NZ709976B2 - Human anti-tau antibodies - Google Patents

Abstract

Provided are novel human tau-specific antibodies as well as fragments, derivatives and variants thereof as well as methods related thereto. Assays, kits, and solid supports related to antibodies specific for tau are also disclosed. The antibody, immunoglobulin chain(s), as well as binding fragments, derivatives and variants thereof can be used in pharmaceutical and diagnostic compositions for tau targeted immunotherapy and diagnosis, respectively. derivatives and variants thereof can be used in pharmaceutical and diagnostic compositions for tau targeted immunotherapy and diagnosis, respectively.

Description

- l - HUMAN ANTi-TAU ANTIBODIES BACKGROUND OF THE INVENTION Field of the Invention The present invention generally relates to novel tau-specific binding molecules, ularly human antibodies as well as fragments, derivatives and variants thereof that recognize the tau protein, including pathologically phosphorylated tau and aggregated forms of tau. In addition, the present invention relates to pharmaceutical and diagnostic compositions sing such binding molecules, antibodies and mimics thereof valuable both as a diagnostic tool to identify tau and toxic tau species in plasma and CSF and also in passive vaccination gies for ng neurodegenerative thies such as mer’s disease (AD), amyotrophic lateral sclerosis/parkinsonism—dementia complex (ALS-PDC), argyrophilic grain dementia (AGD), British type amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with cation, Down’s me, temporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration, Gerstmann—Straussler-Scheinker disease, Hallervorden-Spatz disease, ion body is, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C mP-C), non-Guamanian motor neuron disease With neurofibrillary tangles, Pick’s disease (PiD), postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle only dementia, infarct dementia and ischemic stroke.

Background Art

[0002] n accumulation, modifications and aggregation are pathological aspects of numerous neurodegenerative diseases. Pathologically modified and aggregated tau including hyperphosphorylated tau mers are an invariant rk of tauopathies and correlate with disease severity.

Tau is a microtubule-associated protein expressed in the central nervous system with a primary function to stabilize microtubules. There are six major isoforms of tau ' 2 ' expressed mainly in the adult human brain, which are derived from a single gene by alternative splicing. Under pathological conditions, the tau n becomes hyperphosphorylated, resulting in a loss of tubulin binding and destabilization of ubules followed by the aggregation and deposition of tau in pathogenic neurofibrillary tangles. Disorders related to tau - collectively referred to as neurodegenerative tauopathies - are part of a group of protein misfolding disorders including Alzheimer’s disease (AD), progressive uclear palsy, Pick’s disease, corticobasal degeneration, FTDP—l7 among others. More than 40 mutations in tau gene have been reported to be ated with tary frontotemporal dementia demonstrating that tau gene mutations are ent to trigger neurodegeneration (Cairns et al., Am. J. Pathol. 171 (2007), 227-40). Studies in transgenic mice and cell culture indicate that in AD, tau pathology can be caused by a pathological cascade in which AB lies upstream of tau (Gotz et al., e 293 (2001), 1491—1495). Other finding however point to a dual-pathway model where both cascades function independently of each other (van de Nes et al., Acta Neuropathol. 111 (2006), 126- 138). Immunotherapies targeting the beta-amyloid peptide in AD have produced encouraging results in animal models and shown promise in clinical trials. More recent autopsy data from a small number of subjects suggests that clearance of beta—amyloid plaques in patients with ssed AD may not be sufficient to halt cognitive oration, izing the need for additional therapeutic strategies for AD (Holmes et al., Lancet 372 (2008), 216-223; Boche et al., Acta Neuropathol. 120 (2010), 13-20). In the wake of the s of Abetaebased immunization therapy in transgenic animal models, the concept of active immunotherapy was expanded to the tau protein. Active ation of wild type mice using the tau protein was however found to induce the ion of neurofibrillary tangles, axonal damage and mononuclear infiltrates in the central nervous system, anied by neurologic deficits (Rosenmann et (1]., Arch Neurol. 63 (2006), 1459-1467). Subsequent studies in transgenic mouse lines using active vaccination with phosphorylated tau peptides revealed reduced brain levels of tau aggregates in the brain and slowed progressior: of behavior impairments (Sigurdsson, J. Alzheimers. Dis. 15 (2008), 157-168; Boimel et (11., Exp. Neurol. 224 (2010), 472-485). These findings highlight the potential benefit but also the tremendous risks associated with active immunotherapy approaches ' 3 ' targeting tau. Novel therapeutic strategies are ly needed addressing ogical tau proteins with efficacious and safe therapy.

Passive immunization with human antibodies derived from healthy human subjects which are evolutionarily optimized and affinity matured by the human immune system would provide a ing new therapeutic avenue with a high ility for excellent efficacy and safety.

BRIEF SUMlVlARY OF THE INVENTION The present invention makes use of the tau-specific immune response of healthy human ts for the isolation of natural anti-tau specific human monoclonal antibodies. In particular, experiments performed in accordance with the t invention were successfiJl in the isolation of monoclonal ecific antibodies from a pool of y human subjects with no signs of a egenerative tauopathy. [0006} The present invention is thus directed to human antibodies, antigen-binding fragments and similar antigen-binding molecules which are capable of specifically recognizing tau. By "specifically recognizing tau", "antibody specific to/for tau" and "anti—tau antibody" is meant specifically, generally, and collectively, antibodies to the native form of tau, or aggregated or pathologically modified tau isoforms. ed herein are human antibodies ive for full-length, pathologically phosphorylated and aggregated forms.

[0007] In a particular embodiment of the present invention, the human antibody or antigen-binding fragment f demonstrates the immunological binding characteristics of an antibody characterized by the variable regions VH and/or VL as set forth in Fig. 7.

The antigen-binding fragment of the antibody can be a single chain FV fragment, an F(ab') nt, an F(ab) fragment, and an F(ab')2 fragment, or any other antigen- binding fragment. In a specific embodiment, infra, the antibody or fragment thereof is a human IgG isotype antibody. Alternatively, the antibody is a chimeric human-murine or murinized dy, the latter being particularly useful for diagnostic methods and studies in animals.

[0009] Furthermore, the present invention relates to compositions comprising the antibody of the present invention or active fragments thereof, or agonists and cognate ' 4 ' molecules, or alternately, antagonists of the same and to immunotherapeutic and immunodiagnostic s using such compositions in the prevention, diagnosis or ent of a tauopathy, wherein an effective amount of the composition is administered to a patient in need thereof.

Naturally, the t invention extends to the immortalized human B memory lymphocyte and B cell, respectively, that produces the antibody having the distinct and unique characteristics as defined below.

The t invention also relates to polynucleotides encoding at least a variable region of an immunoglobulin chain of the antibody of the invention. In one embodiment, said variable region comprises at least one complementarity determining region (CDR) of the VH and/or VL of the variable region as set forth in Figure 7.

Accordingly, the present invention also encompasses vectors comprising said polynucleotides and host cells transformed therewith as well as their use for the tion of an antibody and equivalent binding molecules which are specific for tau.

Means and methods for the recombinant tion of antibodies and mimics thereof as well as methods of screening for ing binding molecules, e.g., antibodies, known in the art. However, as bed herein, in particular with respect to therapeutic applications in human the antibody of the present invention is a human dy in the sense that application of said antibody is substantially free of an immune response directed t such antibody otherwise observed for chimeric and even humanized antibodies.

Furthermore, disclosed herein are compositions and methods that can be used to identify tau in samples. The disclosed anti—tau antibodies can be used to screen human blood, CSF, and urine for the presence of tau in samples, for example, by using ELISA-based or surface adapted assay. The methods and compositions disclosed herein can aid in neurodegenerative thies such as Alzheimer's disease diagnosis and can be used to r e progression and therapeutic efficacy.

Hence, it is a particular object of the present invention to provide methods for treating, diagnosing or preventing a neurodegenerative tauopathy such as Alzheimer’s disease, amyotrophic lateral sis/parkinsonism—dementia complex, argyrophilic grain dementia, British type amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary s with calcification, Down’s syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Sträussler-Scheinker e, Hallervorden-Spatz e, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral d angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sing panencephalitis, tangle only dementia, multi-infarct dementia and ischemic stroke. The methods comprise administering an effective concentration of a human antibody or antibody derivative to the subject where the antibody targets tau.

[0015] Further embodiments of the present ion will be apparent from the ption and Examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES Amino acid and nucleotide sequences of the variable region, i.e. heavy chain and lambda light chain of human antibodies NI-105.4E4 (A), NI-105.24B2 (B) and NI-105.4A3 (C). Framework (FR) and complementarity determining regions (CDRs) are indicated with the CDRs being underlined. Due to the cloning strategy the amino acid sequence at the N-terminus of the heavy chain and light chain may potentially contain primer-induced alterations in FR1, which however do not substantially affect the ical activity of the antibody. In order to provide a consensus human antibody, the nucleotide and amino acid sequences of the original clone were aligned with and tuned in accordance with the pertinent human germ line variable region sequences in the database; see, e.g., Vbase //vbase.mrccpe.cam.ac.uk /) hosted by the MRC Centre for Protein ering (Cambridge, UK).

Those amino acids, which are considered to potentially deviate from the consensus germ line sequence due to the PCR primer and thus have been replaced in the amino acid sequence, are indicated in bold.

NI-105.4E4 binds to neurofibrillary tangles (NFT), dystrophic es and neuropil threads in AD brain and human TauP301L expressing mice. .4E4 staining identifies NFTs and neuropil threads in AD brain (A), with no significant binding to tau in the brain of healthy l subject (B). In TauP301L transgenic " 6 ' NFT (E, F mouse (E—I) NI-105.4E4 binds strongly to the pathological tau resembling and H), neuropil threads (E and G) and dystrophic neurites (E and H). In addition, NI- 105.4E4 also identifies tau aggregates at pre-tangle stage (I). NI—105.4E4 binds to NFT, dystrophic neurites and neuropil threads in transgenic mouse expressing human APP with the Swedish and the Arctic on and TauP301L; the arrow marks a beta- amyloid plaque, nded by dystrophic neurites recognized by NI-105.4E4 (J).

Secondary dy only does not give signal both in human AD (C) and healthy control (D).

Tissue amyloid plaque immunoreactivity (TAPIR) assay. Neurofibrillary tangles were d wéth either the anti-phospho-tau antibody AT100 or sera isolated from healthy elderly subjects.

Schematic representation of the Nl-105.4E4 and NI—105.4A3 epitopes and epitopes of commonly used commercially available mouse monoclonal tau dies that comprises two are shown. Human antibody NI-105.4E4 s a unique e linear polypeptides, one of which is located in the microtubule binding domain (R4) of tau which is masked in physiological microtubule-associated tau. Tau-12 (Covance, California, USA), HT7, AT8, AT180 (Thermo Scientific, U.S.A.); PHFl (Lewis et al., Science 293 (2001), 1487-1491).

Human IgG levels in the plasma of mice following intraperitoneal administration of 30 mg/kg NI-105.4E4 or NI—105.4A3 human anti-tau antibody.

Human IgG levels in brain homogenate of mice following intraperitoneal stration of 30 mg/kg .4E4 or NI-105.4A3 human anti-tau antibody.

Amino acid ce of heavy chain and light chain variable regions of (A) NI-105.17C1, (B) NI-105.6C5, (C) NI—105.29G10, (D) NI-105.6L9, (E) NI— 105.40E8, (F) NI-105.48E5, (G) NI-105.6E3, (H) NI-105.22E1, (I) NI—105.26B12, (J) NI-105.12E12, (K) NI—105.60E7, (L) NI-105.14E2, (M) Nl-105.39E2, (N) NI- 105.19C6, and (O) NI-105.9C4 human anti-tau antibodies. Complementarity determining regions (CDRs) are underlined. and chl7Cl(N3lQ) : (A) Binding of ch17C1, chl7Cl(N3lQ) mIgG2a mIgGl Agly to recombinant Tau in an ELISA assay. (B) Comparison of recombinant Tau binding by chl 7C1(N3 IQ) mIgG2a and chl 7C1(N31Q, I48V) mIgG2a in an ELISA assay. ' 7 ' Comparison of recombinant Tau binding by NI-105.40E8 hIgGl and NI- 105.40E8(R1 04W) hIgGl in an ELISA assay.

. Binding of NI-105.40E8, NI-105.48E5, NI-105.6C5 and NI- 105.17C1(I48V) human anti-tau antibodies to pathologically aggregated tau in AD (J: brain and in the brain of transgenic mouse model of tauopathy. Representative images of human anti-tau antibody g to pathological tau aggregates in the brain of Alzheimer’s disease (AD) and in the brain of transgenic mouse of thy (Tg).

Control tissue samples were obtained from mentally healthy subject (Ctr) or wild type mouse brain (Wt).

[0026] . Brain penetration of NI-105.6C5 or NI-105.6E3 human anti-tau antibodies in TauP30lL mice. "tg" indicates representative sections from transgenic s either treated or untreated, and "wt" indicates an untreated non-transgenic animal. Scale bar: 50 pm.

. Effects of chronic treatment of 1L mice with ch4E4(N30Q) and ch17C1(N31Q). Total human tau (A), human pSl99 tau (B), human pT231 tau (C) and human pT181 tau (D) levels in soluble, and insoluble fraction of brain protein ts were quantified with commercial ELISA.

. Soluble and insoluble human tau in TauP301L mice treated with ch17C1(N31Q) and N30Q) detected by Western blots.

[0029] . Average plasma drug concentrations for ch17C1(N31Q) and ch4E4(N30Q) treated animals 24 h after the i.p. administration of the last dose.

Average plasma drug concentrations for ch17C1(N31Q) and N30Q) were 145 and 200 ug/ml, respectively.

. Spatial working memory in lL mice treated with ch17C1(N31Q) and ch4E4(N30Q) was assessed by two-trial Y-maze.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions Neurodegenerative tauopathies are a diverse group of neurodegenerative disorders that share pathologic lesion consisting of intracellular aggregates of a common abnormal ts that are mainly composed of ogically hyperphosphorylated tau in neurons and/or glial cells. Clinical features of the tauopathies are heterogeneous ‘ 8 “ and characterized by dementia and/or motor syndrnmes, The: pragressive accumulation of fiiamentous tau inclusions neuronal degeneration in may cause and glial combination with other deposits as, e. g., beta-amyloid in Alzheimer’s disease or as a sole pathogenic entity as illustrated by mutations in the tau gene that are associated with familial forms of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Because of the heterogeneity of their clinical manifestations a potentially non-exhaustive list of tauopathic diseases can be provided ing Alzheimer’s disease, amyotrophic lateral sclerosis/parkinsonism—dementia complex, argyrophilic grain dementia, British type amyloid angiopatléy, cerebral amyloid angiopathy, corticobasal ration, Creutzfeldt-Jakob disease, ia pugilistica, diffuse neurofibrillary s with calcification, Down’s syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Hallelyorden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic sonism, prion protein cerebral amyloid angiopathy, progressive tical gliosis, ssive supranuclear palsy, subacute sclerosing panencephalitis, tangle only ia, multi- infarct dementia and ic stroke; see for a review, e. g., Lee et al., Annu. Rev.

Neurosci. 24 (2001), 1121-1159 in which Table l catalogs the unique members of tauopathies or Sergeant et al., Bioch. Biophy. Acta 1739 (2005), 179—97, with a list in Figure 2 therein.

In this specification, the terms "tau", is used hangeable to specifically refer to the native monomer form of tau. The term "tau" is also used to generally identify other conformers of tau, for example, oligomers or aggregates of tau. The term "tau" is also used to refer collectively to all types and forms of tau. Due to alternative ng 6 tau ms are t in the human brair; The protein ces for these isoforms are: Isoform Fetal-tau of 352aa MAEFRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGD TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQK GQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSESRTPSLPTPPTR WO 00600 ' 9 " EPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKV QIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEEEVKSEKLDFKDRVQSKIGSLDNIT HVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGS IDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO:1) Isoform Tau-B of 381aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPT EDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTG SDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKS GDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPV PMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPG GGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTD HGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID N022) Isoform Tau-C of 410aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPT EDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGI GDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPFG ATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTP PTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGG KVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLD NITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSS TGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID N023) Isoform Tau-D of 383aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGEETDAGLKAEEAGIGD TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQK GQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTR EPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKV QIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHH VEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKA KTDHGAEIVYKSPVVSG-DTSPRHiSNVSSTGSIDMVDSPQLATLADEVSASLAKQ GL (SEQ ID N024) W0 2014/100600 ' 10 ' 2013/076952 Isoform Tau-E of 412aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPT EDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTG SDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKS GDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPV PMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPG GGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSL DNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVS STGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID N025) Isoform Tau-F of 441aa MAEPRQEFEVMEDHAGTYGLGDRKEEQGGYTMHQDQEGDTDAGLKESPLQTPT PGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGI GDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPG QKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTP PTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMT’DLKNVKSKIGSTENLKHQPGGG KKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNI HHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENA KAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLA KQGL (SEQ ID NO:6)

[0033] The "wild type" tau amino acid sequence is represented by isoform Tau-F of 44laa (SEQ ID NO:6) further also referenced to as "hTau40", "TauF", " or "full-length tau". The amino acid sequence of tau can be retrieved from the literature and pertinent databases; see Goedert et al., Proc. Natl. Acad. Sci. USA 85 (1988), 4051—4055, Goedert et al., EMBO J. ), 393-399, Goedert et al., EMBO J. 9 (1990), 4225-4230 and GenBank UniProtKB/swissprot: locus TAU_HUMAN, accession numbers -2 (Fetal-tau) and P10636-4 to -8 (Isof’orms B to F). {883$} Another striking feature of tau protein is phosphorylation, which occurs at about of 79 potential serine (Ser) and threonine (Thr) phosphorylation sites. Tau is highly phosphorylated during the brain development. The degree of phosphorylation declines it adulthood. Some of the phosphorylation sites are located Wéthin the microtubule binding domains of tau, and it has been shown that an increase of tau phosphorylation negatively regulates the g of microtubules. For example, Ser262 and Ser396, W0 2014/100600 ' 11 ' which lie within or adjacent to microtubule binding motifs, are hyperphosphorylated in the tau proteins of the abnormal paired helical filaments (PHFs), a major component of the brillary tangles (NFTs) in the brain of AD patients. PHFs are filamentous aggregates of tau proteins which are abnormally hyperphosphorylated and can be stained with specific anti-tau antibodies and detected by light microscopy. The same holds true for so called straight tau filaments. PHFs form twisted ribbons consisting of two filaments twisted around one another with a periodicity of about 80nm. These pathological features are commonly referred to as "tau-pathology", “tauopathology” or "tau-related pathology". For a more detailed description of neuropathological features of tauopathies refer to Lee et al., Annu. Rev. Neurosci. 24 (2001), 1121-1159 and Gotz, Brain. Res. Rev. 35 (2001), 266-286, the disclosure content of which is incorporated herein by reference. Physiological tau protein stabilizes ubules in neurons. Pathological phyosphorylation leads to abnormal tau localization and aggregation, which causes ilization of microtubules and impaired cellular ort. Aggregated tau is neurotoxic in vitro (Khlistunova et al., J. Biol. Chem. 281 (2006), 1205-1214). The exact neurotoxic s s unclear, however, as do the mechanism(s) by which they lead to neuronal death. Aggregates of tau can be observed such as as the main component of neurofibrillary tangles (NFT) in many tauopathies, Alzheimer’s disease (AD), Frontotemporal dementias, supranuclear palsy, Pick’s disease, Argyrophilic grain disease (AGD), corticobasal degeneration, FTDP-17, Parkinson’s disease, Dementia pugilistica (Reviewed in n and Petrucelli, M01.

Neurodegener. 4:13 (2009)). Besides these observations, evidence emerges that tau- ed neuronal death can occur ever: in the absence of tangle formation. Soluble phospho—tau species are present in CSF (Aluise et al., Biochim. s. Acta. 1782 (2008), 549—558). Tau ates can it a misfolded state from the outside to the inside of a cell and transfer between co-cultured cells (Frost et al., J. Biol. Chem. 284 (2009), 12845-12852).

In addition to the involvement in neurodegenerative thies, ed alterations in tau phosphorylation during and after ischemia/reperfusion suggest tau playing a crucial role in neuronal damage and clinical pathophysiology of neurovascular disorders sucla as ischemic stroke (Zheng et al., J. Cell. m. 109 (2010), 26-29).

W0 2014/100600 The human au antibodies sed herein specifically bind tau and epitopes thereof and to various conformations of tau and epitopes thereof. For example, disclosed herein are antibodies that specifically bind tau, tau in its full-length, pathologically modified tau isoforms and tau aggregates. As used herein, reference to binds" tau an antibody that "specifically binds", "selectively binds", or "preferentially refers to an antibody that does not bind other unrelated proteins. In one example, a tau antibody sed herein can bind tau or an e thereof and show no binding above about 1.5 times background for other proteins. An antibody that "specifically binds" or tively binds" a tau conformer refers to an antibody that does not bind all conformations of tau, i.e., does" not bind at least one other tau conformer. For example, sed herein are antibodies that can preferentially bind to aggregated forms of tau in AD tissue. Since the human au antibodies of the present invention have been ed from a pool of healthy human subjects exhibiting an tau-specific immune response the tau antibodies of the present invention can also be called "human auto-antibodies" in order to emphasize that those antibodies were indeed expressed by the subjects and have not been isolated from, for example a human immunoglobulin expressing phage library, which hitherto represented one common method for trying to provide human-like antibodies.

It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody," is understood to represent one or more antibodies. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a le ed of monomers (amino acids) linearly linked by amide bonds (also known as e bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, eptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms.

The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, W0 2014/100600 PCT/USZOl3/076952 acetylation, phosphorylation, amidation, derivatization by known protecting/blocking amino acids. groups, proteolytic cleavage, or modification by non-naturally occurring A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by al synthesis.

A ptide of the invention can be of a size of about 3 or more, 5 or more, 10 or more, or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, gh they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as , and ptides which do not possess a defened three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. As used herein, the term glycoprotein refers to a n d to at least or a one carbohydrate moiety that is attached to the protein via an oxygen-containing nitrogen-containing side chain of an amino acid residue, e. g., a serine residue or an asparagine residue.

By an "isolated" polypeptide or a fragment, variant, or tive thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

Also ed as polypeptides of the present invention are fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination f. The terms "fragment," "variant," "derivative" and "analog" when referring to antibodies or dy polypeptides of the present invention include any polypeptides which retain at least some of the antigen-binding properties of the corresponding native binding molecule, antibody, or polypeptide. Fragments of polypeptides of the preSent invention include proteolytic fragments, as well as on fragments, in addition to specific antibody nts discussed elsewhere . Variants of antibodies and antibody polypeptides of the present invention e fragments as described above, and also polypeptides with altered amino acid ces due to amino acid substitutions, W0 2014/100600 PCT/USZOl3/076952 deletions, or insertions. ts can occur naturally or be non-naturally ing.

Non-naturally occurring variants can be produced using art-known mutagenesis techniques. t polypeptides can comprise conservative or non—conservative amino acid substitutions, deletions or additions. Derivatives of tau specific binding molecules, are polypeptides e. g., antibodies and antibody ptides of the present invention, which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion ns. Variant polypeptides can also be referred to herein as "polypeptide analogs". As used herein a "derivative" of a binding molecule or fragment thereof, an antibody, or an antibody polypeptide refers to a subject polypeptide having one or more es chemically derivatized by reaction of functional side Also included as atives" are those a group. peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for e; 5- hydroxylysine can be substituted for ; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

The term "polynucleotide" is intended to encompass a singular nucleic acid as well isolated molecule or as plural nucleic acids, and refers to an nucleic acid uct, A polynucleotide e. g., ger RNA (mRNA) or plasmid DNA (pDNA). bond (e.g. , an can comprise a conventional phosphodiester bond or a non-conventional amide bond, such as found in peptide c acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid ts, e.g., DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native nment.

For example, a recombinant polynucleotide encoding an antibody contained in a vector is considered isolated for the purposes of the present invention. r examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of acids polynucleotides of the present invention. Isolated polynucleotides or nucleic according to the present invention further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a W0 2014/100600 PCT/USZOl3/076952 tory element such as a promoter, ribosome binding site, or a transcription terminator.

As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amirao acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but ribosome binding sites, transcriptional any flanking sequences, for example ers, tern’zinators, introns, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide uct, e.g., on a single vector, or in separate polynucleotide ucts, e. g., on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can se two or more coding regions, e.g., a single vector can tely encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, cleotide, or nucleic acid of the invention can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a binding molecule, an dy, or fragment, variant, or derivative thereof.

Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a logous onal domain.

In certain embodiments, the polynucleotide or raucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally can e a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e. g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory ce(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" or "operably linked" if induction of er function results in the transcription ofmRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be ribed. Thus, a promoter region would be operably ated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcréption of that nucleic acid.

The promoter can be a cell-specific promoter that directs substantial transcription of W0 2014/100600 PCT/USZOl3/076952 the DNA only in predetermined cells. Other transcription control elements, s a ation promoter, for example enhancers, ors, repressors, and transcription signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

A y of ription control regions are known to those skilled in the art.

These include, t limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytorriegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).

Other transcréption control regions include those d from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit B-globin, as well as other expression in eukaryotic cells. Additional sequences capable of controlling gene suitable ription control regions include tissue-specific ers and enhancers interferons or as well as lymphokine-inducible promoters (e.g., ers inducible by interleukins). {9347; Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not d to ribosome binding sites, translation initiation and termination codons, and elements d from picornaviruses (particularly an internal me entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present invention can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic lum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells lly have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full—length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain ' 17 ' embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that ce that retains the ability to direct the secretion of the polypeptide that is operably ated with it.

Alternatively, a heterologous mammalian signal peptide, or a functional tive thereof, can be used. For example, the wild-type leader ce can be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse B- glucuronidase.

Unless stated otherwise, the terms "disorder" and “disease" are used interchangeably herein.

[0051] A "binding le" as used in the context of the present invention relates prémarily to antibodies, and nts thereof, but can also refer to other non-antibody molecules that bind to tau including but not limited to hormones, receptors, ligands, major histocompatibility complex (MHC) molecules, chaperones such as heat shock proteins (HSPs) as well as cell-cell adhesion molecules such as members of the cadherin, intergrin, C-type lectin and immunoglobulin (lg) superfamilies. Thus, for the sake of clarity only and without restricting the scope of the present invention most of the following embodiments are discussed with respect to antibodies and dy-like molecules which represent a specific embodiment. of binding molecules for the development of eutic and diagnostic .

[0052] The terms "antibody" and "immunoglobulin" are used hangeably herein. An antibody or globulin is a tau-binding molecule which comprises at least the variable domain of a heavy chain, and ly comprises at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood; see, e. g., Harlow et 61]., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term "immunoglobulin" comprises var’éous broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, u, or, 8, a) with some subclasses among them (e.g., 71-74). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc. are well characterized and are known to confer functional W0 2014/100600 PCT/USZOl3/076952 specialization. Modified versions of each of these classes and isotypes are readily discernible to the d artisan in View of the instant disclosure and, accordingly, are within the instant invention. All immunoglobulin classes scope of the are clearly within the scope of the t invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule ses two cal light chain polypeptides of molecular weight approximately 23,000 Daltons, and two cal heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by de bonds in a "Y" configuration n the light chains bracket the leeavy chains starting at the mouth of the "Y" and continuing through the variable region.

Light chains are classified as either kappa or lambda (K, A). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail“ portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are ted either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N—terminus at the forked ends of the Y configuration to the C-terrninus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constan " and "variable" are used functionally. In this , it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor g, complement binding, and the like. By convention the numbering the coristant region domains increases as they become more distal from the antigen- binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C—terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

[0056] As ted above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity ining regions (CDRs), of an antibody W0 2014/100600 PCT/USZOl3/076952 combine to form the variable region that defines a three dimensional antigen-binding site. This quaternary antibody structure forms the antigen—binding site present at the end of each arm of the Y. More cally, the antigen-binding site is defined by three CDRs on each of the VH and VL chains. Any antibody or immunoglobulin fragment which contains sufficient ure to specifically bind to tau is denoted herein interchangeably as a "binding fragment" or an "immunospecific fragmen ." In naturally occurring antibodies, an antibody comprises six hypervariable regions, mes called "complementarity determining regions" or "CDRs" present in each antigen-binding domain, which are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three ional configuration in an aqueous environment. The "CDRs" are flanked by four relatively conserved "framework" regions or "FRs" which show less inter-molecular variability. The framework regions largely adopt a B-sheet mation and the CDRs form loops which t, and in some cases form part of, the B-sheet structure. Thus, ork regions act to form a scaffold that es for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.

The antigen-binding domain formed by the oned CDRs defines a surface complementary to the epitope on the reactive n. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprésing the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined; see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US. Department of Health and Human Services, ; and a and Lesk, J. Mol. Biol., 196 (1987), 901—917, which are incorporated herein by reference in their entireties.

