope-binding fragment thereof, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 30; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

Combinations of heavy chain variants and light chain variants are contemplated, such as multiple variants within a light chain and/or multiple variants within a heavy chain, a combination of one variant within a light chain with one or multiple variants within a heavy chain, combinations of multiple variants within the light chain with multiple variants within the heavy chain. The antibody of the present invention preferably contains a light chain selected from the group consisting of

SEQ ID NO: 12 (C10-2 light chain);

SEQ ID NO: 16 (light chain variant N32S);

SEQ ID NO: 17 (light chain variant N32Q);

SEQ ID NO: 18 (light chain variant N34S);

SEQ ID NO: 19 (light chain variant N34Q);

SEQ ID NO: 20 (option N32S, N34S light chain);

SEQ ID NO: 21 (light chain variant N32Q, N34S);

SEQ ID NO: 22 (light chain variant N32Q, N34Q) and

SEQ ID NO: 23 (light chain variant N32S, N34Q); and a heavy chain selected from the group consisting of

SEQ ID NO: 11 (C10-2 heavy chain);

SEQ ID NO: 13 (heavy chain variant D55E);

SEQ ID NO: 14 (heavy chain variant D55Q);

SEQ ID NO: 15 (heavy chain variant D55S);

SEQ ID NO: 24 (heavy chain variant A101T);

SEQ ID NO: 25 (heavy chain variant D55E, A101T);

SEQ ID NO: 26 (heavy chain variant D55Q, A101T) and

SEQ ID NO: 27 (heavy chain variant D55S, A101T).

In one embodiment, the light chain is SEQ ID NO: 12, the heavy chain is selected from the group consisting of SEQ ID NO: 13 (heavy chain variant D55E); SEQ ID NO: 14 (heavy chain variant D55Q); SEQ ID NO: 15 (heavy chain variant D55S); SEQ ID NO: 24 (heavy chain variant A101T); SEQ ID NO: 25 (heavy chain variant D55E, A101T); SEQ ID NO: 26 (heavy chain variant D55Q, A101T) and SEQ ID NO: 27 (heavy chain variant D55S, A101T). In the alternative

- 34 039569 implementation in case the heavy chain is SEQ ID NO: 11, then the light chain is selected from the group consisting of SEQ ID NO: 16 (option N32S light chain); SEQ ID NO: 17 (light chain variant N32Q); SEQ ID NO: 18 (light chain variant N34S); SEQ ID NO: 19 (light chain variant N34Q); SEQ

ID NO: 20 (option N32S, N34S light chain); SEQ ID NO: 21 (light chain variant N32Q, N34S); SEQ ID

NO: 22 (light chain variant N32Q, N34Q) and SEQ ID NO: 23 (light chain variant N32S, N34Q).

A monoclonal antibody or epitope-binding fragment thereof, respectively, contains:

(a) a light chain CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37 and SEQ ID NO: 38;

(b) a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 7; SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; and (f) a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 39.

Embodiments of interest for combinations of variants include those wherein the antibodies are selected from the group consisting of antibody N32S, A101T; antibodies N32Q, A101T; antibodies N32S, D55E; and antibodies N32Q, D55E, preferably antibodies N32S, A101T. As can be seen from the examples, the N32S antibody and the N32S antibody, A101T are the preferred embodiment.

Taken together, the examples show that the antibodies of the present invention, including C10-2, effectively bind to MSD plates coated with P3 antigens from material from subjects with AD. In comparison, commercial antibodies such as PHF-13 have low binding activity. Additionally, PHF-13 showed a significantly higher degree of non-specific binding compared to the antibodies of the present invention. Liquid-phase C10-2 inhibition of Ptau antigen uptake in C10-2 coated plate is effective (IC ₅₀ = 10-20 nM), while it is ineffective for PHF-13 (IC ₅₀ = 500-1000 nM).

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

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5; and (d) a heavy chain selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

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

(a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7;

(c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8; and (d) a light chain selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

The monoclonal antibody or epitope-binding fragment thereof of the present invention typically inhibits P3 from AD subjects in a liquid phase inhibition assay such that the signal is reduced by 50% at a concentration of 100 nM antibody or less, such as 10 to 100 nM antibody, such as 50 nM or less, such as 10 to 50 nM antibody. The monoclonal antibody or epitope-binding fragment thereof of the present invention is typically capable of removing at least 15% of serine-396 phosphorylated tau protein from brain homogenates from AD subjects at approximately 75 ng of antibody as determined by signal tau protein pS396 in Western blot after immunodepletion studies of brain extracts from subjects with Alzheimer's disease.

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

The antibodies and epitope-binding fragments thereof mentioned above, in one embodiment, may further comprise a variant of such light and/or heavy chain CDR1, CDR2, or CDR3 (with no more than 4 amino acid differences, or with no more than 3 amino acid differences, or with no more than 2 amino acid differences, or no more than 1 amino acid difference).

As can be seen from FIG. 18, CDR1 HC, CDR2 HC, CDR3 HC, and CDR3 LC in at least one embodiment are important for binding to the 392-398 region of the tau protein. In one embodiment of the present invention, an antibody of the present invention, or an epitope-binding fragment thereof, comprises: :Y1, H2:Y3 with Y394 tau peptide. In an embodiment, the present invention is directed to an antibody that competes with an antibody further described in

- 35 039569 of this document, for the formation of a network of hydrogen bonds between the solvated ^{p} S396 and L3:T4,

H1:R10, H1:T11, H3:R1, H3:T3; (*) L3:F8 is the C-terminal framework residue flanking CDR L3 (see FIG. 11).

As can be seen from the crystal structure determined by X-ray analysis, the antibody of the present invention binds at two levels of selectivity. The first level of selectivity is for a hyperphosphorylated pathological tau protein, and the second level of selectivity is for a phosphorylated serine residue, where the phosphate of said phosphorylated serine is hydrogen bonded to the side chain of a tyrosine residue one residue away from said phosphorylated serine. Accordingly, an interesting aspect of the present invention is directed to a monoclonal antibody or epitope-binding fragment thereof that is selective for the amino acid motif of a hyperphosphorylated tau protein, the motif consisting of a phosphorylated serine residue and a tyrosine residue located through one residue. Typically, an amino acid motif has the sequence:

Y-X-S(phosphorylated)-P-, where Y is tyrosine,

X is a naturally occurring amino acid,

P is a proline, and

S (phosphorylated) is a serine with a phosphorylated side chain hydroxyl group.

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

Without wishing to be bound by any particular theory, an antibody of the present invention is believed to be selective for an IA amino acid motif, wherein said motif has a conformation that an abnormal tau protein adopts. Accordingly, the IA amino acid motif is typically a sequence that is selectively recognized by an antibody of the present invention. Accordingly, an interesting aspect of the present invention is directed to an antibody or epitope-binding fragment thereof that binds to a phosphorylated tau protein, preferably a hyperphosphorylated tau protein, wherein said antibody or epitope-binding fragment thereof is selective for an IA motif of amino acid residues, wherein R is side chain of a naturally occurring amino acid.

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

- 36 039569

ID

In one aspect of the present invention, the CDR1 region of the HC of the antibody of the present invention, Asp-Arg-Thr-Ile-His (SEQ ID NO: 7) interacts with hyperphosphorylated tau protein IA-ID motifs.

As can be seen from FIG. 18, in this aspect of the present invention, the presence of two consecutive charged residues, namely aspartic acid and arginine in CDR1 HC plays a role in hydrogen bonding to a motif in the target tau protein epitope, where the antibody is selective for the hyperphosphorylated tau amino acid motif. , while the motif consists of a phosphorylated serine residue and a tyrosine residue located through one residue. Accordingly, one aspect of the present invention is directed to an antibody or epitope-binding fragment thereof that binds to a phosphorylated tau protein, preferably a hyperphosphorylated tau protein, wherein said antibody or epitope-binding fragment thereof is selective for an IA-ID motif of amino acid residues, wherein the antibody comprises The CDR1 region of the HC, which contains two consecutive charged amino acid residues, such as aspartic acid and arginine. In a further alternative embodiment of this aspect of the present invention, an antibody or epitope-binding fragment thereof of the present invention binds to a hyperphosphorylated tau protein, wherein said antibody or epitope-binding fragment thereof is selective for an epitope comprising serine-396 and tyrosine394, wherein between the PO4 group and one of a covalent bond is formed between the PO4 group and the other of serine-396 and tyrosine-394, where the antibody contains an HC CDR1 region containing two consecutive charged residues, such as aspartic acid and arginine . The charged amino acid residues of the HC CDR1 region may be selected from the group consisting of arginine, lysine, aspartic acid, and glutamic acid, where at least one of said residues is aspartic acid or arginine, where preferably one charged residue is arginine, and the other is aspartic acid.

In a further embodiment of this aspect of the present invention, one or both of two consecutive charged amino acid residues are flanked by a polar amino acid residue, preferably selected from threonine and tyrosine. Additionally, one or both of one or both of two consecutive charged amino acid residues are flanked by a motif of 3 amino acid residues that is a polar residue-hydrophobic residue-polar residue.

In one embodiment, the HC CDR1 contains a 5-residue motif Polar amino acid

- 37 039569

Hydrophobic amino acid - Polar amino acid - Charged amino acid - Charged amino acid, where at least one of these residues is aspartic acid or arginine, where preferably one charged residue is arginine and the other is aspartic acid. Preferably, the 5 residue motif is Polar Amino Acid - Hydrophobic Amino Acid - Polar Amino Acid - Asp-Arg motif. More preferably, the 5 residue CDR1 motif of the HC is characterized by the sequence Thr-Phe-Thr-Asp-Arg.

Interestingly, the CDR1 of the HC antibody of the present invention contains a Polar amino acid - Hydrophobic amino acid - Polar amino acid - Charged amino acid motif on either side of two consecutive charged amino acid residues involved in hydrogen bonding with a phosphate group involved in the electrostatic interaction between S396 and Y394 . Accordingly, in a preferred embodiment, the HC CDR1 of the present invention comprises an 8 residue palindromic motif Polar amino acid - Hydrophobic amino acid - Polar amino acid - Charged amino acid - Charged amino acid - Polar amino acid - Hydrophobic amino acid - Polar amino acid. Preferably, the 8-residue CDR1 HC motif provides for the Thr-Phe-Thr-Asp-Arg-Polar amino acid-Hydrophobic amino acid-Polar amino acid motif. More preferably, the 8-residue CDR1 HC motif provides for the Thr-Phe-Thr-Asp-Arg-Thr-Ile-His motif.

In one embodiment, the antibody recognizes an epitope within residues 392-398 of a hyperphosphorylated tau protein containing serine-396 and tyrosine-394, where serine-396 is phosphorylated and where the antibody contains an HC CDR1 region containing two consecutive charged amino acid residues. In a preferred embodiment, the antibody recognizes an epitope within residues 392-398 of a hyperphosphorylated tau protein containing serine-396 and tyrosine-394, where serine-396 is phosphorylated and where the antibody contains an HC CDR1 region containing a 5-residue motif Polar amino acid - Hydrophobic amino acid - Polar amino acid Charged amino acid - Charged amino acid. In a preferred embodiment, the antibody recognizes an epitope within residues 392-398 of a hyperphosphorylated tau protein containing serine-396 and tyrosine-394, where serine-396 is phosphorylated and where the antibody contains an HC CDR1 region containing the motif Polar amino acid - Hydrophobic amino acid - Polar amino acid - Charged amino acid - Charged amino acid - Polar amino acid - Hydrophobic amino acid - Polar amino acid.

In a further preferred embodiment, the antibody comprises an HC CDR1 region as defined herein and an HC CDR3 region containing a 6 residue motif containing at least two charged residues.

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

One aspect of the present invention is directed to a method for treating tauopathy using an antibody of the present invention or epitope-binding fragments thereof. Typically, tauopathy is selected from the group consisting of Alzheimer's disease, argyrophilic granule disease (AGD), psychosis, in particular AD-associated psychosis or psychosis in patients with AD, psychiatric symptoms in patients with dementia with Lewy bodies, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART), senile dementia with a predominance of neurofibrillary tangles, dementia boxers, chronic traumatic encephalopathy, stroke, stroke recovery, neurodegeneration associated with Parkinson's disease, chromosome-linked parkinsonism, Lytico-Bodiga disease (Guam's parkinsonism-dementia complex), ganglioglioma and gangliocytoma, meningoangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Huntington's disease, encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lipofuscinosis. More typically, the tauopathy is selected from the group consisting of Alzheimer's disease, argyrophilic granule disease (AGD), psychosis, in particular AD-related psychosis or psychosis in patients with AD, psychiatric symptoms in patients with dementia with Lewy bodies, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD or variants thereof), TBI (traumatic brain injury, acute or chronic), corticobasal degeneration (CBD), and Pick's disease. In particular, taupathies can be selected from Alzheimer's disease, argyrophilic granule disease (AGD), AD-related psychosis, or psychosis in patients with AD and psychiatric symptoms in patients with dementia with Lewy bodies.

Accordingly, a further aspect of the present invention is directed to an antibody of the present invention, or epitope-binding fragments thereof, for use in the treatment of tauopathy. Typically, tauopathy is selected from the group consisting of Alzheimer's disease, argyrophilic granule disease (AGD), psychosis, in particular AD-related psychosis or psychosis in patients with AD, psychiatric symptoms in patients with dementia with Lewy bodies progressive supranuclear

- 38 039569 paralysis (PSP), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic forms), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART), senile tangle-predominant dementia, boxer's dementia, chronic traumatic encephalopathy, stroke, stroke recovery, Parkinson's disease-associated neurodegeneration, chromosome-linked parkinsonism, Lytico-Bodiga disease (Guam's parkinsonism-dementia complex), ganglioglioma and gangliocytoma, meningoangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lipofuscinosis. More typically, the tauopathy is selected from the group consisting of Alzheimer's disease, argyrophilic granule disease (AGD), psychosis, in particular AD-related psychosis, or psychosis in AD patients, AD-related apathy, or apathy in AD patients, psychiatric symptoms in patients with dementia with Lewy bodies, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic), corticobasal degeneration (CBD), and Pick's disease. In particular, taupathies can be selected from Alzheimer's disease, argyrophilic granule disease (AGD), AD-related psychosis or psychosis in AD patients, AD-related apathy or apathy in AD patients, and psychiatric symptoms in patients with body dementia. Levy.

One aspect of the present invention is directed to therapy involving the administration

i) an anti-tau antibody; and ii) a compound selected from the group consisting of

a) a BACE inhibitor;

b) a compound useful in active or passive tau immunotherapy;

c) a compound useful in active or passive immunotherapy for an Ae peptide;

d) NMDA receptor antagonists;

e) an additional tau aggregation inhibitor;

f) an acetylcholinesterase inhibitor;

g) an antiepileptic agent;

h) an anti-inflammatory drug and

i) SSRI.

An additional aspect of the present invention is directed to a composition containing

i) an anti-tau antibody; and ii) a compound selected from the group consisting of

a) a BACE inhibitor;

b) a compound useful in active or passive tau immunotherapy;

c) a compound useful in active or passive immunotherapy for an Ae peptide;

d) NMDA receptor antagonists;

e) an additional tau aggregation inhibitor;

f) additional acetylcholinesterase;

g) an antiepileptic agent;

h) an anti-inflammatory drug and

i) SSRI.

A further aspect of the present invention is directed to a kit containing

i) a composition comprising an anti-tau antibody; and ii) a composition comprising a compound selected from the group consisting of

a) a BACE inhibitor;

b) a compound useful in active or passive tau immunotherapy;

c) a compound useful in active or passive immunotherapy for an Ae peptide;

d) NMDA receptor antagonists;

e) an additional inhibitor of tau aggregation; f) an acetylcholinesterase inhibitor;

g) an antiepileptic agent;

h) an anti-inflammatory drug and

i) an antidepressant.

a) Anti-tau antibody combined with a BACE 1 inhibitor

In a therapy, composition or kit of the present invention, an anti-tau antibody may be combined with a BACE 1 inhibitor. The BACE 1 inhibitor may be a small molecule BACE I inhibitor such as LY2886721, MK-8931, AZD3293, or E2609.

In a further embodiment, the BACE 1 inhibitor is represented by formula I

- 39 039569

where Ar is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, and while Ar is optionally substituted with one or more substituents selected from halogen, CN, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C 1 -C ₆ fluoroalkyl or C 1 -C ₆ alkoxy; and R1 is one or more of hydrogen, halogen, C1- _C3 fluoroalkyl, or C1- _C3 alkyl; and R ₂ is hydrogen or fluorine.

Exemplary compounds of formula I include

(6S)-6-(5-Amino-2-fluorophenyl)-3-fluoro-3,6-bis(fluoromethyl)piperidine-2-thione

An additional suitable BACE 1 inhibitor may be represented by formula II n Ar< .N

Formula II wherein Ar is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, 1,2,4-triazolyl, thiophenyl, thiazolyl, oxazolyl, isoxazolyl, 1,3,4-thiadiazolyl, isothiazolyl, 1 ,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, furazanil and 1,2,4-thiadiazolyl, and where Ar is optionally substituted with one or more of halogen, CN, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ fluoroalkyl or C ₁ -C ₆ alkoxy; R ¹ is C1-C3 alkyl or C1-C3 fluoroalkyl; R ² represents hydrogen, halogen, C1-C ₃ fluoroalkyl or C1-C ₃ alkyl; and R ³ is C ₁ -C ₃ alkyl.

Exemplary BACE 1 inhibitor compounds of formula II include compounds selected from the group consisting of N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2 ,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide; N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5- methoxypyrazine-2-carboxamide; N-(3-((2R,5S)-6amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5- methoxypicolinamide; N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)5 -cyano-3-methylpicolinamide and N-(3-((2R,5R)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl )-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide.

- 40 039569

Other BACE inhibitors may be selected from the formula below:

where Ar is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, and while Ar is optionally substituted with one or more substituents selected from halogen, CN, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C1C ₆ fluoroalkyl or C ₁ -C ₆ alkoxy; and

R ¹ is one or more of hydrogen, halogen, C ₁ -C3fluoroalkyl or C ₁ -C3 alkyl;

R ² represents hydrogen or fluorine, or its corresponding pharmaceutically acceptable salts.

For example, connections such as

A/-(3-((25.55)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -5-methoxypicolinamide;

- 41 039569

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -5-(methoxy-c13)picolinamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -5-cyano-3-methylpicolinamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -5-chloropicolinamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -5-methoxypyrazine-2-carboxamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -2-methyloxazole-4-carboxamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -4-bromo-1-methyl-1 H-imidazole-2-carboxamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -4-methylthiazole-2-carboxamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -2-(difluoromethyl)oxazole-4-carboxamide;

L/-(3-((25.55)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -1-(difluoromethyl)-1H-pyrazole-3-carboxamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -5-(difluoromethyl)pyrazine-2-carboxamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -4-chlorobenzamide;

L/-(3-((28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)4-fluorophenyl) -5-fluoropicolinamide;

N/-(3-((28.5R)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl) 4-fluorophenyl) -5-methoxypicolinamide;

L/-[3-[(28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,5difluorophenyl]-5- fluoropyridine-2-carboxamide;

L/-[3-[(28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,5difluorophenyl]-5- methoxypyridine-2-carboxamide;

L/-[3-[(28.58)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,5difluorophenyl]-5- methoxypyrazine-2-carboxamide;

N/-[3-[(28.5R)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,5difluorophenyl]-5-fluoropyridine -2-carboxamide;

N/-[3-[(28.5R)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,5difluorophenyl]-5-methoxypyridine -2-carboxamide;

- 42 039569

M-[3-[(25.5R)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,5difluorophenyl]-5-methoxypyrazine -2-carboxamide;

L/-[3-[(25.55)-6-amino-5-fluoro-2,5-bis(fluoromethyl)-3,4-dihydropyride in-2-yl]-4fluorophenyl]-5-fluoropyridine- 2-carboxamide;

L/-[3-[(25.55)-6-amino-5-fluoro-2,5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4fluorophenyl]-5-methoxypyridin-2 -carboxamide;

L/-[3-[(25.55)-6-amino-5-fluoro-2,5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4fluorophenyl]-5-methoxypyrazin-2 -carboxamide;

A/-[3-[(25.5/?)-6-amino-5-fluoro-2,5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4fluorophenyl]-5-fluoropyridine- 2-carboxamide;

L/-[3-[(25.5K)-6-amino-5-fluoro-2,5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4fluorophenyl]-5-methoxypyridin-2- carboxamide and

L/-[3-[(25.5/?)-6-amino-5-fluoro-2,5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4fluorophenyl]-5-methoxypyrazin- 2-carboxamide;

or a pharmaceutically acceptable salt of said compounds.

Other BACE inhibitors may be represented by the formula below:

where Ar is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, and isoxazolyl, and wherein Ar is optionally substituted with one or more of halogen, CN, C1- _C6 alkyl, C2- _C6 alkenyl, C2- _C6 alkynyl, C1- _C6 fluoroalkyl or C1- _C6 alkoxy; and

R1 and R2 are independently hydrogen, halogen, C1- _C3 fluoroalkyl or C1- _C3 alkyl;

or its corresponding pharmaceutically acceptable salt.

For example, compounds such as (8)-N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)- 5-chloropicolinamide, (8)-N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5- fluoropicolinamide,

- 43 039569 (3)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-methoxypyrazine-2-carboxamide , (8)-M-(3-(6-Amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-1-(difluoromethyl)-1H-pyrazole -3-carboxamide, (3)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-2-(difluoromethyl) oxazole-4-carboxamide, (8)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-cyanopicolinamide, (8)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-2-methyloxazole-4-carboxamide, (3 )-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-methoxypyrimidine-2-carboxamide, (3)- M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide, (3) -M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4,5difluorophenyl)-2-methyloxazole-4-carboxamide, (8) -M-(3-(6-amino-3,3-difluoro-2-(fluoromethy k)-2D4,5-tetrahydropyridin-2-yl)-4,5difluorophenyl)-5-methoxypyrazine-2-carboxamide, (8)-M-(3-(6-amino-3,3-difluoro-2-( fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4,5difluorophenyl)-5-fluoropicolinamide, (8)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4 ,5-tetrahydropyridin-2-yl)-4,5difluorophenyl)-5-chloropicolinamide, (8)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridine -2-yl)-4,5difluorophenyl)-5-cyanopicolinamide, (3)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl )-4,5difluorophenyl)-5-methoxypicolinamide, (3)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4, 5-difluorophenyl)-5-(methoxy-c13)picolinamide, (8)-N-(3-(6-amino-3Ddifluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4,5difluorophenyl)- 5-cyano-3-methylpicolinamide, (8)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5- (methoxy-c13)picolinamide, (3)-M-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)2 D 4,5-tetrahydropyridin-2-yl)-4-fluorophenyl) -5-bromopicolinamide, (8)-M-(3-(6-amino-3,3-difluoro-2- (fluoromethyl)-2D4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-bromopicolinamide;

or a pharmaceutically acceptable salt of said compounds.

Other BACE compounds may be represented by the formula below

Formula I wherein Ar is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, and wherein Ar is optionally substituted with one or more substituents selected from halogen, CN, C1- _C6 alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C 1 -C ₆ fluoroalkyl or C 1 -C ₆ alkoxy; and

R ¹ represents hydrogen, halogen, C1-C ₃ fluoroalkyl or C1-C ₃ alkyl;

or its corresponding pharmaceutically acceptable salt.

Connections can be

- 44 039569 (8)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5- chlorpicolinamide, (8)-M-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-fluoropicolinamide , (8)-M-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-methoxypyrazin- 2-carboxamide, (8)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-2 -methyloxazole-4-carboxamide, (8)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl )-5-methoxypicolinamide, (8)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl) -5-(difluoromethyl)pyrazine-2-carboxamide, (8)-M-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridine-2 -yl)-4fluorophenyl)-5-cyanopicolinamide, (8)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrahydropyridine-2- yl)-4fluorophenyl)-4-methylthiazole-2-carboxamide, (8)-M-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4,5-tetrag ropyridin-2-yl)-4fluorophenyl)-5-methoxypyrimidine-2-carboxamide, (8)-N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,3,4 ,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-methoxy-3-methylpyrazine-2-carboxamide, (8)-N-(3-(6-amino-2-(difluoromethyl)-3,3- difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-cyano-3-methylpicolinamide, (8)-N-(3-(6-amino-2-(difluoromethyl)-3, 3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-bromopicolinamide, (8)-M-(3-(6-amino-2-(difluoromethyl)-3,3 -difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-(methoxy-c13)picolinamide and (8)-N-(3-(6-amino-2-(difluoromethyl) -3,3-difluoro-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-(methoxy-c13)pyrazine-2-carboxamide or pharmaceutically acceptable salts of these compounds. Other BACE inhibitors may be represented by the formula below:

where Ar is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, and while Ar is optionally substituted with one or more substituents selected from halogen, CN, C 1 -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C 1 -C ₆ fluoroalkyl or C 1 -C ₆ alkoxy; and

R1 is one or more _of hydrogen, halogen, C1-C3 fluoroalkyl, or _C1C3 alkyl;

or its corresponding pharmaceutically acceptable salt.

