BR122022003923B1 - ISOLATED ANTI-TAU ANTIBODIES AND TAU-BINDING FRAGMENTS THEREOF, THEIR USES AND METHOD OF PREPARATION, AS WELL AS POLYNUCLEOTIDES, EXPRESSION VECTORS, COMPOSITIONS, AND IN VITRO METHODS FOR MONITORING THE PROGRESSION OF A NEURODEGENERATIVE TAUTOPATHY AND USING THE LEVEL OF TAUPATOLOGICALLY MODIFIED OR ADDED AS AN INDICATOR OF NEURODEGENERATIVE TAUTOPATHY - Google Patents

Abstract

Human tau-specific antibodies are provided, as well as fragments, derivatives and variants thereof, as well as methods relating thereto. Assays, kits, and solid supports related to tau-specific antibodies 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 and diagnostic immunotherapy, respectively.

Description

BACKGROUND OF THE INVENTION Field of Invention

[001] The present invention generally relates to novel tau-specific binding molecules, particularly human antibodies as well as fragments, derivatives and variants thereof which recognize tau protein, including pathologically phosphorylated tau and aggregated forms of tau. Furthermore, the present invention relates to pharmaceutical and diagnostic compositions comprising such binding molecules, antibodies and mimetics thereof valuable as a diagnostic tool to identify toxic tau and tau species in plasma and CSF and also in passive vaccination strategies. to treat neurodegenerative disorders such as Alzheimer's disease (AD), amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC), argyrophilic granulomatous dementia (AGD), British-type amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration (CBD) , Creutzfeldt-Jakob disease (CJD), dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration, Gerstmann-Straussler- Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guanamanian motor neuron disease with neurofibrillary tangles, Pick disease (PiD ), post-encephalitis 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.

Background of the Technique

[002] Protein accumulation, modifications, and aggregation are pathological aspects of numerous neurodegenerative diseases. Pathologically modified and aggregated tau including hyperphosphorylated tau conformers are an invariable hallmark of tauopathies and correlate with disease severity.

[003] Tau is a microtubule-associated protein expressed in the central nervous system with a primary function of stabilizing microtubules. There are six major isoforms of tau expressed primarily in the adult human brain, which are derived from a single gene by alternative splicing. Under pathological conditions, tau protein becomes hyperphosphorylated, resulting in a loss of tubulin binding and microtubule destabilization followed by aggregation and deposition of tau in pathogenic neurofibrillary tangles. Tau-related disorders - collectively referred to as neurodegenerative tauopathies - are part of a group of protein misfolding disorders, including Alzheimer's disease (AD), progressive supranuclear palsy, Pick's disease, corticobasal degeneration, FTDP-17, among others . More than 40 mutations in the tau gene have been reported to be associated with hereditary frontotemporal dementia demonstrating that mutations in the tau gene are sufficient 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 may be caused by a pathological cascade in which Aß is found upstream of tau (Gotz et al., Science 293 (2001), 1491-1495). Other findings, however, point to a two-way model where the two cascades function independently of each other (van de Nes et al., Acta Neuropathol. 111 (2006), 126-138). Immunotherapies targeting beta-amyloid peptide in AD have produced encouraging results in animal models and have shown promise in clinical trials. The most recent autopsy data from a small number of subjects suggest that clearance of beta-amyloid plaques in AD patients may not be sufficient to halt cognitive deterioration, emphasizing 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). Following the success of Abeta-based immunization therapy in transgenic animal models, the concept of active immunotherapy was expanded to tau protein. Active vaccination of wild-type mice using tau protein has, however, been shown to induce the formation of neurofibrillary tangles, axonal damage, and mononuclear infiltrates in the central nervous system, accompanied by neurological deficits (Rosenmann et al., Arch Neurol. 63 (2006) ), 1459-1467). Further studies in transgenic mouse strains using active vaccination with phosphorylated tau peptides revealed reduced brain levels of brain tau aggregates and delayed progression of behavioral impairment ( Sigurdsson, J. Alzheimers. Dis. 15 (2008), 157-168; Boimel et al., Exp. Neurol., 224 (2010), 472-485 ). These findings highlight the potential benefit but also the tremendous risks associated with tau-targeted active immunotherapy approaches. New therapeutic strategies are urgently needed to address pathological tau proteins with effective and safe therapy.

[004] Passive immunization with human antibodies derived from healthy human subjects that are evolutionarily optimized and have undergone affinity maturation by the human immune system would provide a promising new therapeutic avenue with a high probability of excellent efficacy and safety.

BRIEF SUMMARY OF THE INVENTION

[005] The present invention makes use of the tau-specific immune response of healthy humans for the isolation of natural anti-tau-specific human monoclonal antibodies. In particular, experiments carried out in accordance with the present invention have been successful in isolating tau-specific monoclonal antibodies from a pool of healthy human subjects without signs of a neurodegenerative taupathy.

[006] The present invention, therefore, is directed to human antibodies, antigen-binding fragments and similar antigen-binding molecules that are capable of specifically recognizing tau. "Specifically recognize tau", "specific antibody to/from tau" and "anti-tau antibody" means specifically, generally and collectively, antibodies to the native form of tau, or aggregates or pathologically modified isoforms of tau. Provided here are human antibodies selective for full-length, pathologically phosphorylated and aggregated forms.

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

[008] The antigen-binding fragment of the antibody may be a single-chain Fv fragment, an F(ab' fragment), an F(ab' fragment), and an F(ab')2 fragment, or any other fragment of antigen binding. In a specific embodiment, below, the antibody or fragment thereof is an antibody of the human IgG isotype. Alternatively, the antibody is a human-murine chimeric or murinized antibody, the latter being particularly useful for diagnostic methods and animal studies.

[009] Furthermore, the present invention relates to compositions comprising the antibody of the present invention or active fragments thereof, or agonists and cognate molecules, or alternatively, antagonists thereof, and to methods of immunotherapy and immunodiagnosis using these compositions in the prevention , diagnosing or treating a tauopathy, wherein an effective amount of composition is administered to a patient in need thereof.

[0010] Naturally, the present invention extends to immortalized human memory B lymphocytes and B cells, respectively, which produce the antibody having the distinct and unique characteristics as defined below.

[0011] The present invention also relates to polynucleotides encoding at least one 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 defined below in Figure 7.

[0012] Accordingly, the present invention also includes vectors comprising said polynucleotides and host cells transformed therewith, as well as their use for the production of an antibody and equivalent binding molecules that are specific for tau. Means and methods for recombinantly producing antibodies and mimetics thereof, as well as methods for screening for competing binding molecules, e.g., antibodies, are known in the art. However, as described herein, in particular with regard to therapeutic applications in humans, the antibody of the present invention is a human antibody in the sense that the application of said antibody is substantially free of an immune response directed against such antibodies from another observed for chimeric and even humanized antibodies.

[0013] 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, using an ELISA-based or surface-adapted assay. The methods and compositions disclosed herein can help in neurodegenerative tauopathies as a diagnosis of Alzheimer's disease and can be used to monitor disease progression and therapeutic efficacy.

[0014] Accordingly, it is a particular object of the present invention to provide methods for treating, diagnosing or preventing a neurodegenerative taupathy such as Alzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex, argyrophilic grain dementia, British-type amyloid angiopathy, amyloid angiopathy brain, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle with calcification, Down syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, lobar frontotemporal degeneration, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick type C disease, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease, post-encephalitis parkinsonism, protein cerebral amyloid angiopathy prion, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, multi-infarct dementia, and ischemic stroke. The methods comprise administering an effective concentration of a human or antibody-derived antibody to the subject where the antibody targets tau.

[0015] Other embodiments of the present invention will be apparent from the description and Examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0016] FIG. 1. Amino acid and nucleotide sequences of the variable region, ie heavy chain and lambda light chain, of human antibodies NI-105.4E4 (A), NI-105.24B2 (B) and NI-105.4A3 (C). Structure (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 light chain and heavy chain could potentially contain primer-induced changes in FR1, which, however, do not substantially affect the biological 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 matched with the human germline variable region sequences in the database; see, for example, Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by MRC Center for Protein Engineering (Cambridge, UK). Those amino acids, which are considered to potentially deviate from the consensus germline sequence due to the PCR primer and therefore were substituted in the amino acid sequence, are indicated in bold.

[0017] FIG. 2. NI-105.4E4 binds to neurofibrillary tangles (NFT), dystrophic neurites and neuropil hairs in brain AD and mice expressing human TauP301L. NI-105.4E4 staining identifies NFTs and neuropil hairs in AD brain (A), with no significant binding to tau in healthy control subject brain (B). In TauP301L (E-I) transgenic mice, NI-105.4E4 binds strongly to pathological tau resembling NFT (E, F and H), neuropil wires (E and G) and dystrophic neurites (E and H). Furthermore, NI-105.4E4 also identifies tau aggregates in the pre-entanglement (I) phase. NI-105.4E4 binds to NFT, dystrophic neurites and neuropil hairs in transgenic mice expressing human APP with Swedish and Arctic mutation and TauP301L; the arrow marks a beta-amyloid plaque, surrounded by dystrophic neurites recognized by NI-105.4E4 (J). Secondary antibody only gives no signal in human AD (C) and healthy control (D).

[0018] FIG. 3. Amyloid Plaque Immunoreactivity Assay (TAPIR). Neurofibrillary tangles were stained with the anti-phospho-tau antibody AT100 or sera isolated from healthy elderly subjects.

[0019] FIG. 4. Schematic representation of the NI-105.4E4 and NI-105.4A3 epitopes and commonly used commercially available mouse monoclonal tau antibody epitopes are shown. The human antibody NI-105.4E4 is directed to a unique epitope comprising two 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, USA.); PHF1 ( Lewis et al., Science 293 (2001), 1487-1491 ).

[0020] FIG. 5. Levels of human IgG in the plasma of mice after intraperitoneal administration of 30 mg/kg human anti-tau antibody NI-105.4E4 or NI-105.4A3.

[0021] FIG. 6. Human IgG levels in mouse brain homogenate after intraperitoneal administration of 30 mg/kg human anti-tau antibody NI-105.4E4 or NI-105.4A3.

[0022] FIG. 7. Amino acid sequence of heavy chain and light chain variable regions of human anti-tau antibodies (A) NI-105.17C1, (B) NI-105.6C5, (C) NI-105.29G10, (D) NI-105.6 L9, (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) NI-105.39E2, (N) NI-105.19C6, and (O) NI-105.9C4. Complementarity determining regions (CDRs) are underlined.

[0023] FIG. 8. : (A) Binding of ch17C1, ch17C1(N31Q) mIgG2a and ch17C1(N31Q) mIgG1 Agly to recombinant Tau in an ELISA assay. (B) Comparison of recombinant Tau binding by ch17C1(N31Q) mIgG2a and ch17C1(N31Q, I48V) mIgG2a in an ELISA assay.

[0024] FIG. 9. Comparison of recombinant Tau binding by NI-105.40E8 hIgG1 and NI-105.40E8(R104W) hIgG1 in an ELISA assay.

[0025] FIG. 10. Binding of human anti-tau antibodies NI-105.40E8, NI-105.48E5, NI-105.6C5 and NI-105.17C1(I48V) to pathologically aggregated tau in the AD brain and in the brain of transgenic mouse model of taupathy. Representative images of human anti-tau antibody binding to pathological tau aggregates in Alzheimer's disease (AD) brain and taupathy (Tg) transgenic mouse brain. Control tissue samples were obtained from mentally healthy subjects (Ctr) or wild-type mouse brain (Wt).

[0026] FIG. 11. Brain penetration of human anti-tau antibodies NI-105.6C5 or NI-105.6E3 in TauP301L mice. "tg" indicates sections representing transgenic animals, treated or untreated, and "wt" indicates an untreated non-transgenic animal. Scale bar: 50 µm.

[0027] FIG. 12. Effects of chronic treatment of TauP301L mice with ch4E4(N30Q) and ch17C1(N31Q). Total levels of human tau (A), human pS199 tau (B), human pT231 tau (C) and human pT181 tau (D) in the soluble and insoluble fraction of brain protein extracts were quantified with commercial ELISA.

[0028] FIG. 13. Soluble and insoluble human tau in TauP301L mice treated with ch17C1(N31Q) and ch4E4(N30Q) detected by Western blots.

[0029] FIG. 14. Mean plasma drug concentrations for animals treated with ch17C1(N31Q) and ch4E4(N30Q) 24 h after i.p. of the last dose. Mean plasma drug concentrations for ch17C1(N31Q) and ch4E4(N30Q) were 145 and 200 µg/ml, respectively.

[0030] FIG. 15. Spatial working memory in TauP301L mice treated with ch17C1(N31Q) and ch4E4(N30Q) was assessed by Y-maze from two studies.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0031] Neurodegenerative tauopathies are a diverse group of neurodegenerative disorders that share a common pathological lesion consisting of intracellular aggregates of abnormal filaments that are composed primarily of pathologically hyperphosphorylated tau in neurons and/or glial cells. Clinical features of tauopathies are heterogeneous and characterized by dementia and/or motor syndromes. The progressive accumulation of filamentous tau inclusions can cause neuronal and glial degeneration in combination with other deposits, for example beta-amyloid in Alzheimer's disease, or as a single pathogenic entity, as illustrated by mutations in the tau gene that are associated with forms family members 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 taupathic diseases can be provided, including Alzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex, argyrophilic grain dementia, British-type amyloid angiopathy, cerebral amyloid angiopathy , corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle with calcification, Down syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, lobar frontotemporal degeneration, Gerstmann-Straussler-Scheinker disease, Hallervorden disease -Spatz, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick type C disease, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease, post-encephalitis parkinsonism, prion protein cerebral amyloid angiopathy , progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, multi-infarct dementia, and stroke; see for a review, eg Lee et al., Annu. Rev. Neurosci. 24 (2001), 1121-1159 where Table 1 catalogs the original members of the tauopathies or Sergeant et al., Bioch. Biophy. Acta 1739 (2005), 179-97, with a list in Figure 2.