In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term ”complementarity determining region" ("CDR") to describe the non— contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., US. Dept. of Health and Human Services, "Sequences of ns of Immunological W0 00600 Interest" (1983) and by Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which nce, where the definitions include overlapping or are incorporated herein by subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can ely determine which es comprise a particular hypervariable region or CDR of the human IgG e of antibody given the variable region amino acid sequence of the antibody.

TabEe 1: CDR Definitionsl “WT KabatWrChothia Vm“flusw‘26-32 VH CDR2 W5065 5258 VHCDR3WW95102 “W95102W VLCDRl W3434W2632 ‘ “W__w.“ iNumbe‘iiffé‘SiEii’EDR defiii‘i‘iiSfiE‘iii‘i‘able 1 is éEESEEiifiE‘to the nufiiBEingEBhventic?fi§“‘§et forth by Kabat et al. (see below). [$059] Kabat et al. also defined a numbering system for le domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US. Dept. of Health and Human Services, ”Sequence of Proteins of Immunological Interest" (1983).

Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody or antigen-binding fragment, variant, or derévative f of the present invention are according to the Kabat numbering , which however is W0 2014/100600 PCT/USZOl3/076952 theoretical and may not equally apply every antibody of the present invention. In one embodiment, ing on the position of the first CDR the following CDRs can shifted in either direction.

Antibodies or antigen-binding fragments, immunospecific fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, zed or antibodies, single chain antibodies, e-binding fragments, e. g., Fab, Fab' and F(ab')2, Fd, Fvs, single—chain Fvs , single-chain antibodies, disulfide-linked (dev), fragments comprising either a VL or VH domain, fragments produced by a anti-Id expression library, and anti-idiotypic (anti-Id) antibodies (including, e. g., and are antibodies to antibodies disclosed herein). ScFv molecules are known in the art described, les of the e. g., in US patent 5,892,019. Immunoglobulin or antibody invention car: be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., molecule.

IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or ss of immunoglobulin In one embodiment, the dy of the present invention is not IgM or a derivative thereof with a pentavalent structure. Particular, in specific applications the present invention, ally therapeutic use, Ing are less useful than IgG due to their other bivalent antibodies or corresponding binding molecules since Ing pentavalent structure and lack of affinity maturation often show ific cross- reactivities and very low affinity. [e062] In a particular embodiment, the antibody of the present invention is not a polyclonal antibody, i. e. it substantially consists of one particular antibody species rather than being a mixture ed from a plasma immimoglobulin sample.

Antibody fragments, including single-chain dies, can comprise the variable region(s) alone or in combination with the entirety or a portion of the ing: hinge , CH1, CH2, and CH3 domains. Also included in the invention are tau-birading fragments comprising any combination of variable region(s) with a hinge region, CH1, thereof of the CH2, and CH3 domains. dies or immunospecific fragments birds and mammals. In one present invention can be from any animal origin including embodiment, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e.g. , from sharks).

W0 2014/100600 PCT/USZOl3/076952 In one aspect, the antibody of the present invention is a human monoclonal antibody isolated from a human. Optionally, the framework region of the human antibody is d and adopted in ance with the ent human germ line variable region ces in the database; see, e. g., Vbase (http://vbase.mrc- m.ac.uk/E . by the MRC Centre for Protein Engineeréng (Cambridge, UK). line For example, amino acids considered to potentially deviate from the true germ the cloning sequence could be due to the PCR primer sequences incorporated during human-like antibodies such as single chain process. Compared to artificially generated antibody fragments ) from a phage displayed antibody library or xenogeneic mice the human monoclonal atetibody of the present invention is characterized by (i) being obtained using the human immune response rather than that of animal surrogates, i. e. the antibody has been generated in response to natural tau in its relevant conformatiOn in the human body, (ii) having protected the individual or is at least cant for the presence of tau, and (iii) since the antibody is of human origin the risks of cross-reactivity against self-antigens is minimized. Thus, in accordance with the present ion the terms "human monoclonal antibody”, "human monoclonal autoantibody", "human antibody" and the like are used to denote a tau binding molecule which is of human origin, i.e. which has been ed from a human cell such as a B cell or hybridoma thereof or the cDNA of which has been directly cloned from mRNA of a human cell, for example a human memory B cell. A human antibody is still " even if amino acid substitutions are made in the dy, e. g., to improve binding characteristics. animals Antibodies derived from human immunoglobulin librariées or from transgenic for and not one or more human immunoglobulins that do express endogenous immunoglobulins, as described infia and, for example in, US patent no ,939,598 by Kucherlapati et al., are denoted human-like antibodies in order distinguish them from truly human antibodies of the present invention.

For example, the paring of heavy and light chains of human-like antibodies such from phage display do not as synthetic and semi-synthetic antibodies typically isolated cell. arily reflect the original paring as it occurred in the original human B Accordingly Fab and scFv fragments obtained from recombinant expression ies artificial with all possible as commonly used in the prior art can be considered as being associated effects on immunogenicity and stability.

WO 00600 ‘ 23 ' In contrast, the present invention provides isolated affinity-matured antibodies from selected human subjects, which are characterized by their therapeutic utility and their tolerance in man.

As used herein, the term "murinized antibody" or "murinized immunoglobulin" refers to an antibody comprising one or more CDRS from a human antibody of the invention; and a human framework region that contains amino acid present substitutions and/or deletions and/or insertions that are based on a mouse antibody CDRS is called the "paren " or sequence. The human immunoglobulin providing the "acceptor" and the mouse antibody providing the framework s is called the "donor", Constant regions need not be present, but if they are, they are usually substantially identical to mouse antibody nt regions, 1'. e. at least about 85- 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical. Hence, in some embodiments, a full—length murinized human heavy or light chain immunoglobulin contains a mouse nt region, human CDRs, and a ntially human framework that has a number of "rnurinizing" amino acid substitutions.

Typically, a "murinized antibody" is an antibody comprising a zed variable light chain and/or a murinized le heavy chain. For example, a murinized antibody would not encompass a typical chimeric antibody, e. g., because the entire le region of a chimeric antibody is non-mouse. A modified antibody that has been "murinized" by the s of "murinization" binds to the same antigen as the parent dy that provides the CDRS and is usually less immunogenic in mice, as compared to the parent antibody. {0069] As used herein, the term "heavy chain portion" includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, , and/or lower hinge region) , a CH2 domain, a CH3 , or a variant or fragment thereof. For example, a binding polypeptide for use in the invention can comprise polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 , at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHl domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, CI-H domain, and a CH3 domain. In another embodiment, a polypeptide of the W0 2014/100600 - 24 , PCT/USZOl3/076952 invention comprises a ptide chain comprising a CH3 domain. Further, a binding polypeptide for use in the invention can lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that these s (e. g., the heavy chain portions) can be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.

. In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein, the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer. Alternatively, heavy chain portion-containing monomers of the invention are not identical. For e, each r can comprise a different target binding site, forming, for example, a bispecific antibody or diabody.

In another embodiment, the dies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein are composed of a single polypeptide chain such as scFvs and to be expressed intracellularly (intrabodies) for potential in viva therapeutic and diagnostic ations.

The heavy chain portions of a binding polypeptide for use in the diagnostic and treatment s disclosed herein can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide can comprise a CH1 domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, a heavy chain n can comprise a chimeric hinge d, in part, from an IgGl molecule and, in part, from an lgG4 molecule.

[0073] As used , the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain. In one embodiment, the light chain portion coneprises at least one of a VL or CL domain.

The minimum size of a e or polypeptide epitope for an antibody is thought to be about four to five amino acids. Peptide or polypeptide epitopes can contain least seven, at least nine or between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an e need not be contiguous, and in some cases, may not even be on the same peptide chain. In the present invention, a peptide or polypeptide epitope W0 00600 ' 25 ' PCT/USZOl3/076952 recognized by antibodies of the present invention contains a sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 5 to about 30, ab0ut 10 to about 30 or about 15 to about 30 contiguous or non-contiguous amino acids of tau.

[0075] By "specifically binding", or "specifically recognizing", used interchangeably herein, it is generally meant that a binding molecule, e. g., an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementaréty between the n-binding domain and the epitope. According to this definition, an antibody is said to fically bind" to an epitope when it binds to that e, via its antigen—binding domain more readily than it would bind to a random, ted epitope. A skilled artisan understands that an antibody can specifically bind to, or specifically recognize an isolated polypeptide comprising, or consisting of, amino acid residues corresponding to a linear portion of a non- contiguous e. The term "specificity" is used herein to qualify the relative y by which a certain antibody binds to a certain epitope. For example, antibody "A" can be deemed to have a higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with a higher specificity than it has for related epitope "D“.

Where present, the term "immunological binding characteristics," or other binding characteristics of an antibody wéth an antigen, in all of its tical forms, refers to the specificity, affinity, cross-reactivity, and other binding characteristics of an antibody.

By "preferentially binding", it is meant that the binding molecule, e. g., antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an dy which "preferentially binds" to a given epitope would more likely bind to that e than to a d e, even though such an antibody can cross-react with the related epitope.

By way of non-limiting example, a binding molecule, e. g., an antibody can be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (K13) that is less than the antibody’s KD for the second epitope. In another non-limiting example, an antibody can be considered to bind a first n preferentially if it binds the first epitope with an y that is at least one order of magnitude less than the antibody’s K1) for the second epitope, In another non-limiting W0 2014/100600 PCT/USZOl3/076952 example, an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody’s K1) for the second epitope.

In another non-limiting example, a binding molecule, e. g., an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody’s k(off) for the second e. In another non-limiting example, an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an y that is at least one order of ude less than the antibody’s k(off) for the second epitope. In another non- limiting example, an antibody can be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody’s k(off) for the second epitope.

A binding molecule, e. g., an antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind a tau or a fragment or t thereof with an off rate (k(off)) of less than or equal to 5 x 10'2 sec], 10'2 sec'l, 5 x10'3 sec'1 or '3 sec'lln one embodiment, an antibody of the invention can be said to bind tau or a 104 sec], fragment or variant thereof with an off rate (k(off)) less than or equal to 5 x '4 sec'l, 5 x 10'5 sec'l, or 10'5 sec'l, 5 x 10"5 sec'l, 10'6 sec'l, 5 x 10'7 sec'1 or 10'7 sec‘

[0081] A binding molecule, e.g., an antibody or n-binding fragment, variant, or derivative disclosed herein can be said to bind tau or a fragment or variant thereof wéth an on rate (k(on)) of r than or equal to 103 M'1 sec'l, 5 x 103 M'1 sec'l, 104 M‘1 sec'1 or 5 x 104 M'1 sec'lln one embodiment, an antibody of the invention can be said to bind tau or a fragment or variant thereof with an on rate (k(on)) greater than or equal to 105 M'1 sec'l, 5 x 105 M'1 sec'l, 106 M'1 sec], 5 x 106 M'1 sec‘1 or 107 M'1 sec'l.

A binding molecule, of a e. g., an antibody is said to competitively inhibit g reference antibody to a given epitope if it entially binds to that epitope to the extent that it blocks, to some , g of the reference antibody to the epitope.

Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. An antibody can be said to competitively inhibit binding of the reference antibody to a given e by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%. A d artisan tands that the binding cf an antibody to its epitope can also be competitively inhibited by a binding molecule - 27 ' that is not an antibody. For example, the specific binding of an antibody described herein to tau, e.g., hTau40, can be competitively inhibited by microtubules.

As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope with the CDR of a binding molecule, e.g., an immunoglobulin molecule; see, e. g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor tory Press, 2nd ed. (1988) at pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of globulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen; see, e. g., Harlow at pages 29-34. Avidity is it? related to both the affinity of individual immunoglobulin les in the tion with specific epitopes, and also the valencies of the imn’sunoglobulins and the antigen.

For example, the ction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable ; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. n and Company New York, N Y (1992), and methods described herein. General techniques for ing the affinity of an dy for an antigen include ELISA, RIA, and surface n resonance. The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, e. g., salt concentration, pH. Thus, measurements of affinity and other antigen-binding parameters, e.g, KD, IC50, are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.

[0084] Binding les, e. g., antibodies or antigen-binding fragments, variants or derivatives thereof of the invention can also be described or specified in terms of their cross-reactivity. As used , the term "cross-reactivity" refers to the ability of an dy, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, an antibody is cross reactive if it binds to an epitope other than the one that induced its formation. The structural cross reactive epitope generally contains many of the same complementary features as the inducing epitope, and in some cases, can actually fit better than the original.- W0 2014/100600 ' 28 ' For example, certain antibodies have some degree of cross—reactivity, in that they bind related, but non-identical epitopes, e. g., es with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody can be said to have little or no reactivity if it does not bind epitopes with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody can be deemed "highly specific" for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope. [$5086] Binding molecules, e.g., antibodies or antigen-binding fragments, variants or derivatives thereof of the invention can also be described or specified in terms of their bindirag affinity to tau. In one ment, binding affinities include those with a iation constant or Kd less than 5 X 10'2 M, 10'2 M, 5 x 10'3 M, 10'3 M, 5 x 10'4 M, '4 M, 5 x10'5 M, 10'5 M, 5 x 10'6 M, 10'6 M, 5 x 10'7 M, 10'7 M, 5 x 10'8 M, 10'8 M, 5 x10" x 10'9 M, 10'9 M, 5 x10'10M,10'10M,5 x 10'“ M, 10'11M, 5 x 10'12 M, 10'12M, M, 10'13 M, 5 x 10'14M, mm, 5 x 10‘15 M, or 10'15 M.

As previously indicated, the subunit structures and three ional ration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an globulin heavy chain and the term "CH1 domain" includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain.

The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

As used herein the term "CH2 domain" includes the portion of a heavy chain molecule that extends, e. g. , from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and es 231-340, EU numbering system; see Kabat EA et al. op. cit). The CH2 domain is unique in that it is not closely paired with another . Rather, two N- linked branched ydrate chains are interposed n the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends — 29 - from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues. chain As used herein, the term "hinge region" includes the portion of a heavy le that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N—terminal antigen- into three binding regions to move independently. Hinge regions can be subdivided ct domains: upper, middle, and lower hinge domains; see Roux et al., J.

Immunol. 161 (1998), 4083.

As used herein the term "disulfide bond" includes the covalent bond formed that can between two sulfur atoms. The amino acid cysteine ses a thiol group form a de bond or bridge with a second thiol group. In most naturally occurring the two IgG molecules, the CH1 and CL regions are linked by a disulfide bond and 239 and heavy chains are linked by two disulfide bonds at positions corresponding to 242 using the Kabat numbering system (position 226 or 229, EU numbering ).

[0091] As used herein, the terms "linked", "fused" or "fusion" are used interchangeably.

These terms refer to the joining together of two more elements or components, by "in—frame whatever means including chemical conjugation or recombinant means. An fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) correct to form a continuous longer ORF, in a manner that maintains the translational reading frame of the original ORFs. Thus, a recombinant fusion n a single protein ning two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).

Although the reading frame is thus made continuous hout the fused segments, linker the segments can be physically or spatially separated by, for example, me the CDRS of an immunoglobulin sequence. For example, cleotides encoding variable region can be fused, in-frame, but be separated by'a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long the "fused" CDRs are co-translated as part of a continuous polypeptide.

The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without tion, gene knockdown as well as both ent expression and stable sion.

It includes without limitation transcription of the gene into messenger RNA (mRNA), W0 2014/100600 - 3O - PCT/USZOl3/076952 transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) mRNA into polypeptide(s). If or any other RNA product, and the translation of such the final desired product is a biochemical, expression es the creation of that biochemical and any precursors. Expression of a gene produces a "gene product." As used , a gene product can be either a nucleic acid, e. g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products bed herein further include nucleic acids with post transcriptional modifications, e. g., polyadenylation, or polypeptides with post translational modifications, e. g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

As used herein, the term "sample" refers to any biological material obtained from fluid a subject or patient. In one aspect, a sample can comprise blood, cerebrospinal ("CSF"), or urine. In other aspects, a sample can comprise whole blood, plasma, B cells enriched from blood samples, and cultured cells (e. g., B cells from a subject). A sample can also include a biopsy or tissue sample ing neural tissue. In still other cells. Blood samples aspects, a sample can comprise whole cells and/or a lysate of the In one the can be collected by methods known in the art. aspect, pellet can be ended by vortexing at 4°C in 200 pl buffer (20 mM Tris, pH. 7.5, 0.5% t, 1 mM EDTA, 1 mM PMSF, 0.1M NaCl, IX Sigma se Inhibitor, and IX Sigma Phosphatase Inhibitors 1 and 2). The suspension can be kept on ice for 20 minutes with intermittent vortexing. After spinning at 15,000 X g for 5 minutes at about 4°C, aliquots of supernatant can be Stored at about -70°C.

As used herein, the terms "treat" or "treatment" refer to both therapeutic treatneent slow and prophylactic or preventative measures, wherein the object. is to t or down (lessen) an undesired physiological change or disorder, such as the development of Parkinsonism. Beneficial or desired clinical results e, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not ing) state of disease, delay or slowing of e progression, amelioration or palliation of the disease state, and remission (whether partial or total), r survival detectable or undetectable. "Treatment“ can also mean prolonging as ed to expected al if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the W0 2014/100600 ZOl3/076952 disorder is condition or disorder or those in which the manifestation of the condition or to be prevented. is meant any By "subject" or "individual" or l" or nt" or ,,marnmal,“ subject, particularly a mammalian subject, e. g., a human patient, for whom diagnosis, prognosis, prevention, or therapy is desired.

II. Antibodies The present invention generally relates to human anti-tau antibodies and antigen- invention binding fragments thereof. In one embodiment, an antibody of the present demonstrates the immunological binding characteristics and/or biological ties as outlined for the antibodies illustrated in the Examples. In accordance with the present invention human monoclonal antibodies specific for tau were cloned from a pool healthy human subjects.

In the course of the experiments performed in accordance with the present ion l attempts failed to clone tau specific antibodies but almost always resulted in false-positive clones. In order to circumvent this problem, antibodies conditioned media of human memory B cell cultures were screened in parallel for control binding to recombinant tau protein, PHFTau extracted from AD brain, healthy brain extracts and bovine serum n (BSA). Only B—cell cultures that were BSA were positive for inant tau and/or PHFTau but not control brain t or subjected to antibody cloning. l attempts to isolating to specific antibodies were focused at pools of healthy levels human subjects with high plasnéa binding activity to tau, suggestive of elevated of circulating tau antibodies plasma. Unexpectedly, these attempts failed to e c human memory B cells and the antibodies described in the current invention were isolated from pools of healthy human subjects that were not preselected for high tau plasma reactivity or had low plasma reactivity to tau. [0099'] Due to this measure, several dies could be isolated. Selected antibodies were flirther analyzed for class and light chain subclass determination. Selected relevant antibody messages from memory B cell es are then transcribed by PCR, cloned and combined into expression vectors for recombinant production; see appended Examples. Recombinant expression of the human antibodies in HEK293 or towards CHO cells and the. subsequent characterization of their binding specificities full-length tau, pathologically modified forms thereof on Western Blot and their distinctive binding to pathologically aggregated tau confirmed that for the first time human antibodies have been cloned that are highly specific for tau and recognize distinctive the ogically modified forms of tau protein. [@100] Thus, the present invention generally relates to an isolated naturally occurring human monoclonal anti—tau antibody and binding fragments, derivatives and variants thereof. In one embodiment of the ion, the antibody is capable of specifically binding fiall-length recombinant tau and/or the pathologically ated and/or phosphorylated form (PHFTau) isolated from AD brain under denaturing conditions on Western Blot.

In one embodiment, the present invention is directed to an anti-tau antibody, or antigen-binding fragment, varéant or derivatives thereof, Where the antibody cally binds to the same epitope of tau as a reference antibody selected from the group consisting of Nl-105.17C1, NI-105.6C5, NI-105.29G10, NI-105.6L9, NI-105.40E8, NI- 105.48E5, NI-105.6E3, NI-105.22E1, NI-105.26B12, NI-105.12E12, NI-105.60E7, 105.14E2, NI-105.39E2, .19C6, or NI-105.9C4.

Additional human anti-tau dies are disclosed in U.S. Patent Application Publication No. 087861, the content of which is incorporated herein by reference its entirety.

[0103] In one embodiment, an dy described herein specifically binds to tau at an epitope comprising the amino acid residues selected from the group consisting of: es corresponding to residues 125-131, 397—441, 226-244, 217-227, 37-55, 387—406, 421-427, 9, 1-158, 197-207, 57-67, 1, 313-319, 309-319, and 221-231 of hTau4O (SEQ ID NO:6). In a r embodiment, an antibody described herein specifically binds to tau at an epitope sing the amino acid residues corresponding to es 37-55 and 387-406 of hTau4O (SEQ ID NO:6). In a specific embodiment, tau is hTau40 (SEQ ID NO:6).

In one embodiment, an dy described herein binds to tau at an epitope comprising the microtubule binding domain of tau. In a specific embodiment, an antibody described herein binds to tau at an epitope comprising amino acid residues from the R4 region of tau as depicted in Figure 4. In one embodiment, an antibody described herein competes with microtubules for specific binding to tau. In another embodiment, an antibody described herein has reduced binding affinity to microtubule associated tau W0 2014/100600 PCT/USZOl3/076952 compared to the antibodies binding affinity to tau no associated with microtubules. In a flirther embodiment, an dy bed herein does not bind, or substantially does not bind to tau associated with ubules. In specific ments, the tau protein can native tau protein or recombinant tau protein. In a specific ment, tau is hTau40.

[0105] In one embodiment, a human anti-tau antibody of the present ion can specifically bind pathologically aggregated tau and not substantially bind tau in the physiological form in brain tissue. In addition, a human au antibody of the present invention can be further characterized by its ability to recognize tau at the pre-tangle and/or dystrophic neurites in the stage, in neurofibrillary tangles (NFT), neutropil threads brain. Hence, the present invention provides a set of human tau antibodies with binding specificities, which are thus particularly useful for diagnostic and eutic purposes.

In on, or alternatively, an anti—tau antibody of the present invention preferentially recognizes ogically aggregated tau rather than physiological forms, particular when analyzed according to Examples 4 and 18. In addition, or alternatively, an anti-tau antibody of the present invention binds to disease g mutants of human tau, in particular those described in Example 4. In this context, the binding specificities can in the range of having half maximal effective concentrations (ECSO) of about 100 pM to 100 nM, or an ECSO of about 100 pM to 10nM for ype tau.

Hence, to an anti-tau antibody of the present invention binds preferentially pathological modified forms of tau in brain, e. g. pathological aggregates of tau as exemplified by immunohistochemical staining described in Examples 4 and 18. In binds to another embodiment an anti-tau antibody of the present invention preferentially both recombinant tau and pathologically modified forens of tau as exemplified in Example 2 by n Blot.

[0108] The present ion is also drawn to an antibody, or antigen-binding fragment, variant or derivatives thereof, where the antibody comprises an antigen-binding domain identical to that of an antibody selected from the group consisting of NI—105.17C1, NI- 105.17C1(N31Q), NI-105.6C5, NI-105.29G10, 5.6L9, NI—105.40E8, NI-105.48E5, NI-105.6E3, NI-105.22E1, NI-105.26B12, NI-105.12E12, NI-105.60E7, NI-105.14E2, NI-105.39E2, NI—105.19C6, and NI-105.9C4.

The present invention further exemplifies l such binding molecules, e. g. antibodies and binding fragments thereof, which can be characterized by comprising their variable region, e. g, binding domain at least one complementarity determining region (CDR) of the VH and/or VL variable region comprising any one of the amino acid sequences ed in Fig. 7. The corresponding nucleotide sequences encoding the above-identified variable regions are set forth in Table 4 below. An exemplary set of CDRs of the above amino acid sequences of the VH and/or VL region as depicted in Fig. 7. However, as discussed in the following the person skilled in the art is well aware of the fact that in addition or alternatively CDRs can be used, which differ in their amino acid three or even more amino acids in sequence from those set forth in Fig. 7 by one, two, case of CDR2 and CDR3.

Table 2. Amino acid sequences of the VH region, VH CDR1,VH CDR2, VH CDR2, VL region VL CDR2, VL CDR2, and VL CDR3 of cific antibodies. 2vH/vL A A 2CDRl CDR2 AACDR3 NI-10517C1 SEQ IDN081 I SEQ ID No45ASEQ ID NO.79 SEQ ID NO:80 VLSEQ ID NO46 ‘ SEQ ID N0:822SEQ IDN083 SEQID NO:84 NI-105AiA6AD‘5AAAAAAAA A 2SEQ IDN0482SEQ ID N0:85 NO:86AAA SEQ IDNo87 2 {VL2 SEQIDNo 49 EQIDNO88 SEQIDNO89 SEQIDNO90 NI 10 2 vH2SEQIDN0:50 {SEQ IDNO9 {SEQ ID N9.92 SEQ ID NO:93 2 2 2SEQIDN095 aSEQIDNo96 2 NI-105.6L9 MW]SEQ ID No98 SEQ ID NO99 | SEQ ID S{EQ ID N0102 = : 2 N0:I01 2 NI-105.40E8 vH SEQ ID N0 54 SEQ ID SEQID 8130 IDND;105 2 N0:103 .330104 VL SEQ ID N0 55 SEQ ID 2 SEQ ID SEQ ID NO:108 {N02106 - 2 2N0:107 NI-105.48E5 vH SEQID N0 56 SEQ ID SEQ ID SEQ ID ND111 ‘ N0:109 2 N02110 vL SEQ ID N0:57 SEQ ID SEQ ID 2SEQID NO:114A . N0:112 NO:113 NI-105.6E3 vH SEQ ID N0 58 SEQ ID I SEQ ID SEQ “3 N011” % 2N02115 NO:116 2 vL SEQ ID N0 59 2 SEQ ID SEQ ID SEQ ID NO:120 2 2 2 2N0:118 2NO:119 NI-105.22E1 VH SEQ ID N0:60 SEQ ID 5SEQ ID 2 ‘ N0;121 N0I22 2 SEQ ID N0;123 vL SEQ ID N0;61 {SEQ ID 2SEQID SEQ ID NO:126 .“Wux~“u..4..._._....._...W~._~t«v»--»“unun“ IUXE‘tibody vH NL ICDRl ICDR2 513113 I 1N0124 N0:125 : ‘ I _________ I ‘SEQ .26B12 VHSEQIDN062ISEQID ID ISEQIAADAN0129I N0127 N0:128 " NQ’EISEME W~v~ Wxx‘vvr>1»:__»..u....‘“ SEQIDN0:64 gSEQ ID ~ SEQ ID INO‘130 NO:131 ISEQIQNQIEE A.i2E12 vH gSEQ ID SEQ ID ISEQAID NO:135 . N0133 IN02134 IvL ISEQAIDMNQTEE: SEQ ID '\ W.~~\““.V.rfi...n,,....““W~~“““==»---»n--"n“I SEQ ID ISEQIDNQSI38 NO:13_Q NO:137 g NI—105.66EI..__....§.V...1 SEQIDNQ:67 ESEQID ESEQ ID SEQ IDNQEI4I“ §2N0139 §N02140 ...,“......;.»u+.w-:-M—.—..a...“.mm \‘“—»»: VL SEQID SEQ ID NO:144 u “EAAISEQIDINQSEEISEQ ID INO:143 I N0142 “‘W..Ww.mmm,3.4,“WW._M‘.,.,...,..........mm: N10514E2 VHIWS’EQIBNQEBSEQ ID SEQ ID SEQIDNo:147 NQ145 INO:146 i I: SEQIQNolso I SEQIDANOflo VL SEQID ISEQID §N0I48 ;=N0:149 3 INITIAISE‘EET VH Ali-“SEQ ID N071 ISEQiii" ISEQIISW SEQIDEBHsa‘ NO151 NO:152 i “W““K‘u..».1uu..“n‘ ““W x IvL SEQIDN0172 IEQ ID ISEQ ID SEQIDNQSIEM NO: 154 NO:155 «“-r4.54..»u-.-n.“_‘x gNI-105.19C6 IVHEEQIDN073 SEQIDWSEQIDWSEQIDN0159 ::N0 157 NO:158 I I .,...._.A.._...n‘qu“\wv.r““2““n..nu4W4v4u4-n»»Au-nu ;vL SEQ IDNQ:I4ISEQID“ ISEQ ID SEQ ID N0:162§ = NO160 gN0:161 I VH ISEQID N0:75 EEQ .“‘“—.44—» ..._.n ~ MW“‘— N W...~uu—.—.»u_»“unu INI-105.9C4 %SEQ ID EEEQIDNOIQ? I I I No:_16_3 IN0:164 E LI...:“““WNN‘...,,»......“Hum W.~_m ‘.:..........u...“W._»w,,.,.,:_..:.“m“ VL SEQ ID NO:76 SEQID I SEQ ID I SEQ ID NQEIEEAA" IzNO166 NO:167 =‘ ~~x .9...»w - -§- SEQ ID NO45 ISEQID NO79ISEQ ID NO:80 SEQ ID NO:81 1 W “112.: s NI-105.17C1 I SEQ IDNQ221 ISEQ ID gSEQ IDN083 SEQ ID NO:84 ng I (N31Q) I NO:224 I I IMNN“..gum...“unnuwww“.:.............“mW___m“......_._ least one [0i10] In one embodiment, an antibody of the present invention comprises at CDR comprising, or consisting of an amino acid sequence selected from the group consisting of SEQ IDE? 0: 79-168 and 224.