The connection may be

N-(3-((2R,3S,5S)-6-amino-3,5-diφtorop-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5- fluoropicolinamide,

N-(3-((2R,3S,5S)-6-amino-3,5-diφtorop-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5- methoxypyrazine-2-carboxamide,

N-(3-((2R,3S,5S)-6-amino-3,5-diφtorop-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5- methoxypicolinamide,

N-(3-((2R,3S,5R)-6-amino-3,5-diφtorop-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5- fluoropicolinamide,

N-(3-((2R,3S,5R)-6-amino-3,5-diφtorop-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5- methoxypyrazine-2-carboxamide,

- 45 039569

N-(3-((2E,35,5E)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-2 -methyloxazole-4-carboxamide,

М-(3-((2Е,35,5Е)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -methoxypicolinamide,

М-(3-((2Е,35,5Е)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -(methoxy-c13)picolinamide,

М-(3-((2Е,35,5Е)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -chlorpicolinamide,

М-(3-((2Е,35,5Е)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-1 -methyl-1H-imidazole-2-carboxamide,

М-(3-((2Е,35,5Е)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -(trifluoromethyl)pyrazine-2-carboxamide,

M-(3-((2E,35,5E)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-4 -methylthiazole-2-carboxamide,

М-(3-((2Е,35,5Е)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-2 -(difluoromethyl)oxazole-4-carboxamide,

М-(3-((2Е,35,5Е)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-1 -(difluoromethyl)-1H-pyrazole-3-carboxamide,

М-(3-((2Е,35,5Е)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -(difluoromethyl)pyrazine-2-carboxamide,

M-(3-((2E,35,5E)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-4 -chlorobenzamide,

М-(3-((2Е,35,5Е)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -cyanopicolinamide,

M-(3-((2E,35.5E)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4,5difluorophenyl) -5-(methoxy-c13) picolinamide,

M-(3-((2E,35.55)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -(methoxy-c13)picolinamide,

M-(3-((2E,35.55)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -(methoxy-c13) pyrazine-2-carboxamide,

M-(3-((2E,35.55)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -cyano-3-methylpicolinamide,

M-(3-((2E,35.55)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4,5difluorophenyl) -5-(methoxy-c13) picolinamide,

M-(3-((2E,3E,58)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 -(methoxy-c13)picolinamide,

N-(3-((2R,3S,5S)-6-amino-3,5-diφτop-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5 brompicolinamide or pharmaceutically acceptable salts of these compounds.

b) Anti-tau antibody combined with anti-N3PGLU-A-BETA antibody

In a therapy, composition or kit of the present invention, an anti-tau antibody can be combined with an anti-N3PGLU-A-BETA antibody.

c) An anti-tau antibody combined with a compound useful in active or passive immunotherapy against the Ae peptide

In a therapy, composition or kit of the present invention, an anti-tau antibody may be combined with a compound useful in active or passive Aepeptide immunotherapy.

d) Anti-tau antibody combined with an NMDA receptor antagonist

In a therapy, composition or kit of the present invention, an anti-tau antibody may be combined with NMDA receptor antagonists. The NMDA receptor antagonist may be selected from the group consisting of memantine, namenda, namzaric (memantine/donepezil) and their generic forms. The NMDA receptor antagonist may be selected from an antipsychotic agent. Presynaptic

- 46 039569 Neurons prevent excessive release of glutamate through negative feedback mechanisms, however, such mechanisms are disrupted under conditions of cellular stress, such as in AD. Excess glutamate in the synaptic cleft causes the calcium channels to permanently open, leading to elevated levels of intracellular calcium in neurons, which causes severe neuronal damage and/or death. By antagonizing the NMDA receptor under conditions of excess calcium influx, the antipsychotic reduces excess calcium in neurons, which reduces cell death and improves normal neuronal signaling and thus cognitive function.

e) Anti-tau antibody combined with an additional inhibitor of tau aggregation

In a therapy, composition or kit of the present invention, an anti-tau antibody may be combined with an inhibitor of tau aggregation.

f) Anti-tau antibody combined with an acetylcholinesterase inhibitor

In a therapy, composition or kit of the present invention, an anti-tau antibody may be combined with an acetylcholinesterase inhibitor (AChEI). AChEIs are often used as first line therapy for cognitive symptoms in mild to moderate AD. AChEIs are also widely used for the treatment of moderate to severe AD, including donepezil, which is approved for this subpopulation. AChEIs reduce the cholinergic deficit seen in AD patients and improve the patient's ability to carry out daily activities. In one embodiment, the present invention provides a therapy comprising an anti-tau antibody as defined herein and an acetylcholinesterase inhibitor.

In one embodiment, the AChEI is selected from the group consisting of donepezil, galantamine, and rivastigmine. AChEI may be an oral tablet, jelly, syrup, or other form of oral solution. AChEI can also be a transdermal patch.

g) Anti-tau antibody combined with an antiepileptic agent

In a therapy, composition or kit of the present invention, an anti-tau antibody may be combined with an antiepileptic agent.

h) Anti-tau antibody combined with an anti-inflammatory agent

In a therapy, composition or kit of the present invention, an anti-tau antibody may be combined with an anti-inflammatory agent.

i) Anti-tau antibody combined with an antidepressant

In a therapy, composition or kit of the present invention, an anti-tau antibody may be combined with an antidepressant.

Depression is a common early accompanying symptom of dementia in AD. Tricyclic antidepressants were at one time the treatment of choice for depressive symptoms in AD, but SSRIs have largely replaced these agents. In one embodiment, escitalopram is an antidepressant because it is often prescribed for the treatment of AD due to its favorable side effect profile and minimal drug interactions. In a further embodiment, citalopram or sertraline is an antidepressant as they are also commonly used. In a further embodiment, vortioxetine is an antidepressant because it is associated with improved cognitive function and imaging evidence of neuronal efficacy in patients suffering from MDD. The antidepressant may be selected from the group consisting of escitalopram, sertraline, citalopram, paroxetine, fluoxetine, venlafaxine, trazodone, mirtazapine, vortioxetine and their generic forms.

A further aspect of the present invention is directed to an antibody of the present invention, or epitope-binding fragments thereof, in composition together with a pharmaceutically acceptable carrier, diluent, adjuvant and/or stabilizer. The antibodies of the present invention, or epitope-binding fragments thereof, can be used in therapy for the treatment of tauopathy. Typically, tauopathy is selected from the group consisting of Alzheimer's disease, argyrophilic granule disease (AGD), psychosis, in particular AD-related psychosis, or psychosis in AD patients, AD-related apathy, or apathy in AD patients, psychiatric symptoms. in patients with dementia with Lewy bodies, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic forms), corticobasal degeneration (CBD), Pick's disease, primary age tauopathy (PART), senile dementia with a predominance of neurofibrillary tangles, boxer's dementia, chronic traumatic encephalopathy, stroke, stroke recovery, Parkinson's disease-related neurodegeneration, chromosome-linked parkinsonism, Lytico-Bodiga disease (Guam complex parkinsonism-dementia), ganglioglioma and gangliocytoma, meningoangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lipofuscinosis. More typically, tauopathy is selected from the group consisting of Alzheimer's disease, Argy's disease

- 47 039569 profile grains (AGD), psychosis, in particular, psychosis associated with AD or psychosis in patients with AD, apathy associated with AD or apathy in patients with AD, psychiatric symptoms in patients with dementia with Lewy bodies, progressive supranuclear paralysis (PSP), frontotemporal dementia (FTD or variants thereof), TBI (traumatic brain injury, acute or chronic), corticobasal degeneration (CBD), and Pick's disease. In particular, taupathies can be selected from Alzheimer's disease, argyrophilic granule disease (AGD), AD-related psychosis or psychosis in AD patients, AD-related apathy or apathy in AD patients, and psychiatric symptoms in patients with body dementia. Levy.

The treatment contemplated by the present invention may be long term and the patient may be treated for at least 2 weeks, such as for at least 1 month, 6 months, 1 year or longer.

The antibodies of the present invention may, for example, be monoclonal antibodies obtained by the hybridoma method first described in Kohler et al., Nature 256, 495 (1975), or may be monoclonal antibodies obtained by recombinant DNA or other methods. , or more preferably can be obtained using the new disclosed method. Monoclonal antibodies can also be isolated from antibody phage libraries using the techniques described, for example, in Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. For example, monoclonal antibodies can be obtained from hybridomas derived from mouse spleen B lymphocytes obtained from mice immunized with an antigen of interest, for example, as cells expressing the antigen on the surface, or as a nucleic acid encoding the antigen of interest. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells from immunized humans or non-human mammals such as rats, rabbits, dogs, sheep, goats, primates, and the like.

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

The HuMAb mouse contains a human immunoglobulin gene minilocus that encodes unrearranged sequences for the heavy chain variable and constant domains (μ and γ) and the light chain variable and constant domains (k) of human immunoglobulin, together with targeted mutations that inactivate endogenous loci encoding μ- and k-chain (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, mice show reduced expression of IgM or IgK, and in response to immunization, the introduced transgenes encoding human heavy and light chains undergo class switching and somatic mutation to form high-affinity human IgG monoclonal antibodies of the k-isotype (Lonberg N. et al. (1994 ), supra, reviewed in Lonberg N., Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg N. and Huszar D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding F. and Lonberg N., Ann. N. Y. Acad. Sci 764 536-546 (1995)). The production of HuMAb mice is described in detail in Taylor L. et al., Nucleic Acids Research 20, 62876295 (1992), Chen J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 29122920 (1994), Taylor, L et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., 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 , WO 92/22645, WO 92/03918 and WO 01/09187.

HCo7, HCo12, HCo17 and HCo20 mice have JKD disruption in their endogenous light chain (kappa) genes (described in Chen et al., EMBO J. 12, 811-820 (1993)), CMD disruption in their endogenous heavy chain genes. chains (described in Example 1 of WO 01/14424) and the KCo5 transgene encoding the human kappa light chain (described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)). Additionally, HCo7 mice have the HCo7 transgene encoding the human heavy chain (described in US 5770429), HCo12 mice have the HCo12 transgene encoding the human heavy chain (described in Example 2 of WO 01/14424), HCo17 mice have the HCo17 transgene encoding the human heavy chain (described in Example 2 of WO 01/09187) and HCo20 mice have the HCo20 transgene encoding the human heavy chain. The resulting mice express transgenes encoding human immunoglobulin kappa heavy and light chains in a genetic environment that is homozygous for disruption of endogenous mouse kappa heavy and light chain loci.

In KM mice, the endogenous mouse kappa light chain gene was homozygous disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993), and the endogenous mouse heavy chain gene was homozygous disrupted as described in Example 1 of WO 01/09187. This line

- 48 039569 mice carry the KCo5 transgene encoding the human kappa light chain described in Fishwild et al.,

Nature Biotechnology 14, 845-851 (1996). This mouse line also carries a transchromosome encoding a human heavy chain consisting of the hCF fragment of chromosome 14 (SC20) described in WO

02/43478. Mice HCo12-Balb/c, HCo17-Balb/c and HCo20-Balb/c can be obtained by crossing

HCo12, HCo17 and HCo20 with KCo5[J/K](Balb) as described in WO 09/097006.

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

Splenocytes from these transgenic mice can be used to generate hybridomas that secrete human monoclonal antibodies according to well known techniques. Human monoclonal or polyclonal antibodies of the present invention, or antibodies of the present invention derived from other species, can also be obtained transgenically by obtaining another non-human mammal or plant that is transgenic for immunoglobulin heavy and light chain sequences of interest, and obtaining of which antibodies are in extractable form. With regard to the production of antibodies in mammals transgenically, antibodies can be obtained in the milk of goats, cows or other mammals and extracted from it (see, for example, US 5827690; US 5756687; US 5750172 and US 5741957).

The antibody of the present invention may be of any isotype. The choice of isotype will usually be determined by the desired effector functions such as ADCC induction. Illustrative isotypes are IgG1, IgG2, IgG3 and IgG4. You can use the constant domains of any of the human kappa- or lambda-type light chains. If necessary, the class of the anti-tau antibody of the present invention can be switched using known methods. For example, an antibody of the present invention that was originally an IgM may be class-switched to an IgG antibody of the present invention. Additionally, class switching techniques can be used to convert one IgG subclass to another, for example from IgG1 to IgG2. Thus, the effector function of the antibodies of the present invention can be changed by isotype switching, for example, to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibody for different therapeutic applications. In one embodiment, the antibody of the present invention is an IgG1 antibody, such as an IgG1 κ isotype. An antibody is considered to belong to a particular isotype if its amino acid sequence is most homologous to that isotype compared to other isotypes.

In one embodiment, the antibody of the present invention is a full-length antibody, preferably an IgG antibody, in particular an IgG1 antibody of the κ-isotype. In another embodiment, the antibody of the present invention is an epitope-binding antibody fragment or a single chain antibody.

Antibodies and their epitope-binding fragments can, for example, be prepared by separation into epitope-binding fragments using conventional techniques and the epitope-binding fragments screened for utility in the same manner as described herein for complete antibodies. For example, epitope-binding F(ab') ₂ fragments can be obtained by treating the antibody with pepsin. The resulting epitope-binding F(ab') ₂ fragment can be processed to reduce disulfide bridges to produce epitope-binding Fab' fragments. Epitope-binding Fab fragments can be obtained by treating an IgG antibody with papain; epitope-binding Fab' fragments can be obtained by cleaving an IgG antibody with pepsin. An epitope-binding F(ab') moiety can also be obtained by linking the Fab' described below with a thioether bond or a disulfide bond. An epitope-binding Fab' fragment is an epitope-binding fragment of an antibody obtained by cutting a disulfide bond in the F(ab')2 hinge domain. The epitope-binding Fab' fragment can be obtained by treating the epitope-binding F(ab') ₂ fragment with a reducing agent such as dithiothreitol. An antibody epitope-binding fragment can also be obtained by expressing nucleic acids encoding such epitope-binding fragments in recombinant cells (see, for example, Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimeric gene encoding part of an F(ab')2 epitope-binding fragment may contain DNA sequences encoding a CH1 domain and an H chain hinge domain followed by a stop codon to stop translation to produce such a truncated epitope-binding antibody fragment molecule.

In one embodiment, the anti-tau antibody is a monovalent antibody, preferably the monovalent antibody described in WO 2007059782 (incorporated herein by reference in its entirety), having a hinge deletion. 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 a light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding VL- the region of the selected antigen-specific antibody to the tau protein, and the nucleotide sequence encoding the constant CL region of Ig, where the specified nucleotide sequence encoding the VL region of the selected antigen-specific antibody, and the specified nucleotide sequence encoding the CL region of the Ig, are operably linked with each other, and where, in the case of the IgG1 subtype, the nucleotide sequence encoding the CL region has been modified so that the CL region does not contain any amino acids capable of forming disulfide bonds or covalent bonds with other peptides containing and the identical amino acid sequence of the CL region, in the presence of polyclonal human IgG or when administered to an animal or human; ii) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding a VH region of a selected antigen-specific antibody and a nucleotide sequence encoding a human Ig constant CH region, wherein the nucleotide sequence encoding the CH -region has been modified so that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, does not contain any amino acid residues involved in the formation of disulfide bonds or covalent or stable non-covalent bonds between heavy chains with other peptides containing the identical amino acid sequence of the human Ig CH region, in the presence of a polyclonal human IgG or when administered to an animal or human, where the specified nucleotide sequence encoding the VH region the selected antigen-specific antibody, and the specified nucleotide sequence encoding the CH-region of the specified Ig, functionally linked to each other; iii) providing a cellular expression system for obtaining said monovalent antibody; iv) obtaining said monovalent antibody by co-expressing the nucleic acid constructs from (i) and (ii) in cells of the cellular expression system from (iii).

Similarly, in one embodiment, an anti-tau antibody of the present invention is a monovalent antibody that contains:

(i) the variable domain of an antibody of the present invention described herein, or an epitope-binding portion of said domain, and (ii) an immunoglobulin CH domain or domain containing 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 of the IgG4 subtype, other CH domains, such as the CH3 domain, does not contain any amino acid residues capable of forming disulfide bonds with the identical CH domain or other covalent or stable non-covalent bonds between heavy chains with identical CH-domain in the presence of polyclonal human IgG.

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

In another additional embodiment, the monovalent antibody sequence has been modified such that it does not contain any N-linked glycosylation acceptor sites.

The present invention also includes bispecific antibodies wherein the antibody's tau binding region (e.g., the tau protein binding region of a monoclonal anti-tau antibody) is part of a bivalent or polyvalent bispecific backbone that targets more than one epitope (e.g., a second the epitope may include an active transport receptor epitope such that the bispecific antibody will exhibit improved transcytosis across a biological barrier such as the blood-brain barrier). Thus, in another further embodiment, a monovalent anti-tau Fab can be coupled to an additional Fab or scFv that targets a different protein to form a bispecific antibody. A bispecific antibody may have a dual function, for example, a therapeutic function conferred by the tau-binding domain of the antibody and a transport function by which it can bind to a receptor molecule to enhance transport across a biological barrier such as the blood-brain barrier.

The antibodies and epitope-binding fragments thereof of the present invention also include single chain antibodies. Single chain antibodies are peptides in which the heavy and light chain Fv domains are joined to each other. In one embodiment, the present invention provides a single chain Fv (scFv) wherein the heavy and light chains in the Fv of an anti-tau protein antibody of the present invention are connected via a flexible peptide linker (typically

- 50 039569 approximately 10, 12, 15 or more amino acid residues long) into one peptide chain. Methods for producing such antibodies are described, for example, in US 4946778, 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). A single chain antibody can be monovalent if only one VH and VL is used, bivalent if two VH and VL are used, or polyvalent if more than two VH and VL are used.

Antibodies and their epitope-binding fragments described herein can be modified by including 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 protein selectivity and/or tau protein specificity of the underivatized parent anti-tau antibody. The inclusion of one or more modified amino acids may be advantageous, for example, to increase the serum half-life of the polypeptide, reduce the antigenicity of the polypeptide, or improve the storage stability of the polypeptide. The amino acid(s) are modifiable, eg, co-translationally or post-translationally during recombinant production (eg, N-linked glycosylation in N-X-S/T motifs during expression in mammalian cells), or modifiable 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, geranylgeranylated) 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. The literature presents a variety of sources suitable as a guide for a specialist in the modification of amino acids. Example protocols are found in Walker (1998) Protein Protocols On CD-Rom, Humana Press, Totowa, NJ. The modified amino acid may be selected, for example, 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 their epitope-binding fragments of the present invention can also be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Illustrative polymers and methods of attaching them to peptides are given, for example, in US 4,766,106, US 4,179,337, US 4,495,285, and US 4,609,546. such as from about 2000 to about 20000, for example about 3000-12000 g/mol).

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

In one embodiment, provided are antibodies and epitope-binding fragments of the present invention containing one or more amino acids labeled with radioactive isotopes. Radiolabeled anti-tau antibody can be used for both diagnostic and therapeutic purposes (conjugation to radiolabelled molecules is another possible feature). Non-limiting examples of such labels include, without limitation, bismuth ( ²¹³ Bi), carbon ( ^P C, ¹³ C, ¹⁴ C), chromium ( ⁵¹ Cr), cobalt ( ⁵⁷ Co, ⁶⁰ Co), copper ( ⁶⁴ Cu), dysprosium ( ¹⁶⁵ Dy ), erbium ( ¹⁶⁹ Er), fluorine ( ¹⁸ F), gadolinium ( ¹⁵³ Gd, ¹⁵⁹ Gd), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), germanium ( ⁶⁸ Ge), gold ( ¹⁹⁸ Au), holmium ( ¹⁶⁶ Ho), hydrogen ( ³ H), indium ( ⁿ¹ In, ¹¹² In, ¹¹³ In, ¹¹⁵ In), iodine ( ¹²¹ I, ¹²³ I, ¹²⁵ I, ¹³¹ I), iridium ( ¹⁹² Ir), iron ( ⁵⁹ Fe), krypton ( ^81m Kr), lanthanum ( ¹⁴⁰ La), lutetium ( ¹⁷⁷ Lu), manganese ( ⁵⁴ Mn), molybdenum ( ⁹⁹ Mo), nitrogen ( ¹³ N, ¹⁵ N), oxygen ( ¹⁵ O), palladium ( ¹⁰³ Pd), phosphorus ( ³² P), potassium ( ⁴² K), praseodymium ( ¹⁴² Rg), promethium ( ¹⁴⁹ Rt), rhenium ( ¹⁸⁶ Re, ¹⁸⁸ Re), rhodium ( ¹⁰⁵ Rh), rubidium ( ⁸¹ Rb, ⁸² Rb), ruthenium ( ⁸² Ru, ⁹⁷ Ru), samarium ( ¹⁵³ Sm), scandium ( ⁴⁷ Sc), selenium ( ⁷⁵ Se), sodium ( ²⁴ Na), strontium ( ⁸⁵ Sr, ⁸⁹ Sr, ⁹² Sr), sulfur ( ³⁵ S), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Tl), tin ( ¹¹³ Sn, ¹¹⁷ Sn), xenon ( ¹³³ Xe), ytterbium ( ¹⁶⁹ Yb, ¹⁷⁵ Yb, ¹⁷⁷ Yb), and ttrium ( ⁹⁰ Y), zinc ( ⁶⁵ Zn), and zirconium ( ⁸⁹ Zr). Zirconium ( ⁸⁹ Zr) is of particular interest. Methods for preparing radioactively labeled amino acids and corresponding peptide derivatives are known in the art (see, for example, Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2nd edition, Chafner and Longo, eds., Lippincott Raven (1996 )) and US 4681581, US 4735210, US 5101827, US 5102990 (US RE35500), US 5648471 and US 5697902. -T In HighSpecific-Activity Radioiodination Of Antibodies Using N-Succinimidyl-3-(Thmethylstannyl)Benzoate As An Intermediate, Nucl Med Biol 25(7):659-665 Kurth, M et al (1993) Site -Specific Conjugation Of A Radioiodinated Phenethylamine Derivative To A Monoclonal Antibody Results In Increased Radioactivity Localization In Tumor, J. Med. Chem. 36(9): 1255-1261;

- 51 039569 antibody for targeting tumors, Cancer Res. 50(3 Suppl):857s-861s).

The present invention also provides anti-tau antibodies and epitope-binding fragments thereof labeled with a detectable fluorescent label (such as a rare earth element chelate (e.g., europium chelate)), a fluorescein-type label (e.g., fluorescein, fluorescein isothiocyanate, 5-carboxyfluorescein, 6 -carboxyfluorescein, dichlorotriazinylaminofluorescein), rhodamine type labels (eg ALEXA FLUOR® 568 (Invitrogen), TAMRA® or dansyl chloride), VIVOTAG 680 XL FLUOROCHROME™ (Perkin Elmer), phycoerythrin; umbelliferone, lyssamine; cyanine; phycoerythrin, Texas red, BODIPY FL-SE® (Invitrogen) or equivalent, all of which are suitable for optical detection. Chemiluminescent labels (eg luminol, luciferase, luciferin and aequorin) can be used. Such diagnosis and detection can also be accomplished by linking a diagnostic molecule of the present invention to detectable substances, including, but not limited to, various enzymes, the enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, or complexes with prosthetic groups. such as, but not limited to, streptavidin/biotin and avidin/biotin.

Chemiluminescent labels (eg luminol, luciferase, luciferin and aequorin) can be used. Such diagnosis and detection can also be accomplished by linking a diagnostic molecule of the present invention to detectable substances, including, but not limited to, various enzymes, the enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, or complexes with prosthetic groups. such as, but not limited to, streptavidin/biotin and avidin/biotin. Paramagnetic labels can also be used and are preferably detected by positron emission tomography (PET) or single photon emission computed tomography (SPECT). Such paramagnetic labels include, without limitation, compounds containing paramagnetic ions of aluminum (Al), barium (Ba), calcium (Ca), cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd) , holmium (Ho), iridium (Ir), lithium (Li), magnesium (Mg), manganese (Mn), molybdenum (M), neodymium (Nd), osmium (Os), oxygen (O), palladium (Pd) , platinum (Pt), rhodium (Rh), ruthenium (Ru), samarium (Sm), sodium (Na), strontium (Sr), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti) , tungsten (W), and zirconium (Zi), and, in particular, Co ⁺² , CR ⁺² , Cr ⁺³ , Cu ⁺² , Fe ⁺² , Fe ⁺³ , Ga ⁺³ , Mn ⁺³ , Ni ⁺² , Ti ⁺³ , V ⁺³ and V+4, positron active metals using various types of positron emission tomography and non-radioactive paramagnetic metal ions.

Thus, in one embodiment, an anti-tau antibody or tau-binding fragment thereof of the present invention may be labeled with a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, or an enzyme label. The labeled antibody, or fragment thereof, can be used to detect the presence or measure the amount of said tau protein in the subject's brain. The method may include detecting or measuring the amount of an anti-tau antibody or tau-binding fragment bound to said tau protein by in vivo imaging and may include ex vivo imaging of said anti-tau antibody or tau-binding fragment. a tau protein associated with such a tau protein.

In a further aspect, the present invention provides an expression vector encoding one or more polypeptide chains of an antibody of the present invention or a tau-binding fragment thereof. Such expression vectors can be used to obtain antibodies or their epitope-binding fragments of the present invention in a recombinant way.

The expression vector in the context of the present invention may be any suitable DNA or RNA vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (a nucleic acid sequence containing a suitable set of expression control elements). Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors resulting from the combination of plasmids and phage DNA, and vectors based on viral nucleic acid (RNA or DNA). In one embodiment, the nucleic acid encoding the anti-tau protein antibody is contained in a naked DNA or RNA vector, including, for example, a linear expression element (described, for example, in Sykes and Johnston, Nat Biotech 12, 355-59 (1997)) , a close-packed nucleic acid vector (described for example in US 6,077,835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18 or pUC 118/119, a nucleic acid-based midge vector (described, for example, in Schakowski et al., Mol Ther3, 793-800 (2001)), or presented as a precipitated vector nucleic acid construct, such as a CaPO ₄ precipitated construct (described, for example, in WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 2, 603 (1981)). Such nucleic acid vectors and their use are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

In one embodiment, the vector is suitable for expressing anti-tau antibodies or epitope-binding fragments thereof of the present invention in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509 (1989), pET vectors (Novagen, Madison, WI) etc.

The expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as the factor alpha, alcohol oxidase, and PGH gene promoter (reviewed in F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), Grant et al., Methods in Enzymol 153, 516-544 (1987), Mattanovich, D. et al. Methods Mol. Biol 824, 329-358 (2012), Celik, E. et al. , Biotechnol Adv 30(5), 1108-1118 (2012), Li, P. et al., Appl Biochem Biotechnol 142(2), 105-124 (2007), Boer 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 the expression vector of the present invention, nucleic acids encoding an anti-tau antibody may contain or be associated with any suitable promoter, enhancer, and other expression-enhancing elements. Examples of such elements include strong expression promoters (e.g. human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV and HIV LTR promoters), efficient poly(A) termination sequences, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker and/or a suitable cloning site (eg polylinker). Nucleic acids may also contain an inducible promoter rather than a constitutive promoter such as CMV IE (one of skill in the art will appreciate that such terms actually describe the degree of gene expression under certain conditions).