[0032] In this specification, the term "tau" is used interchangeably to refer specifically to the native monomer form of tau. The term "tau" is also used to generally identify other conformers of tau, for example tau oligomers or tau aggregates. The term "tau" is also used to refer collectively to all types and forms of tau. Due to alternative splicing, 6 isoforms of tau are present in the human brain. The protein sequences for these isoforms are: 352aa Fetal-tau Isoform MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK AEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGK TKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRS GYSSPGSPG TPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKS RLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVT SKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGG GNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSST GSIDMVDSPQ LATLADEVSASLAKQGL (SEQ ID NO:1) 381aa Tau-B Isoform MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK ESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAG HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQ ANATRIPAKTP KTDHGAEIVYKS PVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADE VSASLAKQGL (SEQ ID NO: 2) 410aa Tau-C isoform MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK ESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAA AQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGS DDKKAKGADGKTKIATPRGAAPPGQKGQANA TRIPAKTPPAPKTPPS SGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVR TPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQI VYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIG SLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAE IVYKSPVVSGDT SPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO:3) 383aa Tau-D Isoform MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK AEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGK TKIATPRGAAPPGQKG QANATRIPAKTPPAPKTPPSSGEPPKSGDRS GYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKS RLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQ SKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGG QVEVKSEKLDFKDRVQSKI GSLDNITHVPGGGNKKIETHKLTFRENAK AKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLAD EVSASLAKQGL (SEQ ID NO:4) Tau-E Isoform de 412a MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK ESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAG HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQ ANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSR TPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKS KIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQS KIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVV S GDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO:5) 441aa Tau-E Isoform MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK ESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAA AQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMV SKSKDGTGS DDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPS SGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVR TPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQII NKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLS KVTSKCGSL GNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIE THKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDM VDSPQLATLADEVSASLAKQGL (SEQ ID NO:6)

[0033] The "wild-type" tau amino acid sequence is represented by the Tau-F isoform of 441aa (SEQ ID NO:6) also referred to as "hTau40", "TauF", "Tau-4" or "full-length tau ". The amino acid sequence of tau can be obtained from the relevant literature and databases; see Goedert et al., Proc. Natl. academic Sci. USA 85 (1988), 4051-4055, Goedert et al., EMBO J. 8 (1989), 393-399, Goedert et al., EMBO J. 9 (1990), 4225-4230 and GenBank UniProtKB/swissprot : TAU_HUMAN locus, accession numbers P10636-2 (Fetal-tau) and P10636-4 to -8 (Isoforms B to F).

[0034] Another striking feature of tau protein is phosphorylation, which occurs at about 30 of the 79 potential serine (Ser) and threonine (Thr) phosphorylation sites. Tau is highly phosphorylated during brain development. The degree of phosphorylation declines in adulthood. Some of the phosphorylation sites are located within the microtubule-binding domains of tau, and it has been demonstrated that increased tau phosphorylation negatively regulates microtubule binding. For example, Ser262 and Ser396, which are found within or adjacent to microtubule binding motifs, are hyperphosphorylated in the tau proteins of abnormally paired helical filaments (PHFs), a major component of neurofibrillary tangles (NFTs) in the brain of AD patients. PHFs are filamentous aggregates of tau proteins that are abnormally hyperphosphorylated and can be stained with specific anti-tau antibodies and detected by light microscopy. The same goes for the so-called straight tau filaments. PHFs form distorted loops consisting of two filaments twisted around each other with a periodicity of about 80nm. These pathological features are commonly referred to as "tau pathology", "taupathology" or "tau-related pathology". For a more detailed description of the neuropathologic features of tauopathies, see Lee et al., Annu. Rev. Neurosci. 24 (2001), 1121-1159 and Gotz, Brain. Res. Rev 35 (2001), 266-286, the disclosed content of which is incorporated herein by reference. Physiological tau protein stabilizes microtubules in neurons. Pathologic phosphorylation leads to abnormal tau localization and aggregation, which causes microtubule destabilization and impaired cellular transport. Aggregated tau is neurotoxic in vitro ( Khlistunova et al., J. Biol. Chem. 281 (2006), 12051214 ). The exact neurotoxic species remain unclear, however, as well as the mechanisms by which they lead to neuronal death. Tau aggregates can be observed as the main component of neurofibrillary tangles (NFT) in many tauopathies, such as Alzheimer's disease (AD), Frontotemporal dementia, supranuclear palsy, Pick's disease, argyrophilic grain disease (AGD), corticobasal degeneration, FTDP-17, Parkinson's disease, dementia pugilistica (Reviewed in Gendron and Petrucelli, Mol. Neurodegener. 4:13 (2009)). In addition to these observations, evidence is emerging that tau-mediated neuronal death can occur even in the absence of tangle formation. Soluble phospho-tau species are present in CSF ( Aluise et al., Biochim. Biophys. Acta. 1782 (2008), 549-558 ). Tau aggregates can transmit 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 ).

[0035] In addition to involvement in neurodegenerative tauopathies, observed changes in tau phosphorylation during and after ischemia/reperfusion suggest that tau plays a crucial role in neuronal damage and clinical pathophysiology of neurovascular disorders such as ischemic stroke (Zheng et al. , J. Cell. Biochem. 109 (2010), 26-29).

[0036] The human anti-tau antibodies disclosed herein specifically bind to tau and epitopes thereof and to different conformations of tau and epitopes thereof. For example, disclosed herein are antibodies that specifically bind tau, tau in its full-length, pathologically modified isoforms of tau, and aggregates of tau. As used herein, reference to an antibody that "specifically binds", "selectively binds", or "preferentially binds" to tau refers to an antibody that does not bind to other, unrelated proteins. In one example, a tau antibody disclosed herein can bind tau or an epitope thereof and show no binding above about 1.5 times background for other proteins. An antibody that "specifically binds" or "selectively binds" a tau conformer refers to an antibody that does not bind all conformations of tau, that is, does not bind at least one other tau conformer. For example, disclosed herein are antibodies that can preferentially bind to aggregated forms of tau in AD tissue. Since the human anti-tau antibodies of the present invention were isolated from a pool of healthy human subjects exhibiting a tau-specific immune response, the tau antibodies of the present invention may also be called "human autoantibodies" to emphasize that the tau antibodies of the present invention in fact they were expressed by subjects and were not isolated from, for example, a human immunoglobulin expressing phage library, which until then represented a common method for trying to provide human-type antibodies.

[0037] It should be noted that the term "a" or "an" refers to one or more such 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" may be used interchangeably herein.

[0038] As used herein, the term "polypeptide" is intended to include a singular "polypeptide" as well as plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not make reference to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "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" may be used instead of, or interchangeably with, any of these terms.

[0039] The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by or non-naturally occurring amino acids. A polypeptide may 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 way, including chemical synthesis.

[0040] A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more , 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are called folded, and polypeptides that do not have a defined three-dimensional structure, but can instead adopt a large number of different conformations, are referred to as non-folded. As used herein, the term glycoprotein refers to a protein attached to at least a carbohydrate moiety that is linked to the protein through an oxygen-containing or nitrogen-containing side chain of an amino acid residue, for example, a serine residue or a asparagine residue.

[0041] An "isolated" polypeptide or a fragment, variant, or derivative thereof is intended for a polypeptide that is not in its natural environment. 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 purposes of the invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0042] Also included as polypeptides of the present invention are fragments, derivatives, analogues, or variants of the foregoing polypeptides, and any combination thereof. The terms "fragment", "variant", "derivative" and "analog" when referring to the antibodies or antibody polypeptides of the present invention include any polypeptide that retains at least some of the antigen-binding properties of the antibody molecule, antibody or polypeptide. corresponding native binding. Polypeptide fragments of the present invention include proteolytic fragments as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of antibodies and antibody polypeptides of the present invention include fragments as described above, as well as those polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced using known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of specific tau binding molecules, for example, antibodies and antibody polypeptides of the present invention, are polypeptides that have been altered to display additional characteristics not found in the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogues". As used herein, a "derivative" of a binding molecule or fragment thereof, or an antibody, or an antibody polypeptide refers to a polypeptide having one or more residues chemically derived by the reaction of a functional side group. Also included as "derivatives" are peptides that contain one or more naturally occurring amino acids derived from the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0043] The term "polynucleotide" is intended to include a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct, for example, messenger RNA (mRNA) or plasmid DNA (pDNA ). A polynucleotide may comprise a conventional phosphodiester linkage or an unconventional linkage (for example, an amide linkage, as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more segments of nucleic acid, for example DNA or RNA fragments, present in a polynucleotide. "Isolated" nucleic acid or polynucleotide means a nucleic acid molecule, either DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding an antibody contained in a vector is considered isolated for purposes of the present invention. Other examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides (partially or substantially) in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include those synthetically produced molecules. Furthermore, polynucleotides or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0044] As used herein, a "coding region" is a part of nucleic acid consisting of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered as part of a coding region, but any flanking sequences, e.g., promoters, ribosome binding sites, transcriptional terminators , introns and the like, are not part of a coding region. Two or more coding regions of the present invention may be present on a single polynucleotide construct, for example, on a single vector, or on separate polynucleotide constructs, for example, on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, for example, a single vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. Furthermore, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, fused or non-fused to a nucleic acid encoding a binding molecule, an antibody, or fragment, variant, or derivatives thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0045] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide typically may include a promoter and/or other transcriptional or translational control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, for example 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 regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" or "operably linked" if induction of promoter function results in transcription of the mRNA encoding the desired gene product and if the The nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, in addition to a promoter, for example, enhancers, operators, repressors and transcription termination signals, can be operatively associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed here.

[0046] A variety of transcriptional control regions are known to those skilled in the art and include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but are not limited to, cytomegalovirus promoter and enhancer segments (the promoter immediate early, together with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone and rabbit ß-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (eg, interferon- or interleukin-inducible promoters).

[0047] Likewise, a variety of translation control elements are known to those of skill in the art and include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and picornavirus-derived elements (particularly a site internal ribosome entry point, or IRES, also known as a CITE sequence).

[0048] In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA).

[0049] Coding regions of polynucleotides and nucleic acids of the present invention may be associated with additional coding regions that 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 sequence or secretory leader that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has begun. Those skilled in the art are aware that polypeptides secreted by vertebrate cells generally 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 embodiments, the native signal peptide, for example, an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of such a sequence that retains the ability to direct secretion of the polypeptide that is operatively associated therewith. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be replaced with the human tissue plasminogen activator (TPA) or mouse ß-glucuronidase leader sequence.

[0050] Unless otherwise noted, the terms "disorder", "disease" are used interchangeably herein.

[0051] A "binding molecule" as used herein in the context of the present invention primarily refers to antibodies, and fragments thereof, but may 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 adhesion molecules such as members of cadherin, intergrin, C-type lectin, and immunoglobulin (Ig) superfamilies . Thus, for the sake of clarity only and without restricting the scope of the present invention, most embodiments are discussed in relation to antibodies and antibody-like molecules, which represent a specific embodiment of binding molecules for the development of therapeutic and diagnostic agents.

[0052] The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin is a tau-like molecule that comprises at least one heavy chain variable domain, and normally comprises at least one heavy chain variable domain and one light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood; see, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0053] As will be discussed here in more detail below, the term "immunoglobulin" comprises several broad classes of polypeptides that can be differentiated biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, µ, a, d, e) with some subclasses in between (e.g., Y1-Y4). of this chain that determines the "class" of the antibody IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible by the skilled person in view of the present disclosure and therefore are within the scope of the present invention. All classes of immunoglobulins are clearly within the scope of the present invention, the following discussion will generally be directed towards the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are normally joined by disulfide bonds in a "Y" configuration, where the light chains support the heavy chains starting at the mouth of the "Y" and continuing through the variable region.

[0054] Light chains are classified as kappa or lambda (K, À). Each class of heavy chain can be linked with a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the "tail" parts of the two heavy chains are linked together by covalent or non-covalent disulfide bonds when the immunoglobulins are generated by hybridomas, B cells, or cells. genetically engineered hosts. In the heavy chain, amino acid sequences run from an N-terminus at the forked ends of the Y configuration to a C-terminus at the bottom of each chain.

[0055] The light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. On the other hand, light chain (CL) and heavy chain (CH1, CH2, or CH3) constant domains confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of constant region domains increases as they become more distal from the antigen-binding or amino-terminal site of the antibody. The N-terminal portion is a variable region and 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 indicated above, the variable region allows the antibody to selectively recognize and specifically bind to epitopes on antigens. That is, the VL domain and VH domain, or subset of complementarity determining regions (CDRs), of an antibody 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 specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains. Any antibody or immunoglobulin fragment that contains sufficient structure to specifically bind to tau is referred to interchangeably herein as a "binding fragment" or an "immunospecific fragment".