WO 00600 PCT/U82013/076952 In one embodiment, an dy of the present invention comprises one, two, three, four, five or six CDRs comprising, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 79-168 and 224.

VH CDRl, In one embodiment, an antibody of the t invention comprises a the amino acid VH CDR2, VH CDR3, VL CDRl, VL CDR2, and VL CDR3 comprising 97-102, 103-108, 109—114, sequences, respectively SEQ ID NO: 79-84, 85-90, 91-96, 163-168. 115—120, 121—126, 127-132, 133-138, 139-144, 145-150, 151—156, 157-162, or VH CDRl, VH In one embodiment, an antibody of the present invention comprises a amino acid CDR2, VH CDR3, VL CDRl, VL CDR2, and VL CDR3 comprising the and 84. sequences, respectively, SEQ ID N05: 79, 80, 81, 224, 83, three VH In one embodiment, an antibody of the invention comprises one, two, or CDRs comprising, or consisting of an amino acid sequence selected from the group 121— consisting of SEQ ID NO: 79—81, 85-87, 91-93, 97—99, 103-105, 109-111, 115-117, 123,127-129,133-135,139-141,145-147,151-153,157-159, and 163-165.

[0114] In one embodiment, an antibody of the invention comprises a VH CDRl, ID NO: CDR2, and VH CDR3 comprising the amino acid sequences, respectively, SEQ 79-81, 85-87, 91-93, 97-99, 103-105, 109—111, 115-117, 121-123, 127-129, 133-135, 139-141,145-147,151—153,157-159, or 5. three VL In one embodiment, an antibody of the invention ses one, two, or CDRs comprising, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 82-84, 88-90, 94—96, 100-102, 106-108, 112-114, 0, 224. 124-126, 130-132, 136-138, 142-144,148-150, 154-156,160-162, 8, and In one embodiment, an antibody of the invention comprises a VL CDRl, ID NO: CDR2, and VL CDR3 comprising the amino acid sequences, respectively, SEQ 82-84, 88-90, 94-96, 100—102, 106-108, 112-114, 118-120, 124-126, 2, 136-138, 4, 148—150, 154-156, 160-162, or 166-168. In one embodiment, a VL CDRl, VL ID NO: CDR2, and VL CDR3 comprising the amino acid ces, respectively, SEQ 83, 84, and 224. ing to one embodiment, an antibody of the invention comprises a heavy chain le region comprising a VH CDRl of SEQ ID NO: 79, 85, 91, 97, 103, 109, 115,121,127,133,139,145,151,157, or 163; a VH CDR2 of SEQ ID NO: 80, 86, 92, CDR3 of SEQ ID 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; or aVH 165.

NO: 81, 87, 93, 99,105,111,117,123,129,135,141,147,153,159, or According W0 2014/100600 ZOl3/076952 to another embodiment, an dy comprises a light chain variable region comprésing a VL CDRl of SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160,166 or 224; a VL CDR2 of SEQ ID NO: 83, 89, 95,101,107,113,119,125,131, 137,143, 149, 155, 161, or 167; or a VL CDR3 of SEQ ID NO: 84, 90, 96, 102, 108,114, UI 120, 126, 132, 138, 144, 150, 156, 162, or 168. In another eréibodiment, the antibody comprises a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 79, 85, 91, 97,103,109,115,121,127,133,139,145,151,157, or 163; aVH CDR2 ofSEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; or a VH CDR3 ofSEQ ID NO: 81, 87, 93, 99, 105, 111,117, 123, 129, 135, 141,147, 153,159, CDRl of or 165, and further comprises a light chain variable region comprising a VL SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or 224; aVL CDR2 of SEQ ID NO: 83, 89, 95, 101, 107,113,119, 125, 131, 137,143,149, 1, or 167; or aVL CDR3 ofSEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126,132, 138,144,150,156,162, or168.

[0118] According to one embodiment, an antibody of the invention comprises a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 79, 85, 91, 97, 103, 109, 115,121,127,133,139,145,151,157, or 163; aVH CDR2 of SEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and aVH CDR3 of SEQ ID NO: 81, 8'1”, 93, 99,105, 111,117,123, 129, 135, 141,147,153, 159, or 165. According to another embodiment, an dy comprises a light chain variable region comprising a VL CDRl of SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or 224; a VL CDR2 of SEQ ID NO: 83, 89, 95,101, 107, 113, 119, 125,131, 137, 143, 149, 155, 161, or 167; and a VL CDR3 of SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, or 168. In another embodiment, the antibody comprises a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 79, 85, 91, 97, 103, 109, 115,121, 127, 133, 139, 1,157, or 163; aVH CDR2 of SEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and aVH CDR3 of SEQ ID NO: 81, 87, 93, 99, 105,111,117,123, 129, 135, 141,147, 153, 159, or 165, and r comprises a light chain variable region comprising a VL CDRl ofSEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or 224; a VL CDR2 of SEQ ID NO: 83, 89, 95, 101, 107, 113, 119, 125, 131,137, 143, 149, 155, 161, or 167; and a VL CDR3 of SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150,156,162, or 168. ' 38 ' In one embodiment, an antibody of the invention can comprise a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 79, a VH CDR2 of SEQ ID NO: 80, and VH CDR3 of SEQ ID NO: 81, and can flirther se a light chain le region comprising a VL CDRI of SEQ ID NO: 82, a VL CDR2 of SEQ ID NO: 83, and a VL CDR3 of SEQ ID NO: 84.

In one embodiment, an antibody of the invention can comprise a heavy chain variable region sing a VH CDRl of SEQ ID NO: 79, a VH CDR2 of SEQ ID NO: 80, and VH CDR3 of SEQ ID NO: 81, and can further comprise a light chain variable region comprising a VL CDRl of SEQ ID NO: 224, a VL CDR2 of SEQ ID NO: 83, and a VL CDR3 of SEQ ID NO: 84.

In one embodiment, an dy of the invention can comprise a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 85, a VH CDR2 of SEQ ID NO: 86, and VH CDR3 of SEQ ID NO: 87, and can further comprise a light chain variable region comprising a VL CDRI of SEQ ID NO: 88, a VL CDR2 of SEQ ID NO: 89, and a VL CDR3 of SEQ ID NO: 90.

In one embodiment, an antibody of the invention can comprise a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 91, a VH CDR2 of SEQ ID NO: 92, and VH CDR3 of SEQ ID NO: 93, and can filrther comprise a light chain le region comprising a VL CDRl of SEQ ID NO: 94, a VL CDR2 of SEQ ID NO: 95, and a VL CDR3 of SEQ ID NO: 96.

In one embodiment, an antibody of the invention can comprise a heavy chain variable region sing a VH CDRI of SEQ ID NO: 97, a VH CDR2 of SEQ ID NO: 98, and VH CDR3 of SEQ ID NO: 99, and can further comprése a light chain variable region comprising a VL CDRI of SEQ ID NO: 100, a VL CDR2 of SEQ ID NO: 101, and a VL CDR3 of SEQ ID NO: 102.

In one embodiment, an antibody of the invention can comprise a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 103, a VH CDR2 of SEQ ID NO: 104, and VH CDR3 of SEQ ID NO: 105, and can further se a light chain variable region comprising a VL CDRl of SEQ ID NO: 106, a VL CDR2 of SEQ ID NO: 107, and a VL CDR3 of SEQ ID NO: 108.

In one embodiment, an antibody of the invention can comprise a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 109, a VH CDR2 of SEQ ID NO: 110, and VH CDR3 of SEQ ID NO: 111, and can further comprise a light chain variable region comprising a VL CDRl of SEQ ID NO: 112, a VL CDR2 of SEQ ID NO: 113, and aVL CDR3 of SEQ ID NO: 114.

In one ment, an antibody of the invention can comprise a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 115, a VH CDR2 of SEQ ID NO: 116, and VH CDR3 of SEQ ID NO: 117, and can further se a light chain variable region comprising a VL CDR1 of SEQ ID NO: 118, a VL CDR2 of SEQ ID NO: 119, and a VL CDR3 of SEQ ID NO: 120.

In one embodiment, an antibody of the invention can comprise a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 121, a VH CDR2 of SEQ ID NO: 122, and VH CDR3 of SEQ ID NO: 123, and can further comprise a light chain variable region comprising a VL CDRl of SEQ ID NO: 124, a VL CDR2 of SEQ ID NO: 125, and a VL CDR3 of SEQ ID NO: 126.

In one embodiment, an antibody of the invention can comprise a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 127, a VH CDR2 of SEQ H} NO: 128, and VH CDR3 of SEQ ID NO: 129, and can further comprise a light chain le region comprising a VL CDRl of SEQ ID NO: 130, a VL CDR2 of SEQ ID NO: 131, and a VL CDR3 of SEQ ID NO: 132.

In one embodiment, an antibody of the invention can comprise a heavy chain variable region comprising a VH CDRl of SEQ ID NO: 133, a VH CDR2 of SEQ ID NO: 134, and VH CDR3 of SEQ ID NO: 135, and can further comprise a light chain le region comprising a VL CDRl of SEQ ID NO: 136, a VL CDR2 of SEQ ID NO: 137, and a VL CDR3 of SEQ ID NO: 138.

In one embodiment, an antibody of the invention can comprise a heavy chain variéable region comprising a VH CDRl of SEQ ID NO: 139, a VH CDR2 of SEQ ID NO: 140, and VH CDR3 of SEQ ID NO: 141, and can further comprise a light chain variable region comprising a VL CDRl of SEQ ID NO: 142, a VL CDR2 of SEQ ID NO: 143, and a VL CDR3 of SEQ ID NO: 144.

In one ment, an antibody of the invention can comprise a beavy chain variable region comprising a VH CDRl of SEQ ID NO: 145, a VH CDR2 of SEQ ID NO: 146, and VH CDR3 of SEQ ID NO: 147, and can further comprise a light chain le region comprising a VL CDRl of SEQ ID NO: 148, a VL CDR2 of SEQ ID NO: 149,, and a VL CDR3 of SEQ ID NO: 150. ' 40 ' In one embodiment, an antibody of the invention can comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 151, a VH CDR2 of SEQ ID NO: 152, and VH CDR3 of SEQ ID NO: 153, and can further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 154, a VL CDR2 of SEQ ID? NO: 155, and a VL CDR3 of SEQ ID NO: 156.

In one embodiment, an antibody of the inveration can comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 157, a VH CDR2 of SEQ ID NO: 158, and VH CDR3 of SEQ ID NO: 159, and can further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 166, a VL CDR2 of SEQ ID NO: 161, and a VL CDR3 of SEQ ID NO: 162.

In one embodiment, an antibody of the invention can comprise a heavy chain le region comprising a VH CDR1 of SEQ ID NO: 163, a VH CDR2 of SEQ ID NO: 164, and VH CDR3 of SEQ ID NO: 165, and can further comprise a light chain le region sing a VL CDR1 of SEQ ID NO: 166, a VL CDR2 of SEQ ID NO: 167, and a VL CDR3 of SEQ ID NO: 168.

In one embodiment, the antibody of the present invention is any one of the dies comprising an amino acid sequence of the VH and/or VL region as depicted in Fig. 7 and Table 3. In one embodiment, the antibody of the present invention is characterized by the vation of the cognate pairing of the heavy and light chain as was present in the human B-cell.

Table 3: Amino acid sequences of the VB and VL region of tau specific antibodies. BG — before germlining “ff,dqulhvy(VH)d4 Antbody le light (VL) chains 1 “““W'~ BG VH SWEQMTDMNOM ““Wr VH SEQ. Ifi‘f‘iii6i‘2i3“m NI—105.17C1 V, SEEjT‘iETfiEEZEM SEQTIB’I’NEEEI‘ “ ‘ H : N31Q, I48V VL SEQTID. NO:222 BG VH SEQ: ID. NO:47 NI-105.6C5 ‘Vé WQIDNO48 W\\\‘t\‘-->4 E‘Antibo5§""""W“ “XEifilé‘Zcid 5666;328:251variab‘161162v"§1v11) and? .___ varlable light (VL)1chains .1 VL‘ SEQ‘1DNo‘49 W...» WWVHWSEQ“IDN‘0‘-‘50“W“ NI—105.29G10 WVW SEQ ID. No.51 .‘,‘W~«‘-.-.444.._.“".‘WA WW... SEQ”113“‘N052 “405-6” “ s N‘QEEA ENI-105.40E8 R164W VHW SWDNOZZHW E “ EVL ‘ ‘SEQ.1D‘.‘N0:55 “"““V SEQID“N0‘5‘6“““‘“ ;E NI—105.48E5 ..

ESEQIDNQ“5‘1‘“ E “‘““““VH SEQ“113N6:58 E-NI 105.6E3 1 SEQ113.N059 EN110522E1E $EQ113“No60vv‘E‘SEQID NO;61W“““““““““E SEQ113‘. NO62 ‘N1-10526B12 EEGVL E SEQ11).NO63“W“1‘"..___.

VL ESEQ“.‘IDNO“64““ E ‘ ““ SEQ.1D.N0;65““““““ N1-105.12E12 E EVLWSEQ113N066W“““E .“Wka‘u‘a-r-finu..__.“W~x“‘....»_n.......“,W_.“«m Faun-"nuE SEQ“616?: i 1 05 '60E7 E: NI—l va‘m’m“““W“winESEQID: NO:68m FWVHWSEQIDNC“):63““““NI—105.14E2 VLWESEQ. 113.“N070 :E NI-105.39E2 “‘W‘u‘a-.4.......““ E NI-lOS 19C6 WEVH NO:73 E VL SEQ113“ND;W721“ “ EBCEVH SEQ. ID. No.75 VH SEQ113‘N076 “HOS-9‘34 BGVWSEQwNowW EQ‘.“1“13‘.“NE3‘§1§W WO 00600 ' 42 - In one embodiment, an antibody of the t invention comprises a heavy chain variable region (VH) comprising, or ting of an amino acid sequence selected from the group consisting of SEQ ID NO: 44, 45, 47, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 76, and 220. In one embodiment, an antibody of the present invention comprises a light chain variable region (VL) comprising, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 46, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, and 222. In one embodiment, an antibody of the present ion comprises a heavy chain variable region (VH) comprising, or consisting of an amino acid sequence ed from the group consisting of SEQ ID NO: 44, 45, 47, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 76, and 220, and further comprises a light chain variable region (VL) comprising, or consisting of an amino acid sequence ed from the group ting of SEQ ID NO: 46, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, and 222. In a specific embodiment, the antibody comprises a VH of SEQ ID NO: 45 and a VL of SEQ ID NO: 46; or a VH of SEQ ID NO: 45 and a VL of SEQ ID NO: 221; or a VH of SEQ ID NO: 45 and a VL of SEQ ID NO: 222; or a VH of SEQ ID NO: 48 and a VL of SEQ ID NO: 49; or a VH of SEQ ID NO: 50 and a VL of SEQ ID NO: 51; or a VH of SEQ ID NO: 52 and a VL of SEQ ID NO: 53; or a VH of SEQ ID NO: 54 and a VL of SEQ ID NO: 55; or a VH of SEQ ID NO: 220 and a VL of SEQ ID NO: 55; or a VH of SEQ ID NO: 56 and a VL of 2O SEQ ID NO: 57; or a VH of SEQ ID NO: 58 and a VL of SEQ ID NO: 59; or a VH of SEQ ID NO: 60 and a VL of SEQ ID NO: 61; or a VH of SEQ ID NO: 62 and a VL of SEQ ID NO: 64; or a VH of SEQ ID NO: 65 and a VL of SEQ ID NO: 66; or a VH of SEQ ID NO: 67 and a VL of SEQ ID NO: 68; or a VH of SEQ ID NO: 69 and a VL of SEQ ID NO: 70; or a VH of SEQ ID NO: 71 and a VL of SEQ ID NO: 72; or a VH of SEQ ID NO: 73 and a VL of SEQ ID NO: 74; or a VH of SEQ ID NO: 76 and a VL of SEQ ID NO: 78; or a VH of SEQ ID NO: 44 and a VL of SEQ ID NO: 46; or a VH of SEQ ID NO: 47 and a VL of SEQ ID NO: 49; or a VH of SEQ ID NO: 62 and a VL of SEQ ID NO: 63; or a VH of SEQ ID- NO: 75 and a VL of SEQ ID NO: 77.

Alternatively, the antébody of the present invention is an antibody or antigen- binding fragment, derivative or variant thereof, which competes for binding to tau, such as, for example, hTau40, with at least one of the antibodies having the VH and/or VL region as depicted in Fig. 7 and Table 3. In one embodiment, an antibody of the present invention competes for specific binding to hTau40 with NI-105.17C1, NI-105.6C5, NI- ‘ 43 ' 105.29G10, .6L9, NI-105.40E8, NI-105.48E5, NI-105.6E3, NI—105.22E1, NI- 105.26E:;12, NI-105.12E12, NI-105.60E7, NI-105.14E2, NI-105.39E2, NI-105.19C6, or NI-105.9C4. Those antibodies can be human as well, in particular for therapeutic ations. Alternatively, the antibody is a murine, murénized and chimeric murine- human antibody, which are particularly useful for diagnostic methods and studies in In one embodiment the antibody of the present invention is provided by cultures of single or oligoclonal B—cells that are cultured and the supernatant of the culture, which contains antibodies produced by said B-cells is screened for presence and affinity of anti- tissue tau dies therein. The screening process comprises the steps of a sensitive d plaque immunoreactivity (TAPIR) assay such as described in international application W02004/09503l, the disclosure content of which is incorporated herein by reference; screen on brain sections for binding to PHFTau; screening for binding of a peptide derived from tau of the amino acid sequence ented by SEQ lD NO:6 with phosphate groups on amino acids Ser-202 and Thr-205; on amino acid Thr-231; and/or on amino acids Ser—396 and Ser-404 of said sequence; a screen for g of recombinant human tau of the amino acid sequence represented by SEQ ID N026 and isolating the antibody for which binding is detected or the cell ing said antibody.

As ned above, due to its generation upon a human immune response the human monoclonal antibody of the present invention will recognize epitopes which are of particular pathological relevance and which might not be accessible or less immunogenic in case of immunization processes for the generation of, for example, mouse monoclonal antibodies and in vitro screening of phage display libraries, respectively. Accordingly, it is prudent to stipulate that the epitope of the human anti-tau dy of the present invention is unique and no other antibody which is capable of g to the e recognized by the human monoclonal antibody of the present invention exists. Therefore, the present invention also extends generally to au antibodies and tau binding molecules which compete with the human monoclonal antibody of the present invention for specific binding to tau. The present invention is more specifically directed to an antibody, or antigen-binding fragment, variant or derivatives f, where the antibody specifically binds to the same epitope of tau as a reference antibody ed from the group consisting of NI-105.17C1, .6C5, NI-105.29G10, NI-105.6L9, NI- W0 2014/100600 105.40E8, NI—105.48E5, NI—105.6E3, NI-105.22E1, NI-105.26B12, .12E12, NI- E7, .14E2, NI-105.39E2, .19C6, and NI-105.9C4.

Competition between antibodies is determined by an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such tau. Numerous types of competitive binding assays are known, for as example: solid phase direct or indirect mmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; see Stahli et al., Methods in Enzymology 9 (1983), 242-253; solid phase direct biotin-avidin EIA', see Kirkland et al., J. Immunol. 137 (1986), 619 and Cheung et al., Virology 176 (1990), 546-552; solid phase direct d assay, solid phase direct labeled sandwich A Laboratory Manual, Cold Spring Harbor Press assay; see Harlow and Lane, Antibodies, (1988); solid phase direct label RIA using 1125 label; see Morel et al, Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand. J. Immunol. 32 (1990), 77-82. Typically, such an assay involves the use of purified tau or aggregates thereof bound to a solid surface or cells bearing either of these, an unlabelled test immunoglobulin and a labeled reference immunoglobulin, i. e. the human monoclonal antibody of the present invention.

Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. In one ment, the competitive binding assay is performed under conditions as described for the ELISA assay in the appended Examples. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to the reference antibody an adjacent e sufficiently proximal to the epitope bound by for steric hindrance to occur. Usually, when a competing antibody is present in excess, will t specific binding of a reference antibody to a common n by at least 50% or antigen-binding or 75%. Hence, the present invention is further drawn to an antibody, fragment, variant or derivatives thereof, where the antibody competitively ts a reference antibody selected from the group consisting of NI—105.17Cl,. NI-105.6C5, NI- 105.29G10, NI-105.6L9, NI-105.40E8, NI-105.48E5, NI—105.6E3, NI-105.22E1, NI- 105.26B12, NI-105.12E12, .60E7, NI-105.14E2, NI-105.39E2, NI-105.19C6, or NI-105.9C4 from binding to tau.

In another embodiment, the present ion provides an ed polypeptide comprising, consisting essentially of, or ting of an immunoglobulin heavy chain W0 2014/100600 PCT/USZOl3/076952 variable region (VH), where at least one of VH—CDRs of the heavy chain variable region are at least 80%, 85%, or at least two of the VH-CDRS of the heavy chain le region 90%, 95%, 96%, 97%, 98% or 99% identical to reference heavy chain VH-CDRI, VH- CDR2 or VH-CDR3 amino acid sequences from the antibodies disclosed herein.

Alternatively, the VH-CDRl, VH-CDRZ and VH-CDR3 s of the VH are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% cal to reference heavy chain VH -CDR1, herein.

VH-CDR2 and VH—CDR3 amino acid ces from the antibodies disclosed invention has Thus, according to this embodiment a heavy chain variable region of the shown VH—CDRl, VH-CDR2 and VH~CDR3 polypeptide sequences related to the groups in Fig. 7. While Fig. 7 shows VH-CDRs defined by the Kabat system, other CDR 10 definitions, e.g. , VH-CDRs defined by the Chothia system, are also included in the present invention, and can be easily identified by a person of ordinary skill in the art using data presented in Fig. 7. In one embodiment, the amino acid sequence of the reference VH CDR1 is SEQ ID NO: 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, NO: 80, 86, 157, or 163; the amino acid sequence of the reference VH CDR2 is SEQ ID 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and the amino acid sequence of the reference VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129,135,141,147,153,159, or 165.

In another ment, the present invention provides an isolated polypeptide 2O comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain le region (VH) in which the VH-CERI, VH-CDR2 and 3 regions have polypeptide sequences which are identical to the VH-CDRI, VH-CDR2 and VH-CDR3 groups shown in Fig. 7". In one embodiment, the amino acid sequence of the VH CDRI SEQ ID NO: 79, 85, 91, 97,103,109,115,121,127,133,139,145,151,157, or 163; amino acid sequence of the VH CDR2 is SEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122, VH CDR3 is 128, 134, 140, 146, 152, 158, or 164; and the amino acid ce of the 165.

SEQ ID NO: 81, 87, 93, 99,105,111,117,123,129,135,141,147,153,159, or In r embodiment, the present invention ,provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRI, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2 and 3 for one, two, three, four, five, six, seven, eight, nine, or ten groups shown in Fig. 7, except amino acid amino acid substitutions in any one VH—CDR. IE certain embodiments the substitutions are conservative. In one embodiment, the amino acid sequence of the CDRl is SEQ ID NO: 79, 85, 91, 97, 103,109,115,121,127,133,139,145, 151,157, or 163; the amino acid sequence of the VH CDR2 is SEQ ID NO: 80, 86, 92, 98, 104, 110, the VH 116, 122, 128, 134, 140, 146, 152, 158, or 164; and the amino acid sequence of CDR3 is SEQ ID NO: 81, 87, 93, 99,105,111,117,123,129,135,141,147,153,159, or 165.

In another embodiment, the present ion provides an isolated polypeptide comprising, consisting essentially of, or ting of an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region are at least 80%, 85%, or at least two of the VL-CDRS of the light chain variable region 90%, 95%, 96%, 97%, 98% or 99% identical to reference light chain VL-CDRI, VL- CDR2 disclosed or 3 amino acid sequences from antibodies Alternatively, the VL-CDRl, VL-CDRZ and 3 regions of the VL are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to nce light chain VL-CDRl, herein.

CDR2 and VL-CDR3 amino acid sequences from antibodies disclosed Thus, according to this embodiment a light chain variable region of the invention has VL-CDRl , in Fig.

VL-CDRZ and VL-CDR3 polypeptide sequences related to the polypeptides shown 7. While Fig. 7 shows VL-CDRs defined by the Kabat system, other CDR definitions, also included in the present invention. e. g., VL-CDRS defined by the Chothia , are In one embodiment, the amino acid sequence of the reference VL CDRl is SEQ ID NO: 20 the amino 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or 224; acid ce of the reference VL CDR2 is SEQ ID NO: 83, 89, 95, 101, 107, 113, 119, of the reference 125, 131, 137, 143, 149, 155, 161, or 167; and the amino acid sequence VL CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, or 168.

In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) in which the VL-CDRl, 2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRI, VL-CDR2 and VL-CDR3 the amino acid sequence of the VL CDRl groups shown in Fig. 7. In one embodiment, SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, I36, 142, 148, 154, 160, 166, or 224; the amino acid sequence of the VL CDR2 is SEQ ID NO: 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, or 167; and the amino acid sequence ofthe VL W0 2014/100600 ' 47 ' PCT/USZOl3/076952 CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, or 168.

In another ment, the present invention provides an ed ptide comprising, consisting essentially of, or consisting of an globulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL—CDRl, VL-CDR2 and VL-CDR3 groups shown in Fig. 7, except for one, two, three, four, five, six, seven, eight, nine, or amino acid tutions in any one VL-CDR. In certain ments the amino acid tutions are conservative. In one embodiment, the amino acid sequence of the VL CDRI is SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or 224; the amino acid sequence of the VL CDR2 is SEQ ID NO: 83, 89, 95, 101, 107, 113, 11.9, 125, 131, 137, 143, 149, 155, 161, or 167; and the amino acid sequence of the VL CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, or 168.

[0147] In another embodiment, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) which is. identical to a reference heavy chain variable region shown in Fig. 7 and Table 3. In one embodiment, the amino acid sequence of the reference heavy chain variable regior; comprises SEQ ID NO: 44, 45, 47, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 76, or 220.

In another embodiment, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an globulin heavy chain variable region (VH) having a polypeptide sequence which is identical to a reference heavy chain variable region (VH) sequence shown in Fig. 7 and Table 3, except for one, two, three, four, five, six, seven, eight, nine, or ten aneino acid substitutions. In n embodiments the amino acid substitutions are conservative. In one embodiment, the amino acid sequence of the reference heavy chain variable region sequence comprises SEQ ID NO: 44, 45, 47, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 76, or 220.

According to one embodiment, the invention provides an isolated polypeptide comprising, ting essentially of, or consisting of an immunoglobulin light chain variable region (VL) at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference light chain variable region (VL) amino acid sequence from the antibodies disclosed herein. Thus, according to this embodiment a light chain variable region of ' 48 ' the invention has a ptide sequence related to the light chain le regions shown in Fig. 7 and Table 3. In one embodiment, the amino acid sequence of the reference light chain variable region (VL) comprises SEQ ID NO: 46, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, or 222.

In another embodiment, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) which is identical to a reference light chain le region shown in Fig. 7 and Table 3. In one embodiment, the amino acid ce of the reference light chain variable region comprises SEQ ID NO: 46, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, or 222.

In another ment, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) having a polypeptide sequence which is identical to a nce light chain variable region (VL) sequence shown in Fig. 7 and Table 3, except for one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. In certain embodiments the amino acid substitutions are conservative. In one embodiment, the amino acid sequence of the reference light chain variable region sequence comprises SEQ ID NO: 46, 49, 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, or 222.

[0152] An globulin or its encoding cDNA can be further modified. Thus, in a further embodiment the method of the present invention comprises any one of the step(s) of producing a chimeric antibody, murinized dy, single-chain antibody, Fab- fragment, bi—specific antibody, fiision antibody, labeled antibody or an analog of any one of those. Corresponding methods are known to the person skilled in the art and are described, e.g., in Harlow and Lane odies, A Laboratory Manual", CSH Press, Cold Spring Harbor (1988). When derivatives of said antibodies are ed by the phage display que, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to the same epitope as that of any one of the dies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in international application WO89/09622. Methods for the production of humanized antibodies are described in, e.g., European application EP-Al 0 239 400 and international application WO90/07861. A invention are so- further source of antibodies to be utilized in accordance with the present called xenogeneic antibodies. The general ple for the production of xenogeneic such antibodies in mice is described in, e. g., international antibodies as human-like As sed applications WO91/10741, 2602, 4096 and WO 96/33735. besides complete above, the antibody of the invention can exist in a variety of forms antibodies; including, for example, FV, Fab and F(ab)2, as well as in single chains; see e.g. international application W088/O9344.