In another additional aspect, the present invention relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfectome, that produces an antibody or epitope-binding fragment of the present invention as defined herein, or a bespecifically molecule of the present 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 present invention provides a cell comprising a nucleic acid stably integrated into the cellular genome that contains a sequence encoding an expressible anti-tau antibody of the present invention or an epitope-binding fragment thereof. In another embodiment, the present invention provides a cell containing a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which contains a sequence encoding an expressible anti-tau antibody or epitope-binding fragment thereof of the present invention.

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

In one embodiment, the present invention relates to a formulation, as the term is used herein, that contains an anti-tau antibody as defined herein and that is substantially free of naturally occurring antibodies that are either not are able to bind to tau protein, or do not significantly change the functional characteristics of the anti-tau antibody in the preparation. Thus, such a formulation does not encompass naturally occurring serum or a purified derivative of such serum that contains a mixture of an anti-tau antibody and another antibody that does not alter the performance of the anti-tau antibody in the formulation, where such functional characteristics are (i) virtually no ability to bind to non-phosphorylated tau protein;

(ii) virtually no ability to bind to a tau protein phosphorylated at S404 and not phosphorylated at S396;

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

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

(v) the ability to selectively discriminate between the phosphorylated residues S396 and S404 of the tau protein so that they are substantially unable to bind to the phosphorylated residue 404;

(vi) the ability to bind to hyperphosphorylated tau protein from the brains of people with Alzheimer's disease;

(vii) the ability to discriminate between pathological and non-pathological human tau protein and/or (viii) the ability to specifically reduce the 64 and 70 kDa hyperphosphorylated tau protein bands by at least 90% without reducing the tau protein band 55 kDa

- 53 039569 more than 10% when used as described herein with immunodepleted extracts from rTg4510 transgenic mice, or the ability to specifically reduce S396 phosphorylated hyperphosphorylated tau bands by at least 90% without reducing non-hyperphosphorylated tau bands more than 10% when used as described herein with post-mortem extracts from the brains of people with AD.

The present invention specifically relates to formulations 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) from that of a naturally occurring anti-tau antibody. tau protein, where the specified structural change causes the antibody to tau protein to exhibit significantly altered functional characteristics (i.e., more than 20% difference, more than 40% difference, more than 60% difference, more than 80% more than 100% difference, more than 150% difference, more than 2 times difference, more than 4 times difference, more than 5 times difference, or more than 10 times difference in performance ) compared with the functional characteristics exhibited by the specified naturally occurring antibody to the tau protein; where said functional characteristics are:

(i) virtually no ability to bind to non-phosphorylated tau protein;

(ii) virtually no ability to bind to a tau protein phosphorylated at S404 and not phosphorylated at S396;

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

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

(v) the ability to selectively discriminate between the phosphorylated residues S396 and S404 of the tau protein so that they are substantially unable to bind to the phosphorylated residue 404;

(vi) the ability to bind to hyperphosphorylated tau protein from the brains of people with Alzheimer's disease;

(vii) the ability to discriminate between pathological and non-pathological human tau protein and/or (viii) the ability to specifically reduce the 64 and 70 kDa hyperphosphorylated tau protein bands by at least 90% without reducing the tau protein band 55 kDa greater than 10% when used as described herein with immunodepleted extracts from rTg4510 transgenic mice or the ability to specifically reduce S396 phosphorylated hyperphosphorylated tau bands by at least 90% without reducing non-hyperphosphorylated tau bands by more than than 10% when used as described herein with brain extracts obtained post-mortem from people with AD.

The term substantially free of naturally occurring antibodies refers to the complete absence of such naturally occurring antibodies in such preparations or to the inclusion of such naturally occurring antibodies in such preparations at a concentration at which they do not significantly affect tau-binding properties of these drugs. An antibody is said to be isolated if it has no naturally occurring equivalent or has been separated or purified from components that naturally accompany it.

The term naturally occurring antibodies, as used in relation to such preparations, refers to antibodies (including naturally occurring autoantibodies) that are produced in humans or other animals as a natural consequence of the functioning of their immune systems.

Thus, the formulations of the present invention do not exclude, but in fact explicitly encompass, formulations that contain an anti-tau antibody and a deliberately added additional antibody capable of binding to an epitope not contained in the tau protein. Such formulations specifically include embodiments thereof wherein the formulation exhibits improved efficacy in the treatment of Alzheimer's disease (AD), argyrophilic granule disease (AGD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Additionally, the present invention is directed to formulations that contain an anti-tau antibody or epitope-binding fragments thereof for use in the treatment of psychosis, in particular AD-related psychosis, or psychosis in patients with AD, apathy associated with AD, or apathy in patients with AD and psychiatric symptoms in patients with dementia with Lewy bodies. Additionally, the preparations of the present invention contain an anti-tau antibody or epitope-binding fragments thereof, which can be used in the treatment of stroke, stroke recovery, neurodegeneration associated with Parkinson's disease.

In another additional aspect, the present invention relates to a pharmaceutical composition comprising (i) an anti-tau protein antibody, or an epitope-binding fragment thereof, both of which are defined herein.

- 54 039569 nom document, or a preparation in the sense in which this term is defined in this document, which contains such an antibody to the tau protein or its epitope-binding fragment; and (ii) a pharmaceutically acceptable carrier.

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

Pharmaceutically acceptable carriers or diluents, as well as any other known excipients and excipients, should be suitable for the chosen compound of the present invention and the chosen route of administration. The suitability of carriers and other components of pharmaceutical compositions is determined based on the absence of a significant adverse effect on the desired biological properties of the selected compound or pharmaceutical composition of the present invention (for example, less than a significant effect (10% or less relative inhibition, 5% or less relative inhibition, etc.). n.)) for binding to the epitope.

The pharmaceutical composition of the present invention may also contain diluents, fillers, salts, buffers, detergents (for example, a nonionic detergent such as Tween-20 or Tween-80), stabilizers (for example, sugars or protein free amino acids), preservatives, tissue fixatives , solubilizers and/or other materials suitable for inclusion in the pharmaceutical composition. The diluent is chosen so that it does not affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate buffered saline, Ringer's solution, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may also contain other carriers or non-toxic, non-therapeutic, non-immunogenic stabilizers, and the like. The compositions may also contain large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids, and copolymers (e.g., latex-functionalized sepharose, agarose, cellulose, etc.), amino acid polymers, amino acid copolymers, and lipid aggregates (eg, oil droplets or liposomes).

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and route of administration. The selected dose level will depend on a number of pharmacokinetic factors, including the activity of the specific compositions of the present invention or their amide used, the route of administration, the time of administration, the rate of elimination of the particular compound used, the duration of treatment, other drugs, compounds and/or materials used. in combination with the specific compositions used, age, sex, weight, condition, general health and medical history of the patient being treated, as well as similar factors well known in the medical field.

The pharmaceutical composition can be administered by any suitable route and method, including parenteral, topical, oral or intranasal routes, for prophylactic and/or therapeutic treatment. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. As used herein, the phrases parenteral administration and parenterally administered means methods of administration other than enteral 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, epidural and intrasternal injection and infusion.

Additional suitable routes of administration of the compound of the present invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill in the art.

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

Pharmaceutically acceptable carriers include any suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delay agents, and the like, which are physiologically compatible with the compound of the present invention.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and their suitable blends, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil and sesame oil, carboxymethyl cellulose colloids, gum tragacanth and injectable organic esters such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical field.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the individual preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known from the prior art. Unless any conventional media or agent is incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated.

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

The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, for example (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The pharmaceutical compositions of the present invention may also contain isotonic agents such as sugars, polyhydric alcohols such as mannitol, sorbitol, glycerin, or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may also contain one or more excipients suitable for the chosen route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention may be formulated with carriers that will protect the compound from rapid release, such as in controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or together with wax, or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of the present invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the individual preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known from the prior art. Unless any conventional media or agent is incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated. Additional active compounds may also be included in the compositions.

Pharmaceutical compositions for injection should generally be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier may be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and suitable injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by applying a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. In many cases it will be preferable to include isotonicizing agents, eg sugars, polyhydric alcohols such as glycerol, mannitol, sorbitol, or sodium chloride, in the composition. Long-term absorption of injectable compositions can be achieved by including in the composition an agent that slows down the absorption of the antibody, for example, monostearate salts and gelatin. Sterile injectable solutions can be obtained by placing the active compound in the required amount in an appropriate solvent with one ingredient or combination of ingredients, for example, those listed above, if necessary, followed by sterilizing microfiltration. Typically, dispersions are prepared by placing the active compound in a sterile carrier medium which contains the basic dispersion medium and other necessary ingredients, for example, from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization), which results in a powdered active ingredient plus any

- 56 039569 additional desired ingredient from their solution, previously subjected to sterilizing filtration.

Sterile injectable solutions can be obtained by placing the active compound in the required amount in the appropriate solvent with one ingredient or combination of ingredients listed above, if necessary, followed by sterilizing microfiltration. Generally, dispersions are prepared by placing the active compound in a sterile carrier medium which contains the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization), which results in a powdered active ingredient plus any additional required ingredient from their solution, previously subjected to sterilizing filtration.

Dosing regimens in the above treatments and routes of administration described herein are adjusted to obtain the optimal desired response (eg, therapeutic response). For example, a single bolus dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as determined by the needs of the therapeutic situation. Compositions for parenteral administration may be formulated in unit dosage form for ease of administration and uniformity of dosing. Unit dosage form, as used herein, refers to physically discrete units suitable as unit doses for subjects to be treated; wherein each unit contains a predetermined amount of active compound, calculated to obtain the desired therapeutic effect, together with the necessary pharmaceutical carrier. The technical requirements for unit dosage forms of the present invention are dictated by (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the field of formulation of such an active compound for the treatment of sensitivity in individuals, and are directly dependent on them.

Effective doses and dosing regimens of the antibodies or epitope-binding fragments thereof of the present invention depend on the disease or condition being treated and can be determined by those skilled in the art. On any given day that the dose is given, the dose may be in the range of from about 0.0001 to about 100 mg/kg, and more typically from about 0.01 to about 5 mg/kg of the recipient's body weight. For example, dosages may be 1 mg/kg body weight or 10 mg/kg body weight, or in the range of 1-10 mg/kg body weight. Thus, exemplary dosages include about 0.1 to about 10 mg/kg/body weight, about 0.1 to about 5 mg/kg/body weight, about 0.1 to about 2 mg/kg/body weight , from about 0.1 to about 1 mg/kg/body weight, for example, 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 of ordinary skill in the art can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician may start with doses of an antibody or epitope-binding fragment of the present invention used in a pharmaceutical composition at lower levels than required to achieve the desired therapeutic effect and gradually increase the dose 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 usually depend on the factors described above. Administration may be, for example, intravenous, intramuscular, intraperitoneal or subcutaneous. If desired, the effective daily dose of the pharmaceutical composition may be administered as two, three, four, five, six or more dose portions administered separately at appropriate intervals throughout the day, optionally as unit dosage forms. Although the compound of the present invention can be administered alone, it is preferable to administer the compound as a pharmaceutical composition as described above.

The labeled antibodies or epitope-binding fragments thereof of the present invention can be used for diagnostic purposes to detect, diagnose or monitor diseases or disorders. The present invention provides for the detection or diagnosis of a neurodegenerative or cognitive disease or disorder, including, but not limited to, Alzheimer's disease, argyrophilic granule disease (AGD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD), including: (a) assaying for the presence of pyroglutamylated Ae fragments in cells or tissue samples of the subject using one or more antibodies that specifically bind to the tau protein; and (b) comparing the antigen level to a control level, such as levels in normal tissue samples, wherein an increase in the analyzed antigen level relative to the control antigen level is indicative of the disease or disorder, or indicative of the severity of the disease or disorder.

The antibodies or epitope-binding fragments thereof of the present invention can be used to analyze tau protein or tau protein fragments in a biological sample using immunohistochemical methods well known in the art. Other antibody methods useful for protein detection include immunoassays such as enzyme immunoassay (ELISA) and radioimmunoassay (RIA), as well as analyzes using the Meso Scale Discovery (MSD) platform. Suitable antibody labels can 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 ( ¹²⁵ I, ¹³¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In) and technetium ( ^99t Ts); as well as luminescent labels such as luminol and luciferase; and fluorescent labels such as fluorescein and rhodamine.

The presence of labeled anti-tau antibodies or tau-binding fragments thereof can be detected in vivo for diagnostic purposes. In one embodiment, the diagnosis involves a) administering to a subject an effective amount of such a labeled molecule; b) waiting for a period of time after administration to ensure that the labeled molecule is concentrated at the sites of Ae deposition (if any) and to allow clearing of unbound labeled molecule to the background level; c) determining a background level; and d) detecting a labeled molecule in a subject such that detection of a labeled molecule above background levels is indicative of the subject having a disease or disorder, or indicative of the severity of the disease or disorder. According to such an embodiment, the molecule is labeled with an imaging component suitable for detection with a particular imaging system known to those skilled in the art. Background levels can be determined using various methods known in the art, including by comparing the amount of labeled antibody detected with a standard value predefined for a particular imaging system. Methods and systems that can be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole body scanning such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound.

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

In a further aspect, the present invention provides a monoclonal antibody, or an epitope-binding fragment thereof, as defined herein, for use in the treatment, diagnosis, or imaging of taupathies.

In a further aspect, the present invention provides a monoclonal antibody, or an epitope-binding fragment thereof, as defined herein, for use in the treatment of Alzheimer's disease, argyrophilic granule disease (AGD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD).

In a further aspect, the present invention provides a monoclonal antibody, or epitope-binding fragment thereof, as defined herein, for use in the manufacture of a medicament for the treatment, diagnosis, or imaging of taupathies.

The drug is preferably for the treatment of Alzheimer's disease (AD), argyrophilic granule disease (AGD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), most preferably Alzheimer's disease (AD). The drug is also preferably for the treatment of psychosis, in particular AD-related psychosis or psychosis in AD patients, AD-related apathy or apathy in AD patients, and psychiatric symptoms in patients with Lewy body dementia.

In a further aspect, the present invention provides a method for treating, diagnosing, or imaging Alzheimer's disease or other taupathies in a subject, said method comprising administering a drug based on a monoclonal antibody or epitope-binding fragment thereof, as defined herein, to said subject in an effective amount.

In a preferred embodiment, the treatment is long term and preferably continues for at least 2 weeks, such as for at least 1 month, 6 months, 1 year or longer.

In a further aspect, the present invention provides a kit containing an antibody or fragment thereof as defined herein for use in therapy.

List of Embodiments

1. A monoclonal antibody or epitope-binding fragment thereof capable of specifically binding to the phosphorylated residue 396 of the human tau protein (SEQ ID NO: 1) such that the antibody or epitope-binding fragment thereof does not substantially bind to SEQ ID NO: 1 phosphorylated at residue 404 , in case residue 396 is not phosphorylated.

2. A monoclonal antibody or epitope-binding fragment thereof, according to an embodiment

- 58 039569 1 that inhibit P3 from AD subjects in a liquid phase inhibition assay such that the monoclonal antibody or epitope-binding fragment thereof is characterized

an IC ₅₀ of 100 nM or less, such as 0.1 nM to 100 nM, such as a concentration of 50 nM or less, such as 0.1 nM to 50 nM.

3. A monoclonal antibody or epitope-binding fragment thereof according to any one of embodiments 1 or 2, wherein they are capable of removing at least 15% of serine-396 phosphorylated tau protein from brain homogenates from Alzheimer's disease subjects when containing the antibody, approximately 75 ng as determined by the tau protein pS396 signal in Western blotting after immunodepletion studies of brain extracts from subjects with Alzheimer's disease.

4. Monoclonal antibody or its epitope-binding fragment, containing:

(a) a light chain CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 40 and SEQ ID NO: 46;

(b) a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 4; SEQ ID NO: 41 and SEQ ID NO: 47;

(c) a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 5; SEQ ID NO: 42 and SEQ ID NO: 48;

(d) a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 6; SEQ ID NO: 43; SEQ ID NO: 49; SEQ ID NO: 52 and SEQ ID NO: 55;

(e) a heavy chain CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 7; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 44; SEQ ID NO: 50; SEQ ID NO: 53 and SEQ ID NO: 56; and (f) a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 39; SEQ ID NO: 45; SEQ ID NO: 51; SEQ ID NO: 54 and SEQ ID NO: 57.

5. Monoclonal antibody or its epitope-binding fragment, containing:

(a) a light chain selected from the group consisting of SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22 and SEQ ID NO: 23; and (b) a heavy chain selected from the group consisting of SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26 and SEQ ID NO: 27.

6. Monoclonal antibody or epitope-binding fragment according to embodiments 1 or 5, where:

(a) the light chain is SEQ ID NO: 12; and (b) the heavy chain is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

7. Monoclonal antibody or epitope-binding fragment according to embodiments 1 or 5, where:

(a) the light chain is selected from the group consisting of SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22 and SEQ ID NO: 23; and (b) the heavy chain is SEQ ID NO: 11.

8. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

9. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 13.

10. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

- 59 039569

11. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(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: 8.

12. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 14.

13. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(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: 8.

14. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 30; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

15. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 15.

16. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 30; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

17. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

18. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 16; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

19. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and

- 60 039569 (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

20. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

21. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 17; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

22. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

23. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 33;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

24. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 18; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

25. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 33;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

26. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

27. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 19; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

28. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

- 61 039569 (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

29. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 35;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

30. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 20; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

31. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 35;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

32. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 36;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

33. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 21;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

34. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 36;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

35. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 37;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

36. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 22; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

37. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 37;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of

- 62 039569 (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

38. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

39. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 23; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 11.

40. A monoclonal antibody or epitope-binding fragment thereof according to embodiment 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of:

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

41. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

42. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

43. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising at least one of:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

44. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

45. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 16;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

46. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39; and further comprising at least one of:

- 63 039569 (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of:

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7.

47. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

48. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 17; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

49. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39; and further comprising at least one of:

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of:

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7.

50. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

51. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 16;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 13.

52. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28;

and further comprising at least one of:

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of:

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

53. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

54. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 17;

(b) a heavy chain containing the amino acid sequence of SEQ ID NO: 13.

55. A monoclonal antibody or epitope-binding fragment thereof, according to embodiments

- 64 039569 statements 1 or 4, containing:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 32; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and further comprising at least one of (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of:

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 8.

56. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 33;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

57. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 18; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

58. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 33; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39; and further comprising at least one of:

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of:

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; and (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7.

59. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

60. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 19; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24.

61. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 34; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39;

and further comprising at least one of (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising at least one of (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) A heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7.

62. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

63. A monoclonal antibody or epitope-binding fragment thereof, according to embodiments

- 65 039569 statements 1 or 5, containing:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 25.

64. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

65. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(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: 39.

66. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 26.

67. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(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: 39.

68. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4;

(c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

(d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 30; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

69. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 5, comprising:

(a) a light chain containing the amino acid sequence of SEQ ID NO: 12; and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 27.

70. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1 or 4, comprising:

(a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 3;

(b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5;

and further comprising (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6;

(e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 30; and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39.

71. A monoclonal antibody or epitope-binding fragment thereof according to any one of embodiments 1-5, characterized by selectivity for a hyperphosphorylated tau protein amino acid motif, wherein the motif contains a phosphorylated serine residue and a tyrosine residue located through one residue.

72. A monoclonal antibody or epitope-binding fragment thereof according to embodiment 71, wherein the amino acid motif has the sequence:

-Y -X-S(phosphorylated)-P- where Y is a tyrosine, X is a naturally occurring amino acid,

- 66 039569

P is a proline, and S(phosphorylated) is a serine with a phosphorylated side chain hydroxyl group.

73. A monoclonal antibody or epitope-binding fragment thereof, according to any of the previous embodiments, comprising an Fc region.

74. A monoclonal antibody or epitope-binding fragment thereof according to any of the previous embodiments, further comprising a component to increase the in vivo half-life of the agent.

75. A monoclonal antibody or epitope-binding fragment thereof according to any of the previous embodiments, characterized by specific binding to a human tau protein containing a phosphorylated residue 396, according to the following test criteria: i) the antibody does not substantially bind to non-phosphorylated tau protein; ii) the antibody does not substantially bind to a tau protein phosphorylated at 404 unless it is also phosphorylated at 396; iii) the antibody binds to a tau protein phosphorylated at 396; and iv) the antibody binds to the tau protein if both 396 and 404 are phosphorylated.

76. A monoclonal antibody or epitope-binding fragment thereof according to any of the previous embodiments, the formation of which occurs in response to a diphosphorylated peptide containing at least 18 consecutive amino acid residues, such as at least 20 consecutive amino acid residues, within TDHGAEIVYK ^ip} SPWSGDT ^ip} SPRHL (SEQ ID NO: 2) encompassing residues 386-408 of the 2N4R tau protein.

77. A monoclonal antibody or epitope-binding fragment thereof according to embodiment 76, the formation of which occurs in response to a diphosphorylated peptide containing 18-40, such as 18-30, such as 20-30, consecutive amino acid residues containing TDHGAEIVYK ^{ip }} SPWSGDT ^ip} SPRHL (SEQ ID NO: 2) encompassing residues 386-410 of the 2N4R tau protein.

78. A monoclonal antibody or epitope-binding fragment thereof according to any one of embodiments 1-70, having specificity for phosphorylated tau protein (pTau) from patients affected by AD compared to healthy controls in the appropriate age group, such that said monoclonal antibody or an epitope-binding fragment thereof is characterized by a difference in specificity for phosphorylated tau protein (pTau) from patients affected by AD compared to tau from healthy controls in the corresponding age group, representing more than 50-fold, as, for example, greater than 100-fold increase in specificity for AD-affected material compared to healthy controls in an ELISA-based assay for the detection of phosphorylated tau (pTau) in brain homogenates from AD and healthy controls using a specific scheme 1 E LISA for phosphorylated and multimeric molecules.

79. The monoclonal antibody or epitope-binding fragment thereof according to embodiment 78, having specificity for tau protein from AD-affected material, such that said monoclonal antibody or epitope-binding fragment thereof has a difference in specificity for AD-affected material compared to the material from healthy control subjects in the appropriate age group, representing more than 50-fold, such as more than 100-fold, increase in specificity for AD-affected material compared to material from healthy controls in an ELISA-based assay to detect phosphorylated tau protein (pTau) in brain homogenates from subjects with AD and from healthy controls using a specific ELISA scheme 1 for phosphorylated and multimeric molecules.

80. A monoclonal antibody or epitope-binding fragment thereof according to any of embodiments 1-70, which is formed in response to a diphosphorylated peptide:

TDHGAEIVYK ^ip} SPWSGDT ^ip} SPRHL (SEQ ID NO: 2) encompassing residues 386-410 of the 2N4R tau protein, or an epitope-binding fragment thereof, capable of specifically binding to phosphorylated residue 396 of the human tau protein.

81. An antibody or epitope-binding fragment thereof according to any one of embodiments 1-70 that has been generated or produced in a cell line such as a human cell line, a non-human mammalian cell line, an insect cell line, a yeast or a bacterium, or by recombinant technology.

82. An antibody or epitope-binding fragment thereof according to embodiment 81, produced in a CHO cell line, a HEK cell line, a BHK-21 cell line, a mouse cell line (such as a myeloma cell line), a fibrosarcoma cell line, a PER.C6 cell line, a HKB-11 cells, CAP cell lines, and HuH-7 human cell lines.

83. The monoclonal antibody or epitope-binding fragment thereof according to any one of embodiments 1-70, wherein said monoclonal antibody is expressed by a hybridoma that has been isolated by hybridoma screening using human pathological and non-pathological tau protein to isolate clones that both i) are specific to phospho-

- 67 039569 rylated epitopes of S396, and ii) specifically recognize hyperphosphorylated tau protein from the brain of people with Alzheimer's disease, where these antibodies or their epitope-binding fragments are able to distinguish between pathological and non-pathological human tau protein.

84. A monoclonal antibody or epitope-binding fragment thereof according to any one of embodiments 1-70, wherein the antibody or epitope-binding fragment thereof further comprises a detectable component.

85. The monoclonal antibody or epitope-binding fragment thereof according to embodiment 84, wherein the detectable fragment is a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, or an enzyme label.

86. A formulation comprising an antibody or epitope-binding fragment thereof according to any one of embodiments 1-70, wherein said formulation is substantially free of naturally occurring antibodies that are either unable to bind to the tau protein or do not significantly alter the functional characteristics of the antibody to the tau protein in the preparation, where the specified functional characteristics are selected from the group consisting of:

(i) virtually no ability to bind to non-phosphorylated tau protein;

(ii) virtually no ability to bind to a tau protein phosphorylated at S404 and not phosphorylated at S396;

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

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

(v) the ability to selectively discriminate between phosphorylated residues S396 and S404 of the tau protein so that they are substantially unable to bind to phosphorylated residue 404 or so that they preferentially bind to S396;

(vi) the ability to bind to hyperphosphorylated tau protein from the brains of people with Alzheimer's disease;

(vii) the ability to discriminate between pathological and non-pathological human tau protein and/or (viii) the ability to specifically reduce the 64 and 70 kDa hyperphosphorylated tau protein bands by at least 90% without reducing the tau protein band 55 kDa by more than 10% when used as described in the examples with immunodepleted extracts from rTg4510 transgenic mice.

87. A formulation comprising an antibody or epitope-binding fragment thereof according to embodiments 1-70, wherein said antibody or said epitope-binding fragment thereof has a structural change in its amino acid sequence compared to the structure of a naturally occurring anti-tau protein antibody, where said structural change causes said antibody or fragment showing altered functional characteristics compared to those exhibited by said naturally occurring anti-tau antibody, wherein said functional characteristics are selected from the group consisting of:

(i) virtually no ability to bind to non-phosphorylated tau protein;

(ii) virtually no ability to bind to a tau protein phosphorylated at S404 and not phosphorylated at S396;

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

(iv) the ability to bind to a tau protein phosphorylated in both S396 and S404;

(v) the ability to selectively discriminate between phosphorylated residues S396 and S404 of the tau protein so that they are substantially unable to bind to phosphorylated residue 404 or so that they preferentially bind to S396;

(vi) the ability to bind to hyperphosphorylated tau protein from the brains of people with Alzheimer's disease;

(vii) the ability to discriminate between pathological and non-pathological human tau protein and/or (viii) the ability to specifically reduce the 64 and 70 kDa hyperphosphorylated tau protein bands by at least 90% without reducing the tau protein band 55 kDa by more than 10% when used as described herein with immunodepleted extracts from rTg4510 transgenic mice.