[0057] In naturally occurring antibodies, an antibody comprises six hypervariable regions, sometimes called "complementarity determining regions" or "CDRs" present in each antigen-binding domain, which are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional configurations in an aqueous environment. The "CDRs" are flanked by four relatively conserved (framework) regions or "FRs" that show less intermolecular variability. The framework regions largely adopt a ß-sheet conformation and the CDRs form loops that connect, and in some cases form part of, the ß-sheet structure. Thus, the framework regions act to form a framework that provides for the positioning of the CDRs in the correct orientation by non-covalent, interchain interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes non-covalent binding of an antibody to its cognate epitope. The amino acids comprising the CDRs and framework regions, respectively, can be easily identified by any light or heavy chain variable region determined by one skilled in the art, provided they have been precisely defined; see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporated herein by reference in their entireties.

[0058] In the case where there are two or more definitions of a term that is used and/or accepted within the art, the definition of the term as used herein is intended to include all of these meanings, unless explicitly stated otherwise. 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 light and heavy chain polypeptides. This particular region was described by Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia and Lesk, J. Mol. Biol., 196 (1987), 901-917), which are incorporated herein by reference, where the definitions include overlapping or subsets of amino acid residues when compared against each other. However, the application of any 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 that include the CDRs as defined by each of the references cited above are set forth below in Table 1 as a comparison. The exact residue numbers that a particular CDR includes will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular hypervariable region or CDR of the human IgG subtype of the antibody given the amino acid sequence of the antibody's variable region. Table 1: CDR definitions1 1Numbering of all CDR definitions in Table 1 follows the numbering conventions established by Kabat et al. (see below).

[0059] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One skilled in the art can unambiguously assign this "Kabat Numbering" system to any variable domain sequence, without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system established 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 derivative thereof of the present invention are in accordance with the Kabat numbering system, which, in However, it is theoretical and may not apply to every antibody of the present invention either. In one embodiment, depending on the position of the first CDR, subsequent CDRs can be shifted in either direction.

[0060] 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, murinized or chimeric antibodies, single chain antibodies, fragments epitope-binding domains, e.g. Fab, Fab' and F(ab')2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising the VL domain or VH , fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies disclosed herein). ScFv molecules are known in the art and are described, for example, in US Patent 5,892,019. The immunoglobulin molecules or antibodies of the invention can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, etc.) , or subclass of the immunoglobulin molecule.

[0061] In one embodiment, the antibody of the present invention is not IgM or a derivative thereof with a pentavalent structure. Particularly, in specific applications of the present invention, especially of therapeutic use, IgMs are less useful than IgG and other bivalent antibodies or corresponding binding molecules since IgMs due to their pentavalent structure and lack of affinity maturation often show non-specific cross-reactivities and very low affinity.

[0062] In a particular embodiment, the antibody of the present invention is not a polyclonal antibody, that is, it substantially consists of a particular antibody species rather than being a mixture obtained from a plasma immunoglobulin sample.

[0063] Antibody fragments, including single chain antibodies, may comprise the variable regions alone or in combination with all or a portion of the following: hinge region, CH1, CH2 and CH3 domains. Also included in the invention are tau binding fragments comprising any combination of variable regions or with a hinge region, CH1, CH2 and CH3 domains. Antibodies or immunospecific fragments thereof of the present invention can be from any animal source, including birds and mammals. In one embodiment, the antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be chondrictoid in origin (e.g., from sharks).

[0064] In one aspect, the antibody of the present invention is a human monoclonal antibody, isolated from a human. Optionally, the human antibody framework region is aligned and matched to the pertinent human germline variable region sequences in the database; see, for example, Vbase (http://vbase.mrc-cpe.cam.ac.uk/), hosted by MRC Center for Protein Engineering (Cambridge, UK). For example, amino acids considered to have the potential to deviate from the true germline sequence could be due to PCR primer sequences incorporated during the cloning process. Compared to artificially generated human-type antibodies, such as single-chain antibody fragments (scFvs) from a phage-display antibody library or xenogeneic mice, the human monoclonal antibody of the present disclosure is characterized by (i) being obtained using the human immune response rather than surrogate animals, i.e., the antibody was generated in response to natural tau in its relevant conformation in the human body, (ii) protected the individual or was at least significant for the presence of tau, and (iii) ) since the antibody is of human origin, the risks of cross-reactivity against self-antigens are minimized. Thus, in accordance with the disclosure, the terms "human monoclonal antibody," "human monoclonal autoantibody," "human antibody" and the like are used to denote a tau binding molecule that is of human origin, i.e., that has been isolated from a human cell such as a B cell or hybridoma thereof, or the cDNA from which it has been cloned directly from mRNA from a human cell, for example, a human memory B cell. A human antibody is still "human" even if amino acid substitutions are made to the antibody, for example to improve binding characteristics.

[0065] Antibodies derived from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and which do not express endogenous immunoglobulins, as described below and, for example, in US Patent 5,939,598 by Kucherlapati et al., human-like antibodies are denoted to distinguish them from the truly human antibodies of the present invention.

[0066] For example, the pairing of heavy and light chains of human-type antibodies such as synthetic and semi-synthetic antibodies usually isolated from phage display do not necessarily reflect the original pairing as it occurred in the original human B cell. Therefore, Fab and scFv fragments obtained from recombinant expression libraries, as they are commonly used in the prior art, can be considered artificial with all possible associated effects on immunogenicity and stability.

[0067] In contrast, the present invention provides affinity maturation antibodies isolated from selected human subjects, which are characterized by their therapeutic utility and their tolerability in man.

[0068] As used herein, the term "murinized antibody" or "murinized immunoglobulin" refers to an antibody comprising one or more CDRs of a human antibody of the present invention; and a human framework region that contains amino acid substitutions and/or deletions and/or insertions that are based on a mouse antibody sequence. The human immunoglobulin providing the CDRs is called the "parent" or "acceptor" and the mouse antibody providing the changes in structure is called the "donor". Constant regions need not be present, but if they are, they are generally substantially identical to mouse antibody constant regions, i.e., at least about 85-90%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical. Accordingly, in some embodiments, an entire murine human light or heavy chain immunoglobulin contains a mouse constant region, human CDRs, and a substantially human structure that has a number of "murinized" amino acid substitutions. Typically, a "murinized antibody" is an antibody comprising a murinized variable light chain and/or a murinized variable heavy chain. For example, a murinized antibody would not include a typical chimeric antibody, for example, because the entire variable region of a chimeric antibody is non-murine. A modified antibody that has been "murinized" by the "murinization" process binds to the same antigen as the parent antibody that provides the CDRs and is generally less immunogenic in mice 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 domain (e.g., upper, middle, and/or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment of the same. For example, a linker polypeptide for use in the invention can comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 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 polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a domain CH3. In another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. Furthermore, a linker polypeptide for use in the invention can be devoid of at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set out above, it will be understood by one skilled in the art that these domains (e.g., the heavy chain portions) can be modified in such a way that they vary in the amino acid sequence of the naturally occurring immunoglobulin molecule.

[0070] 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 of a second polypeptide chain of the multimer. Alternatively, monomers containing heavy chain portion of the invention are not identical. For example, each monomer can comprise a different target binding site, forming, for example, a bispecific antibody or diabody.

[0071] In another embodiment, the antibodies, or antigen-binding fragment, variants, or derivatives thereof disclosed herein are composed of a single polypeptide chain such as scFvs and are expressed intracellularly (intrabodies) for therapeutic and diagnostic applications in live potentials.

[0072] The heavy chain portions of a binding polypeptide for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide can comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

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

[0074] The minimum size of a peptide or polypeptide epitope for an antibody is thought to be approximately four to five amino acids. Peptide or polypeptide epitopes can contain at least seven, at least nine, or at least about 15 to 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope 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 recognized by antibodies of the present invention may contain 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 at least 15, at least 20, at least 25, or between about 5 to about 30, about 10 to about 30, or about 15 to about 30 contiguous or non-contiguous amino acids of tau.

[0075] "Specifically binding", or "specifically recognizing", used interchangeably herein, generally means that a binding molecule, for example an antibody binds to an epitope through its antigen-binding domain, and that binding implies a complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope through its antigen-binding domain more quickly than it would bind to a random, unrelated epitope. One skilled in the art understands that an antibody can specifically bind, or specifically recognize, an isolated polypeptide comprising, or consisting of, amino acid residues corresponding to a linear portion of a non-contiguous epitope. The term "specificity" is used here to qualify the relative affinity with which a given antibody binds to a given epitope. For example, the "A" antibody can be said to have a greater specificity for a certain epitope than the "B" antibody, or the "A" antibody can be said to bind the "C" epitope with a greater specificity. than it has for the related epitope "D".

[0076] When present, the term "immunological binding characteristics", or other binding characteristics of an antibody with an antigen, in all its grammatical forms, refers to specificity, affinity, cross-reactivity, and other binding characteristics of an antibody.

[0077] "Preferentially binds," means that the binding molecule, e.g., antibody, specifically binds to an epitope more quickly than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody that "preferentially binds" to a given epitope would be more likely to bind to that epitope than to a related epitope, even though that antibody might cross-react with the related epitope.

[0078] By way of non-limiting example, a binding molecule, for example an antibody can be considered to bind to a first epitope preferentially if it binds to said first epitope with a dissociation constant (KD) that is less than than the KD of the antibody for the second epitope. In another non-limiting example, an antibody can be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least an order of magnitude less than the antibody's KD 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 KD for the second epitope.

[0079] In another non-limiting example, a binding molecule, for example an antibody can be considered to bind a first epitope preferentially if it binds to the first epitope with an off-rate (k(off)) that is less than (k(off)) of the antibody 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 an order of magnitude less than the k(off) of the antibody 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 k(off) of the antibody for the second. epitope.

[0080] A binding molecule, for example, an antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind a tau or a fragment or variant thereof with an off-rate (k( off)) less than or equal to 5 x 10-2 s-1, 10-2 s-1, 5 x 10-3 s-1, or 10-3 s-1. In one embodiment, an antibody of the invention can be said to to bind to a tau or a fragment or variant thereof with an off-rate (k(off)) less than or equal to 5 x 10-4 s-1, 10-4 s-1, 5 x 10-5 s -1, or 10-5 s-1, 5 x 10-6 s-1, 10-6 s-1, 5 x 107 s-1 or 10-7 s-1.

[0081] 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 variant thereof with an association rate (k( on)) greater than or equal to 103 M-1 s-1, 5 x 103 M-1 s-1, 104 M-1 s-1, or 5 x 104 M-1 s-1. invention can be said to bind to tau or a fragment or variant thereof with an association rate (k(on)) greater than or equal to 105 M-1 s-1, 5 x 105 M-1 s-1, 106 M-1 s-1, 5 x 106 M-1 s-1 or 107 M-1 s-1.

[0082] A binding molecule, eg an antibody, is said to competitively inhibit the binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, the binding of the reference antibody with 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 epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%. One skilled in the art understands that the binding of an antibody to its epitope can also be competitively inhibited by a binding molecule that is not an antibody. For example, specific binding of an antibody described herein to tau, e.g., hTau40, can be competitively inhibited by microtubules.

[0083] As used herein, the term "affinity" refers to a measure of the strength of binding of an individual epitope with the CDR of an immunoglobulin molecule, e.g., an immunoglobulin molecule, see, e.g., Harlow et al. al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) on pages 27-28. As used herein, the term "avidity" refers to the general stability of the complex between a population of immunoglobulins and an antigen, ie, the functional combining strength of a mixture of immunoglobulin and antigen; see, for example, Harlow on pages 29-34. Avidity is related to the affinity of individual immunoglobulin molecules in the population for specific epitopes and also the valences of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repetitive epitope structure, such as a polymer, would be a high avidity one. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method; 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. Freeman and Company New York, N Y (1992) and methods described here General techniques for measuring the affinity of an antibody for an antigen include ELISA, RIA, and surface plasmon resonance. The measured affinity of a particular antibody-antigen interaction may vary if measured under different conditions, eg salt concentration, pH. Thus, measurements of affinity and other antigen binding parameters, eg KD, IC50, are preferably done with standardized antibody and antigen solutions, and a standardized buffer.

[0084] Binding molecules, for example antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of parentage 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 cross-reactive epitope usually contains many of the same complementary structural features as the inducing epitope, and in some cases may even fit better than the original.

[0085] For example, certain antibodies have some degree of cross-reactivity, where they bind to related but not identical epitopes, e.g., epitopes 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 an epitope of reference. An antibody can be said to have little or no cross-reactivity if it does not bind to epitopes 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 considered "highly specific" for a given epitope if it does not bind any other analogue, ortholog or homolog of that epitope.

[0086] Binding molecules, for example, antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their binding affinity for tau. In one embodiment, binding affinities can include those with a dissociation constant or Kd of less than 5 x 10-2 M, 10-2 M, 5 x 10-3 M, 10-3 M, 5 x 10-4M , 10-4 M, 5 x 10-5 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 -8M, 5x10-9M, 10-9M, 5x10-10M, 10-10M, 5x10-11M, 10-11M, 5x10-12M, 10-12M M, 5x10-13M, 10-13M, 5x10-14M, 10-14M, 5x10-15M, or 10-15M.

[0087] As previously indicated, the subunit structures and three-dimensional configuration of the constant regions of the various classes of immunoglobulins are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the term "CH1 domain" includes the first constant region domain (most amino terminal) 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.