The antibodies of the present ion or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for and/or example, by using amino acid deletion(s), insertion(s), substitution(s), aridition(s), recombination(s) and/or any other modification(s) known in the art either alone or in combination. s for introducing such ations in the DNA sequence known to the underlying the amino acid sequence of an immunoglobulin chain are well person skilled in the art; see, e.g, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY. and Ausubel, Current Protocols in lar y, Green Publishing Associates and Wiley Interscience, (1994).

Modifications of the antibody of the invention include chemical and/or enzymatic side chain derivatizations at one or more constituent amino acids, including modifications, ne modifications, and N— and C-terrninal modifications including ation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid es, cofactors, and the like. Likewise, the t invention encompasses production of chimeric proteins which comprise the described antibody or some fragment f at the amino terminus fused to heterologous molecule such as an immunostimulatory ligand at the carboxyl terminus; see, e. g., international application WOOD/30680 for corresponding technical details. onally, the t invention encompasses peptides including those containing a binding molecule as described above, for example containing the CDR3 in particular CDR3 region of the variable region of any one of the mentioned antibodies, of the heavy chain since it has frequently been observed that heavy chain CDR3 (HCDR3) is the region having a greater degree of variability and a predominant participation in antigen-antibody interaction. Such peptides can easily be synthesized or produced by recombinant means to produce a binding agent useful according to the aneIEtiOIl. Such methods are well known to those of ordinary skill in the art. Peptides can be synthesized for example, using automated peptide synthesizers which are commercially available. The peptides can also be produced by recombinant techniques by incorporating the DNA sing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide.

[0155] Hence, the t invention s to any g molecule, e. g., an antibody or binding fragment thereof which is oriented towards the human anti—tau antibodies of the present invention and display the ned properties, i. e. which specifically recognize tau. Such antibodies and binding molecules can be tested for their binding specificity and affinity by ELISA and Western Blot and immunohistochemisty as described herein, see, e. g., the Examples. Furthermore, preliminary results of subsequent experiments performed in accordance with the present invention revealed that in one ment, the human ant—tau antibody of the present ion binds primarily to pathologically aggregated tau resembling neurofibrillary tangles (NFT), neuropil threads t on human. brain sections of patients who suffered from Alzheimer’s disease (AD) in addition. Thus, in a particular preferred embodiment of the t invention, the human antibody or g fragment, derivative or variant thereof recognizes tau on human AE brain sections.

As an alternative to obtaining immunoglobulins directly from the culture of immortalized B cells or B memory cells, the immortalized cells can be used as a source of rearranged heavy chain and light chain loci for subsequent expression and/or c manipulation. Rearranged antibody genes can be reverse transcribed from; appropriate mRNAs to produce cDNA. If desired, the heavy chain constant region can be exchanged for that of a different isotype or eliminated altogether. The variable regions can be linked to encode single chain Fv s. Multiple Fv regions can be linked to confer binding ability to more than one target or chimeric heavy and light chain combinations can be employed. Once the genetic material is available, design of analogs as bed above which retain both their ability to bind the d target is straightforward. s for the cloning of antibody variable regions and generation of recombinant antibodies are known to the person skilled in the art and are described, for example, Gilliland et al., Tissue Antigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792. [01%7] Once the appropriate genetic material is obtained and, if desired, modified to encode an analog, the coding sequences, including those that , at a minimum, the variable regions of the heavy and light chain, can be inserted into expression systems host cells. A contained on vectors which can be transfected into standard recombinant mammalian cells variety of such host cells can be used; for efficient processing, however, lines useful for this purpose include, but are can be considered. Typical mammalian cell not limited to, CHO cells, HEK 293 cells, or NSO cells.

[0158] The tion of the antibody or analog is then undertaken by culturing the of the host modified recombinant host under culture conditions appropriate for the growth then recovered by cells and the expression of the coding sequences. The antibodies are include signal isolating them from the e. The expression systems are designed to that the resulting antibodies are secreted into the medium; however, peptides so intracellular production is also possible. ion also relates to a In accordance with the above, the present polynucleotide encoding the antibody or equivalent binding molecule of the present le region of an ion. In one embodiment, the polynucleotide s at least a immunoglobulin chain of the antibody described above. Typically, said variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the VH and/or VL of the le region of the said antibody. domain of the The person skilled in the art will readily appreciate that the le construction of antibody having the above-described variable domain can be used for the fimction. Thus, the other polypeptides or antibodies of desired specificity and biological and antibodies comprising at least one present invention also encompasses polypeptides have CDR of the above-described variable domain and which advantageously in the substantially the same or similar binding properties as the antibody described appended examples. The person skilled in the art knows that binding affinity can be making amino acid tutions within the CDRs or . the enhanced by ariable loops (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917) which partially p with the CDRs as defined by Kabat; see, e. g., Riechmann, et al, Nature wherein one or 332 (1988), 323-327. Thus, the t invention also relates to antibodies or not more than two amino acid more of the mentioned CDRs comprise one or more, in one or both substitutions. In one embodiment, the antibody of the invention comprises set forth in of its immunoglobulin chains two or all three CDRS of the variable s as Fig. l.

Binding molecules, e. g., antibodies, or antigen-binding fragments, variants, or skill in the derivatives thereof of the invention, as known by those of ordinary art, can W0 2014/100600 PCT/USZOl3/076952 comprise a constant region which mediates one or more effector functions. For example, binding of the Cl component of complement to an antibody constant region can activate the complement . Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity. Further, antibodies bind to receptors on various cells via the Fc region, with a PC receptor binding site on the dy Fc region binding to a PC receptor (FCR) on a cell. There are a number of Fc receptors which are specific for different classes of dy, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).

Binding of antibody to Fc receptors on cell surfaces triggers a number of ant and diverse biological responses including ment and destruction of antibody-coated particles, clearance of immune xes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.

[0162] Accordingly, certain embodiments of the present invention include an antibody, or antigen-binding fragment, variant, or derivative f, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as provide desired biochemical characteristics such as reduced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of tau aggregation and deposition, reduced serum half-life, or increased serum ife when compared with a whole, unaltered dy of approximately the same immunogenicity. For example, certain antibodies for use in the diagnostic and treatment methods described herein are domain deleted antibodies which se a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain s. For instance, in certain antibodies, one entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted. In other embodiments, certain antibodies for use in the diagnostic and treatment methods bed herein have a constant region, e. g., an IgG heavy chain nt region, which is altered to ate glycosylation, referred to elsewhere herein as aglycosylated or "agly" antibodies. Such "agly" antibodies can be ed enzymatically as well as by engineering the sus glycosylation site(s) in the constant . While not being bound by theory, it is believed that "agly" antibodies can have an improved safety and stability profile in viva. Methods of producing aglycosylated ' 53 - antibodies, having desired effector function are found for e in international application W02005/018572, which is incorporated by reference in its entirety.

In certain antibodies, or n—binding fragments, ts, or derivatives thereof described herein, the Fc n can be mutated to decrease effector function using techniques known in the art. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor g of the circulating modified antibody thereby increasing tau localization. In other cases it can be that nt region modifications consistent with the instant invention te ment binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the nt region can be used to modify disulfide es or accharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tau localization, biodistribution and serum half-life, can easily be measured and quantified using well know immunological techniques without undue experimentation.

In n antibodies, or antigen-binding fragments, variants, or derivatives thereof described , the Fc portion can be mutated or ged for alternative protein of example by enhancing sequences to increase the cellular uptake of antibodies by way receptor—mediated endocytosis of antibodies via Fc7 receptors, LRP, or Thyl receptors or by ‘SuperAntibody Technology', which is said to enable antibodies to be shuttled into living cells without harming them (Expert Opin. Biol. Ther. (2005), 237-241). For example, the generation of fusion proteins of the antibody binding region and the cognate protein ligands of cell surface ors or bi— or multi-specific antibodies with a specific sequences hiding to tau as well as a cell surface receptor can be engineered using techniques known in the art.

In certain antibodies, or antigen—binding fragments, variants, or derivatives thereof described herein, the Fc portion can be mutated or exchanged for alternative protein increase its blood brain barrier sequences or the antibody can be chemically d to penetration.

[0166] Modified forms of antibodies, or n-binding fragments, variants, or derivatives thereof of the invention can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein. Antibodies, or antigen-binding fragments, variants, or derivatives — 54 - thereof of the invention can be made or manufactured :using techniques that are known in the art. In certain embodiments, antibody molecules or fragments thereof are "recombinantly produced," i.e., are produced using recombinant DNA logy.

Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.

Antibodies, or antigen—binding fragments, variants, or derivatives f of the invention also include derivatives that are modified, e. g., by the nt attachment of such that covalent attachment does not prevent the any type of molecule to the antibody antibody from specifically binding to its cognate epitope. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e. g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic ge, linkage to a ar ligand or known other protein, etc. Any of numerous chemical modifications can be carried out by techniques, inclUding, but not limited to c chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids.

In particular embodiments, dies, or antigen-binding fragments, ts, or derivatives thereof of the invention will not elicit a deleterious immune response in the animal to be treated, e. g., e. g., in a human. In n embodiments, binding molecules, antibodies, or antigen-binding fragments thereof of the invention are deréved from a t, same species from which e. g., a human patient, and are subsequently used in the they are d, e.g., human, alleviating or zing the occurrence of deleterious immune responses.

De-immunization can also be used to decrease the immunogenicity of an antibody.

As used herein, the term "de-immunization" includes alteration of an antibody to modify T cell epitopes; see, e. g, international ations WO98/52976 and WOOD/34317. example, VH and VL sequences from the starting antibody are analyzed and a human T cell epitope "map" from each V region g the location of epitopes in relation to complementarity determining regions (CDRs) and other key residues within the sequence.

Individual T cell epitopes from the T cell epitope map are analyzed in order to fy alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative VH and VL sequences are designed comprising combinations of amino acid substitutions and these ces are subsequently ' 55 ' orated into a of binding ptides, e. g., tau-specific antibodies range or immunosEecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function. lly, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent ds introduced into cell lines for the tion of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of oma, recombinant, and phage display techEologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known iE the art and taught, for example, in Harlow et 61]., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Harnmerling et al., in: onal Antibodies and T-Cell Hybridomas er, N.Y., 563-681 (1981), said references incorporated by reference in their entireties. The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. In certain embodiments, dies of the present invention are derived from human B cells which have been immortalized Via transformation with Epstein-Barr virus, as described herein.

In the well—known hybridoma process r et al., Nature 256 (1975), 495) the relatively short-lived, or mortal, lymphocytes from a mammal, e.g., B cells derived from. line a human subject as described , are fused with an immortal tumor cell (e. g. ,. a myeloma cell line), thus, producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded dy of the B cell. The resulting hybrids are segregated into single genetic strains by selection, dilution, and re- growth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies, which are homogeneous against a d antigen and, in nce to their pure genetic parentage, are termed "monoclonal".

Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that contain one or more substances that inhibit the growth or al of the unfused, w 56 — cell lines parental myeloma cells. Those skilled in the art will iate that reagents, and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established. for Generally, culture medium in which the hybridoma cells are g is assayed production of monoclonal antibodies against the desired antigen. The binding specificity of the monoclonal antibodies ed by hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme—linked immunoabsorbent assay (ELISA) as described herein. Afier hybridoma cells are identified that produce antibodies of the desired specificity, affinity and/or activity, clones can be subcloned by ng dilution procedures and grown by standard methods; Academic Press, pp see, e. g., Goding, Monoclonal Antibodies: Principles and Practice, 59-103 (1986). It will further be appreciated that the monoclonal antibodies ed by the subclones be separated from culture medium, ascites fluid or serum can by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

In another embodiment, lymphocytes can be selected by micromanipulation the le genes isolated. For example, eral blood mononuclear cells can be isolated from an immunized or naturally immune , e.g., a human, and cultured that meet the for about 7 days in vitro. The cultures can be screened for specific IgGs screening criteria. Cells from positive wells can be isolated. Individual Ig-producing cells be ed by FACS or by identifying them in a can complement-mediated and the hemolytic plaque assay. Ig-producing B cells can be anipulated into a tube VH and VL genes can be amplified using, e.g., RT—PCR. The VH and VL genes can cloned into an antibody expression vector and transfected into cells (e. g., eukaryotic or prokaryotic cells) for expression.

Alternatively, antibody-producing cell lines can be selected and cultured using in a variety of techniques well known to the skilled artisan. Such techniques are described le for use laboratory manuals and primary publications. In this t, ques in the invention as described below are described in Current Protocols in Immunology, n et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.

PCT/USZOl3/076952 known Antibody fragments that recognize specific epitopes can be generated by techniques. For example, Fab and F(ab')2 fragments can be produced recombinantly or by proteolytic cleavage of immunoglobulin les, using enzymes such as papain (to e Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab’)2 fragments of the contain the variable region, the light chain constant region and the CHI domain heavy chain. Such fragments are ent for use, for example, in immunodiagnostic procedures ing coupling the immunospecific portions of immunoglobulins to detecting reagents such as radioisotopes.

: Human antibodies, such as bed herein, are particularly desirable for therapeutic use in human patients. Human antibodies of the present invention are isolated, be suspected to be at risk e.g., from y human subjects who because of their age may of developing a tauopathic disorder, e.g., Alzheimer’s e, or a patient with the disorder but with an unusually stable disease course. However, though it is prudent to will. expect that elderly healthy and symptom-free subjects, respectively, more regularly latter can be used have developed protective anti-tau antibodies than younger subjects, the as well as source for obtaining a human antibody of the present ion. This form of a particularly true for younger patients who are predisposed to p a familial tauopathic disease but remain symptom—free since their immune system functions more efficiently than that in older adults.

[0177] In one embodiment, an antibody of the invention comprises at least one heavy or of the light chain CDR of an antibody le. In another embodineent, an antibody another invention comprises at least two CDRs from one or more antibody molecules. In from one or embodiment, an antibody of the invention comprises at least three CDRs an antibody of the invention comprises more antibody les. In another embodiment, In another ment, an at least four CDRs from one or more antibody molecules. antibody of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least six CDRs from one or more dy molecules. Exemplary antibody molecules comprising described herein. at least one CDR that can be ed in the subject antibodies are

[0178] dies of the present invention can be produced by any method known in recombinant art for the synthesis of antibodies, in particular, by chemical synthesis or by expression techniques as described herein. ' 58 ' 2013/076952 {$139} in one embodiment, an antihedy, or antigembindirtg fragment, t, er derivative thereof of the invention comprises a synthetic constant regien wherein etie er more ts are eartiaiiy er entirety deieted {”domairrwdeteted antibodiesft. in certain embodiments compatible modified antibodies will se domain d constructs or ts wherein the entire CH2 domain has been removed (ACH2 constructs). For other embodiments a short connecting peptide can be substituted for the deleted domain to provide flexibility and freedem ef movement fer the variabie regierr. ’i‘hcse skiiied in the art wiii artpreeiate that such ucts are particoiariy preferred due to the reguiatcry ereperties ef the CH3 demain on the eatahotic rate of the airtihedy, Domain deleted ceastructs can be derived using a vecter encoding an igGg human censtant domain, see, sag, intematienai appiieations WSW/$66955 and WOG2I’0S36948A2, This vector is ered te delete the CH2 demain and provide a synthetie vector exeressing a n deleted igfil cetrstant region. in n embodiments, an‘tibedies, er antigen—binding fragments, variants, er derivatives thereof dfthe present inventien are minibedies. Mirrihedies can be made using methods bed in the art, see, e.g., US patent 5,837,821 or international application WO 94/09817.

In one embodiment, an antibody, or antigen—binding fragment, variant, or derivative thereof of the inveetion comprises an globulin heavy chain having deletion or substitution of a few or even a single amino acid as long as it permits association between the monomeric subunits. For example, the mutation of a single amino acid in selected areas of the CH2 domain can be enough to substantially reduce Fc binding and thereby increase tau localization. Similarly, it can be desirable to simply delete that part of one or more constant region domains that control the effector on (e. g. complement binding) to be modulated. Such partial deletions of the constant regions can improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant regions of the disclosed antibodies can be synthetic through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect, the ty provided by a conserved binding site (e. g. Fc binding) can be disrupted while substantially ining the configuration and immunogenic profile of the modified antibody. Yet other embodiments comprise the addition of one enhance desirable or more amino acids to the constant region to characteristics such as effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it can be desirable to insert or replicate specific domains. sequences derived from ed constant region The present invention also es dies that comprise, consist essentially of, varéants (including derivatives) of antibody molecules (e. g., the VH of, or consist thereof regions and/or VL regions) bed herein, which antibodies or fragments immunospecifically bind to tau. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody, including, but reot limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. In one ment, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 10 less than 20 amino acid substitutions, less than 15 amino acid substitutions, amino acid substitutions, less than 5 amino acid tutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH region, VH—CDRI, VH-CDR2, VH-CDR3, VL region, VL- in which CDRl, VL-CDRZ, or VL-CDR3. A "conservative amino acid tution" is one with a the amino acid residue is replaced with an amino acid residue having a side chain similar charge. Families of amino acid residues having side chains with r charges have been defined in the art. These es include amino acids with basic side chains (e. g, lysine, arginine, histidine), acidic side chains (e. g., aspartic acid, glutamic acid), uncharged polar side chains (e. g., e, asparagine, glutamine, serine, threonine, ne, cysteine), nonpolar side chains (e. g., alanine, , leucine, cine, proline, alanine, methionine, tryptophan), beta-branched side chains ( e. g., threonine, valine, isoleucine) and aromatic side chains (e. g., tyrosine, phenylalanine, tryptophan, of the histidine). Alternatively, mutations can be introduced randomly along all or part coding sequence, such as by saturation mutagenesis, and the resultant mutants can screened for biological activity to identify mutants that retain activity (e.g., the ability to bind tau). [0i83] For example, it is possible to introduce mutations only in framework regions or only in CETER regions of an antibody molecule. Introduced mutations can be silent or neutral missense mutations, to bind e. g., have no, or little, effect on an antibody’s ability W0 00600 PCT/USZOl3/076952 antigen, indeed some such mutations do not alter the amino acid sequence ever.

These types Sf mutations can be useful to optimize codon usage, or improve a hybridenia’s antibody production. optimized ceding regiens mending antibodies 0f the invention are sed eisewhere herein. newnentrai present Alternatively, missense mutations can alter an antibody’s ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the franeework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not and test mutant an absolute requirement. One of skill in the art would be able to design molecules with desired properties such as no alteration in antigen-binding activity or alteration in binding activity (e.g., improvements in antigen-binding activity or change in antibody specificity). ing mutagenesis, the encoded protein can routinely be expressed and the functional and/or biological activity of the encoded protein, (e. g., ability to immunospecifically bind at least one epitope of tau) can be determined using techniques described herein or by routinely modifying techniques known in the art. [0E84] Tau binding agents, for example, but not limited to, tau binding antibodies of the present invention can be characterized using vivo in models of any in or Vitro neurodegenerative thies. A skilled artisan y understands that a tau binding (e.g., an antibody) of the invention can be characterized in a mouse model for agent neurodegenerative thies. for example, but not limited to, any one of the following 2O three different animal models for thies can be used to characterize and validate the tau antibodies (and molecules with the binding cities thereof) of the present invention. 1. Transgenic TauP301L mice (line183): expressing human Tau40 with P301L mutation under the murine Thy1.2 promoter (Generation of these transgenic animals is described in Gotz et al., J. Biol. Chem. 276 (2001), 529-534 and in international application , the disclosure content of which is incorporated herein by reference) 2. JNPL3 mice expressing the shortest 4R human tau isoform with P301L mutation under the murine PrP promoter (available from Taconic, , NY, USA).

[0187] 3. P3OISTau (line P819) mice expressing human tau with P3018 mutation under the murine PrP promoter able from the n tory, Bar Harbor, Maine, LLSnA) ' 61 ' A skilled artisan understands that an mental model of neurodegenerative tauopathies can be used in a preventative setting or it can be used in a therapeutic setting.

In a preventative setting, the dosing of animals starts prior to the onset of the neurodegenerative tauopathies or symptoms thereof. In preventative settings, a tau g agent (e.g., antibody) of the invention is evaluated for its ability to prevent, reduce or delay the onset of neurodegenerative tauopathies or symptoms thereof, In a therapeutic setting, the dosing of animals start after the onset of neurodegenerative tauopathies or a symptom thereof. In a eutic setting, a tau g agent (e.g., antibody) of the invention is evaluated for its y to treat, reduce or alleviate the neurodegenerative tauopathies or a symptom thereof. Symptoms of the neurodegenerative tauopathies include, but are not limited to, accumulation of pathological tau deposits, neurofibrillary tangles (NFT), hyperphosphorylated tau polypeptide, insoluble tau fractions in the s, brain, spinal cord, cerebrospinal fluid or serum of the experimental object. A skilled artisan understands that a positive preventative or therapeutic outcome in any animal model of neurodegenerative tauopathies indicates that the particular tau binding agent (e.g., antibody) can be used for tative or therapeutic purposes in a subject other than the experimental model organism, for example, it can be used to treat neurodegenerative tauopathies in a human subject in need thereof

[0189] In one embodiment, a tau binding agent (e.g., an antibody) of the invention can be administered to a tauopathy mouse model and corresponding control Wild type mice. The antibody stered ean be a minimized dy eftlie present inventien er a human— murine chimera {if an antibedy of the present inventien. 'l‘lie tan binding agent. (e.g., an dy) can be stered by any means linemr in the art, fer example, by intraperiteneal, intracraniai, intramuscular, eneus, subcutaneous, anal, and aeresel administration. Experimental animals ean be given 011e, twe, three, liter, live or more doses of tire tau binding agent (e.g., an antibedy) er a. centre} eempesitien, such as PBS. in one embediment, experimental animals Wiil be administered Gm: or twe doses of a tau g agent (e.g., an antibody). See, for example, Example 9. In another embodiment, the animals are chronically dosed with the tau binding agent (e.g., an antibody) over several weeks or months. See, for example, Example 10. A skilled n can readily design a dosing regimen that fits the experimental purpose, for example, dosing regimen for acute studies, dosing regimen for chronic studies, dosing regimen for toxicity studies, dosing n for preventative or therapeutic studies. The ce of tlee tau binding animals, for agent (e.g., dy) in a particular tissue compartment of the mental example, but not limited to, serum, blood, cerebrospinal fluid, brain tissue, can be established using well know methods of the art. See, for example, Example 9 and 10. In one embodiment, a tau binding agent (e.g., antibody) of the invention is capable to the tau penetrate the blood brain barrier. A skilled artisan understands that by adjusting binding agent (e.g., antibody) dose and the dosing frequency, a desired tau binding agent effect (e.g., antibody) concentration can be maintained in the experimental animals. Any of a tan binding agent (e.g., antibody) of the present invention in the tauopathy models or distribution of tau can be assessed by comparing the level, biochemical teristics in the treated and l animals. In one example, the neurofibrillary tangles (NFT) are examined using the silver impregnation technique of Gallyas or by immunostaining with monoclonal mouse antibody AT100 and AT180, which recognize pathologically phosphorylated tau in NFT. The number or frequency of Gallyas-positive neurons and/or ATIOO, AT180 labeled neurons in the brain and spinal cord in antibody d mice control animals can be ined to evaluate the effect of antibody treatment. In one embodiment, an antibody of the present invention is capable of reducing the level, in an animal amount or tration of neurofibrillary tangles in the brain or spinal cord model. The antibody can reduce the level, amount or concentration of neurofibrillary tangles by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. In another embodiment, an antibody of the present invention is capable of reducing the number or frequency of Gallyas—positive neurons in the brain or spinal cord in an animal model, example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. In a further embodiment, an antibody of the present invention is capable of reducing the number or in an frequency of AT100 or AT180 antibody positive neurons in the brain or spinal cord animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. The effect of an antibody of the t invention can also be ed by examining the distribution and biochemical properties of tau following dy stration. In one embodiment, an antibody of the present invention is capable of reducing the amount or concentration of tau protein in the brain or spinal cord of an animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. In another embodiment, an antibody of the present invention is e ofreducing the amount or concentration of insoluble tau protein in the brain or spinal cord of an WO 00600 ' 63 ' 2013/076952 animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. ble tau fraction can be prepared as described, for example, in Example 10 or in t M, Spillantini MG, Cairns NJ, Crowther RA. Neuron 8, 159 (1992). The amount of tau protein in a biological sample can be ined by any method known to one of skill, for example, as described in Example 10. In a further embodiment, an antibody of the present invention can reduce the amount or concentration of hyperphosphorylated tau protein in the brain or spinal cord in an animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more.

Hyperphosphorylated tau can be detected using antibodies specific for pathologically hquoerphosphorylaterl forms ot‘tau, sueh as ATlilll or A3380. An. antibody ofthe present invention can also alter, for example, reduce or inerease, tau concentration in the hiood, serum or cerebrospinal fluid or an animal model, for example, by at least ahout 5%, lil%, %, 30%, 50%, 70%, 90% or more. in one embodiment, the % reduction or increase is relative compared to the level, ntunltter, fiequenoy, amount or tration that existed before treatment, or to the level, number, frequency, amount or concentration that exist in an untreated/control treated subj eet.

In one embodiment, an antibody of the t invention can prevent or delay the onset of at least one symptom of a neurodegenerative tauopathy in a subject. In one ment, an antibody of the present invention can reduce or eliminate at least one symptom of a neurodegenerative tauopathy in a subject. The symptom can be the formation of pathological tau deposits, hyperphosphorylated tau ts, ble tau deposits, neurofibrillary fibers, brillary fibers, pre—tangle phosphor tau aggregates, intraneuronal neurofibrillary tangles or extraneuronal neurofibrillary tangles in the brain or spinal cord of a subject. See, e.g., Augustinack et a1, Acta athol 103:26-35 (2002). The symptom can also be the presence, or elevated concentration or amount, of tau in the serum, blood, urine or cerebrospinal fluid, wherein elevated concentration amount is compared to a healthy subject. The symptom can be a neurological symptom, for example, altered conditioned taste aversion, altered contextual fear conditioning, memory impairment, loss of motor function. In one embodiment, memory impairment is assessed using a two-trial Y-maze task. In a specific ment, the two-trial Y-maze task is performed ntially as described in Example 10. In one embodiment, the at least one symptom is reduced by at least about 5%, 10%, 15%, 20%, 30%, 50%, 70%, or 90%. In another embodiment, the two-trial Y-maze task ratio is significantly higher in an antibody treated t than in a control subject. In a specific embodiment, the two-trial Y-maze task ratio is increased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In another embodiment, the two-trial Y-maze task ratio is at least about two times, three times, four times, five times, ten times, or twenty times higher. The present invention also provides a method of preventing or delaying the onset of at least one m of a neurodegenerative thy in a subject in need thereof, comprising administeréng a therapeutically effective amount of a tau antibody described herein. The present invention further provides a method of reducing or ating least one symptom of a neurodegenerative tauopathy in a subject in need thereof, comprising administeréng a therapeutically effective amount of a tau antibody described herein. In one embodiment, the subject is an experimental organism, such as, but not limited to, transgenic mouse. In one embodiment, the subject is a human.

IiI. Polynucleotides Encoding Antibodies [019i] A polynucleotide encoding an antibody, or antigen-binding fragment, t, or derivative thereof can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, a cleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of single- and double—stranded DNA, DNA that is a mixture of single- and —stranded regions, single- and double-stranded RNA, and RNA that is mixture of single— and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded s. In addition, a cleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of triple- stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding an antibody, or antigen-binding fragment, variant, or tive thereof can also contain one or more modified bases or DNA or RNA nes modified for stability or for other reasons. "Modified" bases include, for example, ated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" es chemically, enzymatically, or metabolically modified forms.

[0192] An isolated polynucleotide ng a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain n or light chain portion) can be created by introducing one or more tide substitutions, W0 2014/100600 ' 65 ' additions or ons into the nucleotide sequence of the globulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR—mediated mutagenesis. In one embodiment, conservative amino acid substitutions are made at one or more non-essential amino acid residues.

As is well known, RNA can be isolated from the original B cells, hybridorna cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography.

Where desirable, mRNA can be isolated from total RNA by standard techniques such as chromatography on oligo dT ose. Suitable techniques are ar in the art. In one embodiment, cDNAs that encode the light and the heavy chains of the antibody can be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well-known methods. FCR can be initiated by consensus constant region primers or by more specific s based on the hed heavy and light chain DNA and amino acid sequences. As discussed above, PCR also can be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries can be screened by sus primers or larger homologous , such as human constant region probes.

DNA, typically d DNA, can be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e. g., in the foregoing references relating to recombinant DNA techniques. Of , the DNA can be synthetic according to the present invention at any point during the isolation process or uent analysis.