88. A pharmaceutical composition comprising a monoclonal antibody or epitope-binding fragment thereof according to any of embodiments 1-70, or a preparation according to any of embodiments 86-87, and a pharmaceutically acceptable carrier.

89. Nucleic acid encoding a monoclonal antibody or epitope-binding fragment according to any of embodiments 1-70 or encoding a chain that is their component.

90. A monoclonal antibody or epitope-binding fragment thereof according to any of embodiments 1-70, or a preparation according to any of embodiments 86-87, or pharmaceutical

- 68 039569 ceutical composition according to embodiment 88 for use in therapy.

91. A monoclonal antibody or epitope-binding fragment thereof according to any of embodiments 1-70, or a preparation according to any of embodiments 86-87, or a pharmaceutical composition according to embodiment 88 for use in the treatment, diagnosis, or imaging of tauopathy.

92. A monoclonal antibody or epitope-binding fragment thereof according to any of embodiments 1-70, or a preparation according to any of embodiments 86-87, or a pharmaceutical composition according to embodiment 88 for use in the treatment of tauopathy selected from the group consisting of Alzheimer's disease, argyrophilic granule disease (AGD), psychosis, in particular AD-related psychosis or psychosis in AD patients, AD-related apathy or apathy in AD patients, psychiatric symptoms in patients with dementia with Lewy bodies, progressive supranuclear palsy (PSP ), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic forms), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART), senile dementia with a predominance of neurofibrillary tangles, dementia boxers, chronic traumatic encephalopathy, stroke, stroke recovery, neurod degeneration associated with Parkinson's disease, chromosome-linked parkinsonism, Lytico-Bodiga disease (Guam complex parkinsonism dementia), ganglioglioma and gangliocytoma, meningoangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, and lipofuscinosis.

93. A monoclonal antibody or epitope-binding fragment thereof according to any one of embodiments 1-70 for use in the treatment of Alzheimer's disease.

94. A monoclonal antibody or epitope-binding fragment thereof according to any of embodiments 1-70, or a preparation according to any of embodiments 86-87, or a pharmaceutical composition according to embodiment 88 for use in the manufacture of a medicament for the treatment, diagnosis, or imaging of tauopathy.

95. A monoclonal antibody or epitope-binding fragment thereof according to any one of embodiments 1-70, or a drug according to any one of embodiments 86-87, or a pharmaceutical composition according to embodiment 88 for use as a drug for the treatment of a disease selected from the group consisting of from Alzheimer's disease, argyrophilic granule disease (AGD), psychosis, in particular AD-related psychosis or psychosis in AD patients, AD-related apathy or apathy in AD patients, psychiatric symptoms in patients with dementia with Lewy bodies, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic forms), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART), senile dementia with a predominance of neurofibrillary tangles, boxer dementia, chronic traumatic encephalopathy, insulin e.g. recovery from stroke, Parkinson's disease-associated neurodegeneration, chromosome-linked parkinsonism, Lytico-Bodiga disease (Guam's parkinsonism-dementia complex), ganglioglioma and gangliocytoma, meningoangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lipofuscinosis.

96. A method of treating, diagnosing, or imaging Alzheimer's disease or other taupathies in a subject, said method comprising administering to said subject a therapeutically effective amount of a monoclonal antibody or epitope-binding fragment thereof according to any one of embodiments 1-70, a drug according to any one of embodiments 8687, or pharmaceutical composition according to embodiment 88.

97. The method of embodiment 96 wherein the treatment is long term.

98. The method of embodiment 97 wherein the long-term treatment is continued for at least 2 weeks, such as for at least 1 month, for at least 6 months, or for at least 1 year.

99. The method according to any one of embodiments 96-98, wherein the subject is a human.

100. A kit containing an antibody or fragment thereof according to any of embodiments 1-70, a preparation according to any of embodiments 86-87, or a pharmaceutical composition according to embodiment 88, for use in therapy.

101. A monoclonal antibody or epitope-binding fragment thereof according to embodiments 1-70, or a preparation or pharmaceutical composition containing said antibody or fragment, for use in detecting the presence or measuring the amount of said tau protein in the brain of a subject.

102. The monoclonal antibody or epitope-binding fragment thereof, formulation or pharmaceutical composition of embodiment 88, wherein said detection or measurement comprises in vivo imaging of said anti-tau antibody bound to said tau protein.

- 69 039569

103. A monoclonal antibody or epitope-binding fragment thereof, preparation or pharmaceutical composition according to embodiments 99-100, wherein said detection or measurement comprises ex vivo imaging of said anti-tau antibody or said fragment thereof associated with said tau protein.

104. A method for removing at least 90% of a hyperphosphorylated tau protein from a coil, wherein said coil contains a hyperphosphorylated tau protein, wherein said method involves contacting the hyperphosphorylated tau protein with a monoclonal antibody or epitope-binding fragment thereof that is selective in against a tau protein having a phosphorylated residue of 396, and as defined in any of embodiments 1-70.

105. A method for delaying the progression of Alzheimer's disease in a patient, wherein said method involves reducing or attenuating the accumulation of pathological tau protein in said patient by administering a monoclonal antibody or epitope-binding fragment thereof, wherein said monoclonal antibody or epitope-binding fragment thereof is selective for tau- a protein having a phosphorylated residue 396 and defined in any of embodiments 1-70.

106. Pharmaceutical composition containing:

i) an anti-tau protein antibody according to embodiments 1-70, and in particular embodiments 17, 18, 44, or 45, and ii) a compound selected from the group consisting of:

a) a BACE inhibitor;

b) a compound useful in active or passive tau immunotherapy;

c) a compound useful in active or passive immunotherapy for an Ae peptide;

d) NMDA receptor antagonists;

e) an additional tau aggregation inhibitor;

e) an acetylcholinesterase inhibitor;

f) an antiepileptic agent;

g) an anti-inflammatory drug and

h) SSRI; and iii) one or more pharmaceutically acceptable excipients.

107. A method of treating Alzheimer's disease, reducing the progression of AD, or reducing the symptoms of AD, comprising therapy comprising administering i) an anti-tau antibody according to embodiments 1-70, and in particular embodiments 17, 18, 44, or 45, and ii) a compound selected from the group consisting of:

a) a BACE inhibitor;

b) a compound useful in active or passive tau immunotherapy;

c) a compound useful in active or passive immunotherapy for an Ae peptide;

d) NMDA receptor antagonists;

e) an additional inhibitor of tau aggregation;

e) an acetylcholinesterase inhibitor;

f) an antiepileptic agent;

g) an anti-inflammatory drug and

h) SSRI.

108. A kit comprising: i) a composition comprising an anti-tau antibody according to embodiments 1-70, and in particular embodiments 17, 18, 44, or 45, and ii) a composition comprising a compound selected from the group consisting of from:

a) a BACE inhibitor;

b) a compound useful in active or passive tau immunotherapy;

c) a compound useful in active or passive immunotherapy for an Ae peptide;

d) NMDA receptor antagonists;

e) an additional tau aggregation inhibitor;

f) an acetylcholinesterase inhibitor;

g) an antiepileptic agent;

h) an anti-inflammatory drug and

i) an antidepressant.

109. The composition of claim 106, the method of claim 107, the kit of claim 108, wherein said BACE1 inhibitor is a small molecule BACE I inhibitor selected from the group consisting of LY2886721, MK-8931, AZD3293, or E2609.

110. The composition according to item 106, the method according to item 107, the kit according to item 108, where the specified BACE1 inhibitor is represented by formula I

- 70 039569

where Ar is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, and wherein Ar is optionally substituted with one or more substituents selected from halo, CN, C1- _C6 alkyl, C2 -C ₆ alkenyl, C 2 -C ₆ alkynyl, C 1 -C ₆ fluoroalkyl or C 1 -C ₆ alkoxy;

R1 is one or more of hydrogen, halogen, C1- _C3 fluoroalkyl, or C1- _C3 alkyl, and

R2 is hydrogen or fluorine.

111. The composition, method or kit according to item 110, where the compound of formula I is selected from the group consisting of

(6S)-6-(5-Amino-2-fluorophenyl)-3-fluoro-3,6-bis(fluoromethyl)piperidine-2-thione.

112. The composition of claim 106, the method of claim 107, the kit of claim 108, wherein said BACE1 inhibitor is represented by formula II

Formula II wherein Ar is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, 1,2,4-triazolyl, thiophenyl, thiazolyl, oxazolyl, isoxazolyl, 1,3,4-thiadiazolyl, isothiazolyl, 1 ,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, furazanil and 1,2,4-thiadiazolyl, wherein Ar is optionally substituted with one or more of halogen, CN, C _g C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ fluoroalkyl or C ₁ -C ₆ alkoxy;

R ¹ is C ₁ -C ₃ alkyl or C ₁ -C ₃ fluoroalkyl;

R ² is hydrogen, halogen, C _g C ₃ fluoroalkyl or C _g C ₃ alkyl and

R ³ is C _g C ₃ alkyl.

- 71 039569

113. The composition of claim 106, the method of claim 107, the kit of claim 108, wherein said BACE1 inhibitor is selected from the group consisting of: N-(3-((2R,5S)-6-amino-3,3 ,5-trifluoro-2,5-dimethyl-2,3,4,5tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide; N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5- methoxypyrazine-2-carboxamide; N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)- 5 methoxypicolinamide; N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2yl)-4-fluorophenyl)-5- cyano-3-methylpicolinamide and N-(3-((2R,5R)-6-amino-3,3,5-trifluoro-2,5-dimethyl2,3,4,5-tetrahydropyridin-2-yl)-4 -fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide.

114. The composition of claim 106, the method of claim 107, the kit of claim 108, wherein said NMDA receptor antagonist is selected from the group consisting of memantine, namenda, namzaric (memantine/donepezil) and generic forms thereof.

115. The composition of claim 106, the method of claim 107, the kit of claim 108, wherein said acetylcholine transferase inhibitor is selected from the group consisting of donepezil, galantamine, and rivastigmine.

116. The composition of claim 106, the method of claim 107, the kit of claim 108, wherein said antidepressant is selected from the group consisting of escitalopram, sertraline, citalopram, paroxetine, fluoxetine, venflaxin, trazodone, mirtazapine, vortioxetine and their generic forms .

117. The composition of claim 106, the method of claim 107, the kit of claim 108 for use in the treatment of tauopathy selected from the group consisting of Alzheimer's disease, argyrophilic granule disease (AGD), psychosis, in particular psychosis due to AD, or psychosis in patients with AD, psychiatric symptoms in patients with Lewy body dementia, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic), corticobasal degeneration (CBD ), Pick's disease, primary age-related tauopathy (PART), tangle-predominant senile dementia, boxer's dementia, chronic traumatic encephalopathy, stroke, stroke recovery, Parkinson's disease-related neurodegeneration, chromosome-linked parkinsonism, Lytico-Bodiga disease ( guam complex parkinsonism-dementia), ganglioglioma and gangliocytoma, meningoangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lipofuscinosis.

118. The composition of claim 106, the method of claim 107, the kit of claim 108 for use in the manufacture of a medicament for the treatment, diagnosis or imaging of tauopathy.

119. The composition of claim 106, the method of claim 107, the kit of claim 108 for the treatment of Alzheimer's disease, argyrophilic granule disease (AGD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), AD psychosis, or psychosis in patients with AD; and psychiatric symptoms in patients with dementia with Lewy bodies.

Examples

Example 1 Immunization of mice with tau peptides phosphorylated at 396/404

C56/BL6 and FVB mice were immunized with 10 μg of phosphorylated taupeptide 386-408 (pS396/pS404) (SEQ ID NO: 2) conjugated to P30 formulated in adjuvant TiterMax.

The mice were females and males from the C56/BL6 and FVB strains. Mice aged 2-3 months were immunized with phosphorylated tau protein 386-408 conjugated to the P30 peptide epitope.

Immunogenic phosphorylated tau peptide 386-408 (pS396/pS404) conjugated to P30 was formulated in TiterMax (400 μg/ml peptide in 1:1 v:v) according to TiterMax supplier's protocol and mice were injected subcutaneously with 20 μg antigenic peptide (100 μl). Control mice were injected with adjuvant alone. All mice immunized with the peptide were boosted with 0.5 μg peptide/TiterMax (10 μg/ml peptide formulated as above and injected) at monthly intervals. Finally, mice were boosted with P30-conjugated phosphorylated tau 386-408 (pS396/pS404) without TiterMax 3 days before splenocyte harvest, followed by splenocyte fusion with SP-2 cells. The resulting primary hybridomas were selected for recloning cycles after they exhibited positive binding to phosphorylated tau protein 386-408 (pS396/pS404) as detected by ELISA and exhibited preferential binding activity to S1 and P3 antigens from brain lysates. from subjects with AD and TG4510 (as described in Example 2B below). Such binding was compared to the binding activity of such antibodies observed in brain lysates from control subjects using dot blot assays and brain lysate coated ELISA or MSD plates.

Example 2A. Obtaining a hybridoma

Mice were boosted with P30-conjugated phosphorylated tau 386-408 (pS396/pS404) without TiterMax 3 days prior to spleen harvest from responder mice, followed by splenocyte fusion with SP-2 cells. Hybridomas were selected for recloning cycles after ELISA showed positive binding to phosphorylated

- 72 039569 tau protein 386-408 (pS396/pS404) and manifestations of preferential antigen binding activity

S1 and P3 from brain lysate from subjects with AD and TG4510 compared to brain lysate from control subjects by dot blot and brain lysate coated ELISA or MSD plates.

Example 2B. Western blot analysis and dot blot analysis of specific antibodies

Biochemical fractionation of tau protein

Human or mouse rTg4510 brain tissue overexpressing human tau with the P301L mutation was homogenized in 10 volumes of Tris buffered saline containing protease and phosphatase inhibitors as follows: 50 mM Tris/HCl (pH 7.4); 274 mM NaCl; 5 mM KCl; 1% mixture of protease inhibitors (Roche); 1% mixture of phosphatase inhibitors I and II (Sigma) and 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma). Homogenates were centrifuged at 27000xg for 20 min at 4°C to obtain fractions of the supernatant (S1) and sediment. Sediment samples were re-homogenized in 5 volumes of high salt/sucrose buffer (0.8 M NaCl, 10% sucrose, 10 mM Tris/HCl, [pH 7.4], 1 mM EGTA, 1 mM PMSF) and centrifuged as described. above. Supernatant samples were collected and incubated with sarcosyl (final concentration 1%; Sigma) for one hour at 37°C followed by centrifugation at 150,000xg for one hour at 4°C to obtain samples of the sarcosyl-insoluble precipitate, herein referred to as the P3 fraction. . The P3 pellet was resuspended in TE buffer (10 mM Tris/HCl [pH 8.0], 1 mM EDTA) to a volume equal to half the original volume used for brain homogenates.

Western blot and dot blot analyzes

Fractionated tissue extracts S1 and P3 were dissolved in SDS sample buffer containing 0.1 M DTT. Heat-treated samples (95°C for 10 min) were separated by gel electrophoresis on 4-12% Bis-Tris SDS-PAGE gels (Invitrogen) and transferred to PVDF membranes (BioRad Laboratories, Hercules, CA) . Dot blot samples were spotted directly onto nitrocellulose membranes (Amersham, Pittsburgh, PA) at known concentrations for all samples. Both western and dot blot membranes were blocked in 5% skimmed milk powder in TBS-Tween (0.5%) pH 7.4 followed by incubation with 1 μg/ml C10-2 overnight at 4°C. The 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). Western blot and dot blot immunoreactivity was quantified and visualized using a LAS-4000 bioimaging analytical system (Fujifilm, Tokyo, Japan) connected to a computer and Multi Gauge v3.1 software (Fujifilm). The protein load was corrected for the volume of the original fractions and can be converted to the original raw tissue weight.

Example 3 Liquid Phase P3 Antigen Uptake Inhibition Assay from Material from AD Subjects

Example 3A.

Objective: To quantify the inhibition of pS396 tau antigen binding from P3 brain material from murine C10.2 AD subjects with human C10-2 and mutated variants. MSD plates coated with mouse C10-2 prior to incubation with P3 from AD subjects or P3 from AD subjects were preincubated with the C10.2 variants of interest. The degree of inhibition was presented as IC ₅₀ values reflecting the apparent binding affinity of the antibody in the liquid phase to antigens. IC ₅₀ values were obtained by fitting to a one-site or two-site binding model using Graph Pad Prism software. A negative control antibody (mouse C10-1) reactive for P(404) tau protein was added for comparison (data not shown).

Way. MSD plates were coated with capture antibody (750 ng/ml mouse C10-2 in carbonate buffer, pH 8.5) overnight at room temperature followed by blocking (30 min in PBS, 3% BSA, 0.1% NP40) and Washing 5 times (PbS, 0.1% BSA, 0.1% NP40). Antibodies at a stepwise concentration (0-1000 nM) were incubated for 60 min with P3 material from AD subjects at room temperature and then incubated for 1 h at room temperature in mouse C10-2 coated MSD plates as described above. The plates were washed 5 times (PBS, 0.1% BSA, 0-.1% NP40) and an anti-total tau antibody (MSD labeled with SULFO-TAG, 1:50) was added to detect captured tau that reflected unexposed inhibition of free tau antigens.

Results. The data showed dose-dependent inhibition of tau uptake by human C10-2 and variants (FIG. 1). Variants C10-2_N32S and C10-2_N32S:A101T showed more pronounced inhibition (IC ₅₀ = 44 and 14 nM, respectively, corresponding to the single-site binding model), while C10-2 was characterized by heterogeneous inhibition, which was reflected by the best fit of the two-site binding model (IC ₅₀ 14 nM/630 nM). Low affinity binding (IC ₅₀ = 630) was predominant because high affinity binding of the antibody (IC ₅₀ = 14 nM) was less than 25% of the total binding. The results are shown in FIG. one.

Example 3B.

Objective: To quantify the binding inhibition of pS396 tau peptides 386-408 by human C10-2 and mutated variants in a liquid phase inhibition assay. The degree of inhibition was presented as IC ₅₀ values reflecting the apparent binding affinity of the antibody. IC ₅₀ values were obtained by fitting to a one-site or two-site binding model using Graph Pad Prism software. A negative control antibody (mouse C10-1) reactive for P(404) tau protein was added for comparison (data not shown).

Way. MSD plates were coated with pS396 tau peptide 386-408 in carbonate buffer, pH 9.5 overnight at room temperature followed by blocking (30 min in PBS, 3% BSA, 0.1% NP40) and washing 5 times (PBS, 0.1% BSA, 0.1% NP40). Tau protein 386-408 pS396 at a stepped concentration (0-1000 nM) was incubated for 60 min with 1 ng/ml antibody at room temperature and then incubated for 1 h at room temperature in MSD plates coated with tau protein 386 -408 pS396 as described above. Plates were washed 5 times (PBS, 0.1% BSA, 0.1% NP40) and anti-total tau (MSD labeled with SULFO-TAG, 1:50) was added to detect bound antibody that reflected uninhibited free antibody . The results are shown in FIG. 2.

Example 4 Immunohistochemical Profiling of Antibodies

fabrics

Mouse. Mouse brain tissues were collected from 8 month old rTg4510 mice. These transgenic mice express human tau mutant (P301L 0N4R) under control of the Tet-Off response element in CamK2 positive neurons and are characterized by marked tau hyperphosphorylation and tangle formation from 6 months of age onwards. Non-transgenic littermates acted as controls. Mouse brain samples were fixed by immersion in 4% paraformaldehyde and embedded in paraffin.

Human. Formalin-fixed and paraffin-embedded human frontal cortex samples were purchased from Tissue Solutions (Glasgow, UK). Tissue from 3 donors diagnosed with end-stage Alzheimer's disease (AD; Braak stage V-VI) were compared with tissue from a non-demented control donor in the appropriate age group.

Immunohistochemical study

Four μm thick sections from mouse and human tissue were cut on a microtome, deparaffinized, and subjected to antigen demasking by microwave treatment of the sections in 10 mM citrate buffer, pH 6, for 10 min. Endogenous peroxidase was blocked with 1% hydrogen peroxide followed by 5% normal porcine serum in PBS, 1% BSA, 0.3% Triton X-100 (PBS-BT). Sections were incubated overnight at 4°C with hC10-2, hC10-2_N32S and hC10-2_N32S_A101T antibodies diluted in PBS-BT over the concentration range indicated in FIG. 1. Sections were washed in PBS, 0.25% BSA, 0.1% Triton X-100 before incubation with biotinylated secondary porcine anti-human immunoglobulin antibody (#B1140; Sigma-Aldrich) at 1:200 for 1 h. a streptavidin-biotin complexation kit (Vector Laboratories, Burlingame, CA) was used in the washes and finally immunoreactivity was visualized with 0.05% diaminobenzidine. The sections were counterstained with hematoxylin to reveal the localization of the nuclei.

results

In 3 brain samples from subjects with AD hC10-2, hC10-2_N32S and hC10-2_N32S_A101T, structures corresponding to pathological tau protein (ie, tangles, neuropil filaments, dystrophic neurites) were labeled. The degree of immunoreactivity was concentration dependent. For example, no visible labeling of glial cells or vessels was detected. In sections from the control sample of the brain, immunoreactivity was not detected. Similarly, all 3 antibodies resulted in the putative pattern for phosphorylated tau in both the hippocampus and cortex from rTg4510 brain samples. No immunoreactivity was detected in brain sections from non-transgenic mice.

Example 5 Staining of structures containing tau protein in rTg4510 mice after i.v. injections

Way:

Ten-month-old mice rTg4510. These transgenic mice express human tau mutant (P301L 0N4R) under control of the Tet-Off response element in CamK2 positive neurons and are characterized by marked tau hyperphosphorylation and tangle formation from 6 months of age onwards. In addition, 10-month-old rTg4510 mice show neurodegeneration in areas of severe pathology. Single-gene transgenic tTA littermates acted as controls. Mice received a single tail vein injection of hC10-2, hC10-2_N32S, or hC10-2_N32S_A101T antibodies at a concentration of 80 mg/kg. Each mouse was

- 74 039569 injection in a volume of 150 µl. Three days after injection, mice were perfused for 2 min with PBS followed by perfusion for 10 min with 4% paraformaldehyde. Brain samples were cryoprotected in 30% sucrose and cut into free-floating cryosections 40 µm thick. Sections were incubated with 5% normal porcine serum in PBS/1% BSA/0.3% Triton X-100 for 20 min, washed in PBS, and finally incubated with AlexaFluor488-conjugated anti-human IgG secondary antibody at 1:200 (no. 709-545-149; Jackson ImmunoResearch Laboratories, West Grove, USA). Hoechst was used for nuclear staining. Sections were washed in PBS, mounted on a glass slide and examined by fluorescence microscopy.

results

I.v. injection of hC10-2, hC10-2_N32S, and hC10-2_N32S_A101T resulted in in vivo binding to target structures in the hippocampus and cortex from age-matched rTg4510 animal brain samples (FIGS. 4-7). The number of positive structures observed varied between individual rTg4510 animals. In tTA control mice, no specific fluorescent signals were detected after injection of any of the three antibodies (FIGS. 4-7).

As a negative control, injection of control human IgG produced no signals in rTg4510 mice (data not shown). The positive signals in rTg4510 brain samples were not easily identified as intracellular staining material and could represent extracellular tau protein material released during the neurodegeneration process. Taken together, these data suggest that hC10-2, hC10-2_N32S, and hC10-2_N32S_A101T antibodies are able to penetrate the brain parenchyma and specifically stain targets in rTg4510 mice in vivo.

Example 6 Characterization of Tau Immunoreactivity in Brain Samples from Subjects with Alzheimer's Disease

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Paraffin-embedded human frontal cortex samples were purchased from Tissue Solutions (Glasgow, UK). Tissues from donors diagnosed with end-stage Alzheimer's disease (AD; Braak stage V-VI) were included.

Immunohistochemical study

Four µm thick sections from human tissues were cut on a microtome, deparaffinized, and subjected to antigen demasking by microwave treatment of the sections in 10 mM citrate buffer, pH 6, for 10 min. Sections were incubated with 5% normal porcine serum in PBS, 1% BSA, 0.3% Triton X-100 (PBS-BT) followed by overnight incubation at 4°C with hC102 or hC10-2_N32S_A101T antibodies diluted in PBS -BT. Sections were washed in PBS, 0.25% BSA, 0.1% Triton X-100. Immunoreactivity was visualized with an AlexaFluor488-conjugated anti-human IgG secondary antibody (1:200; no. 709-545-149, Jackson ImmunoResearch Laboratories, West Grove, USA). In the case of dual immunofluorescence, sections were co-incubated with AT8 (1:500; no. MN1020, ThermoFisher, Waltman, USA) or anti-human total tau E1, a custom made rabbit antibody induced against the N-terminal fragment of tau 19 -33 (Crowe et al., 1991). AT8 and E1 immunoreactivities were visualized respectively with AlexaFluor568-conjugated anti-mouse antibody (1:400; No. A10037, ThermoFisher) and AlexaFluor568-conjugated anti-rabbit antibody (1:400; No. A10042, ThermoFisher). Sections were analyzed by fluorescence microscopy.

results

In AD sections double stained for the N-terminal fragment of total tau and pS396 tau, the population of tangled neurons was labeled with both E1 and hC10-2 or hC10-2_N32S_A101T antibodies (Fig. 7). A number of tau coils were labeled with hC10-2 antibodies only or with hC10-2_N32S_A101T antibodies (FIG. 7, arrows). It has previously been shown that extracellular tau (undetectable coils) is not stained by antibodies to the N-terminal tau fragment (e.g., Bondareff et al., 1990; Braak et al., 1994; Flores-Rodrigues et al., 2015). Thus, tau molecules labeled with hC10-2 or hC10-2_N32S_A101T antibodies alone are likely to represent extracellular undetectable tangles.