[0088] As used herein, the term "CH2 domain" includes the portion of a heavy chain molecule that extends, for example, from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360 , Kabat numbering system; and residues 231-340, EU numbering system; see Kabat EA et al. op. cit) The CH2 domain is the only one that is not closely paired with another domain. In contrast, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.

[0089] As used herein, the term "hinge region" includes the portion of a heavy chain molecule 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 binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains; see Roux et al., J. Immunol. 161 (1998), 4083.

[0090] As used herein, the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group which can form a disulfide bond or bridge with a second thiol group. In naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bridges at positions corresponding to 239 and 242 using Kabat's numbering system (position 226 or 229, EU numbering system).

[0091] As used herein, the terms "attached", "fused" or "fusion" are used interchangeably. These terms refer to the joining of more than two elements or components by any means, including chemical conjugation or recombinant means. An "in-frame merger" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous larger ORF in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein that contains two or more segments that correspond to the polypeptides encoded by the original ORFs (which segments are not normally joined together in this way in nature). Although the reading frame is prepared continuous across the merged segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, while the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0092] The term "expression" as used herein refers to a process by which a gene produces a biochemical, eg, an RNA or polypeptide. The process includes any manifestation of the functional presence of the gene within the cell, including but not limited to gene silencing as well as transient expression and stable expression. This includes, without limitation, transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of such mRNA in polypeptides. If the final desired product is a biochemical, the expression includes the creation of the biochemical and any precursors. Expression of a gene produces a "gene product". As used herein, a gene product can be a nucleic acid, for example, messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript. Gene products described herein further include nucleic acids with post-transcriptional modifications, e.g. polyadenylation, or polypeptides with post-translational modifications, e.g. methylation, glycosylation, addition of lipids, association with other protein subunits, proteolytic cleavage, and others.

[0093] As used herein, the term "sample" refers to any biological material obtained from a subject or patient. In one aspect, a sample may comprise blood, cerebrospinal fluid ("CSF"), or urine. In other aspects, a sample can comprise whole blood, plasma, enriched B cells from blood samples, and cultured cells (e.g., B cells from a subject). A sample may also include a biopsy or tissue sample including neural tissue. In still other aspects, a sample can comprise whole cells and/or a lysate of the cells. Blood samples can be collected by methods known in the art. In one aspect, the pellet can be resuspended by stirring at 4°C in 200 µl buffer (20 mM Tris, pH 7.5, 0.5% Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1M NaCl, IX Sigma Protease Inhibitor, and IX Sigma Phosphatase Inhibitors 1 and 2). The suspension can be kept on ice for 20 minutes using intermittent agitation. After spinning at 15,000 x g for 5 minutes at about 4°C, aliquots of the supernatant can be stored at about -70°C.

[0094] As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the aim is to prevent or delay (lessen) an unwanted physiological change or disorder, such as the development of Parkinsonism. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, decreased extent of disease, stabilized disease state (i.e., not worsening), delayed or slowed disease progression, improved or palliated disease state, and remission (whether partial or total), detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival if not treated. Those in need of treatment include those already with the condition or disorder, as well as those likely to have the condition or disorder, or those in whom the onset of the condition or disorder must be prevented.

[0095] "Subject" or "individual" or "animal" or "patient" or "mammal" is intended to mean any subject, particularly a mammalian subject, for example a human patient, for whom the diagnosis, prognosis, prevention or therapy is desired.

II. antibodies

[0096] The present invention generally relates to human anti-tau antibodies and antigen-binding fragments thereof. In one embodiment, an antibody of the present invention demonstrates the immunological binding characteristics and/or biological properties as described for the antibodies illustrated in the Examples. In accordance with the present invention, human monoclonal antibodies specific for tau were cloned from a pool of healthy humans.

[0097] During experiments performed in accordance with the present invention, initial attempts failed to clone tau-specific antibodies, but almost always resulted in false-positive clones. In order to circumvent this problem, antibodies in conditioned media from human memory B cell cultures were screened in parallel for binding to recombinant tau protein, PHFTau extracted from AD brain, healthy control brain extracts, and bovine serum albumin (BSA). . Only B cell cultures that were positive for recombinant tau and/or PHFTau, but not for control brain extract or BSA were subjected to antibody cloning.

[0098] Initial attempts to isolate specific antibodies focused on pools of healthy human subjects with high plasma binding activity for tau, suggestive of elevated levels of tau antibodies circulating in the plasma. Unexpectedly, these attempts failed to produce tau-specific human memory B cells, and the antibodies described in the present invention were isolated from pools of healthy human subjects who were not prescreened for high tau plasma reactivity or had low tau plasma reactivity. tau.

[0099] Due to this measure, several antibodies can be isolated. The selected antibodies were further analyzed for light chain class and subclass determination. Relevant antibody messages selected from memory B cell cultures are then transcribed by RT-PCR, cloned and combined into expression vectors for recombinant production; see the attached Examples. Recombinant expression of human antibodies in HEK293 or CHO cells and subsequent characterization of their binding specificities for full-length tau, pathologically modified forms thereof on Western blotting, 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 pathologically modified forms of tau protein as distinct.

[00100] Thus, the present invention generally relates to an isolated naturally occurring human anti-tau monoclonal antibody and binding fragments, derivatives and variants thereof. In one embodiment of the invention, the antibody is capable of specifically binding full-length recombinant tau and/or the pathologically aggregated and/or phosphorylated form (PHFTau) isolated from AD brain under denaturing conditions in Western Blot.

[00101] In one embodiment, the present invention is directed to an anti-tau antibody, or antigen-binding fragment, variant or derivative thereof, where the antibody specifically binds to the same tau epitope as a reference antibody selected from the group consisting of NI-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, NI-105.14E2, NI-105.39E2, NI-105.19C6, or NI-105.9C4.

[00102] Additional human anti-tau antibodies are disclosed in US Patent Application Publication 2012/0087861, the contents of which are incorporated herein by reference in their entirety.

[00103] In one embodiment, an antibody described herein specifically binds to tau on an epitope comprising amino acid residues selected from the group consisting of: residues corresponding to 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, and hTau40 NO:6). In another embodiment, an antibody described herein specifically binds to tau on an epitope comprising amino acid residues corresponding to residues 37-55 and 387-406 of hTau40 (SEQ ID NO:6): In a specific embodiment, tau is hTau40 ( SEQ ID NO:6).

[00104] In one embodiment, an antibody 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 shown in Figure 4. In one embodiment, an antibody described herein competes with microtubules for specific binding of tau. In another embodiment, an antibody described herein reduced tau-associated microtubule binding affinity compared to non-microtubule-associated tau antibody binding affinities. In another embodiment, an antibody described herein does not bind, or substantially does not bind, microtubule-associated tau. In specific embodiments, the tau protein can be native tau protein or recombinant tau protein. In a specific embodiment, tau is hTau40.

[00105] In one embodiment, a human anti-tau antibody of the present invention can specifically bind pathologically aggregated tau and does not substantially bind tau in physiological form in brain tissue. Furthermore, a human anti-tau antibody of the present invention can be further characterized by its ability to recognize tau in the pre-tangled phase, in neurofibrillary tangles (NFT), neutropile threads and/or dystrophic neurites in the brain. Therefore, the present invention provides a set of human tau antibodies with binding specificities, which, therefore, are particularly useful for diagnostic and therapeutic purposes.

[00106] Additionally, or alternatively, an anti-tau antibody of the present invention preferentially recognizes pathologically aggregated tau rather than physiological forms, in particular when analyzed in accordance with Examples 4 and 18. Additionally, or alternatively, an anti-tau antibody of the present invention binds to disease causing human tau mutants, in particular those described in Example 4. In this context, binding specificities can be in the range of having half the maximum effective concentrations (EC50) of about 100 pM to 100 nM, or an EC50 of about 100 pM to 10nM for wild-type tau.

[00107] Therefore, an anti-tau antibody of the present invention preferentially binds to pathologically modified forms of tau in the brain, for example, pathological aggregates of tau, as exemplified by immunohistochemical staining described in Examples 4 and 18. In another embodiment , an anti-tau antibody of the present invention preferentially binds the recombinant and pathologically modified form of tau, as exemplified in Example 2 by Western Blot.

[00108] The present invention is also designed for an antibody, or antigen-binding fragment, variant or derivative thereof, where the antibody comprises an antigen-binding domain identical to that of an antibody selected from the group consisting of NI-105.17 C1, NI-105.17C1(N31Q), 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, NI-105.14E2, NI-105.39E2, NI-105.19C6, and NI-105.9C4.

[00109] The present invention also exemplifies several of these binding molecules, for example, antibodies and their binding fragments, which can be characterized by comprising in its variable region, for example, binding domain in at least one complementarity determining region (CDR) of the VH and/or VL variable region comprising any of the amino acid sequences shown 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 from the amino acid sequences above the VH and/or VL region as shown in Fig. 7. However, as discussed below, the person skilled in the art is well aware of the fact that additionally or alternatively CDRs can be used, which differ in their amino acid sequence from those set out in Fig. 7 by one, two, three or more amino acids in the 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 tau-specific antibodies.

[00110] In one embodiment, an antibody of the present invention comprises at least one CDR comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 79-168 and 224.

[00111] In one embodiment, an antibody 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.

[00112] In one embodiment, an antibody of the present invention comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences, respectively, SEQ ID NO: 79-84, 85-90 , 91-96, 97-102, 103-108, 109-114, 115-120, 121-126, 127-132, 133-138, 139-144, 145-150, 151156, 157-162, or 163- 168. In one embodiment, an antibody of the present invention comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences, respectively, SEQ ID NOs: 79, 80, 81, 224, 83, and 84.

[00113] In one embodiment, an antibody of the invention comprises one, two, or three VH CDRs comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 79-81, 85-87, 91-93 , 97-99, 103-105, 109-111, 115-117, 121123, 127-129, 133-135, 139-141, 145-147, 151-153, 157-159, and 163165.

[00114] In one embodiment, an antibody of the invention comprises a VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences, respectively, SEQ ID NO: 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 163-165.

[00115] In one embodiment, an antibody of the present invention comprises one, two, three VL CDRs comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 82-84, 88-90, 94-96 .

[00116] In one embodiment, an antibody of the invention comprises a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences, respectively, SEQ ID NO: 82-84, 88-90, 94-96, 100102, 106- 108, 112-114, 118-120, 124-126, 130-132, 136-138, 142-144, 148-150, 154-156, 160-162, or 166-168. In one embodiment, a VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences, respectively, SEQ ID NO: 83, 84, and 224.

[00117] According to one embodiment, an antibody of the invention comprises a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 79, 85, 91, 97, 103, 109, 115, 121, 127, 133, 139 , 145, 151, 157, or 163; the VH CDR2 of SEQ ID NO: 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; or a VH CDR3 of SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, or 165. In another embodiment, an antibody comprises a light chain variable region comprising a VL CDR1 of SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166 or 224; the 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, 120, 126, 132, 138, 144, 150, 156, 162, or 168. In another embodiment, the antibody comprises a variable region heavy chain comprising a VH CDR1 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, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; or a VH CDR3 of SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, or 165, and further comprises a light chain variable region comprising a VL CDR1 of SEQ ID NO: 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, or 224; the 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, 120, 126, 132, 138, 144, 150, 156, 162, or 168.

[00118] According to one embodiment, an antibody of the invention comprises a heavy chain variable region comprising a VH CDR1 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, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and a VH CDR3 of SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, or 165. In another embodiment, an antibody comprises a light chain variable region comprising a VL CDR1 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 variable region heavy chain comprising a VH CDR1 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, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, or 164; and a VH CDR3 of SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, or 165, and further comprises a light chain variable region comprising a VL CDR1 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.

[00119] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 79, a VH CDR2 of SEQ ID NO: 80, and VH CDR3 of SEQ ID NO: 81 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 82, a VL CDR2 of SEQ ID NO: 83, and a VL CDR3 of SEQ ID NO: 84.

[00120] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 79, a VH CDR2 of SEQ ID NO: 80, and VH CDR3 of SEQ ID NO: 81 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 224, a VL CDR2 of SEQ ID NO: 83, and a VL CDR3 of SEQ ID NO: 84.

[00121] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 85, a VH CDR2 of SEQ ID NO: 86, and VH CDR3 of SEQ ID NO: 87 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 88, a VL CDR2 of SEQ ID NO: 89, and a VL CDR3 of SEQ ID NO: 90.

[00122] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 91, a VH CDR2 of SEQ ID NO: 92, and VH CDR3 of SEQ ID NO: 93 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 94, a VL CDR2 of SEQ ID NO: 95, and a VL CDR3 of SEQ ID NO: 96.

[00123] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 97, a VH CDR2 of SEQ ID NO: 98, and VH CDR3 of SEQ ID NO: 99 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 100, a VL CDR2 of SEQ ID NO: 101, and a VL CDR3 of SEQ ID NO: 102.

[00124] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 103, a VH CDR2 of SEQ ID NO: 104, and VH CDR3 of SEQ ID NO: 105 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 106, a VL CDR2 of SEQ ID NO: 107, and a VL CDR3 of SEQ ID NO: 108.

[00125] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 109, a VH CDR2 of SEQ ID NO: 110, and VH CDR3 of SEQ ID NO: 111 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 112, a VL CDR2 of SEQ ID NO: 113, and a VL CDR3 of SEQ ID NO: 114.