In one embodiment, the present ion provides an isolated polynucleotide comprising, ting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), where at least one of the CDRs of the heavy chain variable region or at least two of the VH-CDRS of the heavy chain variable region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to reference heavy chain VH—CDRI, VH-CDR2, or VH-CDR3 amino acid sequences from the antibodies disclosed herein. Alternatively, the VH-CDEI, VH-CDRZ, or VH-CDR3 regions of the VH are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% cal to reference heavy chain VH-CDRl, VH-CDRZ, and VH-CDR3 amino acid sequences from the antibodies disclosed herein. Thus, according to this embodiment a heavy chain variable region of the invention has VH-CDRI, VH-CDRZ, or VH-CDR3 polypeptide shown in Fig. 7. In one embodiment, the sequences related to the polypeptide sequences amino acid sequence of the reference VH CDRI is SEQ ID NO: 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, or 163; the amino acid sequence ofthe reference VH CDR2 is SEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, NO: 81, 158, or 164; and the amino acid sequence of the reference VH CDR3 is SEQ ID 87, 93, 99,105,111,117,123,129,135,141,147,153,159, or 165.

In one embodiment, the present invention provides an ed polynucleotide comprising, ting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptéde sequences which are identical to the VH-CDRI, VH-CDRZ and VH-CDR3 groups shown in Fig. 7, except for one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in any one VH-CDR. In certain embodiments the amino acid substitutions are conservative. In one embodiment, the amino acid sequence of the VH CDRI is SEQ ID NO: 79, 85, 91, 97, 103, 109, 115, 121, is SEQ 127, 133, 139, 145, 151, 157, or 163; the amino acid sequence of the VH CDR2 ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and amino acid sequence of the VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129,135,141,147,153,159, or 165.

[0197] In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid ng an immunoglobulin light chain variable region (VL), where at least one of the S of variable the light chain variable region or at least two of the s of the light chain region are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to nce dies light chain VL-CDRI, VL-CDRZ, 0r VL-CDR3 amino acid sequences from the disclosed herein. Alternatively, the VL-CDRl, Z, or VL-CDR3 regions of the VL 99% identical to reference light are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or chain I, VL—CDRZ, and VL-CDR3 amino acid sequences from the antibodies sed herein. Thus, according to this embodiment a light chain variable region of ion has VL-CDRl, VL-CDRZ, or VL—CDR3 polypeptide sequences related to the polypeptide sequences shown in Fig. 7. In one embodiment, the amino acid sequence of the reference VL CDRl is SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, CDR2 is 142, 148, 154, 160, 166, or 224; the amino acid sequence of the reference VL SEQ ID NO: 83, 89, 95,101,107, 113, 119, 125, 131, 137, 143, 149, 155, 161, or 167; and the amino acid sequence of the reference VL CDR3 is SEQ ID NO: 84, 90, 96, 102, 4,120,126,132,138,144,150,156, 162, or 168.

In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid ng an immunoglobulin light chain variable region (V1) in which the VL-CDRl, VL-CDRZ and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRI, VL- CDR2 and VL—CDR3 groups shown in Fig. 7, except for one, two, three, four, five, six, seven, eight, nine, or ten amino acid tutions in any one VL-CDR. In certain ments the amino acid substitutions are conservative. in one embodiment, the amino acid sequence of the VL CDRl is SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or 224; the amino acid sequence of the VL CDR2 is SEQ ID NO: 83,89, 95, 101,107,113, 119, 125,131,137, 143, 149,155,161, or 167; and the amino acid sequence of the VL CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120,126,132,138,144,150,156,162, or 168.

In another embodiment, the present invention provides an isolated polynucleotide comprising, ting ially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDRZ, and VH—CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH—CDR2, and VH-CDR3 groups shown in Fig. 7. In one embodiment, the amino acid sequence ofthe VH CDRl is SEQ ID NO: 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, or 163; the amino acid sequence of the VH CDR2 is SEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and the amino acid sequence ofthe VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141,147,153,159, or 165.

In r embodiment, the t invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid ng an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDFi2, and VL—CDR3 regions have polypeptide sequences which are identical to the VL-CDRI, VL- CDR2, and VL-CDR3 groups shown in Fig. 7. In one embodiment, the amino acid sequence ofthe VL CDRl is SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or 224; the amino acid sequence of the VL CDR2 is SEQ ID NO: 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, or 167; and the WO 00600 amino acid sequence ofthe VL CDR3 is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138,144, 150,156, 162, or 168.

As known in the art, "sequence identity" n two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of to polypeptide or one polypeptide or polynucleotide the sequence of a second polynucleotide. When sed , whether any ular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, n, WI 53711). BES’i‘FIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, to find the best segment of homology between two sequences. When using BESTFIT or determine whether a particular sequence is, for any other sequence alignment program to example, 95% identical to a reference sequence ing to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over filll length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference ce are allowed.

In one embodiment of the present invention, the polynucleotide comprises, 2O consists essentially of, or consists of a nucleic acid having a polynucleotide sequence of the VH or VL region of an anti—tau antibody as depicted in Table 4. In this respect, the that the cleotides encoding at least person skilled in the art will readily appreciate the variable domain of the light and/0r heavy chain can encode the variable domain of both immunoglobulin chains or only one.

Table 4: Nucleotide sequences ofthe VH and VL region of tau specific antibodies. BG — before ning may eotiéigéxélifiéfié‘e“;SRWE‘EVETM; and variableulight (VL) chains BG vH SEQ. ID. NO:169 ‘ * .17C1 vH SEQ. ID. NO:170 = [siémdf'iii‘l‘ifiou71 NI-105”,6‘C5 iBG-VH §SEQ.ID.N0:172 W0 2014/100600 ' 69 ' PCT/USZOl3/076952 Antibody Ilm‘m‘EnNucleotidesequences of varlable heavy (VH) and variable light (VL) chams NI-105.6L9 NI-105.40E8 $11111111 NI-105.48E5 I‘N111112 “‘Z‘Nez1z13 NI-105.6E3 ‘ 1111:1111 NI-105.22E1 ‘: . \111117 . 111111111 1111221 1121;;113?31170191 1.111..“ 'N1—1115111137 ...._.m“““ SEQ ED “543192 E...u.....1___......._....m“‘“ NI-105.14E2 i VH SEQ111N13: 191 .39E2 VL SEQ111N11 1111; “""VH .111IN111117 NI-105.19C6 V11‘1W {EE‘VH131111 1111 N11: 1119 NI-105.9C4 ESEQ 113.

In one embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an globulin heavy chain le region at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 95% identical to reference heavy chain VH. In one embodiment, ID NO: amino acid sequence of the reference heavy chain variable region comprises SEQ 44, 45, 47, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 73, 75, 76, or 220.

In one embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region at least 80%, 85%, 90%, 95%, 96%, 97%, the amino 98%, or 99% or 95% identical to reference light chain VL. in one embodiment, NO: 46, 49, acid sequence of the reference light chain variable region comprises SEQ ID 51, 53, 55, 57, 59, 61, 63, 64, 66, 68, 70, 72, 74, 77, 78, 221, or 222. of the The present invention also includes fragments of the polynucleotides invention, as bed elsewhere. Additionally polynucleotides which encode fusion polynucleotides, Fab fragments, and other derivatives, as described herein, are also contemplated by the invention. known in The polynucleotides can be produced or manufactured by any method the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized 17 (1994), 242, oligonucleotides, e. g., as described in Kutmeier et al., BioTechniques which, briefly, involves the synthesis of overlapping ucleotides ning portions of the sequence ng the antibody, annealing and ligating of those oligonucleotides, and then amplification of tEe ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody, or n-binding from a suitable fragment, variant, or derivative f can be generated from nucleic acid is not ble, source. If a clone containing a nucleic acid encoding a particular antibody the dy but the sequence of the antibody molecule is known, a nucleic acid encoding le source (e.g., an antibody cDNA can be chemically synthesized or ed from a library, or a cDNA library ted from, or nucleic acid, preferably polyAJr RNA, isolated from, any tissue or cells expressing the tau-specific antibody, such as hybridoma cells selected to express an antibody) by PCR cation using synthetic s hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e. g., a cDNA clone from a cDNA y that encodes tlee antibody. Amplified nucleic acids ted by PCR can then be cloned into replicable cloning vectors using any d well known in the art.

Once the nucleotide sequence and ponding amino acid sequence of the or derivative thereof is determined, its dy, or antigen-binding fragment, t, i3“; nucleotide sequence can be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for e, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (1990) and Ausubel et al., eds, Current Protocols in Molecular herein Biology, John Wiley & Sons, NY , which are both incorporated by reference in their entireties), to generate antibodies having a different amino acid sequence, example to create amino acid substitutions, deletions, and/or insertions.

IV. Expression of Antibody Polypeptides Following lation of the isolated genetic material to provide antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention, the cleotides encoding the antibodies are typically inserted in an expression vector for introduction into host cells that can be used to produce the desired quantity of antibody.

Recombinant expression of an antibody, or fragment, derivative or analog f, e.g., a herein. heavy or light chain of an antibody which binds to a target molecule is described of an Once a polynucleotide encoding an antibody molecule or a heavy or light chain antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA logy using techniques well known in the art. Thus, methods for preparing a protein by expressing a cleotide herein. s containing an dy encoding nucleotide sequence are described which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic ques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors can include the nucleotide PCT/USZOl3/076952 sequence encoding the constant region of the antibody molecule (see, e. g., international applications WO 07 and WO 89/01036; and US patent no. 5,122,464) and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy or light chain.

[0210] The term "vector" or "expression vector" is used herein to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a host cell. As known to those skilled in the art, such vectors can easily be ed from the group consisting of plasmids, phages, Viruses and retroviruses. In general, vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells. For the purposes of this invention, us expression vector systems can be employed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia Virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others e the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes can be ed by introducing one or more s which allow selection of ected host cells. The marker can provide for prototrophy to an auxotrophic host, biocide resistance (e. g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or uced into the same cell by co-transformation. onal elements can also be needed for l synthesis of mRNA. These elements can include signal and termination sequences, splice signals, as well as transcriptional promoters, enhancers, signals.

[0211] In particular embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain nt region genes (e.g., human heavy and light chain constant region genes) as discussed above. In one embodiment, this is effected using a proprietary sion vector of Biogen IDEC, Inc., referred to as NEOSPLA, disclosed in US patent no. 6,159,730. This vector contains the galovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofclate ase gene and leader level expression of antibodies sequence. This vector has been found to result in very high ' 73 ' transfection in CHO upon incorporation of variable and constant region genes, cells, followed by selection in G418 containing medium and methotrexate amplification. Of course, any sion vector which is capable of eliciting expression in eukaryotic cells can be used in the present invention. Examples of le s include, but are not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEFl/His, piND/GS, MVZ, pSV40/Zeo2, pTRACER—HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI able from Promega, Madison, WI). In general, screening large numbers of transformed cells for those which express suitably high levels if immunoglobulin heavy and light chains is routine experimentation which 1e can be carréed out, for example, by robotic systems. Vector s are also taught in US patent nos. 5,736,137 and 5,658,570, each of which is incorporated by reference in its entirety herein. This system provides for high expression levels, e.g., > 30 pg/cell/day.

Other exemplary vector systems are disclosed e.g., in US patent no. 6,413,777.

In other embodiments the antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be expressed using polycistronic constructs such as those disclosed in US patent application publication no. 2003-0157641 Al and incorporated herein in its entirety. In these expression systems, multiple gene products of interest such as heavy and light chains of antibodies can be produced from a single stronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide vely high levels of antibodies. ible IRES sequences are disclosed in US patent no. 6,193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression systems can be used to effectively produce the full range of antibodies disclosed in the instant application.

More generally, once the vector or DNA sequence encoding a monomeric t of the antibody has been prepared, the expression vector can be introduced into an appropriate host cell. uction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection including lipotransfection using, e. g., Fugene® or lipofectamine, protoplast , calcium phosphate precipitation, cell fusion with enveloped DNA, njection, and infection with intact virus. Typically, plasmid introduction into the host is via standard calcium phosphate co—precipitation method. The host cells harboring the sion construct are grown under conditions appropréate to the production of the light chains and heavy chains,, and assayed for heavy and/0r light chain protein synthesis. - 74 _ Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter is (FACS), immunohistochemistry and the like.

The expression vector is transferred to a host cell by tional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the methods described herein. Thus, the invention includes host cells containing or a heavy or light chain f, a cleotide encoding an antibody of the invention, operably linked to a heterologous promoter. In particular embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains can be c0- expressed in the host cell for sion of the entire immunoglobulin molecule, as detailed below. [0215} The host cell can be co—transfected with two expression vectors of the invention, the first vector vector encoding a heavy chain derived polypeptide and the second encoding a light chain derived polypeptide. The two vectors can contain cal able markers which enable equal expression of heavy and light chain polypeptides.

Alternatively, a single vector. can be used which encodes both heavy and light chain polypeptides. In such situations, the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain; see Proudfoot, Nature 322 (1986), 52; , Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The coding sequences for the heavy and liglet chains can comprise cDNA or genomic DNA.

As used herein, "host cells" refers to cells which harbor vectors constructed using recombinant DNA techniques and ng at least one heterologous gene. In descriptions of processes for ion of antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of antibody unless it is clearly specified otherwise. In other words, recovery of polypeptide from the "cells" culture containing both the can mean either from spun down whole cells, or from the cell medium and the ded cells.

A variety of host-expression vector systems can be utilized to express antibody molecules for use in the methods described herein. Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, s an antibody le of the invention in situ. These include but are not limited to microorganisms such as bacteria W0 2014/100600 (e. g., E. coli, B. subtilis) transformed with inant bacteriophage DNA, plasmid DNA or cosmid DNA sion vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus sion vectors (e. g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors 1:e. g., cauliflower mosaic virus, CaMV; tobacco mosaic Virus, TMV) or transformed with recombinant d expression vectors (e. g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g, COS, CHO, NSC, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters d from the genome of mammalian cells (e. g., metallothionein promoter) or from mammalian viruses tag, the adenovirus late promoter; the vaccinia virus 7.5K promoter). In one embodiment, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant dy le, are used for the expression of a recombinant antibody molecule. For e, mammalian cells such as Chinese Hamster Ovary (CHO) cells, in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies; see, e. g., Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio/Technology 8 (1990), 2.

[E218] Tile host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with ability to determine ular host cell lines which are best suited for the desired gene product to be sed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster , DG44 and DUXBll (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby r kidney), MDCK, W13 8, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse ast), HAK er kidney line), SP2/O (mouse myeloma), P3x63- Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney). In a specific embodiment, host cell lines are CHO or 293 cells. Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.

In addition, a host cell strain can be chosen which modulates the expression of the ed sequences, or modifies and processes the gene product in the c fashion _ 76 _ desired. Such modifications (e.g., ylation) and processing (e. g., ge) of protein products can be important for the function of the n. ent host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host s can be chosen to ensure the correct modification and processing ofthe foreign protein expressed.

To this end, otic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.

For erm, high—yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule can be ered. Rather than using sion s which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e. g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a able marker. ing the introduction of the foreign DNA, engineered cells can be d to grow for 1-2 days in an enriched media, and then are swétched to a selective media. The selectable marker in tlee recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which stably express the antibody molecule. {0221} A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 (1977), 223), hypoxanthine— guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adenine phosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes can be ed in tk-, hgprt- or aprt-cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl. Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072); Clinical resistance to the aminoglycoside G—418 iel et neo, which confers al., Pharmacy 12 (1993), 488—505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, 926- Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993), 932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH - 77 — (1993), 155-215; and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30 (1984), 147. Methods commonly known in the art of recombinant DNA technology which can be used are descrébed in l et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons, NY ; Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, oli et al. (eds), t Protocols in Human Genetics, John Wiley & Sons, NY (1994); re-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification, for a review, see Bebbington and Hentschel, The use of vectors based on in DNA gene amplification for the expression of cloned genes in mammalian cells cloning, Academic Press, New York, Vol. 3. (1987). When a marker in the vector system expressing antibody is able, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is ated with the antibody gene, production of the antibody will also increase; see Crouse et al., Mol. Cell. Biol. 3 (1983), 257.

In vitro production allows scale—up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions in an airlift reactor are known in the art and include homogeneous suspension culture, e.g. e. g. in hollow or in a uous stirrer reactor, or immobilized or entrapped cell culture, fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for e gel filtration, ion-exchange chromatography, chromatography over DEAE—cellulose or (immuno-)affinity tography, e. g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.

Genes encoding antibodies, or antigen-binding fragments, variants, or tives thereof of the invention can also be expressed in non-mammalian cells such as bacteria or insect or yeast or plant cells. Bacteria which readily take up nucleic acids include members of the bacteriaceae, such as strains of Escherichia coli or Salmonella; aceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when sed in bacteria, the heterologous polypeptides typically become part of inclusion bodies. The heterologous W0 2014/100600 PCT/USZOl3/076952 polypeptides must be isolated, purified and then led into functional molecules.

Where tetravalent forms of antibodies are desired, the subunits will then self-assemble into tetravalent antibodies; see, 8.g. , international ation W002/096948.

In bacterial systems, a number of expression vectors can be ageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, s which direct the expression of high levels of fusion protein ts that are readily purified can be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983), 1791), in which the antibody coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors e & Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989), 509); and the like. pGEX s can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be d from lysed cells by adsorption and binding to a matrix of glutathione- agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0226] In addition to prokaryotes, otic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other s are commonly available, e.g., Pichia pastoris. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature 282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al., Gene 10 (1980), 157) is commonly used. This plasmid already ns the TRPl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12).

The ce of the trpl lesion as a teristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes. The virus grows in Spodoptera fiugz‘perda cells. The antibody coding sequence can be cloned individually into nor:— _ 79 _ essential regions (for example the polyhedrin gene) of the Virus and placed under control of an AcNPV er (for example the polyhedrin promoter).

Once an antibody le of the invention has been recombinantly expressed, the whole antibodies, their dimers, individual. light and heavy chains, or other immunoglobulin forms of the present invention, can be purified according to standard procedures of the art, including for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, e. g. um sulfate precipitation, or by any other standard technique for the purification of proteins; see, e.g., Scopes, "Protein Purification", Springer Verlag, NY. (1982). Alternatively, another method for increasing the affinity of antibodies of the ion is disclosed in US patent publication 2002-0123057 A1.

V. Fusion Proteins and Conjugates In certain embodiments, the antibody polypeptide comprises an amino acid associated with an dy. Exemplary sequence or one or more moieties not normally modifications are described in more detail below. For e, a single—chain Fv antibody fragment of the invention can comprise a flexible linker sequence, or can be modified to add a functional moiety (e. g., PEG, a drug, a toxin, or a label such as a fluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and the like)

[0230] An antibody polypeptide of the invention can comprise, consist essentially of, or consist of a fiision n. Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin tau-binding domain with at least one target binding site, and at least one heterologous n, i.e., a portion with which it is not naturally linked in nature. The amino acid sequences can normally exist in separate proteins that are brought together in the fusion polypeptide or they can normally exist in the same protein but are placed in a new arrangement in the fusion ptide. Fusion proteins can be created, for e, by chemical synthesis, or by ng and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

The term ologous" as applied to a polynucleotide or a ptide, means that the polynucleotide or polypeptide is derived from a ct entity from that of the rest of the entity to which it is being compared. For ce, as used herein, a "heterologous polypeptide" to be fused to an antibody, or an antigen-binding fragment, W0 2014/100600 PCT/USZOl3/076952 variant, or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or munoglobulin polypeptide of a different s.

As discussed in more detail elsewhere , antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can r be recombinantly fused to a heterologous polypeptide at the N— or C-terminus or chemically conjugated (including covalent and non—covalent conjugations) to ptides or other compositions. For example, antibodies can be recombinantly fused or conjugated to les useful as labels in detection assays and effector molecules such as logous polypeptides, drugs, radionuclides, or toxins; see, e. g., international applications WO92/08495; WO91/14438; WO89/12624; US patent no. 995; and European patent application EP 0 396 387.

Antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, 1'. e. and can contain amino acids other than the , peptide isosteres, 20 gene-encoded amino acids. Antibodies can be d by natural processes, such as anslational processing, or by chemical modification techniques which are well known in the art. Sue}: modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. ations can occur anywhere in the antibody, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be appreciated that the same type of ation can be present in the same or varying degrees at several sites in a given antibody. Also, a given antibody can contain many types of modifications. Antibodies can be branched, for example, as a result of ubiquitination, and they can be cyclic, with or t branching. Cyclic, branched, and branched cyclic dies can result from posttranslation natural processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, ation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, ion of pyroglutamate, forrnylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, lytic processing, phosphorylation, _ 81 _ prenylation, racemization, selenoylation, ion, transfer-RNA mediated on of amino acids to ns such as arginylation, and ubiquitination; see, e. g., Proteins - Structure And Molecular ties, T. E. Creighton, W. H. Freeman and Company, New York 2nd Ed., (1993); Positranslational Covalent Modification 0f Proteins, B. C.

Johnson, Ed., Academic Press, New York, Meth. pgs. 1-12 (1983); Seifter et al., Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

The present ion also provides for fusion proteins comprising an antibody, or antigen-binding fragment, variant, or derivative f, and a heterologous polypeptide.

In one embodiment, a fusion protein ofthe invention comprises, consists essentially of, or consists of, a polypeptide having the amino acid sequence of any one or more of the VH regions of an dy of the invention or the amino acid sequence of any one or more of the VL regions of an antibody of the invention or fragments or variants thereof, and a heterologous polypeptide sequence. In another embodiment, a fusion protein for use in the diagnostic and ent methods disclosed herein comprises, consists ially of, of any one, two, three of the or consists of a polypeptide having the amino acid sequence VH-CDRs of an antibody, or nts, variants, or derivatives thereof, or the amino acid of an dy, or fragments, variants, or sequence of any one, two, three of the VL-CDRS derivatives thereof, and a heterologous polypeptide sequence. In one embodiment, the fusion protein comprises a polypeptide having the amino acid sequence of a VH-CDR3 of or t thereof, and a an antibody of the present ion, or fragment, derivative, logous polypeptide binds to tau. In sequence, which fusion protein specifically another embodiment, a fusion protein comprises a polypeptide having the amino acid of the invention and the amino acid sequence of at least one VH region of an antibody of at least one VL region of an antibody of the invention or sequence fragments, derivatives or variants thereof, and a heterologous polypeptide sequence. In one embodiment, the VH and VL regions of the fusion protein correspond to a single source antibody (or scFv or Fab fragment) which specifically binds tau. In yet another ment, a fusion n for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or of any one, two, three more of the VH CDRs of an antibody and the amino acid sequence of the VL CDRs of an antibody, or fragments or variants thereof, and a or more heterologous polypeptide sequence. In one embodiment, two, three, four, five, six, or scFv or more of the VH-CDR(s) or VL—CDR(S) correspond to single source antibody (or WO 00600 - 82 — Fab fragment) of the invention. Nucleic acid molecules encoding these fusion proteins are also encompassed by the ion.

Exemplary fusion proteins reported in the literature include fusions of the T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84 (1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531; cker et al., Nature 339 (1989), 68-70; Zettmeissl et 611., DNA Cell Biol. USA 9 (1990), 347-353; and Bym er al., Nature 344 (1990), 667-670); L-selectin g receptor) (Watson et al., J. Cell. Biol. 110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164—167); CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley et al., J. Exp. Med. 173 (l99l),721-730); CTLA-4 (Lisley et al., J. Exp. Med. 174 (1991), 561—569); CD22 (Stamenkovic et al., Cell 66 (1991), 1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991), 2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 489 (1991); and IgE or a (Ridgway and Gorman, J. Cell. Biol. 115 (1991), Abstract No. 1448).

[0236] As discussed elsewhere herein, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be fused to heterologous polypeptides to increase the in Vivo half-life of the polypeptides or for use in immunoassays using methods known in the art. For example, in one embodiment, PEG can be conjugated to the antibodies of the invention to increase their half-life in vivo; see, e. g., Leong et al., Cytokine 16 , 9; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et (1]., m. Soc. Transactions 30 (2002), 512. er, antibodies, or antigen—binding fragments, variants, or derivatives thereof of the invention can be fused to marker sequences, such as a peptide to facilitate their purification or detection. In particular embodiments, the marker amino acid sequence is a hexa-histidine peptide (HIS), such as the tag provided in a pQE vector N, Inc., 9259 Eton Avenue, orth, Calif, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci.

USA 86 (1989), 821-824, for instance, hexa—histidine provides for ient ation of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. , Cell 37 (1984), 767) and the "flag" tag.

Fusion proteins can be prepared using methods that are well known in the art; see for example US patent nos. 5,116,964 and 5,225,538. The precise site at which the fusion W0 2014/100600 PCT/U82013/076952 is made can be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.

Antibodies of the present invention can be used in non—conjugated form or can be conjugated to at least one of a variety of molecules, e. g., to improve the therapeutic properties of the le, to facilitate target detection, or for imaging or therapy of the patient. Antibodies, or n-binding fragments, variants, or derivatives thereof of the ion can be labeled or conjugated either before or after purification, when purification is med. In particular, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the ion can be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, Viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG. ates that are immunotoxins including conventional antibodies have been widely described in the art. The toxins can be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin ns can be produced as fusion proteins. The antibodies of the present invention can be used in a corresponding way to obtain such immunotoxins. Illustrative of such immunotoxins are those described by Byers, Seminars Cell. Biol. 2 , 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.

[0241] Those skilled in the art will iate that ates can also be led using a variety of techniques depending on the selected agent to be conjugated. For example, conjugates with biotin are prepared e. g. by ng a tau binding polypeptide with an ted ester of biotin such as the biotin N-hydroxysuccinimide ester. rly, conjugates with a fluorescent marker can be prepared in the presence of a coupling agent, e. g. those listed herein, or by reaction with an isothiocyanate, or fluorescein— isothiocyanate. Conjugates of the antibodies, or antigen-binding fragments, variants or tives thereof of the invention are prepared in an analogous manner.

The present invention further encompasses antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, demonstrate presence of a neurological disease, to indicate the risk of getting a neurological disease, to r the development or progression of a neurological e, i. e. tauopathic disease as part of a clinical testing procedure to, e,g., determine the efficacy of a given treatment _ 84 _ and/or prevention regimen. Detection can be facilitated by coupling the antibody, or antigen—binding fragment, t, or derivative thereof to a detectable substance.

Examples of detectable substances e various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions; see, e. g., US patent no. 4,741,900 for metal ions which can be ated to antibodies for use as stics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, B-galactosidase, or acetylcholinesterase; examples of suitable prosthetic complexes include streptavidin/biotin and group avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of le ctive material include 125I, 131i, 111In or 99Tc.

An antibody, or antigen-binding fragment, variant, or derivative f also can be ably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, inol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an antibody, or antigen—binding fragment, variant, or tive thereof can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay )" Microbiological Associates Quarterly ation, sville, Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. . 31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); lshikawa, E. et al., (eds.), Enzyme assay, Kgaku Shoin, Tokyo (1981). The enzyme, which is 1Eound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, etric or by visual means. Enzymes which can be used to detectably label the antibody e, but are not limited to, malate _ 85 _ dehydrogenase, staphylococcal nuclease, delta-S-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, , catalase, glucosephosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric s which employ a genic substrate for the enzyme.

Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection can also be accomplished using any of a variety of other immunoassays.

For example, by radioactively labeling the antibody, or antigen—binding fragment, variant, or derivative f, it is possible to detect the antibody through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, 13., ples of Radioimmunoassays, Seventh Training Course on Radioligand Assay ques, The Endocrine Society, (March, 1986)), which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation r, or autoradiography.

An antibody, or antigen-binding nt, variant, or derivative thereof can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or nediaminetetraacetic acid (EDTA).

Techniques for ating various es to an antibody, or antigen-binding nt, variant, or derivative thereof are well known, see, e. g., Arnon et al., "Monoclonal Antibodies For targeting Of Drugs In Cancer Therapy“, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds), pp. 243-56 (Alan R.

Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug ry", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of xic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds), And Future Prospective Of The eutic Use pp. 475-506 (1985); "Analysis, Results, Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds), Academic Press pp. 303-16 (1985), and PCT/USZOl3/076952 Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62 (1982), 119—158.

As ned, in certain embodiments, a moiety that enhances the stability or efficacy of a binding molecule, e.g., a binding polypeptide, e.g, an antibody or immunospecific nt thereof can be conjugated. For example, in one embodiment, PEG can be conjugated to the binding molecules of the ion to increase their half- life in viva. Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et 0]., Biochem. Soc. Transactions 30 (2002), 512.

VI. Compositions and Methods of Use

[0249] The present invention s to compositions comprising the aforementioned tau binding molecule, e.g, antibody or antigen-binding fragment thereof of the present invention or derivative or variant f, or the polynucleotide, vector or cell of the invention. The composition of the present invention can further comprise a pharmaceutically able carrier. Furthermore, the ceutical composition of the present invention can se further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition. For use in the treatment of a tauopathic disease, e.g. , of the Alzheimer’s disease the additional agent can be selected from the group consisting of small organic molecules, anti-tau antibodies, and combinations thereof. Hence, in a particular embodiment the present invention relates to the use of the tau binding molecule, e. g., antibody or antigen-binding fragment f of the present invention or of a binding molecule having substantially the same binding specificities of any one thereof, the polynucleotide, the vector or the cell of the present invention for the preparation of a pharmaceutical or stic composition for prophylactic and therapeutic treatment of a tauopathic e, monitoring the progression of a tauopathic disease or a se to a tauopathic disease treatment in a subject or for determining a subj ect's risk for developing a tauopathic disease. [0250} Hence, in one ment the present invention relates to a method of treating a neurological disorder characterized by abnormal accumulation and/or tion of tau in the brain and the central nervous , respectively, which method ses administering to a subject in need thereof a therapeutically effective amount of any one of the afore-described tau binding molecules, antibodies, polynucleotides, vectors or cells of the instant invention. The term "neurological disorder" includes but is not limited to thic diseases such as Alzheimer’s disease, amyotrophic lateral sclerosis/parkinsonism—dementia complex, argyrophilic grain ia, British type amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt- Jakob disease, ia pugilistica, diffuse brillary tangles with calcification, Down’s syndrome, frontotemporal dementia, frontotemporal ia with parkinsonisrn linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler- Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, ic dystrophy, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical s, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle only dementia, multi-infarct dementia and ischemic stroke. Unless stated otherwise, the terms neurodegenerative, neurological or neuropsychiatric are used interchangeably herein.