Western blot analyzes and immunoprecipitation Experimental procedure and description of the experiment

rTg4510 transgenic mice were used: the human tau protein cDNA with the P301L mutation (4R0N TauP301L) was placed downstream of the tetracycline responder operon (TRE) construct. To activate the transgene, the responder must be co-expressed with an activator construct consisting of a tetracycline conditional gene expression (tTA) system. The tTA activator system was placed downstream of the CaMKIIa promoter, thus restricting TRE expression mainly in forebrain structures. The tau transgene responder was expressed in the FVB/N mouse line (Taconic) and the tTA activator system was maintained in the 129S6 mouse line (Taconic). Their F1 offspring carried the responder and activator transgenes (rTg4510) along with non-transgenic (non-Tg) and single-gene transgenic littermates. Only F1 mice were used for experiments. All mice were mated at Taconic, Denmark and genotyped by tail DNA analysis using pairs of pry5'-GATTAACAGCGCATTAGAGCTG-3' and 5'mers GCATATGATCAATTCAAGGCCGATAAG-3' for the tTA activator transgene and

5-TGAACCAGGATGGCTGAGCC-3' and 5-TTGTCATCGCTTCCAGTCCCCG-3' for the tau mutant responder transgene. The mice were housed in groups and received ad libitum water and food (Brogaarden, Denmark) as well as supplemented substances. The light/dark cycle was 12 hours; room temperature was 21±2° C. and relative humidity was 55%±5%. The experiments were performed in accordance with the Danish legislation on experimental animals (license no. 2014-15-0201-00339).

Mice were sacrificed by displacing the cervical vertebrae in order to preserve the metabolic environment of the brain and prevent the appearance of artifacts that could change the biochemical profiles of the tau protein. Mouse brain samples were cut sagittally along the midline to obtain two hemispheres. The cerebral cortex and right hemisphere hippocampus from each animal were flash frozen on dry ice and stored at -80° C. until use. Frozen human cortex samples from age-matched Alzheimer's disease (AD) patients and healthy controls (HC) were purchased from Tissue Solution (Glasgow, UK). Human brain samples had a similar post-mortem processing time of <6 h and were characterized for amyloid and tau pathology, and selected samples from AD subjects were classified as Braak stages V-VI.

For immunoprecipitation of tau protein from brain lysates, a cross-link immunoprecipitation kit (Thermo Fisher Pierce 26147) was used according to the manufacturer's instructions. Briefly, the antibody was coupled to protein A/G together with agarose, followed by cross-linking of the bound antibody to DSS (disuccinimidyl suberate). Brain homogenate was prepared in Tris buffer (25 mM Tris/HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and a complete mixture of protease and phosphatase inhibitors) and pre-purified overnight at 4°C with control agarose resin. The pre-purified lysate was incubated with antibody cross-linked resin overnight at 4°C followed by eluting the antigen with 50 µl elution buffer (pH 2.8) and immediately centrifuged into sample collection tubes containing 5 µl 1 M Tris , pH 9.5. The immunoprecipitated tau protein was dissolved in SDS sample buffer containing dithiothreitol (DTT, 100 mM), heat treated (95° C. for 10 min) and subjected to Western blotting as described below.

Human tau protein concentrations were measured in brain homogenates and prepurified lysates by total human tau ELISA according to the manufacturer's instructions (Invitrogen).

Tissues were homogenized in 10 volumes of Tris buffered saline (TBS) containing protease and phosphatase inhibitors as follows: 50 mM Tris/HCl (pH 7.4); 274 mM NaCl; 5 mM KCl; 1% mixture of protease inhibitors (Roche); 1% mixture of phosphatase inhibitors I and II (Sigma) and 1 mM phenylmethylsulfonyl fluoride (PMSF).

Homogenates were centrifuged at 27000xg for 20 min at 4°C to obtain fractions of the supernatant (S1) and sediment. Sediment samples were re-homogenized in 5 volumes of high salt/sucrose buffer (0.8 M NaCl, 10% sucrose, 10 mM Tris/HCl, [pH 7.4], 1 mM EGTA, 1 mM PMSF) and centrifuged as described. above. Supernatant samples were collected and incubated with sarcosyl (final concentration 1%; Sigma) for one hour at 37°C followed by centrifugation at 150,000xg for 1 hour at 4°C to give saline and sarcosyl extractables (S3) and insoluble in sarcosyl (P3) fractions. The P3 pellet was resuspended in TE buffer (10 mM Tris/HCl [pH 8.0], 1 mM EDTA) to a volume equal to half the original volume used for brain homogenates. To enrich the S1 fractions with hyperphosphorylated tau protein molecules, part of the S1 fraction was separated by additional centrifugation at 150,000xg for 20 min to obtain supernatant (S1s) and pellet (S1p) fractions. The S1p pellet was resuspended in TBS buffer to a volume equal to one-fifth of the original volume of S1 used. Fractionated tissue extracts S1, S1p and P3 were dissolved in SDS sample buffer containing DTT (100 mM). Heat-treated samples (95°C for 10 min) were separated by gel electrophoresis on 4-12% Bis-Tris SDS-PAGE gels (Invitrogen) and transferred to PVDF membranes (BioRad Laboratories, Hercules, CA) . After blocking with a blocking solution containing 5% skim milk and 0.1% Trion-X100 in TBS, the membranes were incubated with 1 μg/ml hC10.2, hC10-2_N32S, hC10-2_N32S_A101T, or rabbit anti-tau antibody pS396 (Invitrogen) . The membranes were washed and incubated with peroxidase-conjugated anti-human IgG or anti-rabbit antibodies (1:5000; Jackson ImmunoResearch, West Grove, PA). Bound antibodies were detected using an enhanced chemiluminescence system (ECL PLUS kit; Perkin Elmer). Western blot immunoreactivity was quantified and visually analyzed using a LAS-4000 bioimaging analytical system (Fujifilm, Tokyo, Japan) connected to a computer and Multi Gauge v3.1 software.

- 76 039569 (Fujifilm). Approximately 2 μg S1 from mouse, 20 μg S1 from human brain samples, and equal volumes of different fractions (S1, S1p, and P3) were loaded for tau detection with SDS.

PAGE.

Detection of abnormal tau protein by Western blotting

Pooled brain homogenates from three 32-week-old rTg4510 mice and those from non-transgenic (non-Tg) control littermates and pooled cerebral cortex samples from four AD donors and those from four healthy control (HC) donors were separated into soluble (S1) a fraction, a fraction soluble in TBS precipitate (S1p) and insoluble in sarcosyl (P3) fraction. hC10.2, hC10-2_N32S, hC10-2_N32S_A101T were used at 1 μg/mL in Western blot and abnormal tau was detected in samples from rTg4510 mice and AD subjects. The present inventors observed detection of 55 and 64 kDa tau in S1 and 64 and 70 kDa tau in P3 and S1p fractions from 32 week old rTg4510 mice. In addition, three <50 kDa truncated tau bands were observed in the P3 fraction. No signal was detected in S1p and P3 from non-Tg control littermates not expressing the human transgenic tau protein. In the S1 fraction from non-Tg mice, a weak signal was detected, corresponding to a molecule of about 50 kDa, which most likely represented endogenous mouse tau protein phosphorylated at residue S396 (FIGS. 8A-8C). Summarizing, it can be noted that hC10.2, hC10-2_N32S, hC10-2_N32S_A101T ensured the detection of pS396 tau protein, as well as both normal phosphorylated 55 kDa tau molecules and hyperphosphorylated 64 kDa tau molecules in samples from rTg4510 mice. The strongest signal was observed in the case of hC10-2_N32S_A101T.

In fractions S1, S1p and P3 from donors with AD hC10.2, hC10-2_N32S, hC10-2_N32S_A101T provided the detection of a typical AD spot of tau protein and four patterns of pathological tau protein bands (tau protein sizes 54, 64, 69 and 74 kDa). As expected, sarcosyl-insoluble hyperphosphorylated tau molecules isolated from the P3 fraction were the most pronounced, followed by soluble hyperphosphorylated tau molecules enriched in the S1p fraction. No signal was detected in the P3 fractions from the healthy control (HC). Fractions S1 and S1p from HC showed a weak signal corresponding to a molecule of about 55 kDa, which probably represented a normal tau protein phosphorylated at residue S396 (FIGS. 8A-8C). In summary, hC10.2, hC10-2_N32S, hC10-2_N32S_A101T provided detection of the typical AD tau patch and four abnormal tau band patterns representing hyperphosphorylated tau. The strongest signal was observed in the case of hC10-2_N32S_A101T.

Immunoprecipitation of pathological tau protein

To determine the ability of hC10.2, hC10-2_N32S, hC10-2_N32S_A101T to bind to the tau protein under non-denaturing conditions, a tau protein immunoprecipitation (IP) protocol was established in which antibodies to the tau protein are covalently bound via cross-linking to the A/G protein of the resin. and thereby ensure the absence of antibody contamination at IP. In the case of tau protein analysis by SDS-PAGE, the presence of the heavy chains of the antibody used for IP can interfere with the signals, since both proteins are detected with a size of about 50 kDa. The effectiveness of hC10.2, hC10-2_N32S, hC10-2_N32S_A101T in reducing the content of pathological tau protein from the human brain was studied. As antigen, 500 μg of a prepurified lysate from brain homogenates pooled from four AD and HC donors containing 0.1 μg and 0.15 μg of human tau protein (determined by ELISA for human tau) was used as antigen. -squirrel). hC10.2, hC10-2_N32S, hC10-2_N32S_A101T (10 μg) provided a reduction in the content of 54, 64, 69 and 74 kDa tau molecules (four pathological tau protein bands) and a stain characteristic of AD from pre-purified homogenates from subjects with AD (antigen/Ab ratio 1:100) visualized with polyclonal rabbit anti-tau antibody pS396 (FIG. 9). Comparing the intensity of tau bands from brain samples from AD subjects, which was reduced by hC10.2, hC10-2_N32S, hC102_N32S_A101T, with that of a control anti-human IgG antibody and HC brain, it can be concluded that hC10. 2, hC10-2_N32S, hC10-2_N32S_A101T immunoprecipitated the pS396 hyperphosphorylated tau protein exclusively in brain samples from AD subjects and were effective at an antigen/antibody ratio of 1:100.

Cellular and aggregation analysis

HEK293 cells were transiently transfected with human tau-P301L-FLAG in 6-well plates 24 h after seeding, after which they were incubated 24 h later with brain homogenate for 24 h, followed by cell separation and subculture and harvest after an additional 24 h. lysed and sonicated in TBS supplemented with buffer containing 1% Triton X, Phos-stop and complete phosphatase and protease inhibitors (Roche) and ultracentrifuged at 100,000xg for 30 min. The pellet was resuspended in SDS, sonicated and ultracentrifuged for 30 min at 100,000xg. Supernatant samples were analyzed by Western blotting. Cells expressing human tau P301L showed insoluble (SDS fraction, E1/FLAG detection) hyperphosphorylated (pS396 detection) tau when primed with total brain homogenates from rTg4510 tau transgenic mice.

Cells treated with tTA mouse control brain homogenate showed no aggregated hyperphosphorylated human tau protein. Additionally, total cell lysates of HEK293 cells were analyzed using the tau protein aggregation assay from Cisbio. This assay is based on time resolved fluorescence using the same antibody as both the donor (Tb3+-conjugated) and acceptor (d2-conjugated) Ab in FRET. A 10 μl sample was mixed with a 10 μl antibody mixture and incubated for 20 h. The plate was read on a PHERAstar plate reader to evaluate time-resolved fluorescence (measured/integrated after excitation light on, the FRET signal). The assay measures the level of aggregated tau protein in both human sections and rTg4510 mice and primed HEK cells with high specificity and sensitivity. The results are shown in FIG. ten.

Example 7 Tau immunodepletion

Brain extracts from subjects with Alzheimer's disease were prepared from post-mortem frozen prefrontal cortex samples in 10x volume of sterile cold PBS. The tissue was homogenized using a bladed homogenizer followed by sonication at power output mode 2 with 5 x 0.9 s pulses (Branson sonicator). Then the homogenate was centrifuged at 3000 g for 5 min at 4°C. Supernatant samples were aliquoted, flash frozen and stored at -80° C. until use.

μg of antibody (humanized variants C10-2 and 2.10.3, mouse AT8, mn 1020 from Thermo Scientific) was immobilized on Dynabeads magnetic beads in 125 μl of suspension (Dynabeads immunoprecipitation kit with Novex protein G, Cat # 10007D). After thorough washing, the coated granules were mixed with varying amounts of uncoated washed granules. Started with 100% Ab coated beads, corresponding to 5 μg of antibody, decreasing to 100% uncoated beads. The total number of granules was the same in all samples. The beads were mixed with 20 µl of extract from AD subjects and incubated at room temperature for 10 minutes. The magnetic beads were separated from the extract and the extracts were aliquoted, flash frozen and stored at -80° C. until use.

Depletion analysis by Western blotting

Samples were boiled in 1x loading SDS buffer and 100 mM DTT. A volume corresponding to 3 μl of the extracts was loaded onto NuPAGE 4-12% Bis-Tris gel (LifeTech, Novex). After electrophoresis, the proteins were blotted onto an Immobilon-FL PVDF membrane (0.45 μm, IPFL10100, Millipore). The membrane was blocked with blocking buffer SEA (product no. 37527, Thermo). Tau and P-tau levels were assessed in samples using Tau5 (Tau5 is a commercially available anti-tau antibody whose epitope is described to be among amino acids 210-241 of the tau protein. It is a mouse monoclonal to tau- protein Abeam ab80579, 1:2000), mouse C10-2 (1 µg/ml), P-S199/202 (44768G, Invitrogen, 1:1000), P-S422 (ab79415, Abcam, 1:750), human IPN (1 µg/ml). Gapdh and actin were used as loading controls (ab9484, Abeam, 1:2000, A5441, Sigma, 1:20000). Fluorophore-conjugated IgG secondary antibodies (goat anti-human Ig conjugated with IRDye 800CW, goat anti-rabbit Ig conjugated with IRDye 800CW, goat anti-mouse Ig conjugated with IRDye 680, LI-COR Biosciences), and quantified with Odyssey CLx and Image Studio software (LI-COR Biosciences). Individual bands were quantified as well as the signal in whole lanes, and based on this, sigmoidal dose-response curves were built and, if possible, the maximum effect and EC ₅₀ values were estimated.

results

Both 2.10.3 and C10-2 antibodies removed a small fraction of the tau protein from a brain preparation from a subject with Alzheimer's disease. This indicates selectivity for a tau subpopulation in the total tau content. 2.10.3, designed to be specific for the tau protein pS422, removed no more than 24% of the total tau protein, while C10-2 removed no more than 15% of the total tau protein (see Fig. 11) . This can be interpreted to mean that the pS396 subset is a smaller tau subset, with all other factors being equal. Alternatively, the data can be interpreted to mean that the C10-2 antibody is more selective for pS396 than the 2.10.3 antibody is selective for pS422.

Both 2.10.3 and C10-2 removed more than 90% of the tau protein phosphorylated at serine-422, although the amount of antibody required to remove 50% of the pS422 tau protein varied, while achieving one and the same effect required 0.42 µg of antibody in the case of 2.10.3 and 0.27 µg in the case of C10-2 (see Fig. 12). In one embodiment of the present invention, the antibody is specific for an epitope between 386-404 where serine residue 396 of the human tau protein is phosphorylated and where 80% of pS422 tau is removed (in immunodepletion studies using Western blot analysis) at using less than 1 μg of antibody.

C10-2 effectively removed serine-396 phosphorylated tau (maximum effect: 88%, and half of this effect was achieved with 0.30 μg of antibody). 2.10.3 removed a smaller proportion of the tau protein phosphorylated at serine-396 (maximum effect: 60%, and half of this effect was achieved with 0.63 μg of antibody) (see Fig. 13). This indicates that all of the tau protein phosphorylated at serine-422 is also phosphorylated at serine-396, but there is a portion of the hyperphosphorylated tau protein phosphorylated at serine-396 that lacks the phosphorylated serine at position 422. In one embodiment, The antibody of the present invention is specific for an epitope between 386-404 where residue 396 of the human tau protein is phosphorylated and where 80% of pS396 tau is removed (in immunodepletion studies using Western blot analysis) at less than 1 μg antibodies.

A significant proportion of the tau protein removed by C10-2 was also phosphorylated at serine-199/202, as 69% of the tau protein characterized by such phosphorylation was affected by immunodepletion (50% effect with 0.34 μg of antibody). Immuno-depletion with 2.10.3 did not produce a sigmoidal dose-response curve for pS199/202 tau, although a decrease in signal intensity was observed with increasing antibody (maximum 52% reduction with maximum antibody (5 µg) (see FIG. 14) In one embodiment of the present invention, the antibody is specific for an epitope between 386-404 where serine residue 396 of the human tau protein is phosphorylated and where 80% of the P-S199/2O2 tau protein is removed (in immunodepletion studies using western blot analysis) using less than 1 µg of antibody.

These results indicate that C10-2 antibody, which targets phosphorylated serine-396, binds to a larger pool of hyperphosphorylated tau proteins than does antibody 2.10.3, which targets phosphorylated serine at position 422.

When examining individual bands in the western blot pattern after immunodepletion, the band corresponding to 25 kDa was identified as a serine-396 phosphorylated molecule. This fragment was immunodepleted with C10-2, however 2.10.3 and AT8 did not deplete this fragment (see FIG. 15). Thus, C10-2 has the unique property of removing this truncated form of tau from brain extracts from subjects with Alzheimer's disease.

Example 8 Comparison of Options C10-2

All C10-2 variants were equally effective in the immunodepletion assay (see FIG. 16). These results demonstrate that the introduced mutations did not alter functional binding to the brain-specific tau protein of Alzheimer's subjects.

8a) Antibody treatment in primed rTg4510 mice

Transgenic mice expressing the human mutant tau protein (P301L 0N4R) under the control of the Tet-Off response element in SatK2-positive neurons (rTg4510) were used. In this model, tau pathology usually begins to develop at 3 months of age, but when mothers are fed doxycycline during pregnancy and during the first 3 weeks of the calf's life, pathology develops at a later stage (starting at 6 months of age). Mice pretreated with doxycycline were treated for an extended period with mC10-2, hC10-2, 2.10.3 or control antibody at 15 mg/kg/week starting at 2 months of age. At 2.5 months of age, a brain extract from a subject with Alzheimer's disease was infused into the hippocampus. Twenty mice were anesthetized while immobilized in a stereotaxic frame by isoflurane inhalation. The skull was exposed and leveled until the bregma and lambda were at the same level. A hole was drilled in the skull 2 mm lateral (right) and 2.4 mm posterior to the bregma. A 10 µl beveled syringe (SGE) was used to inject the seed material 1.4 mm ventral to the brain surface at the above coordinates. 2 μl of the extracts described in examples 7 were injected by slow infusion at the desired site (1 μl/min) and the syringe was left for 5 minutes before being withdrawn. The wound was closed with sutures and the mice were kept warm while they were awake. Mice were kept for 3 months and then sacrificed and perfused with 4%. Mice were treated with antibody prior to sacrifice, 3 months after priming.

Immunohistochemical study

Fixed brain samples were cut into 35 μm thick coronal sections at NSA 30 and stained every 6th section for the presence of tau tangles (Gallyas silver stain). Positively stained neurons (soma) were counted in the ipsilateral and contralateral sides of the hippocampus of all brain samples. All subregions of the hippocampus were included.

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Counting was performed in eight slices per brain. The results reflect the total number of positive neurons from 8 slices.

Statistical analysis.

The variance differed significantly when comparing groups. In this regard, the non-parametric Kruskal-Wallis test and Dunn's test for multiple comparisons were used.

Results. The extracts caused a seeding effect with the formation of tangle pathology in the ipsilateral hippocampus. Treatment with mC10-2 significantly reduced tangle pathology in the seeded hippocampus by 57% (P<0.05). There was a clear trend indicating that hC10-2 reduced pathology. 2.10.3 was not characterized by such an effect (see Fig. 17).

Example 8b. Anti-priming effects of variants C10.2

Purpose of the study:

It has been suggested that the transcellular distribution of tau protein aggregates contributes to the development of pathology and the topological separation of Alzheimer's disease in the CNS. In vitro seeding models have been developed in which cellular human tau expressed in HEK293 cells is primed with pathological tau aggregates applied extracellularly. The aim of this study was to evaluate and compare the therapeutic effect of human monoclonal antibody C10.2 and variants under seeded conditions in a treatment paradigm. The term primer is intended to mean the introduction of such an amount of hyperphosphorylated tau protein (primer) as to initiate hyperphosphorylation/misfolding of the tau protein expressed in the cells.

Prerequisites determining the relevance of the study

Deposits of extracellular Άβ plaques and the presence of paired helical tau filaments within neurons are hallmarks of Alzheimer's disease. Intraneuronal tau inclusions primarily consist of detergent-insoluble, hyperphosphorylated amyloid forms of tau and are deposited in AD patients in a spatiotemporal pattern indicative of CNS separation of aggregates. It has been experimentally shown that tau protein aggregates spread from cell to cell both in vivo and in vitro by a prion-like mechanism. This presence of extracellular diversity, which affects the spread of the disease, opens up possibilities for immunotherapy with CNS-penetrating antibodies.

The use of tau aggregates/primers has been shown to result in tau aggregation in cells in vitro (Frost et al., 2009, Guo and Lee 2011, Yanamandra et al., 2013). The present inventors developed an in vitro seeding model in which crude brain homogenates from Tg4510 mice (containing aggregated human tau protein) were used to prime the P301L mutated human tau protein 0N4R transiently expressed in HEK293 cells. It is important to note that residual tau aggregates (priming) cannot be detected in pcDNA primed control cells, so all readings indicate cellular tau conversion and do not detect any signal from exogenous seed material. In this assay, the anti-priming effects of various hC10.2 variants were determined.

Options

Antibody C10-2 (hC10.2)

Antibody N32S (hC10.2 N32S)

Antibody N32Q (hC10.2 N32Q)

Antibody N32S, D55E (hC10.2 N32S D55E)

Antibody N32Q, D55E (hC10.2 N32Q D55E)

Antibody N32S, A101T (hC10.2 N32S A101T)

Reference object(s)

Control hIgG1 (B12)

Test system/animals

HEK293 cells transiently transfected with hTau-P301L (0N4R).

Experiment scheme

Seed material.

Homogenates from 12 month old Tg4510 mice (and controls) were homogenized as 10% homogenates in TBS without inhibitors by bead homogenization and sonicated. The homogenates were centrifuged at 21000xg for 15 min and the soluble fraction was used as seed.

Seed action analysis in HEK293.

In this assay, 600,000 HEK293 cells per well were seeded in a 6-well plate on day 0. On day 1, cells were transfected with 4 μg of plasmid DNA using 10 μl of Lipofectamine 2000 according to the manufacturer's protocol. Medium was changed after 4 hours. On day 2, cells were seeded with crude brain homogenate from 12 month old Tg4510 or tTa mice.

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Homogenates containing 40 μg of total protein (approximately 65 ng of total human tau protein in the case of homogenates from Tg4510) were added to the medium in each well. For antibody treatment experiments, homogenates were pre-incubated with or without antibodies overnight at 4°C. Six hours after seeding, medium was changed to low serum medium to reduce cell proliferation and antibody was reapplied.

On day 3 (24 hours after priming), cells were trypsinized for 3 minutes to destroy extracellular primers and replated at 800,000 cells/well in 6-well plates for fractionation experiments, 20,000 cells in 96-well plates for tau aggregation assay α-protein Cisbio and 10,000 cells per 96-well plates to assess antibody uptake using high-throughput imaging. Antibodies were reapplied to each well.

Fractionation of cells

48 hours after seeding, cells for fractionation were harvested by scraping into cold PBS, pelleted and lysed in TBS with 1% Triton-X with phosphatase and protease inhibitors, and sonicated. After ultracentrifugation (100,000xg for 30 min at 4°C), the pellet was resuspended in 1% SDS, sonicated and ultracentrifuged again. Triton-X and SDS soluble fractions were analyzed by Western blotting for total tau (E1) and tau phosphorylated at S396 (D1.2). The D1.2 signal was quantified by immunofluorescence on an Odyssey imaging device.

Cisbio tau aggregation analysis

48 hours after seeding, Cisbio aggregation assay cells were washed in ice-cold PBS and freeze-dried. Cells were lysed in 1% Triton-X with phosphatase/protease inhibitors and benzonase (RNase and DNase) and incubated on an orbital shaker at 650 rpm for 40 min at 4°C. Total cell lysate was analyzed for tau protein aggregates using Cisbio tau aggregation assay; This assay is based on the use of the same donor- and acceptor-bound antibody for FRET measurements using time-resolved homogeneous fluorescence (HTRF®, Cisbio). Monomeric tau can only bind to either the acceptor or donor antibody because they compete for the same binding epitope = no FRET. In contrast, an oligomeric tau protein can bind to both a donor and an acceptor = FRET signal. Total protein was determined in all samples by BCA and the signal-to-noise ratio for the sample was normalized with respect to protein and the data were plotted as relative tau protein aggregation using 8 technical replicates.

Antibody capture

48 hours after priming, antibody capture plates were fixed in 4% paraformaldehyde and 4% sucrose and stained with a secondary antibody to human immunoglobulins. Antibody capture was confirmed using Cellomics.

Data analysis/statistics

Data are presented as pooled data from four independent biological replicates performed and analyzed over two different weeks ± S.E.M. Data were analyzed by one-way ANOVA using Tukey's multiple comparison test, *P<0.05, **P<0.01, ***P<0.001.

Results hC10.2 was characterized by a reducing effect on seeding action and formation of insoluble hyperphosphorylated tau protein by about 40% compared to control. All other antibodies showed a similar or superior effect compared to hC10.2. In particular, the hC10.2 N32S and N32S_A101T variants showed more pronounced effects in terms of reduced tau protein aggregation by 45% and 62%. The N32S_A101T variant showed a significantly more pronounced effect on aggregation compared to hC10.2.

Example 8C. Stability studies of hC10.2 and variants of the present invention

Purpose of the study:

This study was performed to evaluate the main characteristics of the stability of the antibodies of the present invention, focusing on stress conditions to detect any potential differences in the variants.