[00126] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 115, a VH CDR2 of SEQ ID NO: 116, and VH CDR3 of SEQ ID NO: 117 and it may further comprise 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.

[00127] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 121, a VH CDR2 of SEQ ID NO: 122, and VH CDR3 of SEQ ID NO: 123 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 124, a VL CDR2 of SEQ ID NO: 125, and a VL CDR3 of SEQ ID NO: 126.

[00128] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 127, a VH CDR2 of SEQ ID NO: 128, and VH CDR3 of SEQ ID NO: 129 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 130, a VL CDR2 of SEQ ID NO: 131, and a VL CDR3 of SEQ ID NO: 132.

[00129] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 133, a VH CDR2 of SEQ ID NO: 134, and VH CDR3 of SEQ ID NO: 135 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 136, a VL CDR2 of SEQ ID NO: 137, and a VL CDR3 of SEQ ID NO: 138.

[00130] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 139, a VH CDR2 of SEQ ID NO: 140, and VH CDR3 of SEQ ID NO: 141 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 142, a VL CDR2 of SEQ ID NO: 143, and a VL CDR3 of SEQ ID NO: 144.

[00131] In one embodiment, an antibody of the invention may comprise a heavy chain variable region comprising a VH CDR1 of SEQ ID NO: 145, a VH CDR2 of SEQ ID NO: 146, and VH CDR3 of SEQ ID NO: 147 and it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 148, a VL CDR2 of SEQ ID NO: 149, and a VL CDR3 of SEQ ID NO: 150.

[00132] In one embodiment, an antibody of the invention may 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 it may 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.

[00133] In one embodiment, an antibody of the invention may 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 it may further comprise a light chain variable region comprising a VL CDR1 of SEQ ID NO: 160, a VL CDR2 of SEQ ID NO: 161, and a VL CDR3 of SEQ ID NO: 162.

[00134] In one embodiment, an antibody of the invention may comprise a heavy chain variable 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 it may further comprise a light chain variable region comprising 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.

[00135] In one embodiment, the antibody of the present invention is any of the antibodies comprising an amino acid sequence of the VH and/or VL region as shown in Fig. 7 and Table 3. In one embodiment, the antibody of the present invention is characterized by preserving the cognate pairing of the heavy and light chain as it was present in the human B cell. Table 3: Amino acid sequences of the VH, and VL region of tau-specific antibodies. BG - before germline

[00136] In one embodiment, an antibody of the present invention comprises a variable region (VH) heavy chain comprising or consisting 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 invention comprises a heavy chain variable region (VH) comprising, or consisting 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, and further comprises a light chain variable region (VL) comprising or consisting in 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 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 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.

[00137] Alternatively, the antibody of the present invention is an antibody or antigen-binding fragment, derivative or variant thereof, which is capable of binding to tau, such as, for example, hTau40, with at least one of the antibodies having the region VH and/or VL as shown 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-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, NI-105.14E2, NI-105.39E2, NI-105.19 C6 , or NI-105.9C4. Such antibodies can also be human, in particular for therapeutic applications. Alternatively, the antibody is a murine, murinized, and human-murine chimeric antibody, which are particularly useful for diagnostic methods and animal studies.

[00138] In one embodiment, the antibody of the present invention is provided by cultures of single or oligoclonal B cells that are grown and the culture supernatant, which contains antibodies produced by said B cells, is screened for the presence and affinity of anti-tau antibodies the same. The screening process comprises the steps of a tissue-sensitive amyloid plaque immunoreactivity assay (TAPIR), as described in international application WO2004/095031, the contents of the disclosure of which are incorporated herein by reference; screening brain sections for binding with PHFTau; screening for binding a tau-derived peptide of the amino acid sequence represented by SEQ ID NO:6 with phosphate groups on amino acids Ser-202 and Thr-205; on the amino acid Thr-231; and/or amino acids Ser-396 and Ser-404 of said sequence; screening for recombinant human tau binding of the amino acid sequence represented by SEQ ID NO:6 and isolating the antibody to which binding is detected or the cell producing said antibody.

[00139] As mentioned above, due to its generation after a human immune response the human monoclonal antibody of the present invention will recognize the epitopes that are of particular pathological relevance and that may 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. Therefore, it is prudent to stipulate that the epitope of the human anti-tau antibody of the present invention is unique and no other antibody that is capable of binding to the epitope recognized by the human monoclonal antibody of the present invention exists. Therefore, the present invention also extends generally to anti-tau antibodies and tau binding molecules that compete with the human monoclonal antibody of the present invention for specific binding of tau. The present invention is more specifically directed to an antibody, or antigen-binding fragment, variant or derivative thereof, where the antibody specifically binds to the same tau epitope as a reference antibody selected from the group consisting of NI-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-105.60E 7 , NI-105.14E2, NI-105.39E2, NI-105.19C6, and NI-105.9C4.

[00140] Competition between antibodies is determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen such as tau. Various types of competitive binding assays are known, for example: direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase 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), 3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid phase direct labeled assay, solid phase direct labeled sandwich assay; see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988); Solid phase direct labeled RIA using I125 marker; 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 containing these, an unlabeled 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 marker bound to the solid surface or cells in the presence of the test immunoglobulin. Usually, the test immunoglobulin is present in excess. In one embodiment, the competitive binding assay is performed under conditions as described for the ELISA assay in the accompanying Examples. Antibodies identified by the competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound to the reference antibody for steric hindrance to occur. Generally, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50% or 75%. Accordingly, the present invention is further envisioned for an antibody, or antigen-binding fragment, variant or derivative thereof, where the antibody competitively inhibits a reference antibody selected from the group consisting of NI-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, NI-105.14E2 , NI-105.39E2, NI-105.19C6, or NI-105.9C4 to bind tau.

[00141] In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of or consisting of an immunoglobulin heavy chain (VH) variable region, where at least one of the VH-CDRs of the heavy chain variable region or at least at least two of the heavy chain variable region VH-CDRs are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical for amino acid sequences VH-CDR1, VH-CDR2 or reference heavy chain VH-CDR3 of the antibodies disclosed herein. Alternatively, the VH-CDR1, VH-CDR2 and VH-CDR3 regions of the VH are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VH amino acid sequences Reference heavy chain -CDR1, VH-CDR2 and VH-CDR3 of the antibodies disclosed herein. Thus, according to this embodiment, a heavy chain variable region of the invention has VH-CDR1, VH-CDR2 and VH-CDR3 polypeptide sequences related to the groups shown in Fig. 7. While Fig. 7 shows defined VH-CDRs by the Kabat system, other CDR definitions, for example, VH-CDRs defined by the Chothia system, are also included in the present invention, and can be easily identified by a person skilled in the art using the data presented in Fig. 7. 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, 157, or 163; the amino acid sequence of the VH CDR2 reference 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 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.

[00142] In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain (VH) variable region wherein the VH-CDR1, VH-CDR2 and VH-CDR3 regions have polypeptide sequences that are identical to the VH-CDR1, VH-CDR2, and VH-CDR3 groups shown in Fig. 7. In one embodiment, the amino acid sequence of the VH CDR1 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 of the VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, or 165.

[00143] In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain (VH) variable region wherein the VH-CDR1, VH-CDR2 and VH-CDR3 regions have polypeptide sequences that are identical to the VH-CDR1, VH-CDR2, and VH-CDR3 groups shown in Fig. 7 except for one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions on either VH-CDR. In certain embodiments, amino acid substitutions are conservative. In one embodiment, the amino acid sequence of the VH CDR1 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 of the VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, or 165.

[00144] In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain (VL) variable region, where at least one of VL-CDRs of the light chain variable region or at least two of the light chain variable region VL-CDRs are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acid sequences VL-CDR1, VL- Reference light chain CDR2 or VL-CDR3 of antibodies disclosed herein. Alternatively, the VL-CDR1, VL-CDR2 and VL-CDR3 regions of the VL are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VL amino acid sequences Reference light chain -CDR1, VL-CDR2 and VL-CDR3 of the antibodies disclosed herein. Thus, according to this embodiment, a light chain variable region of the invention has VL-CDR1, VL-CDR2 and VL-CDR3 polypeptide sequences related to the polypeptides shown in Fig. 7. While Fig. 7 shows defined VL-CDRs by the Kabat system, other CDR definitions, for example VL-CDRs defined by the Chothia system, are also included in the present invention. In one embodiment, the amino acid sequence of the VL CDR1 reference 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 reference 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 reference is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, or 168.

[00145] In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain (VL) variable region wherein the VL-CDR1, VL-CDR2 and VL-CDR3 regions have polypeptide sequences that are identical to the VL-CDR1, VL-CDR2, and VL-CDR3 groups shown in Fig. 7. In one embodiment, the amino acid sequence of the VL CDR1 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.

[00146] In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain (VL) variable region wherein the VL-CDR1, VL-CDR2 and VL-CDR3 regions have polypeptide sequences that are identical to the VL-CDR1, 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 substitutions on any one VL-CDR. In certain embodiments, amino acid substitutions are conservative. In one embodiment, the VL CDR1 amino acid sequence 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.

[00147] In another embodiment, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain (VH) variable region that is identical to a reference heavy chain variable region shown in Fig. 7 and Table 3. In one embodiment, the reference heavy chain variable region amino acid 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.

[00148] In another embodiment, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain (VH) variable region having a polypeptide sequence that is identical to a heavy chain variable region sequence reference (VH) 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, amino acid substitutions are conservative. In one embodiment, the reference heavy chain variable region amino acid 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.

[00149] According to one embodiment, the invention provides an isolated polypeptide, comprising, consisting 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 (VL) variable region amino acid sequence of the antibodies disclosed herein. Thus, according to this embodiment, a light chain variable region of the invention has polypeptide sequences related to the light chain variable regions shown in Fig. 7 and Table 3. In one embodiment, the amino acid sequence in the light chain variable region reference (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.

[00150] In another embodiment, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain (VL) variable region that is identical to a reference light chain variable region shown in Fig. 7 and Table 3. In one embodiment, the amino acid sequence in 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.

[00151] In another embodiment, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain (VL) variable region having a polypeptide sequence that is identical to a light chain variable region sequence reference (VL) 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, amino acid substitutions are conservative. In one embodiment, the amino acid sequence in 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.

[00152] An immunoglobulin or its encoding cDNA can be further modified. Thus, in another embodiment, the method of the present invention comprises any of the steps of producing a chimeric antibody, murinized antibody, single chain antibody, Fab fragment, bispecific antibody, fusion antibody, labeled antibody, or an analogue of any one. of those. Corresponding methods are known to those skilled in the art and are described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor (1988). When derivatives of said antibodies are obtained through the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to the same epitope as that of any of the described antibodies. here (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 the international application WO89/09622. Methods for producing humanized antibodies are described in, for example, European application EP-A1 0 239 400 and international application WO90/07861. Another additional source of antibodies to be used in accordance with the present invention are so-called xenogeneic antibodies. The general principle for producing xenogeneic antibodies as human-like antibodies in mice is described in, for example, international applications WO91/10741, WO94/02602, WO96/34096 and WO 96/33735. As discussed above, the antibody of the invention can exist in a variety of forms in addition to complete antibodies; including, for example, Fv, Fab and F(ab)2, as well as single chains; see, for example, international application WO88/09344.

[00153] The antibodies of the present invention or their corresponding immunoglobulin chains can be further modified using known conventional techniques, for example, using deletions, insertions, substitutions, additions, and/or recombinations of amino acids and/or any other modifications known in the art alone or in combination. Methods for introducing such modifications to the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are known to those skilled in the art; see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Modifications of the antibody of the invention include chemical and/or enzymatic derivations of one or more constituent amino acids, including side chain modifications, structure modifications, and N- and C-terminal modifications, including acetylation, hydroxylation, methylation, amidation, and the linkage of carbohydrate or lipid fractions, cofactors and the like. Likewise, the present invention includes the production of chimeric proteins comprising the disclosed antibody or some fragment thereof at the amino terminus fused to a heterologous molecule as an immunostimulatory linker at the carboxyl terminus; see, for example, international application WO00/30680 for corresponding technical details.

[00154] Furthermore, the present invention includes peptides including those containing a linker molecule as described above, for example, containing the CDR3 region of the variable region of any of the mentioned antibodies, in particular, CDR3 of the heavy chain since it is frequently observed that the heavy chain CDR3 (HCDR3) is the region with the greatest degree of variability and a predominant participation in the antigen-antibody interaction. Such peptides can easily be synthesized or produced by recombinant means to produce a linker useful according to the invention. Such methods are well known to those skilled in the art. Peptides can be synthesized, for example, using automated peptide synthesizers that are commercially available. Peptides can also be produced by recombinant techniques, incorporating the DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide.

[00155] Therefore, the present invention relates to any binding molecule, for example, an antibody or binding fragment thereof, which is oriented towards the human anti-tau antibodies of the present invention and exhibits the mentioned properties, i.e. that specifically recognize tau. Such antibodies and binding molecules can be tested for their specificity and binding affinity by ELISA and Western Blot and immunohistochemistry as described herein, see, for example, the Examples. Furthermore, preliminary results of subsequent experiments performed in accordance with the present invention revealed that in one embodiment, the anti-human tau antibody of the present invention binds primarily to pathologically aggregated tau, resembling neurofibrillary tangles (NFT), of neuropile present in human brain sections of patients who suffered from Alzheimer's disease (AD) additionally. Thus, in a particular preferred embodiment of the present invention, the human antibody or binding fragment, derivative or variant thereof recognizes tau in sections of human AD brain.