A particular advantage of the therapeutic approach of the present invention lies in the fact that the antibodies of the present ion are derived from B cells or B memory cells from healthy human subjects with no signs of a tauopathic e and thus are, with a certain probability, capable of preventing a clinically st tauopathic disease, or diminishing the risk of the occurrence of the clinically manifest disease, or of delaying the onset or progression of the clinically manifest disease. Typically, the antibodies of the present invention also have y successfully gone through somatic maturation, i.e. the optimization with respect to ivity and iveness in the high affinity g to the target tau molecule by means of somatic variation of the variable regions of the antibody.

The knowledge that such cells in vivo, 2. g. in a human, have not been activated by means of related or other physiological proteins or cell structures in the sense of an autoimmunological or allergic reaction is also of great l importance since this es a corisiderably increased chance of successfully living through the clinical test phases. So to speak, efficiency, acceptability and tolerability have already been demonstrated before the preclinical and clinical development of the prophylactic or therapeutic antibody in at least one human subject. It can thus be expected that the human antistau antibodies of the present invention, both its target structure-specific efficiency as therapeutic agent and its decreased probability of side effects significantly increase its clinical probability of success.

W0 2014/100600 - 88 — PCT/USZOl3/076952 The present invention also provides a pharmaceutical and diagnostic, respectively, pack or kit comprising one or more containers filled with one or more of the above described ients, tag, anti~tau dy, binding fragment, derivative or varé‘ant thereof, cleotide, vector or cell of the present inven‘ticu. Asscciated with such centainertfis} can be a nutice in the term prescrébed by a gctrerttinentai agency regifiating the manufacture, use or sale of pharmaceuticais er binicgicai preducts, which natice reflects apprcvai by the agency ci‘manutacture, use at sate for human administration, in additicn or alternatively the kit comprises reagents and/or instructions fer use in appropriate stic assays. The composition, e. g. kit of the present invention is of course particularly suitable for the risk assessment, diagnosis, prevention and treatment of a disorder which is accompanied with the presence of tau, and in ular applicable for the treatment of Alzheimer’s disease (AD), amyotrophic l sclerosis/parkinsonism— ia complex (ALS-PDC), argyrophilic grain dementia (AGD), British type d angiopatliy, al amyloid angiopathy, corticobasal degeneration (CBD), Creutzfeldt- Jakob disease (CJD), dementia pugilistica, e neurofibrillary tangles with calcification, Down’s syndrome, frontotereporal ia, frontotemporal dementia with sonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, vorden-Spatz disease, inclusion body myositis, multiple system y, myotonic dystrophy, Niemann—Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease (PiD), postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle only dementia, multi-infarct dementia and ischemic stroke.

The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art; see for example Remington: The e and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0 306472. Examples of suitable ceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose.

Administration of the suitable compositions can be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or _ 89 _ intradermal administration or spinal or brain ry. Aerosol formulations such as nasal solutions of the active agent with spray formulations include puréfled aqueous or other preservative agents and isotonic agents. Such formulations are adjusted to a pH and isotonic state ible with the nasal mucous membranes. Formulations for rectal or vaginal ad-ministration can be presented as a itory with a suitable carrier.

Furthermore, s the present invention includes the now standard (though fortunately infrequent) procedure of drilling a small hole in the skull to administer a drug of the t invention, in one aspect, the binding molecule, especially antibody or antibody based drug of the present invention can cross the blood-brain barrier, which allows for intravenous or oral administration.

The dosage regimen will be determined by the attending ian and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon surface area, age, the particular compound many factors, including the patient's size, body to be administered, sex, time and route of administration, l , and other drugs being stered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 pg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the dosage can range, e.g, from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e. g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bedy weight or Within the range of l- mg/kg, or at least 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the invention. Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule ined by empirical analysis. An exemplary treatment s administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 . Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on ate days or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls Within the ranges indicated. Progress can be monitored by periodic assessment. Preparations for parenteral administration include e s or non—aqueous solutions, suspensions, W0 2014/100600 and emulsions. Examples of non-aqueous solvents are propylene , polyethylene glycol, vegetable oils such as olive oil, and able organic esters such as ethyl .

Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solutiore, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.

Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating , and inert gases and the like. Furthermore, the pharmaceutical composition of the invention can comprise further agents such as dopamine or psychopharmacologic drugs, depending on the intended use of the pharmaceutical ition.

Furthermore, in a particular embodiment of the present invention the pharmaceutical composition can be formulated as a vaccine, for example, if the ceutical composition of the invention ses an au antibody or binding nt, derivative or variant thereof for passive immunization. As mentioned in the background section, phosphor-tau species have been reported ellularly in plasma and CSF (Aluise et al., Biochim. Biophys. Acta. 1782 (2008), 549—558) andstudies in transgenic mouse lines using active vaccination with phosphorylated tau peptides revealed reduced brain levels of tau aggregates in the brain and slowed progression of behavior impairments (Sigurdsson, J. mers Dis. 15 (2008), 157-168; Boimel et 0]., Exp Neurol. 224 (2010), 472-485). Accordingly, it is t to expect that passive immunization with human anti-tau antibodies and equivalent tau binding molecules of the present ion would help to circumvent several adverse effects of active immunization therapy concepts as already discussed in the background section.

Therefore, tlee present anti-tau antibodies and their equivalents of the present invention will be particularly useful as a vaccine for the tion or amelioration of tauopathic diseases such as AD, ALS-PDC, AGD, CBD, CJD, FTD, FTDP-17, NP-C, PiD, PSP or other thies as ned before. {0258] In one embodiment, it can be beneficial to use recombinant bispecific or multispecific constructs of the antibody of the present invention. For a nce see Fischer and Léger, Pathobiology 74 (2007), 3-14. Such bispecific molecule might be designed to target tau with one binding arm and another pathologic entity such as AB or alphamsynuclein or a different pathological conformation of tau with a second binding - 91 _ arm. Alternatively the second binding arm can be designed to target a n present the blood—brain-barrier to facilitate antibody penetration into the brain.

In one embodiment, it can be beneficial to use recombinant Fab (rFab) and single chain fragments (scFvs) of the antibody of the present invention, which might more readily penetrate a cell membrane. For example, Robert et 611., Protein Eng. Des. Sci. (2008) Oct 16; 81741-0134, published online ahead, describe the use of chimeric recombinant Fab (rFab) and single chain nts (scFvs) of monoclonal dy WO- 2 which recognizes an epitope in the N-terminal region of AB. The engineered fragments fibréls were able to (i) t amyloid fibrillization, (ii) disaggregate preformed ABl—42 and (iii) inhibit ABl-42 oligomer-mediated neurotoxicity in vitro as ntly as the whole IgG le. The perceived advantages of using small Fab and scFV engineered antibody formats which lack the effector function include more efficient passage across the blood-brain barrier and minimizing the risk of triggering atory side reactions. rmore, besides scFV and -domain dies retain the binding specificity of full-length antibodies, they can be sed as single genes and intracellularly in mammalian cells as intrabodies, with the potential for alteration of the folding, interactions, modifications, or subcellular localization of their targets; see for review, e.g, Miller and Messer, Molecular Therapy 12 (2005), 394—401.

In a different approach Muller et 411., Expert Opin. Biol. Ther. (2005), 237-241, describe a technology platform, so-called 'SuperAntibody Technology', which is said to enable antibodies to be shuttled into living cells without g them. Such cell- penetrating antibodies windows. The open new diagnostic and therapeutic term 'TransMabs' has been coined for these antibodies.

In a r embodiment, co-administration or sequential administration of other dies useful for treating a tauopathic disease can be desirable. In one embodiment, the additional antibody is comprised in the pharmaceutical composition of the present invention. Examples of antibodies which can be used to treat a subject include, but are not limited to, antibodies targeting beta—amyloid, alpha—synuclein, TDP—43 and SOD-l.

In a further embodiment, co-administration or sequential administration of other neuroprotective agents useful for treating a thic disease can be desirable. In one embodiment, the additional agent is comprised in the pharmaceutical composition of the present invention. es of neuroprotective agents which can be used to treat a subject include, but are not limited to, an acetylcholinesterase inhibitor, a glutamatergic - 92 — receptor antagonist, kinase inhibitors, HDAC inhibitors, anti—inflammatory , divalproex sodium, or any combination thereof. Examples of other neuroprotective agents that can be used concomitant with pharmaceutical composition of the present invention are described in the art; see, e. g. international application W02007/011907. In one embodiment, the additional agent is ne or a dopamine receptor agonist.

A therapeutically effective dose or amount refers to that amount of the active ingredient sufficient to rate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be ined by standard pharmaceutical procedures in cell cultures or experimental s, e. g., ED50 (the dose therapeutically effective in 50% of the tion) and LDso (the dose lethal to 50% of the tion). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be sed as the ratio, LDso/EDSO. In one embodiment, the therapeutic agent in the composition is present in an amount sufficient to e or preserve normal behavior and/or cognitive properties in case of AD, ALS-PDC, AGD, CBD, CJD, FTD, FTDP-l7, NP-C, PiT), PSP or other tauopathic diseases as mentioned before.

From the foregoing, it is evident that the present invention encompasses any use of a tau binding molecule comprising at least one CDR of the above described antibody, in particular for diagnosing and/or treatment of a tauopathic disease as mentioned above, particularly Alzheimer’s disease. In one embodiment, said binding molecule is an antibody of the present invention or an immunoglobulin chain thereof. In addition, the present invention relates to anti-idiotypic antibodies of any one of the mentioned antibodies described hereinbefore. These are antibodies or other binding molecules which bind to the unique antigenic peptide sequence located on an antibody's variable region near the antigen-binding site and are useful, e. g., for the detection of anti-tau antibodies in sample of a subject.

In another ment the present invention relates to a diagnostic ition comprising any one of the above described tau g molecules, antibodies, antigen- binding nts, cleotides, vectors or cells of the invention and optionally suitable means for detection such as reagents conventionally used in immuno or nucleic 3O acid based diagnostic s. The antibodies of the invention are, for example, suited for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of immunoassays which can utilize the antibody of the invention are itive and non-competitive immunoassays in either a direct or indirect format.

W0 00600 ' PCT/USZOl3/076952 Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay), flow cytometry and the Western blot assay. The antigens and antibodies of the ion can be bound to many different carriers and used to isolate cells cally bound thereto. Examples of well known carriers include glass, polystyrene, polyvinyl chloride, opylene, polyethylene, polycarbonate, dextran, nylon, amyloses, l and modified celluloses, polyacrylamides, es, and magnetite. The nature of the carrier can be either e or insoluble for the es of the invention. There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and inescent compounds; see also the embodiments discussed hereinabove.

By a further embodiment, the tau binding molecules, in particular antibodies of the present invention can also be used in a method for the diagnosis of a disorder in an individual by obtaining a body fluid sample from the tested individual which can be a blood , a lymph sample or any other body fluid sample and contacting the body fluid sample with an antibody of the instant invention under conditions enabling the formation of antibody-antigen xes. The level of such complexes is then determined by methods known in the art, a level significantly higher than that formed in a control sample indicating the disease in the tested individual. In the same manner, the specific antigen bound by the antibodies of the invention can also be used. Thus, the present invention relates to an in vitro immunoassay comprising the binding molecule, e. g., antibody or antigen-binding fragment thereof of the invention.

In this context, the present invention also s to means specifically designed for this purpose. For example, an antibody-based array can be used, which is for example loaded with antibodies or equivalent antigen-binding les of the present invention which specifically recognize tau. Design of microarray imniunoassays is summarized in Kusnezow et al., Mol. Cell mics 5 , 1681-1696. Accordingly, the present invention also relates to microarrays loaded with tau binding molecules identified in accordance with the present invention.

In one embodiment, the present invention relates to a method of diagnosing a tauopathic disease in a subject, the method comprising determining the presence of tau and/or pathologically modified and/or aggregated tauin a sample from the subject to be _ 94 _ diagnosed with at least one antibody of the present invention, an tau binding fragment thereof or an nding molecule having substantially the same binding specificities of modified and/or aggregated tau is any one thereof, wherein the presence of pathologically indicative of a egenerative tauopathy and an increase of the level of the pathologically modified and/or aggregated tau in comparison to the level of the physiological tau forms is indicative for progression of a neurodegenerative tauopathy in said subject.

The subject to be diagnosed can be asymptomatic or nical for the disease. In one embodiment, the control subject has a tauopathic disease, for example, AD, ALS— PDC, AGD, CBD, CJD, FTD, FTDP—17, NP-C, PiD, PSP or other tauopathies as mentioned before, wherein a rity between the level of pathologically modified and/or aggregated tau and the reference standard indicates that the subject to be diagnosed has a tauopathic disease. Alternatively, or in on as a second control the l subject does not have a tauopathic disease, wherein a difference between the level tau and/or of pathologically modified and/or aggregated tau and the reference standard indicates that the subject to be diagnosed has a tauopathic disease. In one embodiment, the subject to be diagnosed and the control subject(s) are age-matched. The sample to be analyzed can be any body fluid suspected to contain pathologically modified and/or aggregated tau, for example a blood, CSF, or urine sample.

[0270] The level tau and/or of ogically modified and/or ated tau can be assessed by any suitable method known in the art comprising, e. g., analyzing tau by one or more ques chosen from Western blot, imrnunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent activated cell sorting , two—dimensional gel electrophoresis, mass spectroscopy (MS), - assisted laser desorption/ionization-time of flight-MS (MALDI-TOF), surface—enhanced laser desorption ionization—time of flight (SELDI-TOF), high mance liquid chromatography (HPLC), fast n liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS), and laser densitometry. In one embodiment, said in vivo imaging of tau comprises positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).

Methods of sing a tauopathic disease such as mer's disease, monitoréng a thic disease progression, and monitoring a tauopathic disease _ 95 - treatment using antibodies and related means which can be adapted in accordance with the present ion are also described in ational applications WO93/08302, WO94/13795, WO95/17429, W096/04309, WO2002/0-62851 and WO2004/Ol6655.

Similarly, antibody based detection methods for tau are described in international application W02005/080986, the disclosure content of all being incorporated herein by nce. Those s can be applied as described but with a tau c antibody, binding fragment, derivative or variant ofthe present invention. [0272} In a r aspect the present invention also relates to es having an epitope of tau specifically recognized by any antibody of the present invention. In one embodiment, such peptide comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of: residues 125-131, 397—441, 226-244, 217—227, 37-55, 387-406, 421—427, 427-439, 1-158, 197-207, 57-67, 355-441, 313-319, 309-319, 221-231 of SEQ ID NO:6, and any combination thereof, and a modified amino acids are sequence thereof in which one, two, three, four, five, six, seven or more substituted, deleted and/or added, wherein the peptide is recognized by any antibody of the present invention. {@273} In one embodiment of this invention such a peptide can be used for diagnosing a neurodegenerative tauopathy in a subject, sing a step of determining the ce of an antibody that binds to a peptide in a biological sample of said subject, and being used for sis of a tauopathy in said subject by measuring the levels of antibodies which ize the above descrtébed peptide of the present invention and comparing the measurements to the levels which are found in healthy subjects of comparable age and gender. An elevated level of measured antibodies specific for said peptide of the t invention would be indicative for diagnosing a thy in said t. The peptide of the present invention can be formulated in an array, a kit and composition, respectively, as described hereinbefore.

[E274] These and other embodiments are disclosed and encompassed by the description and es of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the t invention can be retrieved from public libraries and databases, using for example onic devices. For example the public database "Medline" can be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, - 96 _ such as those of the European Bioinformatics Institute (EBI), which is part of the an Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search s. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective ing and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and lar Biology, Oxford University Press, 1997, revised 2000 and ted 2003, ISBN 0 19 850673 2. Several documents are cited throughout the text of this specification. Full bibliographic citations can be found at the end of the specification immediately preceding the claims. The contents of all cited references (including literature references, issued patents, published patent ations as cited throughout this application and manufacturer's specifications, ctions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any nt cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to the following specific es which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLES The examples which follow further illustrate the invention, but should not be construed to limit the scope of the invention in any way. The experiments in the following Examples are illustrated and bed with respect to antibodies NI-105.4E4, NI- 105.24B2 and Nl-105.4A3 as cloned, i.e. the framework 1 (FRI) Ig-Variable regions without being adjusted to the germ line (GL) sequences of human le heavy and light chains; see Figure 1.

Material and methods Detailed descriptions of conventional methods, such as those employed herein can be found in the cited literature; see also "The Merck Manual of Diagnosis and y" Seventeenth Ed. edited by Beers and Berkow (Merck & Co., Inc. 2003) and US. Patent _ 97 _ Application Publication No. 2012/0087861, the content of which is orated herein by reference in its entirety.

The practice of the present ion will employ, unless ise indicated, conventional ques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. For further elaboration of general ques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell biology and tissue culture; see also the references cited in the es. General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. ook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligoreucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Harnes and Higgins eds. 1984); ription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in lar Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., eds.); and Recombinant DNA Methodology (Wu, ed., Academic Press). Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To lar Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and ell, eds., 1986). Protein Methods (Bollag et al., John Wiley & Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., Academic Press 1995); Immunology Methods Manual (Leflcovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). ts, cloning vectors and kits for genetic manipulation ed to in this disclosure are available from commercial vendors such as BioRad, Stratagene, rogen, Sigma- Aldrich, and ClonTech. General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); free Media (Kitano, hnology 17 (1991), 73); Large Scale _ 98 - Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251); ting ation from cDNA , Herzel et al. , CHAOS 11 , 98-107. “dagger...._r..__.r_._».__._.“nmmmm-----------------------------------------

[0280] Unless indicated otherwise below, identification of tau-specific B cells and molecular g of anti-tau antibodies displaying specificity of interest as well as their recombinant expression and functional characterization has been or can be generally med as described in the Examples and Supplementary Methods section of international application published as W02008/081008, the disclosure content of which is incorporated herein by reference in its entirety. See also U.S. Patent Application Publication No. 2012/0087861, the content of which is incorporated herein by reference in its entirety. A new method for identification of ecific B cells and molecular cloning of tau antibodies displaying specificity of interest as well as their recombinant expression and functional characterization is ed within this application. As described above in one embodiment of the present invention cultures of single or oligoclonal B-cells are cultured and the supernatant of the culture, which contains antibodies produced by said s is ed for presence and affinity of new anti-tau antibodies therein. The screening process comprises the steps of a sensitive tissue amyloid plaque immunoreactivity (TAPIR) assay such as described in international application , the sure content of which is incorporated herein by reference, and shown in Figure 3; screen on brain extracts for binding to PHFTau as descrébed in Example 2; screening for binding of a peptide derived from tau of the amino acid sequence represented by SEQ ID NO:6 with phosphate groups on amino acids Ser- 202 and Ser—205; on amino acid Thr-231; and/or on amino acids Ser-396 and Ser-404 of said ce as analogously described in Example 3 with non-phosphorylated peptides due to the epitope confirmation ments for antibody NI-105.4E4; a screen for binding of full-length tau of the amino acid sequence represented by SEQ ID N016 and isolating the antibody for which binding is detected or the cell producing said antibody as described in international patent WO2008/081008 and as bed in Example 1.

W0 2014/100600 PCT/USZOl3/076952 Euéikraruigianflssa Recombireant human Tau40 was purchased from rPeptide (Bogart, GA, USA).

PHFTau was extracted from AD brain.

Isolation of paired helical ts containing pathologically phosphorylated tau filaments (PHFTau) was performed following the method by Goedert et a1. (Goedert et al., Neuron 8 (1992), 159-168) with modifications. One gram of AD brain tissue was cut into 5mm pieces with all visible blood vessels removed. The tissue was washed with 40 ml ice cold washing on (lOOmM Tris pH 7.4, 6 mM EGTA, 1 mM Na3VO4 and 1 mM NaF) for three times followed by homogenization with 20 ml lysis buffer (lOmM Tris pH 7.4, 0.8M NaCl, 1mM EGTA, 1 x protease inhibitor cocktail, 1 mM Na3VO4, lmM NaF, lmM AEBSF, 10% sucrose). The homogenate was centrifuged at 4°C at ’000xg for 20 min. Supernatant was collected with addition of N-lauroyl sarcosinate (Sigma, Switzerland) to 1% (w/V). After two hours incubation at 37°C with shaking, the supernatant was then centrifuged at 4°C at lOO’OOOxg for one hour. The pellet was collected and re-suspended in PBS. After ng out possible inating globulins with protein A magnetic beads, the PHFTau suspension was stored at - 80°C before use. A control extract from healthy control human brain tissue was prepared accordingly.

Lhamnrbnanfihndsanegnna ELISA: 96 well half area microplates (Corning) were coated with recombinant Tau protein (rPeptide, , USA) at a standard concentration of l 11ng in ate ELISA coating buffer (pH 9.6) overnight at 4°C. For PHFTau screening, 96 well Immobilizer Microplates (Nunc, k) were coated with PHFTau extracted from human AD brain at 1:100 dilutions in carbonate ELISA coating buffer (pH9.6) overnight at 4°C. Plates were washed in PBS-T pH 7.6 and non-specific binding sites were blocked for 2 hrs at RT with PBS-T ning 2% BSA (Sigma, Buchs, Switzerland). B cell conditioned medium was transferred from memory B cell culture plates to ELISA plates and incubated for one hour at RT. ELISA plates were washed in PBS-T and then incubated with horse radish peroxidase (HRP)-conjugated donkey anti-human IgG (Fcy fragment specific) polyclonal antibodies (Jackson ImmunoResearch, Newmarket, UK). After W0 2014/100600 — 10G — ZOl3/076952 washing with PBS-T, binding of human antibodies was determined by measurement of HRP activity in a standard colorimetric assay.

MULTI-ARRAY® micro rlate in‘r {02843 Standard 96 well 10-Spot MULTI-SPOT plates (Meso Scale Discovery, USA) were coated with 30 ug/ml rTau (rPeptide), PHFTau brain extract and healthy control brain t in PBS. Non-specific g sites were d for 1 hr at RT with PBS—T containing 3% BSA followed by incubation with B cell conditioned medium for 1 hr at RT. Plates were washed in PBS-T and then incubated with SULFO-Tag conjugated anti- human polyclonal antibody (Meso Scale Discovery, USA). After washing with PBS-T, bound of antibody was detected by electrochemiluminescence measurement using a SECTOR Imager 6000 (Meso Scale Discovery, USA). hBflganarghnunsrnlauannhgdka Samples containing memory B cells were obtained from healthy human subjects.

Living B cells of selected memory B cell cultures are harvested and mRNA is prepared.

Immunoglobulin heavy and light chain sequences are then obtained using a nested PCR approach.

A combination of primers representing all sequence families of the human immunoglobulin germline oire are used for the amplifications of leader peptides, V- segments and J-segments. The first round amplification is performed using leader peptide-specific primers in 5’-end and constant region-specific primers in 3’-end (Smith et al., Nat Protoc. 4 (2009), 372-384). For heavy chains and kappa light chains, the second round amplification is performed using V-segment-specific primers at the 5’-end ar‘2d J-segment—specific primers at the 3’end. For lambda light , the second round amplification is performed using V-segment-specific primers at the 5’-end and a C- region-specific primer at the 3’end (Marks er al., Mol. Biol. 222 (1991), 581-597; de Haard et al., J. Biol. Chem. 26 , 18218-18230).

Identification of the antibody clone with the desired specificity is performed by re- screening on ELISA upon recombinant expression of te antibodies. Recombinant expreSSion of complete human IgG1 antibodies or chimeric IgG2a antibodies is achieved upon insertion of the le heavy and light chain sequences "in the correct reading frame" into expression s that ment the variable region sequence with a sequence encoding a leader peptide at the 5’-end and at the 3’-end with a sequence encoding the appropriate constant (s). To that end the primers ned restriction sites designed to tate cloning of the variable heavy and light chain sequences into antibody expression vectors. Heavy chain immunoglobulins are sed by inserting the immunoglobulin heavy chain RT-PCR product in frame into a heavy chain expression vector g a signal peptide and the nt domains of human immunoglobulin gamma 1 or mouse immunoglobulin gamma 2a. Kappa light chain immunoglobulins are expressed by inserting the kappa light chain RT—PCR-product in frame into a light chain expression vector providing a signal peptide and the constant domain of human kappa light chain immunoglobulin Lambda light chain immunoglobulins are expressed by ing the lambda light chain RT—PCR—product in frame into a lambda light chain expression vector providing a signal e and the constant domain of human or mouse lambda light chain immunoglobulin.

Functional recombinant monoclonal antibodies are obtained upon co-transfection into HEK293 or CHO cells (or any other appropriate recipient cell line of human or mouse origin) of an Ig- heavy—chain expression vector and a kappa or lambda Ig—light— chain expression vector. Recombinant human monoclonal antibody is subsequently purified from the ioned medium using a standard Protein A column purification.

Recombinant human monoclonal antibody can produced in unlimited quantities using either transiently or stably transfected cells. Cell lined producing recombinant human monoclonal antibody can be established either by using the Ig-expression vectors directly or by re—cloning of Ig-Variable regions into different expression vectors. Derivatives such as F(ab), F(ab)2 and scFv can also be generated from these Ig-variable regions.

Antibodies {mag} Mouse monoclonal uman tau antibody Tau12 (Covance, California, USA.) and mouse monoclonal tau antibody AT180 (Thermo Scientific, USA.) were used according to manufacturer’s protocol. Recombinant human tau antibodies NI-105.4E4, Nl-105.24BZ and NI-105.4A3 are described in U.S. Patent Application Publication No. 2012/0087861, the content of which is orated herein by reference in its ty.

Recombinant human tau antibodies NI-105.17C1, NI-105.17C1(N31Q), NI-105.6C5, NI- 105.29G10, NI—105.6L9, NI-105.40E8, NI-105.48E5, NI-105.6E3, .22El, NI- 105.26Bl2, NI-105.12E12, NI—105.60E7, Nl—105.14E2, NI-105.39E2, NI-105.19C6, and _ 102 ..

NI-105.9C4 are antibodies of this invention. They were expressed in HEK293 or CHO cells, purified from conditioned media and were directly used in subsequent applications unless otherwise stated.

Direct ELISA

[0290] 96 well microplates (Costar, Corning, USA) were coated with recombinant Tau protein (hTau40, rPeptide, Bogart, USA) diluted to a concentration of l ug/ml in carbonate ELISA coating buffer (SOmM, pH9.6) at 4°C overnight. Non-specific g sites were blocked for 2 hr at RT wéth PBS ning 2% BSA , Buchs, Switzerland) and 0.5% Tween20. Binding of human antibodies of the present invention was determined using HRP conjugated goat anti-human IgG Fey (Jackson immunoResearch, Newmarket, UK), followed by measurement of HRP activity in a standard colorimetric assay. ECso values were estimated by a non-linear regression using GraphPad Prism software (San Diego, USA).

WWestemBlottiinasztgtndnstaiuins

[0291] PHFTau and recombinant hTau40 were resolved by gradient SDS-PAGE (NuPAGE 4-12%; Invitrogen, Basel, Switzerland) followed by electroblotting on nitrocellulose membranes. After blocking the non-specific binding with 2% BSA at room temperature for one hour, blots were ted overnight with primary human anti-tau antibodies or Tau12 (mouse monoclonal antibody, Covance, California, USA), followed by a HRP—conjugated goat anti-human IgGFcy (for human primary antibodies) or a HRP-conjugated goat anti-mouse IgG secondary antibody.

Blots were developed using ECL and ImageQuant 350 ion (GE Healthcare, Otelfingen, Switzerland).

PHFTau extraction from AD brain,

[0293] ion of paired l filaments containing pathologically phosphorylated tau filaments (PHFTau) was performed following the method by Goedert et a]. (Goedert et al., Neuron 8 (1992), 8) with ations. One gram of AD brain tissue was cut into 5mm pieces with all visible blood vessels removed. The tissue was washed with 40 ml ice cold g solution (IOOmM Tris pH 7.4, 6 mM EGTA, 1 mM Na3VO4 and 1 mM NaF) for three times followed by homogenization with 20 ml lysis buffer (IOmM W0 2014/100600 PCT/USZOl3/076952 Tris pH 7.4, 0.8M NaCl, lmM EGTA, 1 x protease inhibitor cocktail, 1 mM Na3VO4, lmM NaF, lmM AEBSF, 10% sucrose). The nate was centrifuged at 4°C at ’000xg for 20 min. Supernatant was collected with addition of N-lauroyl sarcosinate (Sigma, Switzerland) to 1% (w/V). After two hours incubation at 37°C wth shaking, the supernatant was then centrifuged at 4°C at 0xg for one hour. The pellet was collected and resuspended in PBS. After clearing out possible contaminating globulins with proteir: A magnetic beads, the PHFTau suspension was stored at — 80°C before use. A control extract from y control human brain tissue was ed accordingly.