Study scheme

All samples were initially prepared under the same initial conditions (buffer, concentration and level of aggregation). This allows you to eliminate differences in the original properties that could potentially affect the subsequent manifestation of the behavior of the sample. The control was stored at -80°C and analyzed in parallel with stressed samples (40°C). Samples were removed from 40° C. conditions at various time points and finally analyzed simultaneously by SEC-UPLC, peptide mapping by liquid chromatography-mass spectrometry (LCMS) and differential scanning fluorimetry (DSF).

Options

Antibody C10-2 (hC10.2)

Antibody N32S (hC10.2 N32S)

Antibody N32Q (hC10.2 N32Q)

Antibody N32S, D55E (hC10.2 N32S D55E)

Antibody N32Q, D55E (hC10.2 N32Q D55E)

Antibody N32S, A101 (hC10.2 N32S A101T)

Antibody A101T (hC10.2 A101T)

Antibody D55E (hC10.2 D55E)

Antibody N32Q, A101T (hC10.2 N32Q A101T)

reference object

Antibody samples stored at -80°C during the study were used as a reference.

Introductory information

Aspects of the stability of a monoclinal antibody are important for both aspects of manufacture, final storage, and formulation of the drug substance. In this study, the present inventors stressed antibodies using freeze-thaw, high temperature, and low pH cycles to determine if there were clear problems in any of the candidates.

Materials and methods

For studies on deamidation, samples were placed in vials with a sample amount of 1 ml and incubated at 40°C. At predetermined time points, the sample was transferred to -80° C. and stored until analysis.

Research on deamidation

Deamidation of Asn residues was investigated at the peptide level in order to obtain detailed information regarding the actual residues in question. Thus, the mAb was digested with porcine trypsin after reduction and alkylation (ioacetic acid) using standard protocols. The peptides were separated on a 1.7 μm CSHT130 C18 column and injected into a MS XEVO QTOF (Waters) device operating in MSe mode. The same parameters were used in all series. Peptide maps generated across all time points were analyzed in BiopharmaLynx and quantified under the following limitations: only peptides identified as positive for deamidation were included, fragments generated by fragmentation in the ion source were not included. The % deamidation of each identified peptide was monitored over time.

Summary of the SEC Method

Aggregation was determined by SEC chromatography on an ACQUITY UPLC® BEH200 SEC 1.7 µm, 4.6x150 mm column in Acquity UPLC with a TUV (Waters) detector. Samples in a volume of 20 µl adjusted to 100 µg/ml (diluted in running buffer: PBS Gibco - Invitrogen No. 14190-094 + 0.1 M NaCl) were loaded onto the column at 0.4 ml/min and separated for 6 min using isocratic elution.

Data was analyzed with MassLynx and AUC was used to quantify levels of aggregation.

Low pH experiment hC10.2 was loaded onto a protein G column and eluted at pH 2.8 using standard conditions. The sample was stored at pH 2.8 and aliquots were removed at 0, 15, 30, 60, 120 and 180 minutes and neutralized before desalting in PBS. Samples were analyzed for binding affinity, aggregation and deamidation.

Characterization of hC10.2 under stress conditions.

Deamidation at 40°C and 28 days.

Deamidation of Asn was observed in several minor and three major regions covered by the peptides LC:T2, HC:T36 and HC:T25. Their level of deamidation increased with time, while being respectively 75%, 38% and 28% at the end point. In contrast to the HC:T36 and T25 peptide, based on in silico analysis (motif sequence), it was assumed that the Asn residues (LC N32 and N34) in the LC:T2 peptide are not subject to deamidation, however, in an unexpected way in the actual experiment, they clearly differed from other Asn residues in hC10.2. The increased level of deamidation of this peptide correlates with reduced binding activity to the diphosphorylated peptide and suggests a mechanistic relationship between deamidation and IC ₅₀ . This observation also suggests that altering Asn 32 and/or 34 residues in LC hC10.2 could reduce the level of deamidation in these regions.

When analyzing the variants, it is clearly seen that a simple modification of Asn32 by the type of replacement or by

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Ser or on Gln completely prevents the deamidation reaction at Asn34 (Fig. 20). In addition, the authors of the present invention did not detect any deamidation of Gln32 in variants with this mutation.

Deamidation at low pH

In this study, the authors of the present invention analyzed hC10.2 in the same way as described above for the study of stability at 40°C. Only in this case was a mixture of trypsin and Lys-C (Promega product) used to optimize the digestion of lysine residues. No additional sites or degree of deamidation were observed and again the most prominent peptides were LC-T2, HC-T25 and HC-T36. In addition, no change in IC ₅₀ was observed in samples treated at low pH.

SEC Analysis

The level of aggregation was below 3% in all preparations, and no change in the degree of aggregation was observed within 28 days at 40°C.

Example 9 Isolation of Tau Primers in rTg4510 Mice

The rTg4510 mice are transgenic for two genes, co-expressing the 4R0N tau protein with the P301L mutation in the human MART gene downstream of the tetracycline responder operon (TRE) and an activator construct consisting of the tetracycline conditional gene expression (tTA) system. The P301L mutation is a dominant mutation resulting in frontotemporal dementia with chromosome 17-linked parkinsonism (FTDP-17). In rTg4510 mice, hTau P301L expression induces tau protein pathology, including early tangle and neurofibrillary tangle (NFT) formation, neuronal loss, and behavioral abnormalities, in an age-dependent manner. NFTs are aggregates of tau protein within neurons, consisting of paired helical filaments (PHF), twisted ribbons or straight filaments, which are insoluble in detergents and contain mostly hyperphosphorylated tau protein. Hyperphosphorylation means that tau is phosphorylated at more sites than tau from the brain of a healthy adult, and that a higher than normal percentage of tau molecules is phosphorylated at a given site (>8 mol phospho/mol tau protein). The anatomical distribution of NFT is a post-mortem histopathological signature that correlates with cognitive decline in Alzheimer's disease (AD) and memory loss in normal aging and mild cognitive impairment. The distribution pattern of NFT in the brains of AD patients is highly hierarchical and has been divided into six stages. The gradual occurrence of NFT changes in the brain was also confirmed biochemically and classified into ten stages according to the affected area. Characterization of various tau protein molecules from brain samples from AD subjects by biochemistry was initially based on a fractionation protocol using 1% sarcosyl to isolate insoluble tau protein. Based on this method, sarcosyl-insoluble hyperphosphorylated tau molecules isolated in the sarcosyl-insoluble precipitate fraction (P3) were determined as tau PHF and considered as the biochemical equivalent of NFT. Buffer-soluble hyperphosphorylated tau molecules isolated into a soluble fraction (S1) were defined as non-fibrillar oligomeric tau molecules and considered as the biochemical equivalent of early tau coils. In rTg4510 mice, the normal monomeric phosphorylated and non-phosphorylated human transgenic 4R0N tau protein is present in the soluble fraction (S1) and visualized as 55 kDa tau molecules on SDS-PAGE. The hyperphosphorylated tau protein 4R0N with the P301L mutation was displayed as a 64 kDa and 70 kDa mobility-shifted tau protein in the soluble (S1) fraction and exclusively in the Tris buffered saline (TBS) soluble precipitate (S1p) fraction and the insoluble fraction. in the sarcosyl precipitate (P3). Sarcosyl-insoluble 64 kDa and 70 kDa tau molecules isolated in P3 are tau fibrils (tau PHF) and the biochemical equivalent of NFT. Buffer-soluble tau molecules of 64 kDa and 70 kDa in S1 and enriched in the TBS-soluble precipitate (S1p) fraction represent an oligomeric tau protein and the biochemical equivalent of early tau coils. The 70 kDa tau protein molecules are specific for the P301L mutant and are also found in brain samples from FTDP-17 patients. Brain tissue from 40 week old rTg4510 mice was homogenized in 10 volumes of TBS containing 50 mM Tris/HCl (pH 7.4), 274 mM NaCl and 5 mM KCl. Homogenates were centrifuged at 27000xg for 20 min. at 4°C to obtain fractions of the supernatant (S1) and sediment (P1). The fraction S1 extractable in TBS was separated by centrifugation at 150,000 x g for 1 hour at 4°C into supernatant (S1s) and sediment (S1p) fractions. The S1p pellet was resuspended in 10 mM Tris/HCl [pH 8.0] to a volume equal to one-fifth of the original S1 volume. P1 pellet was re-homogenized in 5 volumes of high salt/sucrose buffer (0.8 M NaCl, 10% sucrose, 10 mM Tris/HCl, [pH 7.4]) and centrifuged at 27,000 x g for 20 min. at 4°C. Supernatant samples were collected and incubated with sarcosyl (final concentration 1%; Sigma) for 1 h at 37°C followed by centrifugation at 150,000xg for 1 h at 4°C to obtain samples of the sarcosyl-insoluble precipitate, termed in this document fraction P3. The P3 pellet was resuspended in 10 mM Tris/HCl [pH 8.0] to a volume equal to half the original volume used for brain homogenates. Pathological tau protein from mouse brain rTg4510, characterized on SDS-PAGE as hyperphosphorylated tau protein of 64 and 70 kDa, was present exclusively in fractions S1p and P3 as TBS-soluble oligomeric tau protein and sarcosyl-insoluble fibrillar tau protein respectively. Fractions of S1s contained normal unphosphorylated and phosphorylated tau protein, displayed on SDS-PAGE as a 55 kDa tau protein. Pathological tau protein, soluble oligomeric and insoluble fibrillar, isolated in fractions S1p and P3, respectively, was used as primers of hyperphosphorylated tau protein to recruit endogenous monomeric human tau protein to form aggregated tau protein inclusions and to induce tau seeding action. -protein in cultures of primary cortical neurons isolated from rTg4510 mice.

Analysis of seeding action in culture of primary neurons isolated from rTg4510 mice

Mouse cortical neurons (CTX) were isolated from E14-16 day old rTg4510 mouse embryos. Transgenic for one gene, the tTA activator, mice were mated at a certain time with transgenic for one gene, the mutant tau protein responder, mice. Pregnant females were sacrificed 14-16 days after conception and the embryos were genotyped with brain DNA using primer pairs 5'-GATTAACAGCGCATTAGAGCTG-3' and 5-GCATATGATCAATTCAAGGCCGATAAG-3' for the _tTA activator transgene and 5'-TGAACCAGGATGGCTGAGCC-3' and 5'-TTGTCATCGCTTCCAGTCCCCG-3' for the tau mutant responder transgene, with isolated embryonic cortex samples stored in Hibernate E without calcium chloride (BrainBits LLC) at 4°C. Cortical samples from rTg4510 mouse embryos were taken and dissociated neurons plated on 100 μg/ml poly-L-lysine coated dishes at a density of 0.13x106 cells/cm ² (420,000 cells/ml, 100 μl/well, 96 well plate) and cultured in glia-conditioned neurobasal medium supplemented with 2% antioxidant supplement B-27, 0.5 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin (all solutions from Gibco-BRL Invitrogen). Glia-conditioned neurobasal medium was obtained using confluent and non-proliferating cultures of primary mouse astrocytes after 24 h of incubation. Neurons were fed after 4 days in vitro (DIV) by replacing half of the medium with fresh glia-conditioned neurobasal medium and then fed every seventh day. On DIV4, after feeding, the neuronal culture was treated with 1 μM cytosine-arabinoside to stop cell proliferation. The proportion of glial cells in cultures was less than 10%, as determined by antibody to glial fibrillar acidic protein (GFAP) on DIV15. CTX from rTg4510 mice expressed endogenous mouse tau and transgenic human tau 4R0N. CTX from rTg4510 mice contained only normal human monomeric non-phosphorylated and phosphorylated tau protein, displayed on SDS-PAGE as 55 kDa tau bands; at the same time, hyperphosphorylated tau protein molecules (64 and 70 kDa) were absent in native CTX from rTg4510 mice. On DIV7, tau seeding was induced by incubating CTX with pathological tau as either soluble oligomeric or insoluble fibrillar hyperphosphorylated tau isolated in S1p and P3 fractions, respectively. Full media change was performed on DIV11 to prevent continuous uptake of pathological tau seedings, and tau seeding activity in CTX was measured on DIV15. Tau seeding action was characterized by recruitment of monomeric soluble human tau protein to form aggregated and hyperphosphorylated tau inclusions that appear on SDSPAGE as mobility shifted tau bands at higher molecular weights of 64, 70 and 140 kDa. Both primers of hyperphosphorylated tau protein, soluble oligomeric or insoluble fibrillar from the S1p and P3 fractions, respectively, equally induced tau seeding activity in CTX. To study the effect of anti-tau antibodies on tau priming, CTX was treated with a mixture of 0.1 µl of the P3 fraction or 0.2 µl of the S1p fraction (containing 0.2 ng of total human tau protein) isolated from 40-week-old rTg4510 mice, and 10 μg of antibody (hC10.2, hC10.2_N32S, hC10.2_A101T_N32S, control human IgG) or phosphate buffered saline (PBS). The mixture of tau seed and antibody was pre-incubated for 2 hours at 4° C. prior to addition to CTX. On DIV11, complete medium replacement was performed with fresh glia-conditioned neurobasal medium. At DIV15, neurons were lysed in ice-cold Triton lysis buffer (1% Triton X-100 in 50 mM Tris, 150 mM NaCl (pH 7.6) with 1% protease inhibitor mix (Roche), 1% protease inhibitor mix I and II (Sigma) and 0.2% benzonase (Sigma)) with shaking at 200 rpm for 45 min at 4° C. and lysates were used in an in vitro aggregation assay of Cisbio and protein content was determined using a bicinchoninic acid assay ( BCA) according to the manufacturer's instructions. At the heart of Cisbio's tau aggregation assay

- 84 039569 is time resolved fluorescence using the same antibody as both donor (Tb3+ conjugated) and acceptor (d2 conjugated) antibodies in fluorescence resonance energy transfer (FRET) and was performed according to the manufacturer's instructions. Briefly, a 9 μl sample was mixed with a 9 μl antibody mixture and incubated for 20 h. The plate was read on a PHERAstar plate reader to evaluate time-resolved fluorescence (measured/integrated after excitation light on, the FRET signal). This assay measured tau aggregation of both tau oligomers and fibrils in brain material from rTg4510 mice and in neuronal lysates from tau-primed CTX isolated from rTg4510 embryos with high specificity and sensitivity. The results can be seen in Fig. 21. Incubation of P3 or S1p primers with human control IgG antibody resulted in a similar tau aggregation signal from seeded CTX as incubation of P3 or S1p primers with PBS. Signals from PBS and human control IgG antibody were averaged and taken as 100% tau protein aggregation. Anti-tau antibodies hC10.2, hC10.2N32S, and hC10.2A101T-N32S significantly reduced tau aggregation signals induced by both P3 and S1p tau seeding by DIV15. The results of 2 separate experiments were summarized. The hC10.2 antibody reduced P3 and S1p induced tau seeding by 23%. The hC10.2N32S and hC10.2A101T-N32S variants reduced P3 and S1p induced tau seeding by 41-53% and 48-60%, respectively. Variants hC10.2N32S and hC10.2A101T-N32S were superior to hC10.2 in terms of reducing the seeding effect on tau in CTX from rTg4510, induced by seeding hyperphosphorylated tau from S1p or P3, consisting of oligomeric or fibrillar tau, respectively.

Example 10 Dose-dependent staining of rTg4510 tau protein structures after i.v. hC10-2_N32S_A101T injections

Way:

Twelve-month-old rTg4510 mice.

These transgenic mice express human tau mutant (P301L 0N4R) under control of the Tet-Off response element in Satk2 positive neurons and are characterized by marked tau hyperphosphorylation and tangle formation from 6 months of age onwards. In addition, 12-month-old rTg4510 mice show neurodegeneration in areas of severe pathology. Mice received a single i.v. injection into the tail vein of antibodies hC10-2_N32S at doses of 80, 20, 8 mg/kg and 0.8 mg/kg. Each mouse was injected with a volume of 100 microliters. Three days after injection, mice were perfused for 2 min with PBS followed by perfusion for 10 min with 4% paraformaldehyde. Brain samples were cryoprotected in 30% sucrose and cut into free-floating cryosections 40 microns thick. Sections were incubated with 5% normal porcine serum in PBS/1% BSA/0.3% Triton X-100 for 20 min, washed in PBS, and finally incubated with AlexaFluor488-conjugated anti-human IgG secondary antibody at 1:200 (no. 709-545-149; Jackson ImmunoResearch Laboratories, West Grove, USA). Sections were washed in PBS, mounted on a glass slide and examined by fluorescence microscopy.

results

I.v. hC10-2_N32S injection resulted in in vivo binding to target structures primarily in the hippocampus and cortex from age-matched rTg4510 animal brain samples (FIG. 1). The number of positive structures observed varied between individual rTg4510 animals. The positive signals in rTg4510 brain samples were not easily identified as intracellular staining material and could represent extracellular tau protein material released during the neurodegeneration process. By semi-quantitative evaluation, the highest signals (fluorescence intensity and number of positive structures) were detected in all mice dosed at 20 and 80 mg/kg (Table 6). Labeled structures were observed at 8 mg/kg in 3 out of 4 mice, however at a clearly lower level than at 20 and 80 mg/kg. Intravenous injection at a dose of 0.8 mg/kg did not lead to the appearance of signals above the background level with the imaging method used. Taken together, these data suggest that hC10-2_N32S antibodies are able to dose-dependently penetrate the brain parenchyma and specifically stain targets in rTg4510 mice in vivo (FIG. 22).

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Table 6

Tg4510 i.v. injection Signal Intensity(according to relevant hC10-2_N32S fluorescence age towards n= 80 mg/kg) 3 80 mg/kg + y 3/3 H—ΐ—h four 20 mg/kg + at 4/4 ++-+-+ four 8 mg/kg + y 3/4 + four 0.8 mg/kg -y 4/4 0

Fluorescently labeled tau protein structures detected in brain sections after i.v. hC10-2_N32S injections. Semiquantitative assessment: +++ strong; ++ moderate; + weak; 0 is not detected.

Example 11 Tau Seed Action to Generate Tau Pathology

Getting a seed

Frozen cortical tissue samples from AD donors were obtained from Tissue Solutions (Glasgow, UK). The brain tissue was weighed and homogenized using a blade homogenizer in sterile cold PBS (10 times the volume of the brain sample). The homogenate was then subjected to ultrasonication on a Branson ultrasonicator with output power set to 2 with 5 x 0.9 s pulses. The homogenate was centrifuged at 3000 g for 5 min at 4°C and the supernatant was aliquoted, flash-frozen and stored at -80°C until use.

Animals used in the study

Transgenic mice expressing the human mutant tau protein (P301L 0N4R) under the control of a tetracycline controlled transactivator element in CamKII positive neurons (rtg4510) were used. In this model, the pathology usually begins to develop at the age of 34 months. When mothers are fed doxycycline during pregnancy and during the first 3 weeks of the baby's life, the pathology develops at a later stage (from the age of 6 months). The doxycycline pre-treated mice used in these studies were 2.5 months old at the time of priming.

Antibody treatment

Antibodies were obtained at a concentration of 1.5 mg/ml in sterile PBS and aliquoted with enough antibody for one dosing time point. Aliquots were stored at 4°C until use. Starting at 2 months of age until sacrificing at 5.5 months of age, mice received a weekly IP dose of antibody (15 mg/kg equal to 10 ml/kg). Antibodies used: control human B12-IgG1, hC10-2, hC10-2_N32S and hC10-2_N32S_A101T.

Stereotactic injection

Immediately after receiving the 3rd dose of antibody, mice were anesthetized while immobilized in a stereotaxic frame by inhaling isoflurane. The skull was exposed and leveled until the bregma and lambda were at the same level. A hole was drilled in the skull 2 mm lateral (right) and 2.4 mm posterior to the bregma. A 10 µl 26 gauge beveled syringe (SGE) was used to inject the seed material 1.4 mm ventral to the brain surface.

Two μl of the material was injected by slow infusion at the desired site (0.5 μl/min.) and after that the syringe was left for another 5 min before withdrawal. The wound was closed and stitched, and the mice were warmed during recovery from anesthesia. Mice were then housed for 3 months prior to perfusion fixation with 4% paraformaldehyde.

Histology

Fixed brain samples were processed at Neuroscience Associates (Knoxville, TN) using MultiBrain® technology. No more than 25 mouse brain samples were co-embedded into one block and cut frozen into 35 µm slices in the frontal plane across the entire brain. Every 6th section was stained with Gallyas silver stain to detect neurofibrillary tangles. Sections were mounted on a glass slide, covered with a coverslip, and neurons (soma) positive for Gallyas silver staining were counted in all brain samples in the hippocampus from the injection side. All subregions of the hippocampus were included. Counting was performed in eight slices per brain. The results reflect the total number of positive neurons from 8 slices.

Result

All of hC10-2, hC10-2_N32S and hC10-2_N32S_A101T significantly reduced the priming effect of tau tangles in the injected hippocampus (univariate anova and Tukey's multiple comparison test). hC10-2: 50%, hC10-2_N32S: 47% and hC10-2_N32S_A101T: 60% compared to control treated mice (FIG. 23).

- 86 039569

Sequence listing < 110> X. LUNDBECK A/C < 120> ANTIBODIES SPECIFIC TO HYPERPHOSPHORYLATED TAU PROTEIN < 130> 1049-WO-PCT < 160> 57 < 170> Patentin version 3.5 < 210> 1 < 211> 441 < 212 > PROTEIN < 213> Artificial <220>

<223> Human tau protein <400> 1

Met 1 Al a Glu Pro Arg 5 Gln G1u Phe Glu Wai 10 Met G1u asp Hi s Al a 15 gly Thr Tyr gly Leu 20 gly asp Arg Lys Asp 25 Gln gly gly Tyr Thr 30 Met Hi s Gln asp Gln 35 Glu gly asp Thr Asp 40 Al a gly Leu Lys Glu 45 Ser Pro Leu Gln Thr 50 Pro Thr Glu asp Gly 55 Ser Glu Glu Pro Gly 60 Ser Glu Thr Ser Asp 65 Al a Lys Ser Thr Pro 70 Thr Al a Glu asp Vai 75 Thr Al a Pro Leu Vai 80 asp G1u gly Al a Pro 85 gly Lys Gln Al a Al a 90 Al a G1n Pro Hi s Thr 95 Glu lie Pro Glu Gly 100 Thr Thr Al a Glu Glu 105 Al a gly lie gly Asp 110 Thr Pro Ser Leu Glu 115 asp Glu Al a Al a Gly 120 Hi s Vai Thr G1n Al a 125 Arg Met Vai Ser Lys 130 Ser Lys asp gly Thr 135 gly Ser asp asp Lys 140 Lys Al a Lys gly Al a 145 asp gly Lys Thr Lys 150 lie Al a Thr Pro Arg 155 gly Al a Al a Pro Pro 160 gly G1n Lys gly Gln 165 Al a Asn Al a Thr Arg 170 lie Pro Al a Lys Thr 175 Pro Pro Al a Pro Lys 180 Thr Pro Pro Ser Ser 185 gly Glu Pro Pro Lys 190 Ser gly

- 87 039569

asp Arg Ser 195 gly Tyr Ser Ser Pro 200 gly Ser Pro gly Thr 205 Pro gly Ser Arg Ser 210 Arg Thr Pro Ser Leu 215 Pro Thr Pro Pro Thr 220 Arg Glu Pro Lys Lys 225 Vai Al a Vai Vai Arg 230 Thr Pro Pro Lys Ser 235 Pro Ser Ser Al a Lys 240 Ser Arg Leu gin Thr 245 Al a Pro Vai Pro Met 250 Pro asp Leu Lys Asn 255 Val Lys Ser Lys lie 260 gly Ser Thr Glu Asn 265 Leu Lys Hi s gin Pro 270 gly gly gly Lys Vai 275 gin lie lie Asn Lys 280 Lys Leu asp Leu Ser 285 Asn Val gin Ser Lys 290 cys gly Ser Lys Asp 295 Asn lie Lys Hi s Val 300 Pro gly gly gly Ser 305 Vai gin lie Vai Tyr 310 Lys Pro Vai asp Leu 315 Ser Lys Val Thr Ser 320 Lys cys gly Ser Leu 325 gly Asn lie Hi s Hi s 330 Lys Pro gly gly Gly 335 gin Vai G1u Vai Lys 340 Ser Glu Lys Leu Asp 345 Phe Lys asp Arg Val 350 gin Ser Lys lie Gly 355 Ser Leu asp Asn lie 360 Thr Hi s Vai Pro Gly 365 gly gly Asn Lys Lys 370 lie Glu Thr Hi s Lys 375 Leu Thr Phe Arg G1 and 380 Asn Al a Lys Al a Lys 385 Thr asp Hi s gly Al a 390 G1 U lie Vai Tyr Lys 395 Ser Pro Val Val Ser 400 gly asp Thr Ser Pro 405 Arg Hi s Leu Ser Asn 410 Vai Ser Ser Thr Gly 415 Ser lie asp Met Vai 420 asp Ser Pro gin Leu 425 Al a Thr Leu Al a Asp 430 Glu Val Ser Al a Ser 435 Leu Al a Lys G1n Gly 440 Leu

<210> 2 <211> 23 <212> PROTEIN <213> Artificial

- 88 039569 <220>

<223> tau protein residues 386-408 (pS396, pS404) <400> 2

Thr Asp 1 > His Gly Ala Glu 5 lie Vai Tyr Lys 10 Ser Pro Vai Vai Ser Gly 15 Asp Thr Ser Pro Arg His 20 Leu <210> <211> <212> <213> 3 eleven PROTEIN Artificial <220> <223> CDR1 light chain C10-2 <400> 3 Gin Ala Ser 1 Gin Asp Thr 5 Ser lie Asn Leu 10 Asn <210> <211> <212> <213> four 7 PROTEIN Artificial <220> <223> CDR2 light chain C10-2 <400> four Gly Ala Ser 1 Asn Leu Glu 5 Thr <210> <211> <212> <213> 5 9 PROTEIN Artificial <220> <223> CDR3 light chain C10-2 <400> 5 Leu G1r । His Thr Tugh Leu Pro Phe Thr

5 < 210> 6 < 211> 5 < 212> PROTEIN < 213> Artificial <220>

< 223> CDR1 heavy chain C10-2 < 400> 6

Asp Arg Thr lie His

5

- 89 039569

<210> <211> <212> <213> 7 fifteen PROTEIN Artificial <220> <223> CDR2 heavy chain C10-2 <400> 7 Pro Gly Asp Gly Ser Thr Lys Tyr Ser Gin Lys Phe Gin Gly