[00156] As an alternative to obtaining immunoglobulins directly from cultured immortalized B cells or memory B cells, the immortalized cells can be used as a source of rearranged heavy chain and light chain locus for subsequent expression and/or genetic manipulation. Rearranged antibody genes can be reverse transcribed from appropriate mRNAs to produce cDNA. If desired, the heavy chain constant region can be replaced with one of a different isotype or deleted entirely. The variable regions can be linked to encode single chain Fv regions. Multiple Fv regions can be linked to confer the ability to bind to more than one target or combinations of chimeric heavy and light chains and can be employed. Once the genetic material is available, the design of analogues as described above that retain their ability to bind to the desired target is straightforward. Methods for cloning antibody variable regions and generating recombinant antibodies are known to one 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.

[00157] Once the appropriate genetic material is obtained and, if desired, modified to encode an analog, coding sequences, including those encoding, at a minimum, the light and heavy chain variable regions, can be inserted into expression systems contained in vectors that can be transfected into standard recombinant host cells. A variety of these host cells can be used; for efficient processing, however, mammalian cells can be considered. Typical mammalian cell lines useful for this purpose include, but are not limited to, CHO cells, HEK 293 cells, or NSO cells.

[00158] The production of the antibody or analogue is then carried out by culturing the modified recombinant host under appropriate culture conditions for the growth of host cells and the expression of the coding sequences. The antibodies are then recovered by isolating them from the culture. Expression systems are designed to include signal peptides so that the resulting antibodies are secreted into the medium; however, intracellular production is also possible.

[00159] In accordance with the above, the present invention also relates to a polynucleotide encoding the antibody or equivalent binding molecule of the present invention. In one embodiment, the polynucleotide encodes at least one variable region of an immunoglobulin chain of the above-described antibody. Usually, said variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the VH and/or VL variable region of said antibody.

[00160] The person skilled in the art will readily understand that the antibody variable domain having the variable domain described above can be used for the construction of other polypeptides or antibodies of desired specificity and biological function. Thus, the present invention also includes the polypeptides and antibodies comprising at least one CDR of the variable domain described above and which advantageously have substantially the same or similar binding properties as the antibody described in the appended examples. One skilled in the art knows that binding affinity can be enhanced by making amino acid substitutions in the CDRs or hypervariable loops (Chothia and Lesk, J. Mol. Biol 196 (1987), 901917) that partially overlap with the CDRs as defined by Kabbat; see, for example, Riechmann, et al, Nature 332 (1988), 323327. Thus, the present invention also relates to antibodies wherein one or more of the CDRs mentioned comprise one or more or no more than two amino acid substitutions. In one embodiment, the antibody of the invention comprises in one or both of its immunoglobulin chains two or all three CDRs from the variable regions as set out in Fig. 1.

[00161] The binding molecules, for example the antibodies, or antigen-binding fragment, variants or derivatives thereof of the invention, as known to those skilled in the art, may comprise a constant region that mediates one or more effector functions. For example, binding of complement component C1 to an antibody constant region can activate the complement system. Complement activation is important in the opsonization and lysis of pathogen cells. Complement activation also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. In addition, antibodies bind to receptors on various cells through the Fc region, with an Fc receptor site in the Fc region of the antibody binding to an Fc receptor (FcR) on a cell. There are several Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of antibodies to Fc receptors on cell surfaces elicits a number of different and important biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called cytotoxicity). dependent cell-mediated antibody, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[00162] Likewise, certain embodiments of the present invention include an antibody, or antigen-binding fragment, variant, or derivative thereof, wherein at least a fraction of one or more of the constant region domains has been deleted or altered another way to provide desired biochemical characteristics such as reduced effector functions, the ability not to covalently dimerize, increased ability to localize to the site of tau aggregation and deposition, reduced serum half-life, or increased serum half-life when compared to a whole, unaltered antibody of approximately the same immunogenicity. For example, certain antibodies for use in the diagnostic and treatment methods described herein are domain-deleted antibodies that comprise a polypeptide chain similar to an immunoglobulin heavy chain, but lacking at least a portion of one or more heavy chain domains. For example, in certain antibodies, an 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 described herein have a constant region, for example, an IgG heavy chain constant region, that is altered to eliminate glycosylation, referred to elsewhere herein as aglycosylated antibodies or "agly". Such "agly" antibodies can be prepared enzymatically as well as by engineering consensus glycosylation sites in the constant region. While not being bound by theory, it is believed that "agly" antibodies may have an improved safety and stability profile in vivo. Methods for producing aglycosylated antibodies having desired effector function are found, for example, in international application WO2005/018572, which is incorporated herein by reference in its entirety.

[00163] In certain antibodies, or antigen-binding fragment, variants or derivatives thereof described herein, the Fc portion may be mutated to decrease effector function using known techniques. For example, deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the modified circulating antibody, thereby increasing tau localization. In other cases, it may be that constant region modifications, consistent with this invention, moderate complement binding and thereby reduce the serum half-life and nonspecific association of a conjugated cytotoxin. Still other constant region modifications can be used to modify disulfide bonds or oligosaccharide moieties that allow for enhanced localization due to greater antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as localization, biodistribution and serum half-life of tau, can be easily measured and quantified using well-known immunological techniques without undue experimentation.

[00164] In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion may be mutated or exchanged for alternative protein sequences to increase cellular uptake of antibodies, by way of example, potentiating endocytosis mediated by antibody receptors via FCY, LRP, or Thy1 receptors or by 'SuperAntibody Technology', which is said to allow antibodies to be transported 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 receptors or bi- or multispecific antibodies with a specific sequence binding tau as well as a cell surface receptor can be designed. using known techniques.

[00165] In certain antibodies, or antigen-binding fragment, variants or derivatives thereof described herein, the Fc portion may be mutated or replaced with alternative protein sequences or the antibody may be chemically modified to enhance its penetration of the blood-brain barrier.

[00166] Modified forms of antibodies, or antigen-binding fragment, variants or derivatives thereof of the invention may be prepared from the full-length precursor or parental antibodies using techniques known in the art. Exemplary are discussed in more detail here. Antibodies, or antigen-binding fragment, variants or derivatives thereof of the invention can be prepared or manufactured using techniques which are known in the art. In certain embodiments, antibody molecules or fragments thereof are "recombinantly produced," that is, they are produced using recombinant DNA technology. Exemplary techniques for preparing antibody molecules or fragments thereof are discussed in more detail elsewhere herein.

[00167] Antibodies, or antigen-binding fragment, variants or derivatives thereof of the invention also include derivatives that are modified, for example, by covalently linking any type of molecule to the antibody such that covalent linkage does not prevent the antibody from bind specifically to its cognate epitope. For example, but not by way of limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, binding of a cellular ligand or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, the derivatives may contain one or more non-classical amino acids.

[00168] In particular embodiments, antibodies, or antigen-binding fragment, variants or derivatives thereof of the invention will not provoke a deleterious immune response in the animal to be treated, for example, in a human being. In certain embodiments, the binding molecules, e.g., antibodies, or antigen-binding fragments thereof of the invention are derived from a patient, e.g., a human patient, and are further used in the same species from which they are derived, e.g. , human, attenuating or minimizing the occurrence of deleterious immune responses.

[00169] Deimmunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term "deimmunization" includes altering an antibody to modify T cell epitopes; see, for example, international applications WO98/52976 and WO00/34317. For example, VH and VL sequences from the starting antibody are analyzed and a human T cell epitope "map" of each V region showing the location of the 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 identify alternative amino acid substitutions with a low risk of altering the activity of the final antibody. A range of alternative VH and VL sequences is designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, e.g. tau specific antibodies or immunospecific fragments thereof for use in diagnostic and treatment methods disclosed here, which are then tested for function. Typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified human V and C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for full-length antibody production. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.

[00170] Monoclonal antibodies can be prepared using a wide variety of known techniques, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas Elsevier, N.Y., 563-681 (1981), said references incorporated herein by reference in their entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced using hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryote, prokaryote, or phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced using hybridoma technology. In certain embodiments, antibodies of the present invention are derived from human B cells that have been immortalized through transformation with the Epstein-Barr virus, as described herein.

[00171] In the known process of hybridoma (Kohler et al., Nature 256 (1975), 495) relatively short-lived, or deadly, lymphocytes from a mammal, e.g., B cells, derived from a human subject as described herein , are fused with an immortal tumor cell line (e.g., a myeloma cell line), thereby producing hybrid cells, or "hybridomas," which are immortal and capable of producing the genetically encoded B-cell antibody. The resulting strains are separated into single genetic strains by selection, dilution and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies, which are homogeneous against a desired antigen and, in reference to their pure genetic ancestry, are termed "monoclonal".

[00172] The hybridoma cells thus prepared are propagated and grown in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Those skilled in the art will appreciate that these reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standard protocols are well established. Generally, the culture medium in which the hybridoma cells are growing is analyzed for the production of monoclonal antibodies against the desired antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) as described herein. Once hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, clones can be subcloned by limiting dilution procedures and grown by standard methods see, e.g., Goding, Monoclonal Antibodies: Principles and Practice , Academic Press, pp 59-103 (1986). It will also be appreciated that the monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid or serum by conventional purification procedures, such as, for example, protein A chromatography, hydroxylapatite, gel electrophoresis, dialysis or affinity chromatography. .

[00173] In another embodiment, lymphocytes can be selected by micromanipulation and the variable genes isolated. For example, peripheral blood mononuclear cells can be isolated from an immunized or naturally immune mammal, for example a human, and cultured for about 7 days in vitro. Cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated. Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay. Ig-producing B cells can be micromanipulated in a tube and the VH and VL genes can be amplified using, for example, RT-PCR. The VH and VL genes can be cloned into an antibody expression vector and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.

[00174] Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to those skilled in the art. These techniques are described in a variety of laboratory manuals and leading publications. In this regard, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is incorporated herein by reference in its entirety, including supplements.

[00175] Antibody fragments that recognize specific epitopes can be generated using known techniques. For example, Fab and F(ab')2 fragments can be produced by recombinant or proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). . F(ab')2 fragments contain the variable region, light chain constant region, and heavy chain CH1 domain. These fragments are sufficient for use, for example, in immunodiagnosis procedures involving the coupling of portions of immunospecific immunoglobulins for the detection of reagents such as radioisotopes.

[00176] Human antibodies, such as those described here, are particularly desirable for therapeutic use in human patients. The human antibodies of the present invention are isolated, for example, from healthy human subjects who, because of their age, are suspected to be at risk of developing a taupatic disorder, for example Alzheimer's disease, or a patient with the disorder, but with an exceptionally stable disease course. However, although it is prudent to expect that healthy and symptom-free elderly individuals, respectively, will more regularly develop protective anti-tau antibodies than younger individuals, the latter can also be used as a source for obtaining a human antibody. of the present invention. This is particularly true for younger patients who are predisposed to develop a familial form of a taupatic disease, but remain symptom-free as their immune systems function more efficiently than in older adults.

[00177] In another embodiment, an antibody of the invention comprises at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an antibody of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antibody of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an 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 antibody molecules. Exemplary antibody molecules comprising at least one CDR that can be included in the subject's antibodies are described herein.

[00178] Antibodies of the present invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques as described herein.

[00179] In one embodiment, an antibody, or antigen-binding fragment, variant or derivative thereof of the invention comprises a synthetic constant region in which one or more domains are partially or completely deleted ("domain-deleted antibodies"). In certain embodiments, compatible modified antibodies will comprise domain-deleted constructs or variants in which the entire CH2 domain has been removed (?CH2 constructs). For other embodiments, a short linker peptide can be substituted for the deleted domain to provide flexibility and freedom of movement for the variable region. Those of skill in the art will appreciate that such constructs may be particularly preferred due to the regulatory properties of the CH2 domain on the catabolic rate of the antibody. Domain-deleted constructs can be derived using a vector encoding a human IgG1 constant domain; see, for example, international applications WO02/060955 and WO02/096948A2. This vector is designed to delete the CH2 domain and provide a synthetic vector expressing a domain-deleted IgG1 constant region.

[00180] In certain embodiments, antibodies, or antigen-binding fragment, variants, or derivatives thereof of the present invention are minibodies. Minibodies can be prepared using methods described in the art, see, for example, US Patent 5,837,821 or International Application WO 94/09817.

[00181] In one embodiment, an antibody or antigen-binding fragment, variant, or derivative thereof of the invention comprises an immunoglobulin heavy chain having deletion or replacement of some or even a single amino acid, while allowing association between subunits monomeric. For example, single amino acid mutation in selected areas of the CH2 domain may be sufficient to substantially reduce Fc binding and thus increase tau localization. Likewise, it may be desirable to simply delete that part of one or more constant region domains that control the effector function (eg, 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's constant region domain intact. Furthermore, as alluded to above, the constant regions of the disclosed antibodies can be modified by mutating or substituting one or more amino acids that improve the profile of the resulting construct. In this regard, the activity provided by a conserved binding site (eg, Fc binding) can be disrupted while substantially maintaining the configuration and immunogenic profile of the modified antibody. Still other embodiments may comprise adding one or more amino acids to the constant region to enhance desirable characteristics such as effector function or provide increased binding for the cytotoxin or carbohydrate. In these embodiments it may be desirable to insert or reproduce specific sequences derived from selected constant region domains.