.Iahnentidjs synthesis,““““““““““““““ -—.a ——-—-- A peptide corresponding to amino acids 333-346 of hTau40 (333GGGQVEVKSEKLDF346) which includes the e of NI—105.4E4 identified by Pepspot g (amino acids 337-343) was synthesized by Schafer—N (Copenhagen, Denmark). An onal cysteine was added to the C-terminus to allow for covalent g to Immobilizer Microplates (Nunc, Denmark). A second peptide corresponding to amino acids 226-239 of human tau (226VAVVRpTPPKSPSSA239), the cognate e of the commercially available mouse monoclonal tau antibody AT180 (Therrno Scientific, USA) was synthesized accordingly and used as control.

Transgenic mice

[0295] Three different animal models for tauopathies are used to validate the tau antibodies (and molecules with the binding specificities thereof) of the present invention. {£32953 1. Transgenic TauP301L mice (linel83): expressing human Tau40 with P301L mutation under the murine Thy1.2 promoter (Generation of these transgenic animals is described in Gotz et al., J. Biol. Chem. 276 (2001), 529-534 and in international application , the disclosure content of which is incorporated herein by reference) 2. JNPL3 mice expressing the shortest 4R human tau isoform with P301L mutation under the murine PrP promoter (available from Taconic, Hudson, NY, USA). 3. P3OISTau (line P819) mice expressing human tau with P3018 mutation under the murine PrP er (available from the Jackson Laboratory, Bar Harbor, Maine, U.S.A). _ 104 _ Tauopathies mouse models and corresponding wild type mice are kept under standard housing conditions on a reversed 12h212h light/dark cycle with free access to food and water. The treatment groups are balanced for age and gender.

Example 1 Validation of target and binding specificity of human tau-antibodies To validate tau as a recognized target of isolated antibodies direct ELISA assays were performed as described above. For the exemplary recombinant human .4A3 antibody 96 well microplates (Costar, Corning, USA) were coated with recombinant human tau (hTau40, rPeptide, Bogart, USA) diluted to a tration of 3 ug/ml or with BSA in carbonate ELISA coating buffer (pH 9.6) and binding efficiency of the antibody was tested. The ary NI-105.4A3 antibody specifically bound to human tau by ELISA. No binding was ed to BSA .

For a determination of the half maximal effective concentration (EC50) of the exemplary antibodies NI-105.4E4 andNI-105.24B2 additional direct ELISA ments with varying antibody concentrations were performed. 96 well microplates (Costar, Corning, USA) were coated with recombinant human tau (hTau40, rPeptide, Bogart, USA) diluted to a concentration of 1 ng/ml (for the assay with NI-105.4E4Antibody), or of 3 pg/ml (for the assay with Nl-105.24B2 Antibody) in carbonate ELISA coating buffer and binding ncy of the antibody was tested. The EC50 values were estimated by a 2O non-linear regression using GraphPad Prism re. Recombinant human-derived antibody NI-105.4E4 bound to hTau40 with high y in the low nanomolar range at 2.4 nM EC50 . NI—105.24B2 bound to hTau40 with high y in the low nanomolar range at 6.6 nM EC50. [03021 The half maximal effective concentration (EC50) of the exemplary antibody NI- 105.4A3 was also ined using direct ELISA experiments. ELISA plates were coated with recombinant human tau (hTau40, lug/m1), PHFTau ( 1:100) and control preparation (1:100), and incubated with varying antibody concentrations. NI-105.4A3 bound to rTau with high y in the low nanomolar range at 1.4 nM EC50. NI—105.4A3 binds to PHFTau with high affinity in the low nanomolar range at 1.2 nM ECso.

W0 2014/100600 - 105 — PCT/USZOl3/076952 Example 2 inant human antibodies g is to recombinant tau and ogical tau extracted from AD brain To determine the binding capacity of NI-105.4E4 and Nl-105.24B2 to pathological tau species extracted from AD brain. SDS—PAGE and n Blot analysis was performed as described in detail above. Blots were incubated overnight with primary antibodies NI-105.4E4 (human), NI-105.24B2 (human) or Tau12 (mouse monoclonal dy, Covance, California, USA), followed by a HRP-conjugated goat anti-human IgGFcy (for human antibodies) or a HRP—conjugated goat anti-mouse IgG secondary antibody.

Recombinant antibodies NI-105.4E4 ared NI-105.24B2 recognized recombinant hTau40 as well as pathologically modified PHFTau extracted from AD brain on Westem blot. The control antibody Tau12 recognized both tau species as well .

Additionally, as discussed in Example 1 above, the half maximal effective tration (EC50) of the exemplary antibody NI-105.4A3 was determined in direct ELISA ments using PHFTau. NI-105.4A3 bound to PHFTau with high affinity in the low nanomolar range at 1.2 nM EC50 .

Mapping ofthe NT-105.4E4 and NI-105.4A3 binding epitope on hTau40

[0306] A peptide array of 118 peptide sequences covering the full-length hTau4O (amino acids 1-441) with an overlap of 11 amino acids n two adjacent peptides was d on a nitrocellulose membrane (JPT Peptide Technologies GmbH, Berlin, Germany). Immunolabeling of antibodies as well as membrane regeneration were carried out according to manufacturer’s instructions. To rule out non-specific binding of the ion antibody, the membrane was first probed by HRP-conjugated goat anti-human IgG omitting the primary antibody. After regeneration the membrane was probed with 100 nM recombinant NI-105.4E4 antibody. Bound antibody was detected using ECL and ImageQuant 350 detection (GE Healthcare, Otelfingen, Switzerland). _ 106 _ Two groups of adjacent peptide spots (peptide 83, 84 and 85; peptide 96 and 97) were specifically identified by NI105.4E4, when compared to the detection antibody only.

The sequences covered by these two groups of peptides correspond to amino acids 329- 351 and 387-397 of hTau40. These data suggested that Nl-105.4E4 recognized a discontinuous e sing two linear sequences: one within the R4 microtubule binding domain and another in the inal domain.

The sequence shared by peptides 83-85 comprises amino acid es 337-343 of hTau40. The Pepspot (JPT) data suggested that NI-105.4E4 recognized an epitope in hTau that comprises amino acids 3 of human tau. This region is located within the ubule g domain of tau and is conserved among all neuronal human tau isoforms as well as across other species including mouse and rat.

As this domain is bound to microtubules in physiological microtubule—associated tau, NI—105.4E4 is expected to entially target the pathologically relevant pool of tau that is detached from the microtubules.

[0310] To determine key residues within the NI-105.4E4 binding peptides, e scanning was performed to substitute each residue with e one at a time. The alanine residues in the original sequence (A384 and A390) were substituted to proline and glycine. Spots 35-50 and 51-68 are the original peptides (spot 35 and spot 51) and their alanine substituted variants,. Alanine scan suggested V339, E342, D387, E391 and K395 were necessary for NI-105.4E4 binding.

An additional experiment has been performed by testing the binding of NI- 105.4E4 to tau es. Direct ELISA showed that .4E4 specifically recognized a peptide corresponding to amino acid 333-346 of , which ns the amino acid residues 337-343 identified by Pepspot mapping. No cross-reactivity of NI-105.4E4 was observed to the control peptide covering the AT180 epitope. Vice versa, AT180 recognized its cognate epitope containing peptide but failed to bind to the NI—105.4E4 specific peptide. Species-specific secondary antibodies did not bind to any of the peptides. Together, direct ELISA with coated peptides confirmed that NI-105.4E4 specifically recognized a peptide containing the amino acid residues 337-343 of human tau identified by Pepspot mapping.

To grossly map the .4A3 binding epitope on hTau40, four tau domain polypeptides (Tau domain 1, domain 11, domain III and domain IV) were produced. DNA fragments, synthesized using GeneArt® (Invitrogen), which encode each Tau domain with 6xHis tagged at the N-terminus were cloned into the pRSET-A expression vector (Invitrogen), were ected into E. Coli BL21 (DE3) (New England Biolabs). The sions of the His-tagged Tau domains were induced by 0.5mM IPTG for six hours before bacteria were lysed with lysozyme with sonication. The lysate was boiled for five minutes before being further purified wéth Ni-NTA Superflow Columns (Qiagen). The eluted gged Tau domains were coated on ELISA plates or loaded on polyacrylamide gel for Western Blot. These sequentially overlapping tau domain polypeptides covered the full lengeh of . Puréfied tau domains were coated on ELISA plate and the binding of NI—105.4A3 was tested. NI-105.4A3 binds only to tau domain I and the full length hTau40, indicating the epitope was within the N—terminal part of the hTau40 (aa1-136). n blot confirmed the specific binding of NI—105.4A3 to tau domain 1. NI—105.4A3 epitope mapping with PepSpot (JPT) technology identified amino acids Q35-Q49 of the human Tau40. To ine key es within the epitope for NI-105.4A3 binding, alanine scanning was performed to substitute each residue with alanine one at a time. The alanine residue in the original sequence (A41) was substituted With glycine or proline. Alanine scan showed that D40, A41 and K44 are key residues for NI-105.4A3 binding.

Example 4 Assessment of the binding ofNI-105.4E4 to physiological forms as well as pathological aggregates of tau AD brain tissues and in human tau transgenic mice Neurofibrillary tangles (NFT) composed of hyperphosphorylated tau filaments are a neuropathological hallmarks of AD. Hyperphosphorylated tau filaments are also the major ents of dystrophic neurites and neuropil threads, both of which are common neuropathological features in AD. Overexpression of human tau ning the al P301L tau on in mice induces NFT formation at six months of age (Gotz et al., 2001a).

To assess the binding of recombinant human tau antibody to physiological forms as well as pathological aggregates of tau, immunohistological stainings were med in AD brain tissues and in TauP301L transgenic mice with the exemplary NI—105.4E4 antibody of this invention. _ 108 _ Mice were perfused with 20 m1 100 mM /6 mM EGTA (pH7.4) at room temperature under deep anesthesia. Brains were taken out and immersed in 4% PFA in PBS (pH 7.4) at 4°C overnight for fixation ed by embedding in n. For human tissue, paraffin blocks of brain tissues from AD and healthy control subjects were used. DAB staining was cariéed out following standard protocols. As positive control, mouse monoclonal antibody Tau-12 (Covance, California, USA.) was used. HRP- conjugated detection antibodies without primary antibodies were also included.

Recombinant human antibody NI-105.4E4 identified numerous NFTs and neuropil threads in AD brain (Figure 2A), which were absent in healthy control brain (Figure 2B). Secondary antibody alone did not give s in both AD (Figure 2C) and control brain (Figure 2D). In P301L tau enic mouse brain, Nl—105.4E4 b0und strongly to the pathological tau resembling NFT (Figure 2E, F and H), neuropil threads (Figure 2E and G) and dystrophic es (Figure 2E and H). In addition, Nl-105.4E4 also identified tau aggregates at pre-tangle stage e 21). In the brain of transgenic mice overexpressing both human P301L tau and human APP with Swedish and Arctic ons, NI-105.4E4 bound cally to dystrophic neurites surrounding beta- amyloid plaques (Figure 2]).

Example 5 In vivo tests of the antibodies of the present ion

[0317] As already bed above studies in transgenic mouse lines using active vaccination with phosphorylated tau peptides revealed reduced brain levels of tau aggregates in the brain and slowed progression of behavior impairments (Sigurdsson, J.

Alzheimers Dis. 15 (2008), 157-168; Boimel et all, Exp. Neurol. 224 , 472-485).

However, active vaccination may not be particularly useable in humans because a significant fraction of the elderly population is expected to be non-responders t0 vaccination. Furthermore, the potential side effects associated wéth a tau-directed immune response can be difficult to l. Tau binding molecules of the present invention can be reasonably expected to achieve similar reductions in brain levels of tau aggregates as bed anve for the mouse antibodies, e of their similar binding specificities against pathologically tau species. However, because of the evolutionarély optimization and affinity maturation within the human immune system antibodies of the present invention e a valuable therapeutic tool due to being isolated from healthy human subjects with high probability for excellent safety profile and lack of immunogenicity.

Confirmation of these expected therapeutic effects can be provided by test methods as described in the above mentioned experiments with mouse antibodies. In particular, the antibodies to be screened can be applied on diverse possible routes to the animals such as intraperitoneal antibody injection, intracranial injection, intraventricular brain infusion and tested for treatment effects. Either of the above mentioned application possibilities can be also used after prior brain injection of beta-amyloid preparations into the brain of tau enic mice to evaluate ent effects on beta amyloid-induced tau pathology.

Evaluation of the treatment effects can be performed by hemical methods comprising quantification of Gallyas positive cells , total human tau staining, brain burden of phosphorylated tau and/or a biochemical determination of brain soluble and insoluble tau and phosphor—tau levels upon sequential brain extraction. Further on, behavior testing of the treated mice can be performed, e. g., conditioned taste aversion or contextual fear conditioning for a confirmation of the therapeutic effects of the antibodies of the present invention (Pennanen, Genes Brain Behav. 5 , 369-79, Pennanen Neurobiol Dis. 15 (2004), 500-9.) Example 6 Chimerization of antibodies Nl-105.4E4 and NI-105.4A3 with mouse IgG2a nt domains [03,19] In order to generate dies with reduced immunogenicity for use in chronic treatment studies, mouse chimeric versions of antibodies NI—105.4E4 and NI-105.4A3 were generated using recombinant DNA technology. A mouse IgG2a/lambda isotype was selected for these chimeric dies, in order to generate a le which bound with high affinity to mouse Fc-gamma receptors, and was therefore capable of inducing an immune effector response. The amino acid sequences of the ic NI—105.4E4 4") and chimeric NI—105.4A3 ("ch4A3") heavy and light chain constructs are shown below. _ 1 10 _ Table 5: Amino acid sequences of chimeric "NI—1 05.4E4 (ch4E4) and chimeric NI—105.4A3 (ch4A3) inmhuech4E4 aEVQLVESGGGLVQPGGSLKLSCAASGFNFNISAIHWVRQASGkéiEWVGR‘ ‘ iIRSKSHNYATLYAASLKGRFTLSRDDSRNTAYLQMSSLQTEDMAVYYCTVi heavy chain : iLSANYDTFDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLV lggzg) iKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSi t ITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPK iSEQ IDNO: 20 2 ~ iIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYi iNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP' iQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEP iVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG inmnuech4E4 ESYELTQEPE§§V§EEQTARISCFGDTLPKQYTYWYQQKPGQAPVLVIYKD ’ iTERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCLSADNSATWVFGG i light chain , EGTKVTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDEYPGVVTVDWK lmnbmfi iVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEG ‘ EHTVEKSLSRADCS SEQ ID NO: 21 g Example 7 l of consensus N—Iinked glycosylation site from ch4E4 heavy chain (mouse IgG2a) A consensus N-linked glycosylation site was identified in the CDRI region of the NI—105.4E4 heavy chain. Upon mammalian (CHO) cell expression, the predicted N- glycosylation site (Asn-30) was fully occupied by glycan, as trated by mass spectrometry. In order to eliminate N-glycosylation in this region and reduce product heterogeneity, Asn-30 of the heavy chain of ch4E4 was d to Gln (Table 4). When produced and purified from CHO cells, the modified antibody bound to recombinant tau with ~4—fold higher apparent binding affinity relative to the al, glycosylated antibody.

Table 6: Amino acid sequences of mature ch4E4(N30Q) heavy chain (mouse IgG2a). Substituted Gln e is in bold, underlined. imam EVQLVEsGGGLre‘fiaeg‘m‘é‘emé‘é‘fifi‘igiémwvtars:5&3vaGR i N30Q) I RSKSHNYATLYAASLKGRFTLSRDDSRNTAYLQMSSEQTEDMfiRVYYCTV i LSANYDTFDYWGQGTLVTVS SAKTTAPSVYPLAPVCGDTTGS SVTLGCLV i heavy chain KGYFPEPVTLTWNSGSLS SGVHTFPAVLQS DLYTLSSSVTVT SSTWPSQS ti... ITCNVAHPASSTKVDKKIEPRGPTIKPCPPQECPAPNLLGGPSVFI FP Pia; ]' (mouse IgGié)M§f"iKD\/LMISLSPtvrevaDVSEDDPDVQISWFVNqu‘v‘fiT'AQTQTHREDY NSTLRVVSALP-EQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP SEQID N022 gQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEP. " l:VLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG Example 8 Production of aglycosylated chimeric NI-105.4E4(N30Q) (ch4E4(N3OQ) mIgGl Agly) A mouse chimeric aglycosylated variant of germlined NI-105.4E4 was produced ("ch4E4(N3OQ) IgGl-Agly") in order to evaluate the relationship between dy effector function and activity. For the heavy chain (SEQ ID 214), the variable domain of NI-105.4E4(N30Q) (SEQ ID NO: 43) was fused to a mouse IgGl heavy chain constant region containing an Asn to Gln mutation to eliminate the consensus Fc glycosylation site. The heavy chain variable region contained the N30Q change in order to eliminate the sus osylation site in CDRl (Example 7). The light chain was the ch4E4 lambda light chain described above (SEQ ID 21).

Example 9 Acute brain penetration study ofhuman 4E4 and 4A3 Human NI-105.4E4 and NI—105.4A3 germlined antibodies were produced by transient transfection of CHO cells and d by y purification. The endotoxin levels were controlled and were all bellow 1 EU/rng. TauP301L mice were intraperitoneally injected with 30 mg/kg NI—105.4E4 (n=7), 4A3 (n=7) antibody or equal volume of PBS (n=7) at day 1 and day 4. At day 5, mice were perfused under anesthesia with PBS containing 1 Unit/m1 heparin. Blood, brain and spinal cord were collected for es. Right hemisphere of the brain was frozen at -80°C, left hemisphere of the brain and the spinal cord were post fixed in 10% neutralized formalin at 4°C for two days before being ed in paraffin block and sectioned. Plasma was stored at —80°C in aliquots.

Brain protein extraction: frozen right hemisphere was weighed and homogenized in 5 volumes (5 mL/g of wet tissue) of a solution containing 50 mM NaCl, 0.2% _ 112 - diethylamine, protease inhibitors (Roche stics GmbH) and phosphatase inhibitor (Roche Diagnostics GmbH). Samples were then erred to polycarbonate tubes and added another 5 volume of homogenization solution, and kept on ice for 30 min. Soluble fraction was then collected after centrifugation at 100,000 g, 4°C for 30 min. This soluble fraction was used in human IgG assay. The pellet was re—suspended in 3 volumes ofPBS with protease and phosphatase inhibitor. After centrifugation at 16,000 g, 4°C for 30min, supernatants and pellets were stored separately at -80°C for further insoluble tau tion. Pellets further extracted with modified sarcosyl extraction (Goedert M, Spillantini MG, Cairns NJ, Crowther RA. Neuron 8, 159 (1992)).

[0324] Human IgG-specific sandwich ELISA: 2 ug/ml of goat anti-human IgG Fab (Jackson) in 50 mM carbonate ELISA coating buffer (pH9.6) was used as capture antibody. Half-area l microtiter plates was coated with 30 ill/well with capture antibody at 4°C overnight. The plate was then washed 4 times with PBS containing 0.1% Tween 20 before incubating with 50 l PBS containing 2% BSA at room temperature for one hour. Soluble fractions of brain extracts, plasma samples and antibody standard (4A3) were diluted in PBS containing 2% BSA and 0.1% Tween 20. 30 ul of the diluted s were added into each well and incubated at room temperature for one hour. The plate was then washed with 200 ul/well PBS containing 0.1% Tween for four times before incubated with HRP—conjugated donkey anti-human Fcy (Jackson, diluted at 1:10,000 in PBS ning 2% BSA and 0.1% Tween 20) at room temperature for one hour. The plate was then washed with 200 ul/well PBS containing 0.1% Tween 20 for four times before adding 20 til/well TMB (1:20 in 10 mM citrate solution pH=4.l). The on was then stopped by adding 10 u] 1M H2SO4to each well.

Antibody rd curve was obtained from serial dilutions of NI-105.4A3. Antibody concentrations in plasma and brain s were calculated according to the standards.

Brain human IgG level was then converted to ug antibody/gram fresh brain tissue (assuming 1g/10 ml) as indicated in Figure 6.

High levels of human IgG were detected in the plasma of all NI-105.4E4 and NI— 105.4A3 d mice. In contaast, no human IgG was detected in the plasma of PBS d mice (Figure 5). Significant amount of human IgG was detected in brain homogenates of4E4 and 4A3 treated mice (Figure 6). - 113 — Example 10 Chronic study with chimeric Nl-105.4E4 and Nl—105.4A3 Chimeric NI-105.4E4 and NI-105.4A3 containing the varéable domains of the original human antibody and the constant regions of mouse IgGZa can be ed by transient transfection of CHO cells and purified by affinity ation. The endotoxin levels in each batch of the antibodies wll be controlled and kept below 1 Eng. Gender balanced TauP301L mice at age of 7.5-8 months wéll be intraperitoneally injected with 10 mg/kg, 3 mg/kg of antibody solution, or equal volume of PBS control. Each treatment gafoup will have 20-25 mice. The ent will be d out once a week for 26 weeks.

Alternatively, the treatment will be carried out twice a week for 13 weeks. Body weight will be monitored every two weeks. Mice wéll be perfused under anesthesia at the end of the treatment period. Brain, spinal cord and blood will be ted. Half brain and spinal cord can be post-fixed in 10% formalin for three days before being embedded in paraffin block. 4-6 mm thick ns cut from these tissue blocks can be used for immunohistochemistry studies. The other half brain will be weighted and deep frozen at - 80°C for mical analyses.

Drug effects will be evaluated by comparing the level of neurofibrillary tangles (NFT) and the level of tau with different solubility characteristics in treated and control samples. NFT can be visualized by Gallyas silver impregnation (F s Acta MOIphol.

Acad. Sci. Hung 19.1 (1971)), or by immunostaining with monoclonal mouse dy ATIOO and AT180, which recognize pathologically phosphorylated tau in NFT. The number or frequency of Gallyas-positive neurons and/or ATlOO, AT180 labeled neurons in the brain and spinal cord in antibody treated mice and control animals can be determined to evaluate the effect of antibody treatment.

[0328] e and insoluble tau can be extracted following the brain protein extraction ol described herein. Alternatively, soluble and insoluble tau can be extracted with modified sarcosyl extraction (Goedert M, Spillantini MG, Cairns NJ Crowther RA.

Neuron 8, 159 (1992)). Briefly, frozen brain is homogenized in 10 volumes (wt/vol) of 10 % sucrose homogenate buffer consisting of 10 mM Tris-HCI (pH 7.4), 0.8 M NaCl, 1 mM EGTA, 1mM Na3VO4, 1 mM NaF, 1mM AEBSF, protease inhibitors (Roche Diagnostics GmbI-l) and phosphatase inhibitor (Roche Diagnostics GmbH). The WO 00600 _ 1 14 _ homogenate is spun for 20 min at 20,000g, and the supernatant retained. The pellet is homogenized in 10 volumes of homogenization buffer and centrifuged for a second time.

The supematants can be pooled together, and N-lauryl—sarkosinate (Sigma) is added to 1% l) final concentration, and incubated at 37°C with 300 rpm rotation for 1.5 hour, followed by centrifugation at 0 g for 1 h. The supernatant is collected as sarcosyl soluble fraction and the pellet of 1 g brain tissue is re-suspended in 0.2 ml 50 mM TriS’HCI (pH 7’.4) as PHF fraction.

The levels of soluble and insoluble tau will be ed with commercially available Tau ELISA kits (Invitrogen). In addition, brain protein extracts will be separated with 4-12% Bis-Tris SDS-PAGE followed immunoblotting with Tau12 (human tau), AT8 (pS202/pT205), ATIOO (pT212/p8214), AT180 (pT231) and E178 (pS396) antibodies. uantitative analysis will be performed with measuring the integrated density of each sample against standards of known quantities of tau. {0330] Additionally, behavioral tests can be performed as ted in Example 5, above.

For e, improvement of working memory in antibody treated TauP301L mice can be tested using a two-trial Y-maze task (e.g., Pennanen, Genes Brain Behav. 5 (2006), 369- 79, which is herein incorporated by reference in its entirety). The three arms of the maze are 22cm long, 5 cm wide and 15 cm deep. Black and white abstractive clues are placed on a black curtain surrounding the maze. Experiments are ted wéth an ambient light level of 6 lux during the dark phase. Each experiment comprises a ng session and an observation session. During the training session, a mouse is assigned to two of the three arms (the start arm and the second arm), which can be freely explored during 4 min, with no access to the third arm (the novel arm). The mouse is then removed from the maze and kept in a holding cage for 1.5-5 min, while the maze is thoroughly cleaned with 7’0‘5’9 ethanoi to remove any Olfactory eiues. The mouse is then put back again in the maze fer observation with ali three arms accessible for 4 min. The sequence ef entries, the number of entry to each arm and the time spent in each arm is recorded. From that the ratio of time spent in the nevei third arm over the average of time spent in the other two arms (start arm and second arm) is calculated and compared among different treatment wild type mice. Rodents groups in tauopathy mouse model and ponding l typically prefer to investigate a new arm of the maze rather than returning to one that was previously visited. Effects of the antibodies can be monitored in regard of regaining this preference by treated tauopathy model mice in comparison to non-discriminative behavior WO 00600 — 115 - of untreated mice due to their disorder-related working memory impairment. Therefore, a ratio close to 1 indicates impaired working memory. A higher ratio indicates better working . Impaired working memory in TauP301L mice is considered to be due to tau pathology resulting from the overexpression of human tau. ore a U2 significantly higher ratio observed in anti-tau antibody treated TauP301L mice than in the control TauP301L mice will indicate that the anti-tau antibody has therapeutic effect on tau ogy.

Example 11 Identification ofhuman anti-tau antibodies.

[0331] Recombinant human tau antibodies NI-105.17C1, NI—105.6C5, .29G10, .6L9, NI-105.4OE8, NI-105.48E5, NI-105.6E3, NI-105.22E1, NI-105.26B12, NI- 105.12E12, NI—105.60E7, .14E2, NI-105.39E2, NI-105.19C6, and NI-105.9C4 were isolated according to the methods described herein. The target and binding specificity of these human tau-antibodies were validated as described above. A summary of the findings is provided in Table 5. All antibodies used except NI-105.17C1 were germlined.

Table 7. In Vitro characterization of human anti-tau antibodies.

Antlbody ECso TauECSO [nM]/ PHFTau Bmdmg reglon Phosphorylation = ELISA ELISA reamed“ E-NI 1056C5 E0033 E004 N0 E33 E .NP. WY“ 217-227 N0 E:NI 105.48E5 E>500 E132 pS396, pS46 37-55; 387-406 unconfirmed W .__ .....

Phosphorylation at ENI—105.60E7 E018 E 197—207 either 198,199 202 orE : a .

. . EMMWWWM..“1.1...“...w.“Wm““wfl....................................'1 . ..............______.1.E205 dlsrupts bmdlng ....

: \ ENI-105 14E2 E0.65 57; 67 355-441 313319 ‘-.-.».».».».».».».».»...._...A...1.“....n..eee“‘“uW...~e«««““-,.,......

... . .:-. . -.-. . WEI".1:1::1:...-.......1:3.00 ‘309-319 ENe E—NI105.9C4 E221-231 1-111deaES23:1 disrupts *..blnding region on hTau40 Was Identlfied by combinedapproaches of PepSPOTs tau— fragments western blot and ELISA, tau—peptide ELESA and Alanine scanning. **2 whether antibody binding requires phosphorylation at certain amino acids on tau n was verified by comparing the binding of antibody to rTau, PHFTau, PHFTau dephosphorylated by calf intestine atase, rTau in vitro phosphorylated by GSK3B or GSK3B /CDK5/p35, and phosphorylated tau peptides on PepSPOTs and direct ELISA.

Example 12 Chimerization of human dies with mouse IgGZa constant domains.