10 15 < 210> 8 < 211> 6 < 212> PROTEIN < 213> Artificial <220>

< 223> CDR3 heavy chain C10-2 < 400> 8

Arg Gly Ala Met Asp Tyr 1 5 < 210> 9 < 211> 214 < 212> PROTEIN < 213> Mouse C10-2 light chain < 400> 9

Asp Vai 1 gin Met lie 5 gin Ser Pro Ser Ser 10 Leu Ser Al a Ser Leu 15 gly asp lie Vai Thr 20 Met Thr cys gin Al a 25 Ser gin gly Thr Ser 30 lie Asn Leu Asn T rp 35 Phe gin gin Lys Pro 40 gly Lys Al a Pro Lys 45 Leu Leu lie Tyr Gly 50 Al a Ser Asn Leu G1 to 55 asp gly Vai Pro Ser 60 Arg Phe Ser gly Ser Arg 65 Tyr gly Thr Asp 70 Phe Thr Leu Thr lie 75 Ser Ser Leu Glu Asp 80 Glu Asp Met Al a Thr 85 Tyr Phe cys Leu Gin 90 Hi s Thr Tyr Leu Pro 95 Phe Thr Phe gly Ser 100 gly Thr Lys Leu Glu 105 lie Lys Arg Al a Asp 110 Al a Al a ProThr Vai 115 Ser lie Phe Pro Pro 120 Ser Ser Glu G1n Leu 125 Thr Ser gly G1 y Al a Ser Vai Vai cys Phe Leu Asn Asn Phe Tyr Pro Lys asp lie

- 90 039569

130 135 140

Asn Vai Lys Trp Lys lie Asp Gly

145 150

Ser Glu Arg Gin Asn Gly Vai Leu

155 160

Asn Ser Trp Thr Asp Gin Asp Ser

165

Lys Asp Ser Thr Tyr Ser Met Ser

170 175

Ser Thr Leu Thr Leu Thr Lys Asp

180

Glu Tyr Glu Arg His Asn Ser Tyr

185 190

Thr Cys Glu Ala Thr His Lys Thr

195 200

Ser Thr Ser Pro lie Vai Lys Ser 205

Phe Asn Arg Asn Glu Cys

210 < 210> 10 < 211> 439 < 212> PROTEIN < 213> Mouse heavy chain C10-2 < 400> 10

Gin Vai Gin Leu Gin Gin Ser Asp Ala Glu Leu Vai Lys Pro Gly Ala

1015

Ser Vai Lys lie Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg 20 2530

Thr lie His Trp Vai Lys Gin Arg Pro Glu Gin Gly Leu Glu Trp lie 35 4045

Gly Tyr lie Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Asn Phe 50 5560

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 7580

Met Gin Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Vai Tyr Phe Cys 85 9095

Ala Arg Arg Gly Ala Met Asp Tyr Trp Gly Gin Gly Thr Ser Vai Thr

100 105110

Vai Ser Ser Ala Lys Thr Thr Pro Ser Vai Tyr Pro Leu Ala Pro

115 120125

Gly Ser Ala Ala Gin Thr Asn Ser Met Vai Thr Leu Gly Cys Leu Vai

130 135140

Lys Gly Tyr Phe Pro Glu Pro Vai Thr Vai Thr Trp Asn Ser Gly Ser

145 150 155160

- 91 039569

Leu Ser Ser Gly Vai His Thr Phe

165

Pro Ala Vai Leu Gin Ser Asp Leu

170 175

Tyr Thr Leu Ser Ser Ser Vai Thr

180

Vai Pro Ser Ser Thr Trp Pro Ser

185 190

Glu Thr Vai Thr Cys Asn Vai Ala

195 200

His Pro Ala Ser Ser Thr Lys Vai

205

Asp Lys Lys lie Vai Pro Arg Asp

210 215

Cys Gly Cys Lys Pro Cys lie Cys

220

Thr Vai Pro Glu Vai Ser Ser Vai

225 230

Phe lie Phe Pro Pro Lys Pro Lys

235 240

Asp Vai Leu Thr lie Thr Leu Thr

245

Pro Lys Vai Thr Cys Vai Vai Vai

250 255

Asp lie Ser Lys Asp Asp Pro Glu

260

Vai Gin Phe Ser Trp Phe Vai Asp

265 270

Asp Vai Glu Vai His Thr Ala Gin

275 280

Thr Gin Pro Arg Glu Glu Gin Phe

285

Asn Ser Thr Phe Arg Ser Vai Ser

290 295

Glu Leu Pro lie Met His Gin Asp

300

Trp Leu Asn Gly Lys Glu Phe Lys 305 310

Cys Arg Vai Asn Ser Ala Ala Phe

315 320

Pro Ala Pro lie Glu Lys Thr lie

325

Ser Lys Thr Lys Gly Arg Pro Lys

330 335

Ala Pro Gin Vai Tyr Thr lie Pro

340

Pro Pro Lys Glu Gin Met Ala Lys 345 350

Asp Lys Vai Ser Leu Thr Cys Met

355 360 lie Thr Asp Phe Phe Pro Glu Asp 365 lie Thr Vai Glu Trp Gin Trp Asn

370 375

Gly Gin Pro Ala Glu Asn Tyr Lys

380

Asn Thr Gin Pro lie Met Asp Thr 385 390

Asp Gly Ser Tyr Phe Vai Tyr Ser

395 400

Lys Leu Asn Vai Gin Lys Ser Asn

405

Trp Glu Ala Gly Asn Thr Phe Thr

410 415

Cys Ser Vai Leu His Glu Gly Leu

420

His Asn His His Thr Glu Lys Ser

425 430

- 92 039569

Leu Ser His Ser Pro Gly Lys

435 <210>

<211>

<212>

<213>

445 PROTEIN Artificial <220>

<223>

Heavy chain of humanized C10-2 <400>

Gin 1

Vai

gin

Leu

Wai 5

gin

Ser

gly

Al a

Glu 10

Vai

Vai

Lys

Pro

Gly 15

Al a

Ser

Vai

Lys lie 20

Ser cys

Lys

Al a

Ser 25

gly

Tyr

Thr

Phe

Thr 30

asp

Arg

Thr lie

Hi s 35

T rp

Vai

Arg

G1n

Al a 40

Pro

gly

gin

gly

Leu 45

Glu

T rp lie

gly

Tyr 50 lie

Tyr

Pro

gly

Asp 55

gly

Ser

Thr

Lys

Tyr 60

Ser

gin

Lys

Phe

Gin 65

gly

Arg

Al a

Thr

Leu 70

Thr

Al a

asp

Thr

Ser 75

Al a

Ser

Thr

Al a

Tyr 80

Met

G1u

Leu

Ser

Ser 85

Leu

Arg

Ser

Glu

Asp 90

Thr

Al a

Vai

Tyr

Tyr 95 cys

Al a

Arg

Arg

Gly 100

Al a

Met

asp

Tyr

T rp 105

gly

gin

gly

Thr

Ser

110

Vai

Thr

Vai Ser Ser Ala 115 Ser Thr Lys Gly 120 Pro Ser Vai Phe Pro 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Al a Al a Leu gly

Leu cys

130

135

140

Al a

Pro

Leu

Vai

Lys 145

asp

Tyr

Phe

Pro

Glu

150

Pro

Vai

Thr

Vai

Ser

155

trp

Asn

Ser

gly

Al a 160

Leu

Thr

Ser

Leu

Tyr

Ser

Leu

180

gly

Vai 165 Hi s Thr Phe Pro Al a 170 Vai Leu gin Ser Ser 175 Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu

gly

gly

185

190

Thr

G1n

Thr

195

Tyr lie cys

Asn

Vai

200

Asn

Hi s

Lys

Pro

Ser

205

Asn

Thr

Lys

Vai

Asp 210

Lys

Arg

Vai

Glu

Pro

215

Lys

Ser cys

asp

Lys 220

Thr

Hi s

Thr cys

- 93 039569

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Vai Phe Leu

225 230 235240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu

245 250255

Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys

260 265270

Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys

275 280285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Vai Vai Ser Vai Leu

290 295300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315320

Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys

325 330335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360365

Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440445 <210> 12 <211> 214 <212> PROTEIN <213> Artificial <220>

<223> Humanized C10-2 light chain <400> 12

Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

10 15

- 94 039569

asp Arg Vai Thr 20 Met Thr cys gin Al a 25 Ser gin asp Thr Ser 30 lie Asn Leu Asn T rp 35 Phe gin gin Lys Pro 40 gly Lys Al a Pro Lys 45 Leu Leu lie Tugh Gly 50 Al a Ser Asn Leu G1 to 55 Thr gly Vai Pro Ser 60 Arg Phe Ser gly Ser 65 Arg Ser gly Thr Asp 70 Phe Thr Leu Thr lie 75 Ser Ser Leu gin Pro 80 Glu asp Met Al a Thr 85 Tyr Tyr cys Leu Gin 90 Hi s Thr Tyr Leu Pro 95 Phe Thr Phe gly Ser 100 gly Thr Lys Leu Glu 105 lie Lys Arg Thr Vai 110 Al a Al a Pro Ser Vai 115 Phe lie Phe Pro Pro 120 Ser asp Glu G1n Leu 125 Lys Ser gly Thr Al a 130 Ser Vai Vai cys Leu 135 Leu Asn Asn Phe Tyr 140 Pro Arg Glu Al a Lys 145 Vai gin T rp Lys Vai 150 asp Asn Al a Leu gin 155 Ser gly Asn Ser gin 160 Glu Ser Vai Thr Glu 165 gin asp Ser Lys Asp 170 Ser Thr Tyr Ser Leu 175 Ser Ser Thr Leu Thr 180 Leu Ser Lys Al a Asp 185 Tyr Glu Lys Hi s Lys 190 Vai Tyr Al a cys Glu 195 Vai Thr Hi s G1n Gly 200 Leu Ser Ser Pro Vai 205 Thr Lys Ser Phe Asn 210 Arg gly Glu cys

<210> 13 <211> 444 <212> PROTEIN <213> Artificial <220>

<223> Humanized C10-2 heavy chain variant D55E <400> 13

Gin Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Vai Lys Pro Gly Ala 15 10 15

Ser Vai Lys lie Ser Cys Lys Ala Ser Gly Tug Thr Phe Thr Asp Arg

- 95 039569

Thr lie His Trp Val 35

Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp lie 40 45

gly

Tyr 50 lie

Tyr

Pro

gly

G1 to 55

gly

Ser

Thr

Lys

Tyr 60

Ser

gin

Lys

Phe

Gin 65

gly

Arg

Al a

Thr

Leu 70

Thr

Al a

asp

Thr

Ser 75

Al a

Ser

Thr

Al a

Tyr 80

Met

G1u

Leu

Ser

Ser 85

Leu

Arg

Ser

Glu

Asp 90

Thr

Al a

Val

Tyr

Tyr 95 cys

Al a

Arg

Arg

Gly 100

Al a

Met

asp

Tyr

T rp 105

gly

gin

gly

Thr

Ser

110

Val

Thr

Val

Ser

Ser

115

Al a

Ser

Thr

Lys

Gly 120

Pro

Ser

Val

Phe

Pro

125

Leu

Al a

Pro

Ser

Ser

130

Lys

Ser

Thr

Ser

Gly 135

gly

Thr

Al a

Al a

Leu

140

Glycys

Leu

Val

Lys 145

asp

Tyr

Phe

Pro

Glu

150

Pro

Val

Thr

Val

Ser

155

trp

Asn

Ser

gly

Al a 160

Leu

Thr

Ser

gly

Val

165

Hi s

Thr

Phe

Pro

Al a 170

Val

Leu

gin

Ser

Ser

175

gly

Leu

Tyr

Ser

Leu

180

Ser

Ser

Val

Val

Thr

185

Val

Pro

Ser

Ser

Ser

190

Leu

gly

Thr

G1n

Thr

195

Tyr lie cys

Asn

Val

200

Asn

Hi s

Lys

Pro

Ser

205

Asn

Thr

Lys

Val

Asp 210

Lys

Arg

Val

Glu

Pro

215

Lys

Ser cys

asp

Lys 220

Thr

Hi s

Thr cys

Pro 225

Pro cys

Pro

Al a

Pro 230

G1u

Leu

Leu

gly

Gly 235

Pro

Ser

Val

Phe

Leu

240

Phe

Pro

Pro

Lys

Pro

245

Lys

asp

Thr

Leu

Met

250 lie

Ser

Arg

Thr

Pro

255

Glu

Val

Thr cys

Val

260

Val

Val

asp

Val

Ser

265

Hi s

Glu

asp

Pro

Glu

270

Val

Lys

Phe

Asn

T rp 275

Tyr

Val

asp

gly

Val

280

Glu

Val

Hi s

Asn

Al a 285

Lys

Thr

Lys

Pro

Arg 290

Glu

Glu

gin

Tyr

Asn

295

Ser

Thr

Tyr

Arg

Val

300

Val

Ser

Val

Leu

- 96 039569

Thr Vai Leu His Gin Asp Trp

305 310

Leu Asn Gly Lys Glu Tyr Lys Cys Lys

315 320

Vai Ser Asn Lys Ala Leu Pro

325

Ala Pro lie Glu Lys Thr lie Ser Lys

330 335

Ala Lys Gly Gin Pro Arg Glu

340

Pro Gin Vai Tyr Thr Leu Pro Pro Ser

345 350

Arg Glu Glu Met Thr Lys Asn

355

Gin Vai Ser Leu Thr Cys Leu Vai Lys 360 365

Gly Phe Tyr Pro Ser Asp lie

370 375

Ala Vai Glu Trp Glu Ser Asn Gly Gin 380

Pro Glu Asn Asn Tyr Lys Thr

385 390

Thr Pro Pro Vai Leu Asp Ser Asp Gly

395 400

Ser Phe Phe Leu Tyr Ser Lys

405

Leu Thr Vai Asp Lys Ser Arg Trp Gin

410 415

Gin Gly Asn Vai Phe Ser Cys

420

Ser Vai Met His Glu Ala Leu His Asn

425 430

His Tyr Thr Gin Lys Ser Leu

435

Ser Leu Ser Pro Gly 440 <210> 14 <211> 444 <212> PROTEIN <213> Artificial <220>

< 223> Variant D55Q heavy < 400> 14

Gin Vai Gin Leu Vai Gin Ser 1 5 Chain Humanized C10-2

Gly Ala Glu Vai Vai Lys Pro Gly Ala 10 15

Ser Vai Lys lie Ser Cys Lys 20

Ala Ser Gly Tyr Thr Phe Thr Asp Arg

thirty

Thr lie His Trp Vai Arg Gin 35

Ala Pro Gly Gin Gly Leu Glu Trp lie 40 45

Gly Tyr lie Tyr Pro Gly Gin

55

Gly Ser Thr Lys Tyr Ser Gin Lys Phe 60

Gin Gly Arg Ala Thr Leu Thr 65 70

Ala Asp Thr Ser Ala Ser Thr Ala Tyr 75 80

Met Glu Leu Ser Ser Leu Arg

Ser Glu Asp Thr Ala Vai Tyr Tyr Cys

- 97 039569

90 95

Al a Arg Arg Gly 100 Al a Met asp Tyr T rp 105 gly gin gly Thr Ser 110 Val Thr Vai Ser Ser 115 Al a Ser Thr Lys Gly 120 Pro Ser Val Phe Pro 125 Leu Al a Pro Ser Ser 130 Lys Ser Thr Ser Gly 135 gly Thr Al a Al a Leu 140 gly cys Leu Val Lys 145 asp Tyr Phe Pro Glu 150 Pro Vai Thr Val Ser 155 trp Asn Ser gly Al a 160 Leu Thr Ser gly Vai 165 Hi s Thr Phe Pro Al a 170 Val Leu gin Ser Ser 175 gly Leu Tyr Ser Leu 180 Ser Ser Vai Vai Thr 185 Val Pro Ser Ser Ser 190 Leu gly Thr G1n Thr 195 Tyr lie cys Asn Val 200 Asn Hi s Lys Pro Ser 205 Asn Thr Lys Vai Asp 210 Lys Arg Vai Glu Pro 215 Lys Ser cys asp Lys 220 Thr Hi s Thr cys Pro 225 Pro cys Pro Al a Pro 230 G1u Leu Leu gly Gly 235 Pro Ser Val Phe Leu 240 Phe Pro Pro Lys Pro 245 Lys asp Thr Leu Met 250 lie Ser Arg Thr Pro 255 Glu Vai Thr cys Vai 260 Vai Vai asp Val Ser 265 Hi s Glu asp Pro Glu 270 Val Lys Phe Asn T rp 275 Tyr Vai asp gly Val 280 Glu Val Hi s Asn Al a 285 Lys Thr Lys Pro Arg 290 Glu Glu gin Tyr Asn 295 Ser Thr Tyr Arg Val 300 Val Ser Val Leu Thr 305 Vai Leu Hi s gin Asp 310 trp Leu Asn gly Lys 315 G1u Tyr Lys Cys Lys 320 Vai Ser Asn Lys Al a 325 Leu Pro Al a Pro lie 330 Glu Lys Thr lie Ser 335 Lys Al a Lys gly gin 340 Pro Arg G1u Pro gin 345 Val Tyr Thr Leu Pro 350 Pro Ser Arg G1u Glu 355 Met Thr Lys Asn gin 360 Val Ser Leu Thr cys 365 Leu Val Lys

- 98 039569

Gly Phe Tug Pro Ser Asp lie Ala

370 375

Vai Glu Trp Glu Ser Asn Gly Gin

380

Pro Glu Asn Asn Tyr Lys Thr Thr

385 390

Pro Pro Vai Leu Asp Ser Asp Gly

395 400

Ser Phe Phe Leu Tyr Ser Lys Leu

405

Thr Val Asp Lys Ser Arg Trp Gin

410 415

Gin Gly Asn Vai Phe Ser Cys Ser

420

Val Met His Glu Ala Leu His Asn

425 430

His Tyr Thr Gin Lys Ser Leu Ser

435 440

Leu Ser Pro Gly <210> 15 <211> 444 < 212> PROTEIN < 213> Artificial <220>

< 223> Variant D55S heavy flail < 400> 15

Gin Vai Gin Leu Vai Gin Ser Gly 1 5 and humanized C10-2

Ala Glu Val Val Lys Pro Gly Ala

fifteen

Ser Vai Lys lie Ser Cys Lys Ala 20

Ser Gly Tyr Thr Phe Thr Asp Arg 25 30

Thr lie His Trp Vai Arg Gin Ala 35 40

Pro Gly Gin Gly Leu Glu Trp lie 45

Gly Tyr lie Tyr Pro Gly Ser Gly

55

Ser Thr Lys Tyr Ser Gin Lys Phe 60

Gin Gly Arg Ala Thr Leu Thr Ala 65 70

Asp Thr Ser Ala Ser Thr Ala Tyr

80

Met Glu Leu Ser Ser Leu Arg Ser 85

Glu Asp Thr Ala Val Tyr Tyr Cys

95

Ala Arg Arg Gly Ala Met Asp Tyr

100

Trp Gly Gin Gly Thr Ser val Thr

105 110

Vai Ser Ser Ala Ser Thr Lys Gly

115 120

Pro Ser Val Phe Pro Leu Ala Pro

125

Ser Ser Lys Ser Thr Ser Gly Gly

130 135

Thr Ala Ala Leu Gly Cys Leu Val

140

Lys Asp Tyr Phe Pro Glu Pro Vai

Thr Val Ser Trp Asn Ser Gly Ala

- 99 039569

145 150 155160

Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly

165 170175

Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly

180 185190

Thr Gin Thr Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys

195 200205

Vai Asp Lys Arg Vai Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Vai Phe Leu 225 230 235240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu

245 250255

Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys

260 265270

Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys

275 280285

Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Vai Vai Ser Vai Leu

290 295300

Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315320

Vai Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys

325 330335

Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser

340 345350

Arg Glu Glu Met Thr Lys Asn Gin Vai Ser Leu Thr Cys Leu Vai Lys

355 360365

Gly Phe Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin

370 375380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly

385 390 395400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin

405 410415

Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn

420 425430

- 100 039569

His Tyr Thr Gin Lys Ser Leu Ser

435 440

Leu Ser Pro Gly <210> 16 <211> 214 <212> PROTEIN <213> Artificial <220>

< 223> Light chain option N32S < 400> 16

Asp Vai Gin Met Thr Gin Ser Pro 1 5 Humanized C10-2

Ser Ser Leu Ser Ala Ser Val Gly

fifteen

Asp Arg Vai Thr Met Thr Cys Gin 20

Ala Ser Gin Asp Thr Ser lie Ser 25 30

Leu Asn Trp Phe Gin Gin Lys Pro 35 40

Gly Lys Ala Pro Lys Leu Leu lie 45

Tyr Gly Ala Ser Asn Leu Glu Thr

55

Gly Val Pro Ser Arg Phe Ser Gly 60

Ser Arg Ser Gly Thr Asp Phe Thr 65 70

Leu Thr lie Ser Ser Leu Gin Pro

80

Glu Asp Met Ala Thr Tyr Tyr Cys 85

Leu Gin His Thr Tyr Leu Pro Phe

95

Thr Phe Gly Ser Gly Thr Lys Leu

100

Glu lie Lys Arg Thr Val Ala Ala

105 110

Pro Ser Vai Phe lie Phe Pro Pro

115 120

Ser Asp Glu Gin Leu Lys Ser Gly

125

Thr Ala Ser Vai Vai Cys Leu Leu

130 135

Asn Asn Phe Tyr Pro Arg Glu Ala

140

Lys Vai Gin Trp Lys Vai Asp Asn

145 150

Ala Leu Gin Ser Gly Asn Ser Gin

155 160

Glu set val Thr Glu Gin Asp set

165

Lys Asp set Thr Tyr set Leu set

170 175

Ser Thr Leu Thr Leu Ser Lys Ala

180

Asp Tyr Glu Lys His Lys Val Tyr

185 190

Ala Cys Glu Val Thr His Gin Gly

195 200

Leu Ser Ser Pro Val Thr Lys Ser

205

Phe Asn Arg Gly Glu Cys

- 101 039569

210 <210> 17 <211> 214 <212> PROTEIN <213> Artificial <220>

<223> Light chain variant N32Q of humanized C10-2 <400> 17

Asp Val 1 Gln Met Thr Gln 5 Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 gly AspArg Val Thr 20 Met Thr cys Gln Al a 25 Ser Gln Asp Thr Ser thirty lie Gln Leu Asn T rp 35 Phe Gln Gln Lys Pro 40 gly Lys Ala Pro Lys Leu 45 Leu lie Tyr Gly fifty Al a Ser Asn Leu G1u 55 Thr gly Val Pro Ser 60 Arg Phe Ser gly Ser Arg 65 Ser gly Thr Asp 70 Phe Thr Leu Thr Lie Ser 75 Ser Leu Gln Pro 80 Glu Asp Met Al a Thr Tyr 85 Tyr cys Leu Glin 90 His Thr Tyr Leu Pro 95 Phe Thr Phe gly Ser 100 Gly Thr Lys Leu Glu 105 lie Lys Arg Thr Val 110 Al a Al a Pro Ser Val 115 Phe lie Phe Pro Pro 120 Ser asp Glu Gln Leu Lys 125 Ser gly Thr Ala 130 Ser Val Val Cys Leu 135 Leu Asn Asn Phe Tyr 140 Pro Arg Glu Al a Lys Val 145 Gln T rp Lys Val 150 asp Asn Al a Leu Gln Ser 155 Gly Asn Ser Gln 160 Glu Ser Val Thr Glu Gln 165 asp Ser Lys Asp 170 Ser Thr Tyr Ser Leu 175 Ser Ser Thr Leu Thr 180 Leu Ser Lys Al a Asp 185 Tyr G1u Lys His Lys 190 Val Tyr Ala Cys Glu 195 Val Thr His G1n Gly 200 Leu Ser Ser Pro Val Thr 205 Lys Ser

Phe Asn Arg Gly Glu Cys

210

- 102 039569 <210> 18 <211> 214 <212> PROTEIN <213> Artificial <220>

< 223> Light chain option N34S < 400> 18

Asp Vai Gin Met Thr Gin Ser Pro 1 5 Humanized C10-2

Ser Ser Leu Ser Ala Ser Vai Gly

fifteen

Asp Arg Vai Thr Met Thr Cys Gin 20

Ala Ser Gin Asp Thr Ser lie Asn 25 30

Leu Ser Trp Phe Gin Gin Lys Pro

40

Gly Lys Ala Pro Lys Leu Leu lie 45

Tyr Gly Ala Ser Asn Leu Glu Thr

55

Gly Vai Pro Ser Arg Phe Ser Gly 60

Ser Arg Ser Gly Thr Asp Phe Thr 65 70

Leu Thr lie Ser Ser Leu Gin Pro

80

Glu Asp Met Ala Thr Tyr Tyr Cys

Leu Gin His Thr Tyr Leu Pro Phe

95

Thr Phe Gly Ser Gly Thr Lys Leu

100

Glu lie Lys Arg Thr Vai Ala Ala

105 110

Pro Ser Vai Phe lie Phe Pro Pro

115 120

Ser Asp Glu Gin Leu Lys Ser Gly

125

Thr Ala Ser Vai Vai Cys Leu Leu

130 135

Asn Asn Phe Tyr Pro Arg Glu Ala

140

Lys Vai Gin Trp Lys Vai Asp Asn

145 150

Ala Leu Gin Ser Gly Asn Ser Gin

155 160

Glu Ser Vai Thr Glu Gin Asp Ser

165

Lys Asp Ser Thr Tyr Ser Leu Ser

170 175

Ser Thr Leu Thr Leu Ser Lys Ala

180

Asp Tyr Glu Lys His Lys Vai Tyr

185 190

Ala Cys Glu Vai Thr His Gin Gly

195 200

Leu Ser Ser Pro Vai Thr Lys Ser

205

Phe Asn Arg Gly Glu Cys

210 <210> 19 <211> 214 <212> PROTEIN

- 103 039569 <213> Faux <220>

<223> Light chain variant N34Q of humanized C10-2 <400> 19

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

1015

Asp Arg Val Thr 20 Met Thr Cys G1n Ala Ser 25 gin asp Thr Ser 30 lie Asn Leu Gin Trp Phe Gin Gin Lys Pro Gly Lys Al a Pro Lys Leu Leu lie