[00182] The present invention also provides antibodies comprising, consisting essentially of, or consisting of variants (including derivatives) of antibody molecules (e.g., VH regions and/or VL regions) described herein, wherein antibodies thereof bind immunospecifically to tau. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding an antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions. In one embodiment, 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 20 amino acid substitutions, less than 15 amino acid substitutions of amino acids, less than 10 amino acid substitutions, less than 5 amino acid substitutions, 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-CDR1, VH-CDR2, VH-CDR3, VL region, VL-CDR1, VL-CDR2, or VL-CDR3. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with another amino acid residue having a similarly charged side chain. Families of amino acid residues having similarly charged side chains have been defined in the art. These families 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. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be randomly introduced along all or part of the coding sequence, as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (e.g., ability to bind to tau).

[00183] For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. The introduced mutations can be silent or neutral missense mutations, for example, they have little or no effect on an antibody's ability to bind antigen, in fact, some of these mutations do not change the amino acid sequence in any way. These types of mutations can be useful for optimizing codon usage, or improving antibody production of a hybridoma. Codon-optimized coding regions encoding antibodies of the present invention are disclosed elsewhere herein. Alternatively, non-neutral missense mutations can alter an antibody's ability to bind antigen. The location of the most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in a CDR, although this is not an absolute requirement. One skilled in the art would be able to design and test mutant molecules with desired properties such as no change in antigen binding activity or change in binding activity (e.g., improvements in antigen binding activity or change in antibody specificity). After mutagenesis, the encoded protein can be routinely expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecifically bind to at least one tau epitope) can be determined using the techniques described here or routinely modifying known techniques.

[00184] Tau binding agents, for example but not limited to tau binding antibodies of the present invention can be characterized using any in vivo or in vitro models of neurodegenerative tauopathies. One skilled in the art readily understands that a tau binding agent (e.g., an antibody) of the invention can be characterized in a mouse model for neurodegenerative tauopathies. For example, among others, any of the following three different animal models for tauopathies can be used to characterize and validate the tau antibodies (and molecules with their binding specificities) of the present invention.

[00185] 1. TauP301L transgenic mice (strain 183): expressing human Tau40 with P301L mutation under the murine Thy1.2 promoter (The generation of these transgenic animals is described in Gotz et al., J. Biol. Chem. 276 (2001) , 529-534 and in international application WO 2003/017918, the contents of which are incorporated herein by reference)

[00186] 2. JNPL3 mice expressing the shorter 4R human tau isoform with P301L mutation under the murine PrP promoter (available from Taconic, Hudson, NY, USA).

[00187] 3. P301STau mice (PS19 strain) expressing human tau with P301S mutation under murine PrP promoter (available from Jackson Laboratory, Bar Harbor, Maine, USA).

[00188] An expert understands that an experimental model of neurodegenerative tauopathy can be used in a preventive setting or can be used in a therapeutic setting. In a preventive setting, dosing of animals begins prior to the onset of neurodegenerative tauopathies or symptoms thereof. In preventive settings, a tau binding 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, dosing of animals begins after the onset of neurodegenerative tauopathies or a symptom thereof. In therapeutic settings, a tau binding agent (e.g., antibody) of the invention is evaluated for its ability to treat, reduce, or ameliorate neurodegenerative tauopathies or symptoms thereof. Symptoms of neurodegenerative tauopathies include, but are not limited to, accumulation of pathological tau deposits, neurofibrillary tangles (NFT), hyperphosphorylated tau polypeptide, insoluble tau fractions in neurons, brain, spinal cord, cerebrospinal fluid or serum of the experimental subject. One skilled in the art understands that a positive preventive or therapeutic result in any animal model of neurodegenerative taupathy indicates that the particular tau binding agent (e.g., antibody) can be used for preventive or therapeutic purposes in a subject other than the target organism. experimental model, for example, can be used to treat neurodegenerative tauopathies in a human subject in need thereof.

[00189] In one embodiment, a tau binding agent (e.g., an antibody) of the invention can be administered to a mouse model of taupathy and corresponding control wild-type mice. The administered antibody can be a murine antibody of the present invention or a human-murine chimera of an antibody of the present invention. The tau binding agent (e.g., an antibody) may be administered by any means known in the art, for example, by intraperitoneal, intracranial, intramuscular, intravenous, subcutaneous, oral, and aerosol administration. Experimental animals may be administered one, two, three, four, five, or more doses of a tau binding agent (e.g., an antibody) or a control composition such as PBS. In one embodiment, experimental animals will be administered one or two doses of a tau binding agent (e.g., an antibody). See, for example, Example 9. In another embodiment, animals are chronically dosed with the tau binding agent (e.g., an antibody) over several weeks or months. See, for example, Example 10. A specialist can readily design a dosing schedule that fits the experimental purpose, e.g., dosing schedule for acute studies, dosing schedule for chronic studies, dosing schedule for toxicity studies, dosing schedule for of dosage for preventive or therapeutic studies. The presence of the tau binding agent (e.g., an antibody) in a particular tissue compartment of the experimental animals, e.g., inter alia, serum, blood, CSF, brain tissue, can be established using methods well known in the art. See, for example, Example 9 and 10. In one embodiment, a tau binding agent (e.g., an antibody) of the invention is capable of penetrating the blood brain barrier. One skilled in the art understands that by adjusting the dose of tau binding agent (e.g., an antibody) and the frequency of dosing, a desired concentration of tau binding agent (e.g., an antibody) can be maintained in experimental animals. . Any effect of a tau-binding agent (e.g., an antibody) of the present invention on models of taupathy can be assessed by comparing the level, biochemical characteristics, or distribution of tau in treated and control animals. In one example, neurofibrillary tangles (NFT) are examined using the Gallyas silver staining technique or by immunostaining with mouse monoclonal antibodies AT100 and AT180, which recognize pathologically phosphorylated tau in NFT. The number or frequency of Gallyas positive neurons and/or AT100, AT180 labeled neurons in the brain and spinal cord in antibody-treated mice and control animals can be determined to assess the effect of antibody treatment. In one embodiment, an antibody of the present invention is capable of reducing the level, amount or concentration of neurofibrillary tangles in the brain or spinal cord in an animal 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 positive Gallyas neurons 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. In another embodiment, an antibody of the present invention is capable of reducing the number or frequency of AT100 or AT180 antibody positive neurons 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. The effect of an antibody of the present invention can also be evaluated by analyzing the distribution and biochemical properties of tau after administration of the antibody. 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 capable of reducing the amount or concentration of insoluble 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. The insoluble tau fraction can be prepared as described, for example, in Example 10 or in Goedert M, Spillantini MG, Cairns NJ, Crowther RA. Neuron 8, 159 (1992). The amount of tau protein in a biological sample can be determined by any method known to one skilled in the art, for example as described in Example 10. In another embodiment, an antibody of the present invention is capable of reducing the amount or concentration of hyperphosphorylated 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. Hyperphosphorylated tau can be detected using antibodies specific for pathologically hyperphosphorylated forms of tau, such as AT100 or AT180. An antibody of the present invention can also alter, for example, reduce or increase the concentration of tau in the blood, serum or cerebrospinal fluid of an animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more. In one embodiment, the % reduction or increase is relative compared to the level, number, frequency, amount, or concentration that existed before treatment, or to the level, number, frequency, amount, or concentration that existed in an untreated subject. /treated with control.

[00190] In one embodiment, an antibody of the present invention can prevent or delay the onset of at least one symptom of a neurodegenerative tauopathy in a subject. In one embodiment, an antibody of the present invention can reduce or eliminate at least one symptom of a neurodegenerative disorder in a subject. The symptom may be the formation of pathological tau deposits, hyperphosphorylated tau deposits, insoluble tau deposits, neurofibrillary fibers, pre-tangled tau phosphorus aggregates, intraneuronal neurofibrillary tangles, or extraneuronal neurofibrillary tangles in a subject's brain or spinal cord. See, for example, Augustinack et al, Acta Neuropathol 103:26-35 (2002 ). The symptom can also be the presence, or elevated concentration or amount, of tau in serum, blood, urine or cerebrospinal fluid, where the amount of elevated concentration is compared to a healthy individual. The symptom may be a neurological symptom, for example, altered conditioned taste aversion, altered contextual fear conditioning, impaired memory, loss of motor function. In one embodiment, memory impairment is assessed using a two-test Y-maze task. In a specific embodiment, the two-test Y-maze task is performed substantially 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-test Y-maze task ratio is significantly greater in an antibody-treated subject than in a control subject. In a specific embodiment, the two-test 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-test Y-maze task ratio is at least about twice, three times, four times, five times, ten times, or twenty times greater. The present invention also provides a method of preventing or delaying the onset of at least one symptom of a neurodegenerative tauopathy in a subject in need thereof, comprising administering a therapeutically effective amount of a tau antibody described herein. The present invention also provides a method of reducing or eliminating at least one symptom of a neurodegenerative tauopathy in a subject in need thereof, comprising administering 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, a transgenic mouse. In one embodiment, the subject is a human being. III. Polynucleotides Encoding Antibodies

[00191] A polynucleotide encoding an antibody, or antigen-binding fragment, variant or derivative thereof may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding an antibody, or antigen-binding fragment, variant or derivative thereof may be composed of single-stranded or double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single-stranded RNA or double-stranded, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. Additionally, a polynucleotide encoding an antibody, or antigen-binding fragment, variant or derivative thereof may be composed of triple-stranded regions comprising RNA or DNA or RNA and DNA. A polynucleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof may also contain one or more modified bases or modified DNA or RNA structures for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms.

[00192] An isolated polynucleotide encoding an unnatural variant of an immunoglobulin-derived polypeptide (e.g., an immunoglobulin heavy chain portion or immunoglobulin light chain portion) can be created by introducing one or more substitutions, additions, or nucleotide deletions in the immunoglobulin nucleotide sequence 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.

[00193] As is known, RNA can be isolated from parent B cells, hybridoma 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 using standard techniques such as oligo dT cellulose chromatography. Proper techniques are familiar. In one embodiment, cDNAs encoding the light and heavy chains of the antibody can be prepared, simultaneously or separately, using reverse transcriptase and DNA polymerase, in accordance with known methods. PCR can be primed by consensus constant region primers or more specific primers based on heavy and light chain DNA and published amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones that encode antibody light and heavy chains. In this case, libraries can be selected by consensus primers or larger homologous probes, such as human constant region probes.

[00194] DNA, usually a plasmid DNA, can be isolated from cells using known techniques, restriction maps and sequenced in accordance with standard, known techniques set out in detail, for example, in the above references relating to recombinant DNA techniques. Of course, the DNA can be synthetic according to the invention at any point during the isolation process or further analysis.

[00195] In one embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain (VH) variable region, wherein at least one of the CDRs of the immunoglobulin variable region heavy chain or at least two of the heavy chain variable region VH-CDRs are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the VH-CDR1 amino acid sequences , heavy chain reference VH-CDR2, or VH-CDR3 of the antibodies disclosed herein. Alternatively, the VH-CDR1, VH-CDR2, or VH-CDR3 regions of the VH are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 of the reference heavy chains of antibodies disclosed herein. Thus, according to this embodiment, a heavy chain variable region of the invention has VH-CDR1, VH-CDR2, or VH-CDR3 polypeptide sequences related to the polypeptide sequences shown in Fig. 7. In one embodiment, the sequence of amino acid reference VH CDR1 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 reference 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 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.

[00196] In one embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), wherein the regions VH-CDR1, VH-CDR2 and VH-CDR3 have polypeptide sequences that are identical to the VH-CDR1, VH-CDR2 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, amino acid substitutions are conservative. In one embodiment, the amino acid sequence of the VH CDR1 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 of the VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, or 165.

[00197] In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain (VL) variable region, where at least one of the VL-CDRs of the region light chain variable region or at least two of the light chain variable region VL-CDRs are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VL amino acid sequences -CDR1, VL-CDR2, or light chain reference VL-CDR3 of the antibodies disclosed herein. Alternatively, the VL-CDR1, VL-CDR2, or VL-CDR3 regions of the VL are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences of Light chain reference VL-CDR1, VL-CDR2, and VL-CDR3 from the antibodies disclosed herein. Thus, according to this embodiment, a light chain variable region of the invention has VL-CDR1, VL-CDR2, or VL-CDR3 polypeptide sequences related to the polypeptide sequences shown in Fig. 7. In one embodiment, the sequence of amino acid reference VL CDR1 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 reference 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 reference is SEQ ID NO: 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, or 168.

[00198] In another 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 (VL) in which the regions VL-CDR1, VL-CDR2 and VL-CDR3 have polypeptide sequences that are identical to the VL-CDR1, 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 substitutions in any one VL-CDR. In certain embodiments, amino acid substitutions are conservative. In one embodiment, the VL CDR1 amino acid sequence 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.

[00199] In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) in which the regions VH-CDR1, VH-CDR2 , and VH-CDR3 have polypeptide sequences that are identical to the VH-CDR1, VH-CDR2, and VH-CDR3 groups shown in Fig. 7. In one embodiment, the amino acid sequence of the VH CDR1 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 of the VH CDR3 is SEQ ID NO: 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, or 165.

[00200] In another 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 (VL) in which the regions VL-CDR1, VL-CDR2 , and VL-CDR3 have polypeptide sequences that are identical to the VL-CDR1, VL-CDR2, and VL-CDR3 groups shown in Fig. 7. In one embodiment, the amino acid sequence of the VL CDR1 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.