In order to generate antibodies with reduced genicity for use in chronic treatment studies, mouse chimeric versions of dies NI-105.17C1 ("ch17C1"), NI- 105.6C5 ("ch6C5"), .40E8 ("ch40E8"), and NI-105.6E3 ("ch6E3") were generated using recombinant DNA technology. A mouse IgGZa/lambda isotype was ed for these ic antibodies, in order to generate a molecule which bound with high affinity to mouse Fc-gamma receptors and was therefore capable of inducing are immune effector response. The amino acid sequences of ch17C1, ch6C5, ch40E8, ch40E8(R104W), and ch6E3 heavy and light chain constructs are shown below. hlvcnmmmbdywq1Dmm "‘"eh3155‘héWfifiBfiéfigfiéjiWWWIDNO:205 »»»»»»»»»»» “0116135llght chaln (mouse lambda) ESEQ ID NO266“ “61140138heavychain (mouse IgG2a) “ESEQ: ID NO:207 E”ch40138(R104Wheavy chain (mouse IgG2a ‘sM3Q1D NO:208 1 01'{3‘1A205m;001b—‘I;1—i-(IQ!E: O W .... 5‘1N;r—I-52 A.—1%0‘CL3’2 ch6E3 heavy chain(mouse IgG2a)W SEQ1D NO210 33 llgh’t Chaln(m0useW““““““““““WWWWSEQ1DNo:211 35S‘ WO 00600 - 1 17 — 2013/076952 Example 13 Elimination of CDR glycosylation site in NI-105.17C1 light chain.

A consensus ed glycosylation site was identified in the CDRl region of the NI-105.17C1 light chain. Upon mammalian (CHO) cell expression, the predicted N glycosylation site (Asn-31) was fully occupied by glycan, as demonstrated by mass spectrometry. In order to eliminate N-glycosylation in this region and improve product heterogeneity, Asn-31 of the light chain of ch17C1 was changed to Gln (see sequence below). When produced and purified from CHO cells, the modified antibody (ch17C1(N31Q) mi'gGZa) bound to recombinant tau with similar nt binding affinity relative to the original, glycosylated antibody (see Figure 8A). The NI- 105.17C1(N31Q) light chain variable region comprises the amino acid sequence of SEQ ID NO:221. ‘ch17C1(N31Q)Welambda) SEQIDN0212 humanN110517C1(N31Q) VL ‘ SEQIDN0221_ e 14 tion of antibodies with reduced effector on.

Antibody variants ning mutations Within the consensus N—glycosylation site in the heavy chain Fc domain were generated. These variants, designated “Agly”, were designed to generate anti-tau antibodies with reduced immune effector function. The amino acid sequences of Agly variants of the tau antibodies are provided below. ch4A3-mIgG1Aglyheavycha1nSEQ ID N0:213 ch4E4(N30Q)-m1g5ifi§i§ii€§€9Efiaifi"""”“”"‘"EmsiéEiifimfio‘E‘iz14 cthhamSEQlDNOZlS ch17WychamSEQlDN0216 - l 18 - Example 15 Comparison of Binding Activity of chl 7C1-mIgG2a and ch17C1-mIgGl-Agly.

The relationship between antibody effector function and activity was assessed for ClEl7Cl Agly (Figure 8A). The ch17C1 dy comprised the ch17C1 heavy chain (SEQ ID NO:203), and the ch17C1 light chain (SEQ ID NO:204) The ch17C1(N31Q) mIgGZa antibody comprised the ch17C1 heavy chain (SEQ ID ), and the ch17C1(N31Q) light chain (SEQ ID NO:212), which incorporates the N31Q mutation in CDRl to eliminate the CDR glycosylation site. The ch17C1(N31Q) mIgGl Agly antibody comprised the ch17C1-mIgG1-Agly heavy chain (SEQ ID ), wherein the variable domains of 17C1 are fused to a mouse IgGl heavy chain containing an Asn to Gln mutation at position 294 (Kabat residue 297) to ate the consensus Fe glycosylation site, and the ch17C1 (N3 1Q) light chain (SEQ ID ). When produced and purified from CHO cells, ch17C1(N3 1 Q) mIgGl Agly bound to recombinant tau with similar apparent binding affinity relative to the original, glycosylated antibody (see Figure 8A).

Example 16 Comparison of Valine vs. cine at position 48 ofthe 17C1 light chain.

: In the process of generating the mouse chimeric IgG2a version of germlined antibody NI—105.17C1, residue 48 of the light chain was also d from valine to ‘ isoleucine. To confirm that this suEstitution did not affect the binding y of NI- Cl, a mouse chimeric IgG2a version of NI—105.17C1 with valine at position 48 was prepared. When produced and purified from CHO cells, ch17C1(N31Q, I48V) mIgGZa antibody bound to recombinant tau with similar apparent binding affinity relative to the ch17C1(N31Q) mIgG2a (see Figure8B). The ch17C1(N31Q) mIgG2a antibody comprised the ch17C1 heavy chain (SEQ ID NO:203), and the ch17C1(N31Q, I48V) light chain (SEQ ID NO:217).

(N31Q148WSEQIDNOZI7 lambda) huTnWVWLSEQIDNozzz - 1 19 - e 17 Comparison of Arg vs. Trp at on 104 ofNI-105.40E8. [173371 Antibody NI-105.4OE8, which is selective for the phosphorylated form of tau found in human paired helical filaments (PHF), contains an unusual arginine residue at position 104 of the NI—105.40E8 VH. lly this on within the human immunoglobulin oire is occupied by a tryptophan e. A form of the NI- 105.40E8 heavy chain, NI-l05.40E8(R104W)-hIgGl, was generated in which residue Arg104 was replaced with tryptophan. When produced and purified from CHO cells, NI— 105.40E8(R104Wj—hIgG1 antibody bound to humar‘e PHF tau with similar apparent binding affinity relative to NI—105.40E8-hIgGl (see Figure 9). The light chain of the two antibodies was identical.

"Ni-1‘65fibfiéfifiifiktwyhlgG1, heavy SEQ 115N0218 Echain {“hfiihiih“““i§iI-105.40E8 light cha1n(humanSEQIDN5219 lambda) humanN110540E8(R104W)VH1SEQIDN022OM Example 18 Human anti-tau antibodies bind to pathologically aggregated tau in AD brain and in the brain of transgenic mouse model oftauopathy.

Brain tissue samples obtained from Alzheimer's disease and control patients, as well as from the brain of transgenic mouse of tauopathy and wild-type control were stained with the germlined human au antibodies provided herein. Representative images of germlined human .40E8, NI—105.48E5, NI-105.6C5 and NI-105.17C1 anti-tau antibodies binding to pathological tau aggregates in the brain of Alzheimer’s disease (AD) and in the brain of transgenic mouse of tauopathy (Tg) are shown in Figure . None of these antibodies bind to normal tan in mentally healthy subject (Ctr) or wild type mouse brain (Wt). The different patterns among these antibodies reflected their differences in epitope specificity and binding affinity.

Example 19 Brain penetration of antibodies in TauP301L mice.

[0339] Animals and Antibody treatments: Human NI-105.6C5, NI-105.4GE8 and NI- 105.6E3 anti—tau antibodies were produced by transient ection of CHO cells and d using standard s. A humanized antibody with no cross-reactivity to mouse antigens was used as an isotype control (hIgGl). In the first experiment, half of the injected NI-105.6C5, NI—105.6E3 and hIgGl antibodies were labeled with Cy3 (GE Healthcare, PA13105) wéth an approximate antibody: Cy3 ratio of 1:3. Cy3-labeling did not change the antibody g as confirmed by ELISA and immune-staining with TauP301L brain (data not shown). In the second experiment, unlabeled NI-105.6C5 and .4OE8 anti-tau antibodies were used. 1L mice between 18-22 months of age received two doses of 30 mg/kg NI-105.6C5, NI-105.6E3 or human IgG1 isotype control hIgGl via i.p. injection within seven days. Tissue samples were collected from three mice of each d group at time points of one day, eight days and 22 days post the second dosing. In the second experiment, TauP301L mice were ip injected with 30 mg/kg h4OE8 and h6C5 twice within seven days and tissue samples were collected from those mice one day post the second injection.

For tissue sample collection, mice were deeply anaesthetized with ketamine/xylazine before blood was collected through the right atrium. CSF was then collected by cistema magna re. Brain and spine were subsequently collected following perfusion for 2 min with ice cold PBS, containing 10 UI/ml heparin and five minutes with 10% neutralized formalin through the left ventricle. The brain was fixed in % lized formalin for another 3 h at 4°C, following immersion in 30% sucrose for 48 h. The brain was then frozen in dry ice and subsequently sectioned into 30 pm thick coronal section series. The section series were stored at -20°C in antifreeze solution containing 1M e, 37.5% ethylene glycol in 50 mM sodium phosphate buffer pH7.4 _ 121 _ with 0.025% sodium azide before use. The spine was post fixed in 10% neutralized formalin for two days and embedded in paraffin blocks. iasazg Immunohistochemistry: Coronal ns of 30 um thickness were probed with biotinylated donkey anti-human IgG (H+L) by free floating staining. Free-floating sections were washed in Tris—Triton pH7.4 (SOmM Tris, 150 mM NaCl, 0.05% Triton X- 100), incubated in 1% H202 PBS for 30 min, and incubated with a ng solution containing 2% normal goat— and horse serum in Tris-Triton with additional 0.2% Triton X-100 for 1 h at room temperature. The sections were then incubated with biotinylated donkey anti-human IgG (H+L) (Jackson research Labs, 709149) at 1:200 in blocking solution for 16 h at 4°C with agitation at 100 rpm to detect neuronal human IgG.

The —bound biotinylated antibody was visualized by peroxidase chromogenic reaction using the Vectastain Elite ABC kit (Vector Laboratories, PK6100, 1:100). The tic reaction was stopped with ice cold PBS and the sections were washed in PBS 3 times. The sections were then mounted on glass slides and air dried over night before they were counterstained with hemalum solution to visualize the nuclei (Carl Roth GmbH + Co., T8651). After dehydration steps, the slides were covered with coverslips before being scanned with the s dotSlide 2.1 virtual microscopy system.

Human antibodies were detected in the brains of TauP301L mice, which had ed either human au antibodies or hlgGl control antibody via i.p. injection, but not in TauP301L and wild type mice without antibody treatment (Fig. 11). However, neuronal staining was only observed in the hippocampi of NI-105.6C5, NI-105.6E3 and NI-105.40E8 anti-tau antibody treated mice, but not in hIgGl treated mice. Neuronal staining with anti-tau antibodies was readily detectable one day post injection, less pronounced at eight days post injection, and was undetectable at 22 days post ion (data not shown). TauP301L mice produce high levels of transgenic human tau in the hippocampal formation, and the hippocampus is one of the earliest regions which p neurofibrillary tangles. Thus, peripherally injected anti-tau antibodies not only entered the brain but also likely entered into neurons which contained high levels of tau.

Example 20 Effects of chronic treatment of TauP301L mice with ch4E4(N30Q) and chl7C1(N31Q). {0344] s and Antibody treatments: Chimeric NI-105.4E4(N30Q) ("ch4E4(N30Q)") and chimeric NI-105.17C1(N31Q) ("ch17C1(N31Q)") containing the variable domains of the human dy and the constant regions of mouse IgG2a were produced by transient transfection of CHO cells and purified using standard methods.

Gender balanced TauP301L mice at ages of 7.5-8 months were weekly given 10 mg/kg ch17C1(N31Q) (n=20), 10 mg/kg ch4E4(N30Q) (n=20) or an equal volume of PBS (nreZO) through intraperitoneal injection. Body weight was monitored every two weeks. No significant weight loss was observed. Two mice from the PBS group and one mouse from the chl7C1(N31Q) treated group died prematurely. Mice were anaesthetized one day after the 25th treatment for tissue collection. Blood was collected through the orbital sinus. Brain and spine were uently ted following perfusion for 2 min with ice cold PBS, containing 10 UI/ml heparin, through the left ventricle. The left half brain was then weighed, rozen in dry ice and stored at -80°C before use. The réght half of the brain and the spine were post-fixed in lized 10% formalin at 4°C for two days followed by further e in PBS before being processed to paraffin embedded brain and spinal cord blocks.

[0346] Brain protein extraction: Brain protein was sequentially extracted based on the solubility. The left half brain was first homogenized in 10 times w/v of 50 mM NaCl ning 0.2 % diethylamine, 1X protease inhibitor (Roche Diagnostics GmbH) and 1 X phosphatase inhibitor (Roche Diagnostics GmbH). After 30 min incubation on ice, the nate was centrifuged at 100,000 g at 4°C for 30 min. Subsequently, the supernatant was collected and defined as the e fraction. The remaining pellet was homogenized in 12 times w/v of 10 % sucrose lysis buffer containing 10 mM Tris 7.4, 0.8M NaCl, lmM EGTA, 1X phosphatase inhibitor,1X protease inhibitor,1 mM Na3VO4, 1 mM NaF and 1 mM AEBSF. After 30 min tion on ice, the homogenate was centrifuged at 20,500 g at 4°C for 20 min. 95% of the supernatant was carefully collected for sarcosyl extraction. The pellet was stored at -80°C.N—1aury1—sarcosinate was added to the supernatant (1% (w/v) final concentration). Following 1 h incubation at 37°C with W0 2014/100600 PCT/USZOl3/076952 agitation at 220 rpm, the solution was centrifuged at 100,000g at 4°C for one hour. The supernatant was collected and defined as the sarcosyl soluble on. The pellet was left to dry at room temperature for 30 min, then dissolved in 50 mM Tris pH 7.4 (20% v/w initial brain weight) and defined as the PHF ble fraction, which was stored at -80°C until use.

ELISA measurements Human total tau and phosphorylated tau in three brain protein ons were fied with commercial ELISA kits (Life Technologies) following the manufacturer’s protocol. Total human tau, human tau phosphorylated at Threonine 231 (pT231 tau), human tau phosphorylated at Serine 199 (pSl99 tau) and human tau orylated at Threonine 181 (pT181) tau were detected. s of the soluble fraction were standardized to 1 mg/ml based on the total protein content measured with BCA protein assay (Pierce) with 50 mM Tris pH7.4. Aliquots of the standardized samples were prepared for ELISA measurements and Western blotting. To e solubilized PHF insoluble fraction for ELISA, 10 ul PHFTau was ted with 10 pl 8M guanidine hydrochloride at room temperature for one hour followed by addition of 180 pl 50 mM Tris 7.4. ELISA measurements were d out following standard protocols. The tau levels in each sample measured by ELISA were normalized to initial brain weight for final is.

End point plasma drug levels were measured using a sandwich ELISA. Briefly, 3 ug/ml rTau (rPeptide) (SEQ ID NO:6) in 100 nM carbonate ELISA coating buffer (pH9.6) was incubated in Costar half-area ELISA plates at 4°C overnight. The plates were blocked with 3% BSA in PBS at room temperature for one hour. Plasma samples were diluted in 3% BSA in PBS containing 0.1% Tween®20 to 1:200, 1:400 and 1:800.

Serial dilutions of ch17C1(N31Q) and ch4E4(N30Q) were used to generate standard curves. After one hour incubation, the plates were washed 4 times with PBS containing 0.1% Tween®20 followed by a one hour incubation with donkey anti-mouse IgGFcy- HRP (1:10,000). After washing, the bound antibody was further determined by a rd colorimetric assay. Standard curves were generated by dal curve fit with GraphPad Prism 5.

[0349] Western blot: Protein samples of the three fractions were heated at 70°C for 10 min in 4X NuPAGE® LDS sample buffer (Life Technologies) and an equal amount of total protein from each sample was electrophoresed on a NuPAGE® 4-12% (w/v) gel.

Following semi-dry transfer of protein to PVDF membrane, the membrane was blocked — 124 — in 3% BSA containing 0.1% Tween-20 in TBS and subsequently probed with different anti-tau antibodies at 4°C overnight. Peroxidase—conjugated ary antibodies were then incubated at room temperature for 1 h following 4 washes with TBST.

Subsequently, the bound antibodies were detected by enhanced chemiluminescence (ECL) (Pierce). Densitometric analysis of immunoblots was performed with the National utes of Health Image] program.

Two-trial Y maze: Mice were tested for short-term spatial memory using a two- trial Y-maze test. The arms of the maze were 35 cm long, 5 cm wide and 10 cm deep.

Abstractive cues were placed on the n surrounding the maze. Experiments were conducted wéth an ambient light level of 6 lux mice were . During the re phase, assigned to two arms (the start arm and one other arm), which can be freely explored during 4 min, without access to the third arm (new arm), blocked by a door made of the same material as the maze. Mice were then removed from the maze and kept in the holding cage for 2 min, when the maze was cleaned with 50% ethanol. During the test please, mice were placed at the end of the start arm and allowed to freely explore all. three arms during 4 min. The test phase was recorded with the TSE VideoMot2 software for Video tracking and analysis of animal or (TSE Systems, Bad Homburg, Germany).

The number of arm entries and time spent in the new arm were recorded. The average of number of arm entries and time spent in the other two arms that were open during the training session was calculated. A ratio n the number of arm entries into (or time spent in) the new arm and the average of the other two arms were calculated. Wild type control animals which do not have a deficit in spatial working memory will typically have a ratio n 1.5 and 2 in this test. Pennanen et al., Genes Brain Behav 5(5):369-79 (2006).

[0351] Data analysis: ELISA data were log teansformed to meet the normality assumption for the two-way analysis of variance. The ence was considered significant when p<0.05. ch4E4(N30Q) significantly d soluble human tau in TauP301L mice: Human tau levels in the DEA-soluble, sarcosyl-soluble, and insoluble fractions of brain n extracts were quantified by ELISA. The majority of the human tau was found in the DEA-soluble fraction (data not shown). Total human tau (hTau) was reduced in the DEA-soluble fraction from ch17Cl(N3lQ) and ch4E4(N30Q) treated mice compared with that of PBS treated mice (29% reduction on e in ch17Cl(N3lQ) and 37% in - 125 _ ch4E4(N30Q), Fig. 12A). Reductions were also seen in phosphorylated tau (pT231, pSl99 and pT181) in the DEA-soluble fraction (Fig. 12B, C and D respectively). We have previously observed lower human tau expression in female 1L mice than their male counterparts. Therefore, to accurately analyze the data we used two-way ANOVA with gender and treatment as the two variables. A gender effect was confirmed with a p<0.01 in all soluble human tau measurements (total human tau, pSl99, pT181 and pT231 human tau). There was no interaction between gender and ent (0.49 <p< 0.91 in all e human tau measurements). Importantly, there was a significant treatment effect in DEA-soluble human tau 5 for hTau, p8199 and pT231, and p=0.06 for pT181). The treatment effect was inantly driven by ch4E4(N30Q).

When compared with PBS control, ch4E4(N3OQ) significantly reduced hTau (p<0.05), pSl99 (p<0.01), pT23l (p<0.01) and pTl81 (p<0.05). ELISA measurements using the sarcosyl-insoluble fraction showed a high variability among animals, and no sigraificant gender effect was observed. No significant treatment effect was observed in the sarcosyl- insoluble on. Similarly, no cant treatment effect was observed by ELISA in the yl-soluble fraction. n blots using human tau-specific monoclonal antibody Tau12 showed full length human tau, as a single band at 62 kDa, as the major tau immunoreactive component in the DEA-soluble fraction. A clear reduction of the full length human tau was ed in majority of the mice treated with ch4E4(N30Q) and ch17Cl(N31Q). In the sarcosyl insoluble fractior’a, a 64 kDa band and several other higher molecular weight bands were observed, as well as smaller molecular weight bands presumably corresponding to human tau fragments. Densitometric analysis showed a high degree of variability among individual animals, which prevented any quantitative comparison.

However, there was an overall qualitative ion in all human tau proteins in the sarcosyl insoluble fraction detected by Tau12 in ch17Cl(N31Q) and ch4E4(N30Q) treated mice (representative Western blot shown in Fig. 13).

Plasma drug levels: Mice were treated with ch17Cl(N31Q) and ch4E4(N30Q) at mg/kg weekly through i.p. injection. To assess the drug exposure, plasma samples were collected 24 h after the last treatment. Plasma drug levels were measured with ELISA using rTau as capture agent. The average plasma levels of ch17Cl(N31Q) and N30Q) were 200 ug/ml and 145 ug/ml, respectively, suggesting both antibodies had good blood exposure (Fig. 14). - 126 _ Antibody treatment and spatial memory: An earlier study suggested deficits in spatial reference memory, which is hippocampus—dependent, in TauP301L mice (Pennanen et at, Genes Brain Behav 5(5):369—79 (2006)). The two-trial Y maze was reported as a sensitive test to detect deficits in short—term spatial memory in tau transgenic mice (Troquier et al., Curr Alzheimer Res. 9(4):397—405 (2012)). During the exposure phase, all groups explored the maze equally, ng a similar amount of time in each available arm (data not shown). No differences were found comparing distance moved.

During the test phase, PBS treated TauP301L mice made almost equal number of entries in the new arm as the average of the other two arms explored during the exposure phase, =1.18), suggesting a poor spatial working memory in PBS treated TauP301L mice.

Both ch17C1(N31Q) and ch4E4(N30Q) treated mice showed a preference for the new arm ve to the other two arms, and they made more visits to the new arm than the e of the other two arms (ratio=150 for ch17C1(N31Q) and 1.30 for ch4E4(N30Q)). The ratio of new arm entey in ch17C1(N31Q) and ch4E4(N30Q) treated TauP301L mice compared with that ofthe PBS d group is shown in Fig. 15.

The present invention is not to be limited in scope by the specific embodiments bed which are intended as single rations of individual aspects of the invention, and any compositions or methods which are flinctionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such ations are intended to fall within the scope of the appended claims.

Definitions of specific embodiments of the invention as claimed herein .

According to a first embodiment of the invention, there is provided an isolated anti-tau antibody or tau-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), n the VH comprises VH complementarity determining regions : VHCDR1; VHCDR2; and VHCDR3, and the VL comprises VL CDRs: VLCDR1; VLCDR2; and VLCDR3, wherein: the VHCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:85, the VHCDR2 ses the amino acid sequence as set forth in SEQ ID NO:86 and the VHCDR3 comprises the amino acid sequence as set forth in SEQ ID NO:87; and the VLCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:88, the VLCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:89 and the VLCDR3 ses the amino acid as sequence as set forth in SEQ ID NO:90.

According to a second embodiment of the invention, there is ed an anti-tau antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:215 or SEQ ID NO:205 and the light chain comprises the amino acid sequence of SEQ ID NO:206.

According to a third ment of the invention, there is provided a pharmaceutical ition comprising the anti-tau antibody or tau-binding fragment of the first or second embodiment, and a pharmaceutically acceptable carrier.

According to a fourth embodiment of the invention, there is provided a vector or s comprising an isolated polynucleotide or polynucleotides comprising a nucleotide sequence or nucleotide sequences encoding the anti-tau antibody or tau-binding fragment of the first or second embodiment.

According to a fifth embodiment of the ion, there is provided an isolated host cell comprising the vector or vectors of the fourth embodiment.

According to a sixth embodiment of the invention, there is provided a method of preparing an anti-tau antibody or the tau-binding nt thereof, the method comprising: culturing the host cell of the fifth embodiment in a cell culture; and isolating the anti-tau dy or tau-binding fragment thereof from the cell culture.

According to a seventh embodiment of the present invention, there is provided a use of the anti-tau antibody or the tau-binding fragment of the first or second embodiment or the pharmaceutical ition of the third embodiment in the cture of a medicament for treating a neurodegenerative tauopathy.

According to an eighth ment of the present ion, there is provided a use of the anti-tau antibody or the tau-binding fragment thereof of the first or second embodiment or the pharmaceutical composition of the third embodiment in the manufacture of a medicament for treating abnormal accumulation or deposition of tau in the central nervous system in a human subject in need thereof.

Claims (23)

What we claim is:

1. An isolated anti-tau antibody or tau-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain le domain (VL), wherein the VH comprises VH complementarity determining regions : VHCDR1; VHCDR2; and VHCDR3, and the VL comprises VL CDRs: VLCDR1; VLCDR2; and VLCDR3, wherein: the VHCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:85, the VHCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:86 and the VHCDR3 comprises the amino acid sequence as set forth in SEQ ID NO:87; and the VLCDR1 comprises the amino acid sequence as set forth in SEQ ID NO:88, the VLCDR2 comprises the amino acid sequence as set forth in SEQ ID NO:89 and the VLCDR3 ses the amino acid as sequence as set forth in SEQ ID NO:90.

2. The au dy or tau-binding fragment f of claim 1, wherein VHCDR1 consists of the amino acid sequence as set forth in SEQ ID NO:85, VHCDR2 consists of the amino acid sequence as set forth in SEQ ID NO:86 and VHCDR3 consists of the amino acid sequence as set forth in SEQ ID NO:87; and the VLCDR1 consists of the amino acid sequence as set forth in SEQ ID NO:88, the VLCDR2 consists of the amino acid sequence as set forth in SEQ ID NO:89 and the VLCDR3 consists of the amino acid sequence as set forth in SEQ ID NO:90.

3. The anti-tau antibody or tau-binding fragment thereof of claim 2, wherein: (a) the VH is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:48; (b) the VH is identical to the amino acid sequence set forth in SEQ ID NO:48; (c) the VH is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:47; (d) the VH is identical to the amino acid sequence set forth in SEQ ID NO:47; (e) the VL is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:49; (f) the VL is identical to the amino acid sequence set forth in SEQ ID NO:49; (g) the VH is at least 90% cal to the amino acid sequence set forth in SEQ ID NO:48 and the VL is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:49; (h) the VH is identical to the amino acid sequence set forth in SEQ ID NO:48 and the VL is identical to the amino acid sequence set forth in SEQ ID NO:49; (i) the VH is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:47 and the VL is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:49; or (j) the VH is identical to the amino acid sequence set forth in SEQ ID NO:47 and the VL is identical to the amino acid sequence set forth in SEQ ID NO:49.

4. The au antibody or tau-binding fragment of claim 1, wherein the VH comprises the amino acid sequences as set forth in SEQ ID NO:47 and the VL comprises the amino acid sequence as set forth in SEQ ID NO:49.

5. The anti-tau antibody or tau-binding fragment of claim 1, wherein the VH comprises the amino acid sequence as set forth in SEQ ID NO:48 and the VL comprises the amino acid sequence as set forth in SEQ ID NO:49.

6. The anti-tau antibody or tau-binding fragment of any one of claims 1 to 5, which comprises a human IgG1 heavy chain constant .

7. The anti-tau antibody or tau-binding fragment of any one of claims 1 to 5, which comprises a human lambda light chain constant region.

8. The anti-tau antibody or tau-binding nt of any one of claims 1 to 5, which comprises a human IgG1 heavy chain constant region and a human lambda light chain nt region.

9. The anti-tau antibody or tau binding fragment of claim 1, wherein the antibody is a human dy or a humanized antibody.

10. The anti-tau dy or tau binding fragment of any one of claims 1 to 5, wherein the tau-binding fragment is a single chain Fv fragment (scFv), an F(ab') fragment, an F(ab) fragment or an F(ab')2 fragment.

11. The au antibody or tau binding fragment of claim 1, which is an anti-tau antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid ce of SEQ ID NO:215 or SEQ ID NO:205 and the light chain comprises the amino acid sequence of SEQ ID NO:206.

12. A ceutical composition comprising the anti-tau antibody or tau-binding fragment of any one of claims 1-11, and a pharmaceutically acceptable carrier.

13. A vector or vectors comprising an ed polynucleotide or polynucleotides comprising a nucleotide sequence or nucleotide sequences encoding the anti-tau antibody or tau-binding fragment of any one of claims 1-11.

14. The vector or s of claim 13, wherein the polynucleotide or polynucleotides comprise a sequence of nucleotides ng a VH that comprises the nucleotides as set forth in SEQ ID NO:172 or SEQ ID NO:173 and a sequence of nucleotides encoding a VL that ses the nucleotides as set forth in SEQ ID NO:174.

15. An isolated host cell comprising the vector or vectors of claim 13 or 14.

16. A method of preparing an anti-tau antibody or the nding fragment thereof, the method comprising: culturing the host cell of claim 15 in a cell culture; and isolating the anti-tau dy or tau-binding fragment thereof from the cell culture.

17. The method of claim 16, further comprising formulating the anti-tau antibody or ding nt thereof into a sterile pharmaceutical composition suitable for administration to a human subject.

18. Use of the anti-tau dy or the tau-binding fragment of any one of claims 1-11 or the pharmaceutical composition of claim 12 in the manufacture of a medicament for treating a neurodegenerative tauopathy.

19. The use of claim 18, wherein the neurodegenerative tauopathy is selected from the group consisting of Alzheimer’s e, amyotrophic lateral sclerosis/parkinsonism–dementia complex, argyrophilic grain dementia, British type d athy, cerebral amyloid angiopathy, corticobasal ration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down’s syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick’s disease, postencephalitic sonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, ssive supranuclear palsy, subacute sclerosing panencephalitis, Tangle only dementia, multi-infarct dementia and ischemic stroke.

20. The use of claim 18, wherein the neurodegenerative tauopathy is mer’s disease.

21. The use of claim 18, wherein the neurodegenerative tauopathy is frontotemporal dementia with parkinsonism linked to chromosome 17 or frontotemporal lobar ration.

22. The use of claim 18, wherein the neurodegenerative tauopathy is progressive supranuclear palsy.

23. Use of the anti-tau antibody or the tau-binding fragment thereof of any one of claims 1- 11 or the pharmaceutical composition of claim 12 in the manufacture of a medicament for treating al accumulation or deposition of tau in the central nervous system in a human subject in need thereof.