40 45

Tyr Gly Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 5560

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gin His Thr Tyr Leu Pro Phe 85 9095

Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys Arg Thr Val Ala Ala

100 105110

Pro Ser Val Phe lie Phe Pro Ser Asp Glu Gin Leu Lys Ser Gly

115 120125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

145 150 155160

Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185190

Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200205

Phe Asn Arg Gly Glu Cys

210 <210> 20 <211> 214 <212> PROTEIN <213> Artificial <220>

- 104 039569 <223> Light chain variant N32S, N34S of humanized C10-2 <400> 20

Asp Vai Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 15 1015

Asp Arg Vai Thr Met Thr Cys Gin Ala Ser Gin Asp Thr Ser lie Ser 20 2530

Leu Ser Trp Phe Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 4045

Tug Gly Ala Ser Asn Leu Glu Thr Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gin His Thr Tyr Leu Pro Phe 85 9095

Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys Arg Thr Vai Ala Ala

100 105110

Pro Ser Vai Phe lie Phe Pro Ser Asp Glu Gin Leu Lys Ser Gly

115 120125

Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135140

Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

145 150 155160

Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr

180 185190

Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser

195 200205

Phe Asn Arg Gly Glu Cys

210 <210> 21 <211> 214 <212> PROTEIN <213> Artificial <220>

<223> Light chain variant N32Q, N34S of humanized C10-2 <400> 21

- 105 039569

asp 1

Val

gin

Met

Thr 5

gin

Ser

Pro

Ser

Ser 10

Leu

Ser

Al a

Ser

Val 15

gly

asp

Arg

Val

Thr 20

Met

Thr cys

gin

Al a 25

Ser

gin

asp

Thr

Ser 30 lie

gin

Leu

Ser

T rp 35

Phe

gin

gin

Lys

Pro 40

gly

Lys

Al a

Pro

Lys 45

Leu

Leu lie

Tyr

Gly 50

Al a

Ser

Asn

Leu

G1u

Thr

gly

Val

Pro

Ser 60

Arg

Phe

Ser

gly

Ser 65

Arg

Ser

gly

Thr

Asp 70

Phe

Thr

Leu

Thr lie 75

Ser

Ser

Leu

gin

Pro 80

Glu

asp

Met

Al a

Thr 85

Tyr

Tyr cys

Leu

Gin 90

Hi s

Thr

Tyr

Leu

Pro 95

Phe

Thr

Phe

gly

Ser

100

gly

Thr

Lys

Leu

Glu

105 lie

Lys

Arg

Thr

Val

110

Al a

Al a

Pro

Ser

Val

115

Phe lie

Phe

Pro

Pro

120

Ser

asp

Glu

G1n

Leu

125

Lys

Ser

gly

Thr

Al a 130

Ser

Val

Valcys

Leu

135

Leu

Asn

Asn

Phe

Tyr 140

Pro

Arg

Glu

Al a

Lys 145 Val Gin Trp Lys Val 150 Asp Asn Al a Leu gin 155 Ser gly Asn Ser Gin 160 Glu Ser Val Thr Glu gin Asp Ser Lys asp Ser Thr Tyr Ser Leu Ser

165

170

175

Ser

Thr

Leu

Thr

180

Leu

Ser

Lys

Al a

Asp 185

Tyr

Glu

Lys

Hi s

Lys 190

Val

Tyr

Al a

Cys

Glu

195

Val

Thr

Hi s

G1n

Gly 200

Leu

Ser

Ser

Pro

Val

205

Thr

Lys

Ser

Phe

Asn

210

Arg

gly

Glu cys < 210> 22 <211> 214 < 212> PROTEIN < 213> Artificial <220>

<223> Light chain variant N32Q, N34Q of humanized C10-2 <400> 22

Asp Val Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

- 106 039569

Asp Arg Vai Thr Met Thr Cys Gin Ala Ser Gin Asp Thr Ser lie Gin 20 2530

Leu Gin Trp Phe Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 4045

Tyr Gly Ala Ser Asn Leu Glu Thr Gly Vai Pro Ser Arg Phe Ser Gly 50 5560

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 7580

Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gin His Thr Tyr Leu Pro Phe 85 9095

Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys Arg Thr Vai Ala Ala

100 105110

Pro Ser Vai Phe lie Phe Pro Ser Asp Glu Gin Leu Lys Ser Gly

115 120125

Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135140

Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

145 150 155160

Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr

180 185190

Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser

195 200205

Phe Asn Arg Gly Glu Cys

210 <210> 23 <211> 214 <212> PROTEIN <213> Artificial <220>

<223> Light chain variant N32S, N34Q of humanized C10-2 <400> 23

Asp Vai Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Gly 15 10 15

Asp Arg Vai Thr Met Thr Cys Gin Ala Ser Gin Asp Thr Ser lie Ser

- 107 039569

Leu Gin Trp Phe

Gin Gin Lys Pro

Gly Lys Ala Pro

Lys Leu Leu lie 45

Tyr Gly Ala Ser 50

Asn Leu Glu Thr

Gly Val Pro Ser 60

Arg Phe Ser Gly

Ser Arg Ser Gly 65

Thr Asp Phe Thr 70

Leu Thr lie Ser

Ser Leu Gin Pro

Glu Asp Met Ala

Thr Tyr Tyr Cys 85

Leu Gin His Thr

Tyr Leu Pro Phe 95

Thr Phe Gly Ser

100

Gly Thr Lys Leu

Glu lie Lys Arg 105

Thr Val Ala Ala

110

Pro Ser Val Phe

115 lie Phe Pro Pro

120

Ser Asp Glu Gin

Leu Lys Ser Gly 125

Thr Ala Ser Val

130

Val Cys Leu Leu

135

Asn Asn Phe Tyr

140

Pro Arg Glu Ala

Lys Val Gin Trp 145

Lys Val Asp Asn

150

Ala Leu Gin Ser

155

Gly Asn Ser G1 n

160

Glu Ser Val Thr

Glu Gin Asp Ser 165

Lys Asp Ser Thr

170

Tyr Ser Leu Ser

175

Ser Thr Leu Thr

180

Leu Ser Lys Ala

Asp Tyr Glu Lys 185

His Lys Val Tyr

190

Al a Cys G1u Val

195

Thr His Gin Gly

200

Leu Ser Ser Pro

Val Thr Lys Ser 205

Phe Asn Arg Gly Glu Cys

210

<210> <211> <212> <213> 24 444 PROTEIN Artificial <220> <223> Heavy chain variant A101T of humanized C10-2 <400> 24 Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Val Lys Pro Gly Ala

10 15

Ser Val Lys lie Ser Cys 20

Lys Ala Ser Gly Tyr Thr 25

Phe Thr Asp Arg 30

- 108 039569

Thr lie Hi s 35 T rp Val Arg G1n Ala Pro Gly 40 gin gly Leu 45 Glu T rp lie Gly Tyr fifty lie Tyr Pro Gly Asp 55 Gly Ser Thr Lys Tyr 60 Ser gin Lys Phe Gin Gly 65 Arg Al a Thr Leu Thr 70 Ala Asp Thr Ser 75 Al a Ser Thr Al a Tyr 80 MetGlu Leu Ser Ser 85 Leu Arg Ser Glu Asp 90 Thr Al a Val Tyr Tyr 95 cys Ala Arg Arg Gly 100 Thr Met Asp Tyr Trp Gly 105 gin gly Thr Ser 110 Val Thr Val Ser Ser 115 Al a Ser Thr Lys Gly Pro Ser 120 Val Phe Pro 125 Leu Al a Pro Ser Ser 130 Lys Ser Thr Ser Gly 135 Gly Thr Ala Al a Leu 140 gly cys Leu Val Lys Asp 145 Tyr Phe Pro Glu Pro 150 Val Thr Val Ser 155 trp Asn Ser gly Al a 160 Leu Thr Ser gly Val 165 His Thr Phe Pro Ala 170 Val Leu gin Ser Ser 175 gly Leu Tyr Ser Leu 180 Ser Ser Val Val Thr Val 185 Pro Ser Ser Ser 190 Leu gly Thr Gin Thr 195 Tyr lie Cys Asn Val Asn His 200 Lys Pro Ser 205 Asn Thr Lys Val Asp 210 Lys Arg Val GluPro 215 Lys Ser Cys asp Lys 220 Thr Hi s Thr cys Pro Pro 225 cys Pro Al a ProGlu 230 Leu Leu Gly Gly 235 Pro Ser Val Phe Leu 240 PhePro Pro Lys Pro 245 Lys Asp Thr Leu Met 250 lie Ser Arg Thr Pro 255 Glu Val Thr cys Val 260 Val Val Asp Val Ser His 265 Glu asp Pro Glu 270 Val Lys Phe Asn T rp 275 Tyr Val Asp Gly Val Glu Val 280 Hi s Asn Al a 285 Lys Thr Lys Pro Arg 290 Glu Glu gin Tyr Asn 295 Ser Thr Tyr Arg Val 300 Val Ser Val Leu Thr Val Leu Hi s gin Asp Trp Leu Asn Gly Lys G1u Tyr Lys cys Lys

- 109 039569

305 310 315320

Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys

325 330335

Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser

340 345350

Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys

355 360365

Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin

370 375380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin

405 410415

Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425430

His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly

435440 <210> 25 <211> 444 <212> PROTEIN <213> Artificial <220>

<223> Variant D55E, A101T humanized C10-2 heavy chain <400> 25

Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Val Lys Pro Gly Ala 15 10 15

Serval Lys lie Ser 20 cys Lys Ala Ser 25 Gly Tyr Thr Phe Thr 30 asp Arg Thr lie His Trp Val Arg G1 n Al a Pro Gly Gin gly Leu Glu T rp lie

40 45

gly Tyr lie 50 Tyr Pro Gly G1 and 55 Gly Ser Thr Lys Tyr 60 Ser Gin Lys Phe gin Gly Arg Al a Thr Leu Thr Ala Asp Thr Ser Al a Ser Thr Al a Tyr

70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

- 110 039569

Ala Arg Arg Gly Thr 100 Met Asp Tyr T rp 105 gly gin gly Thr Ser 110 Val Thr Val Ser Ser Al a Ser Thr Lys gly Pro Ser Val Phe Pro Leu Al a Pro

115

120

125

Ser

Ser

130

Lys

Ser

Thr

Ser

Gly 135

gly

Thr

Al a

Al a

Leu

140

Glycys

Leu

Vai

Lys Asp 145 Tyr Phe Pro Glu 150 Pro Val Thr Val Ser 155 trp Asn Ser gly Al a 160 Leu Thr Ser gly Val Hi s Thr Phe ProAla Val Leu gin Ser Ser gly

165

170

175

Leu

Tyr

Ser

Leu

180

Ser

Ser

Vai

Vai

Thr

185

Vai

Pro

Ser

Ser

Ser

190

Leu

gly

Thr

G1n

Thr

195

Tyr lie cys

Asn

Val

200

Asn

Hi s

Lys

Pro

Ser

205

Asn

Thr

Lys

Vai

Asp 210

Lys

Arg

Vai

Glu

Pro

215

Lys

Ser cys

asp

Lys 220

Thr

Hi s

Thr cys

Pro

225

Pro cys

Pro

Al a

Pro

230

G1u

Leu

Leu

gly

Gly 235

Pro

Ser

Vai

Phe

Leu

240

Phe

Pro

Pro

Lys

Pro

245

Lys

asp

Thr

Leu

Met

250 lie

Ser

Arg

Thr

Pro

255

Glu

Vai

Thr cys

Val

260

Vai

Vai

asp

Vai

Ser

265

Hi s

Glu

asp

Pro

Glu

270

Vai

Lys

Phe

Pro

Arg 290

Asn

Trp Tyr 275 Val asp Gly Val 280 Glu Val Hi s Asn Al a 285 Glu Glu gin Tyr Asn Ser Thr Tyr Arg Val Val

Lys

Ser

295

300

Thr

Vai

Lys

Leu

Thr

305

Vai

Leu

Hi s

gin

Asp 310

trp

Leu

Asn

gly

Lys

315

G1u

Tyr

Lys cys

Lys

320

Val

Ser

Asn

Lys

Al a 325

Leu

Pro

Al a

Pro lie

330

Glu

Lys

Thr lie

Ser

335

Lys

Al a

Lys

gly

gin

340

Pro

Arg

G1u

Pro

gin

345

Val

Tyr

Thr

Leu

Pro

350

Pro

Ser

Arg

G1u

Glu

355

Met

Thr

Lys

Asn

gin

360

Val

Ser

Leu

Thr cys 365

Leu

Val

Lys

gly

Phe

Tyr

Pro

Ser

asp lie

Al a

Val

Glu

T rp

G1u

Ser

Asn

gly

gin

- 111 039569

370 375380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser Asp Gly

385 390 395400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435440 <210> 26 <211> 444 <212> PROTEIN <213> Artificial <220>

<223> Variant D55Q, A101T humanized C10-2 heavy chain <400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 15 1015

Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Arg 20 2530

Thr lie His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp lie 35 4045

Gly Tyr lie Tyr Pro Gly Gln Gly Ser Thr Lys Tyr Ser Gln Lys Phe 50 5560

Gln Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 7580

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 9095

Ala Arg Arg Gly Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

100 105110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155160

- 112 039569

Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly

165 170175

Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly

180 185190

Thr Gin Thr Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys

195 200205

Vai Asp Lys Arg Vai Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Vai Phe Leu

225 230 235240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu

245 250255

Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys

260 265270

Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys

275 280285

Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Vai Vai Ser Vai Leu

290 295300

Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315320

Vai Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys

325 330335

Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser

340 345350

Arg Glu Glu Met Thr Lys Asn Gin Vai Ser Leu Thr Cys Leu Vai Lys

355 360365

Gly Phe Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin

370 375380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly

385 390 395400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin

405 410415

Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn

420 425430

His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly

- 113 039569

435 440 <210> 27 <211> 444 <212> PROTEIN <213> Artificial <220>

<223> Variant D55S, A101T humanized C10-2 heavy chain <400> 27

Gin Vai Gin Leu Vai Gin Ser Gly 1 5

Ala Glu Vai Vai Lys Pro Gly Ala

fifteen

Ser Vai Lys lie Ser Cys Lys Ala 20

Ser Gly Tyr Thr Phe Thr Asp Arg 25 30

Thr lie His Trp Vai Arg Gin Ala 35 40

Pro Gly Gin Gly Leu Glu Trp lie 45

Gly Tyr lie Tyr Pro Gly Ser Gly

55

Ser Thr Lys Tyr Ser Gin Lys Phe 60

Gin Gly Arg Ala Thr Leu Thr Ala 65 70

Asp Thr Ser Ala Ser Thr Ala Tyr

80

Met Glu Leu Ser Ser Leu Arg Ser 85

Glu Asp Thr Ala Vai Tyr Tyr Cys

95

Ala Arg Arg Gly Thr Met Asp Tyr

100

Trp Gly Gin Gly Thr Ser Vai Thr

105 110

Vai Ser Ser Ala Ser Thr Lys Gly

115 120

Pro Ser Vai Phe Pro Leu Ala Pro

125

Ser Ser Lys Ser Thr Ser Gly Gly

130 135

Thr Ala Ala Leu Gly Cys Leu Vai

140

Lys Asp Tyr Phe Pro Glu Pro Vai

145 150

Thr Vai Ser Trp Asn Ser Gly Ala

155 160

Leu Thr Ser Gly Vai His Thr Phe

165

Pro Ala Vai Leu Gin Ser Ser Gly

170 175

Leu Tyr Ser Leu Ser Ser Vai Vai

180

Thr Vai Pro Ser Ser Ser Leu Gly

185 190

Thr Gin Thr Tyr lie Cys Asn Vai

195 200

Asn His Lys Pro Ser Asn Thr Lys

205

Vai Asp Lys Arg Vai Glu Pro Lys

210 215

Ser Cys Asp Lys Thr His Thr Cys

220

- 114 039569

Pro 225 Pro cys Pro Al a Pro 230 G1u Leu Leu gly Gly 235 Pro Ser Val Phe Leu 240 Phe Pro Pro Lys Pro 245 Lys asp Thr Leu Met 250 lie Ser Arg Thr Pro 255 Glu Vai Thr cys Vai 260 Vai Vai asp Vai Ser 265 Hi s Glu asp Pro Glu 270 Val Lys Phe Asn T rp 275 Tyr Vai asp gly Vai 280 Glu Val Hi s Asn Al a 285 Lys Thr Lys Pro Arg 290 Glu Glu gin Tyr Asn 295 Ser Thr Tyr Arg Val 300 Val Ser Val Leu Thr 305 Vai Leu Hi s gin Asp 310 trp Leu Asn gly Lys 315 G1u Tyr Lys cys Lys 320 Vai Ser Asn Lys Al a 325 Leu Pro Al a Pro lie 330 Glu Lys Thr lie Ser 335 Lys Al a Lys gly gin 340 Pro Arg G1u Pro gin 345 Val Tyr Thr Leu Pro 350 Pro Ser Arg G1u Glu 355 Met Thr Lys Asn gin 360 Vai Ser Leu Thr cys 365 Leu Val Lys gly Phe 370 Tyr Pro Ser asp lie 375 Al a Vai Glu T rp G1 and 380 Ser Asn gly gin Pro 385 G1u Asn Asn Tyr Lys 390 Thr Thr Pro Pro Val 395 Leu asp Ser asp Gly 400 Ser Phe Phe Leu Tyr 405 Ser Lys Leu Thr Val 410 asp Lys Ser Arg T rp 415 gin gin gly Asn Vai 420 Phe Ser cys Ser Vai 425 Met Hi s G1u Al a Leu 430 Hi s Asn Hi s Tyr Thr 435 gin Lys Ser Leu Ser 440 Leu Ser Pro gly

< 210> 28 < 211> 15 < 212> PROTEIN < 213> Artificial <220>

<223> Humanized C10-2 heavy chain CDR2 variant D55E <400> 28

Pro Gly Glu Gly Ser Thr Lys Tyr Ser Gin Lys Phe Gin Gly Arg 15 10 15

- 115 039569 <210> 29 <211> 15 <212> PROTEIN <213> Artificial <220>

<223> Humanized C10-2 heavy chain CDR2 variant D55Q <400> 29

Pro Gly Gin Gly Ser Thr Lys Tyr Ser Gin Lys Phe Gin Gly Arg 15 10 15 < 210> 30 <211> 15 < 212> PROTEIN < 213> Artificial <220>

<223> Humanized C10-2 heavy chain CDR2 variant D55S <400> 30

Pro Gly Ser Gly Ser Thr Lys Tyr 1 5

Ser Gin Lys Phe Gin Gly Arg

15 <210> 31 <211> 11 <212> PROTEIN <213> Artificial <220>

< 223> N32S CDR1 light variant < 400> 31

Gin Ala Ser Gin Asp Thr Ser lie 1 5 chain humanized C10-2

Ser Leu Asn < 210> 32 < 211> 11 < 212> PROTEIN < 213> Artificial <220>

< 223> N32Q CDR1 light variant < 400> 32

Gin Ala Ser Gin Asp Thr Ser lie 1 5 chain humanized C10-2

Gin Leu Asn < 210> 33 < 211> 11 < 212> PROTEIN < 213> Artificial <220>

< 223> N34S CDR1 light variant < 400> 33

Gin Ala Ser Gin Asp Thr Ser lie 1 5 chain humanized C10-2

Asn Leu Ser

- 116 039569 <210> 34 <211> 11 <212> PROTEIN <213> Artificial <220>

<223> Light chain N34Q CDR1 variant of humanized C10-2 <400> 34

Gin Ala Ser Gin Asp Thr Ser lie Asn Leu Gin 15 10 <210> 35 <211> 11 <212> PROTEIN <213> Artificial <220>

<223> Variant N32S, N34S light chain CDR1 humanized C10-2 <400> 35

Gin Ala Ser Gin Asp Thr Ser lie Ser Leu Ser 15 10 < 210> 36 < 211> 11 < 212> PROTEIN < 213> Artificial <220>

<223> Variant N32Q, N34S light chain CDR1 humanized C10-2 <400> 36

Gin Ala Ser Gin Asp Thr Ser lie Gin Leu Ser 15 10 < 210> 37 < 211> 11 < 212> PROTEIN < 213> Artificial <220>

<223> Variant N32Q, N34Q light chain CDR1 humanized C10-2 <400> 37

Gin Ala Ser Gin Asp Thr Ser lie Gin Leu Gin

10 < 210> 38 < 211> 11 < 212> PROTEIN < 213> Artificial <220>

<223> Variant N32S, N34Q light chain CDR1 humanized C10-2 <400> 38

Gin Ala Ser Gin Asp Thr Ser lie Ser Leu Gin

- 117 039569

one 5 10 <210> <211> <212> <213> 39 6 PROTEIN Artificial <220> <223> Heavy chain variant A101T CDR3 of humanized C10-2 <400> 39

Arg Gly Thr Met Asp Tyr

one 5 <2Yu> <211> <212> <213> 40 6 PROTEIN Artificial <220> <223> IMGT SYU. 2LC CDR1 <400> 40

Gin Asp Thr Ser lie Asn

one 5 <2Yu> <211> <212> <213> 41 four PROTEIN Artificial <220> <223> IMGT SYU. 2LC CDR2 <400> 41

Gly Ala Ser 1

<2Yu> <211> <212> <213> 42 9 PROTEIN Artificial <220> <223> IMGT SYU. 2LC CDR3 <400> 42

Leu Gin His Thr Tyr Leu Pro Phe Thr 1 5

<2Yu> <211> <212> <213> 43 eight PROTEIN Artificial <220> <223> IMGT SYU. 2 NS CDR1 <400> 43

- 118 039569

Gly Tyr Thr Phe Thr Asp Arg Thr

one 5 <210> <211> <212> <213> 44 8 PROTEIN Artificial <220> <223> IMGT CIO.2HC CDR2 <400> 44

lie Tyr Pro Gly Asp Gly Ser Thr

one 5 <210> <211> <212> <213> 45 8 PROTEIN Artificial <220> <223> IMGT CIO.2HC CDR3 <400> 45

Ala Arg Arg Gly Ala Met Asp Tyr

one 5 <210> <211> <212> <213> 46 6 PROTEIN Artificial <220> <223> IMGT N32S/ N32S,A101T LC CDR1 <400> 46

Gln Asp Thr Ser lie Ser

one 5 <210> <211> <212> <213> 47 four PROTEIN Artificial <220> <223> IMGT N32S/N32S,A101T LC CDR2 <400> 47

Gly Ala Ser 1

<210> <211> <212> <213> 48 9 PROTEIN Artificial <220> <223> IMGT N32S/N32S,A101T LC CDR3 <400> 48

- 119 039569

Leu Gin His Thr Tyr Leu Pro Phe Thr 1 5 < 210> 49 < 211> 8 < 212> PROTEIN < 213> Artificial <220>

< 223> IMGT A101T/N32S,A101T HC CDR1 < 400> 49

Gly Tyr Thr Phe Thr Asp Arg Thr

5 < 210> 50 < 211> 8 < 212> PROTEIN < 213> Artificial <220>

< 223> IMGT A101T/N32S,A101T HC CDR2 < 400> 50 lie Tyr Pro Gly Asp Gly Ser Thr

5 < 210> 51 < 211> 8 < 212> PROTEIN < 213> Artificial <220>

< 223> IMGT A101T/N32S,A101T HC CDR3 < 400> 51

Ala Arg Arg Gly Thr Met Asp Tyr

5

<210> 52 <211> 7 <212> PROTEIN <213> Artificial <220> <223> CDR1 HC C10.2/N32S according to Chotia <400> 52 Gly Tyr ’ Thr Phe Thr Asp Arg one 5 <210> 53 <211> 6

<212> PROTEIN <213> Artificial <220>

<223> CDR2 HC C10.2/N32S according to Chotia

- 120 039569 <400> 53

Tyr Pro Gly Asp Gly Ser

one 5 <210> <211> <212> <213> 54 6 PROTEIN Artificial <220> <223> CDR3 NS C10.2/N32S according to Chotia <400> 54

Arg Gly Ala Met Asp Tyr

one 5 <210> <211> <212> <213> 55 7 PROTEIN Artificial <220> <223> CDR1 HC A101T/N32S, A101T according to Chotia <400> 55

Gly Tyr Thr Phe Thr Asp Arg

one 5 <210> <211> <212> <213> 56 6 PROTEIN Artificial <220> <223> CDR2 NS A101T/N32S, A101T according to Chotia <400> 56

Pro Gly Asp Gly Ser

one 5 <210> <211> <212> <213> 57 6 PROTEIN Artificial <220> <223> CDR3 NS A101T/N32S, A101T according to Chotia <400> 57

Arg Gly Thr Met Asp Tyr 1 5

1049-WO-PCT

Claims (6)

1. A pharmaceutical composition containing a monoclonal antibody that specifically binds to a phosphorylated serine residue 396 of the human tau protein (2N4R isoform), or its epitope-binding fragment, and a pharmaceutically acceptable carrier, wherein the monoclonal antibody or its epitope-binding fragment contains: (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31; (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 4; (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 5; (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 6; (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7; and - 121 039569 (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 39. 2. Pharmaceutical composition according to claim 1, wherein the monoclonal antibody or its epitope-binding fragment contains: (a) a light chain containing the amino acid sequence of SEQ ID NO: 16; (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 24. 3. The pharmaceutical composition of claim 2, wherein the epitope-binding fragment of said monoclonal antibody is a Fab' or Fab fragment. 4. Pharmaceutical composition according to claims 1-3, containing an antibody or epitope-binding fragment thereof, as defined in claims 1-3, which were obtained in a cell line or using recombinant technology. 5. The pharmaceutical composition of claim 4, wherein the cell line is a human cell line, a non-human mammalian cell line, an insect cell line, a yeast or a bacteria. 6. Pharmaceutical composition according to claim 4, containing an antibody or epitope-binding fragment thereof, as defined in claims 1-3, obtained in a CHO cell line, a HEK cell line, a BHK21 cell line, a mouse cell line (such as a myeloma cell line), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines, and HuH-7 human cell lines.