[00201] As known in the art, "sequence identity" between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide. When discussed herein, whether any particular 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, Madison, WI 53711). BESTFIT 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 any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention, the parameters are of course defined such that the percentage of identity is calculated over the entire 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 sequence are allowed.

[00202] In one embodiment of the present invention, a polynucleotide comprises, 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 shown in Table 4. In this regard, a one skilled in the art will understand that polynucleotides encoding at least the light and/or heavy chain variable domain can encode the variable domain of both immunoglobulin chains or just one. Table 4: Nucleotide sequences of the VH and VL region of BG tau-specific antibodies - before germline

[00203] In one embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or 95% identical to reference VH heavy chains. In one embodiment, the reference heavy chain variable region amino acid 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.

[00204] 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%, 98% or 99% or 95% identical to reference VL light chains. In one embodiment, the amino acid sequence in 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.

[00205] The present invention also includes fragments of the polynucleotides of the invention, as described elsewhere. Additional polynucleotides encoding fusion polynucleotides, Fab fragments, and other derivatives, as described herein, are also contemplated by the invention.

[00206] The polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides, for example, as described in Kutmeier et al., BioTechniques 17 (1994), 242, which, briefly, involves synthesizing overlapping oligonucleotides that contain portions of the sequence encoding the antibody, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[00207] Alternatively, a polynucleotide encoding an antibody, or antigen-binding fragment, variant, or derivative thereof can be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (e.g., a library). of antibody cDNA, or a cDNA library generated from nucleic acid, preferably polyA+ RNA, isolated from any tissue or cells expressing the tau-specific antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using primers hybridisable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, for example, a cDNA clone from a cDNA library encoding the antibody. The PCR-generated amplified nucleic acids can then be cloned into replicable cloning vectors using any method known in the art.

[00208] Once the nucleotide sequence and corresponding amino acid sequence of the antibody, or antigen-binding fragment, variant or derivative thereof is determined, its nucleotide sequence can be manipulated using methods known in the art for sequence manipulation of nucleotides, for example, recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (See, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), the contents of which are incorporated herein by reference in their entireties), to generate antibodies with a different amino acid sequence, e.g., to create amino acid substitutions, deletions, and/or insertions . IV. Expression of Antibody Polypeptides

[00209] After manipulation of the isolated genetic material to provide antibodies, or antigen-binding fragment, variant, or derivative thereof of the invention, the polynucleotides encoding the antibodies are usually inserted into an expression vector for introduction into host cells that can be used to produce the desired amount of antibody. Recombinant expression of an antibody, or fragment, derivative or analogue thereof, for example, a light or heavy chain of an antibody which binds to a target molecule is described herein. Once a polynucleotide encoding an antibody molecule or an antibody light or heavy chain or portion thereof (preferably containing the heavy or light chain variable domain) of the invention has been obtained, the vector for producing the molecule antibodies can be produced by recombinant DNA technology using well known techniques. Thus, methods for preparing a protein by expressing a polynucleotide comprising an antibody-encoding nucleotide sequence are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors comprising antibody-encoding sequences and appropriate transcriptional and translational control signals. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, therefore, 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 sequence encoding the constant region of the antibody molecule (see, for example, international publication WO 86/05807 and WO 89/01036; and US Patent 5,122,464) and the variable domain of the antibody can be cloned into a vector for expression of the entire heavy or light chain.

[00210] 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 in the art, such vectors can readily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the present invention will comprise a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and/or replicate in prokaryotic or eukaryotic cells. For purposes of the present invention, numerous expression vector systems can be employed. For example, one class of vector utilizes DNA elements that are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells that have integrated the DNA into their chromosomes can be selected for by introducing one or more markers that allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (eg, antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can also be directly linked to DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be required for optimal mRNA synthesis. These elements can include signal sequences, recombination signals, as well as transcriptional promoters, enhancers and termination signals.

[00211] In particular embodiments, the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human heavy and light chain constant region genes), as discussed above. In one embodiment, this is accomplished using a proprietary expression vector from Biogen IDEC, Inc., also known as NEOSPLA, disclosed in US Patent 6,159,730. This vector contains the cytomegalovirus promoter/enhancer, mouse beta globin major promoter, SV40 origin of replication, bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, dihydrofolate gene and leader sequence reductase. This vector has been shown to result in very high level expression of antibodies upon incorporation of the variable and constant region genes, transfection into CHO cells, followed by selection in medium containing G418 and methotrexate amplification. Of course, any expression vector that is capable of eliciting expression in eukaryotic cells can be used in the present invention. Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, CA) and pCI plasmid (available from Promega, Madison, WI). In general, screening large numbers of transformed cells for those that express appropriately high levels of immunoglobulin light and heavy chains is routine experimentation that can be performed, for example, by robotic systems. Vector systems are also taught in US Patent Nos. 5,736,137 and 5,658,570, each of which is incorporated herein by reference in its entirety. This system provides high levels of expression, eg > 30 pg/cell/day. Other exemplary vector systems are disclosed, for example, in US Patent 6,413,777.

[00212] In other embodiments, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be expressed using polycistronic constructs such as those disclosed in patent application publication no. 2003-0157641 A1 and incorporated herein in their entirety. In these expression systems, various gene products of interest such as antibody heavy and light chains can be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to deliver relatively high levels of antibodies. Compatible IRES sequences are disclosed in US Patent 6,193,980, which is also incorporated herein. Those of skill in the art will appreciate that such expression systems can be used to effectively produce the full range of antibodies disclosed in the present application.

[00213] More generally, once the vector or DNA sequence encoding a monomeric subunit of the antibody has been prepared, the expression vector can be introduced into an appropriate host cell. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those skilled in the art. These include, but are not limited to, transfection, including lipotransfection using, for example, Fugene® or lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. Typically, the introduction of the plasmid into the host is via the calcium phosphate co-precipitation method. Host cells harboring the expression construct are grown under conditions appropriate for the production of light chains and heavy chains, and analyzed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[00214] The expression vector is transferred into a host cell by conventional 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 a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter. In particular embodiments for expressing double-chain antibodies, vectors encoding both the heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

[00215] The host cell can be cotransfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers that allow equal expression of heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. In these 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; Kohler, Proc. Natl. academic Sci. USA 77 (1980), 2197 . The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[00216] As used herein, "host cells" refers to cells that harbor vectors constructed using recombinant DNA techniques and that encode at least one heterologous gene. In descriptions of methods for isolating antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to designate the source of an antibody, unless otherwise clearly specified. In other words, recovery of polypeptide from "cells" can mean from whole cells spun down, or from cultured cells containing medium and suspended cells.

[00217] A variety of host expression vector systems can be used to express antibody molecules for use in the methods described herein. These host expression systems represent vehicles by which coding sequences of interest can be produced and further purified, but also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. . These include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody-encoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody-encoding sequences; insect cell systems infected with recombinant virus (e.g., baculovirus) expression vectors containing antibody-encoding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., plasmid Ti) containing antibody-encoding sequences; or mammalian cell systems (e.g., COS, CHO, NOS, BLK, 293, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or from mammalian viruses (eg, 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 expression of the whole recombinant antibody, are used for expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese Hamster Ovary (CHO) cells, together with a vector such as the human cytomegalovirus intermediate early gene promoter element is an effective expression system for antibodies; see, for example, Foecking et al., Gene 45 (1986), 101; Cockett et al., Bio/Technology 8 (1990), 2.

[00218] The host cell line used in protein expression is often of mammalian origin; those skilled in the art are credited with the ability to determine the specific host cell lines that are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (Human Cervical Carcinoma), CVI (Monkey Kidney Line), COS (a CVI derivative with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney strain), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes) and 293 (human kidney). In a specific embodiment, host cell lines are CHO or 293 cells. Host cell lines are commonly available and are commonly available from commercial services, the American Tissue Culture Collection, or from published literature.

[00219] Furthermore, a host cell strain can be chosen such that it modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific way desired. These modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important for protein function. Different host cells have specific characteristics and mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic cells which possess the cellular machinery for the proper handling of primary transcription, glycosylation and phosphorylation of the gene product can be used.

[00220] In the long term, the production of recombinant proteins with high yield, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be engineered. Rather than using expression vectors comprising 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 selection marker. After the introduction of foreign DNA, the engineered cells are allowed to grow for 1-2 days in an enriched medium and then placed in a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that, in turn, can be cloned and expanded into cell lines. This method can be advantageously used to design cell lines that stably express the antibody molecule.

[00221] A variety of selection systems can be used, including but not limited to 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 genes (Lowy et al., Cell 22 (1980), 817) can be employed in tk, hgprt, or aprt cells, respectively. Also, antimetabolic resistance can be used as a basis for 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); neo, which confers resistance to the aminoglycoside G-418 Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu, Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191217; TIB TECH 11 (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 that can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol., 150:1, (1981), which are incorporated herein by reference in their entireties.

[00222] The expression levels of an antibody molecule can be increased by vector amplification, for review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987). When a marker in the vector system expressing an antibody is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Once the amplified region is associated with the antibody gene, antibody production will also increase; see Crouse et al., Mol. Cell. Biol. 3 (1983), 257.

[00223] In vitro production allows scaling to generate a large amount of the desired polypeptides. Techniques for growing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, for example, in an air transport reactor or continuous stirring reactor, or immobilized or trapped cell culture, by for example, in hollow fibers, microcapsules, in agarose microspheres or ceramic cartridges. If necessary and/or desired, the polypeptide solutions can be purified by the usual chromatography methods, for example, gel filtration, ion exchange chromatography, chromatography on (immuno)affinity chromatography or DEAE-cellulose, for example, after preferential biosynthesis of a synthetic hinge region polypeptide either before or shortly after the HIC chromatographic step described herein.

[00224] Genes encoding antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be expressed in non-mammalian cells, such as bacterial or insect or yeast or plant cells. Bacteria that readily take up nucleic acids include members of the Enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillus, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. It will be further appreciated that, when expressed in bacteria, heterologous polypeptides normally become part of inclusion bodies. Heterologous polypeptides must be isolated, purified, and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits then self-assemble into tetravalent antibodies; see, for example, international application WO02/096948.

[00225] In bacterial systems, a number of expression vectors can be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when a large amount of such a protein must be produced, for the generation of pharmaceutical compositions from an antibody molecule, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, among others, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983), 1791), in which the antibody coding sequence can be individually linked to the vector in frame with the region lacZ coding so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989), 5503-5509); and others. pGEX vectors can also be used to express foreign polypeptides as glutathione S-transferase (GST) fusion proteins. In general, these fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding onto the glutathione-agarose granule matrix 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 product of the target gene can be released from the GST moiety.

[00226] In addition to prokaryotes, eukaryotic 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 strains are commonly available, for example 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 contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, e.g. ATCC No. 44076 or PEP4-1 (Jones, Genetics 85 (1977), 12). The presence of the trpl lesion as a feature of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[00227] In an insect system, the nucleopolyhedrovirus Autographa californica (AcNPV) is normally used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells The antibody coding sequence can be individually cloned into non-essential regions (eg, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (eg, the polyhedrin promoter).

[00228] Once an antibody of the invention has been expressed recombinantly, whole antibodies, their dimers, individual heavy and light chains, or other forms of immunoglobulin of the present invention, can be purified according to standard procedures in 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., ammonium sulfate precipitation, or by any other standard technique for purifying proteins; See, for example, Scopes, "Protein Purification", Springer Verlag, N.Y. (1982). Alternatively, another method for increasing the affinity of antibodies of the invention is disclosed in patent publication US 20020123057 A1. V. Fusion Proteins and Conjugates

[00229] In certain embodiments, the antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, a single-chain Fv antibody fragment of the invention may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a tag such as fluorescent, radioactive, enzyme , nuclear magnetic, heavy metal and the like)

[00230] An antibody polypeptide of the invention may comprise, consist essentially of, or consist of a fusion protein. Fusion proteins are chimeric molecules comprising, for example, an immunoglobulin tau binding domain with at least one target binding site, and at least one heterologous portion, i.e., a portion with which it is not naturally bound in nature. The amino acid sequences can normally exist in separate proteins that are joined together in the fusion polypeptide, or they can normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[00231] The term "heterologous" as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from an entity distinct from the rest of the entity to which it is being compared. For example, as used herein, a "heterologous polypeptide" to be fused to an antibody, or an antigen-binding fragment, variant, or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide. immunoglobulin from another species.

[00232] As discussed in more detail elsewhere herein, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may further be recombinantly fused to a heterologous polypeptide at the N or C terminus or chemically conjugated ( including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies can be recombinantly fused or conjugated to molecules useful as markers in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins; see, for example, International Applications WO92/08495; WO91/14438; WO89/12624; US patent 5,314,995; and European patent application EP 0 396 387.

[00233] Antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be composed of amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isoesters, and may contain amino acids other than those 20 amino acids encoded by the gene. Antibodies can be modified by natural processes, such as post-translational processing, or by known chemical modification techniques. These modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in the antibody, including the peptide backbone, amino acid side chains, and the amino or carboxyl terminus, or in moieties such as carbohydrates. It will be appreciated that the same type of modification can be present to varying or equal degrees at various sites in a given antibody. Furthermore, a given antibody may contain many types of modifications. Antibodies can be branched, for example as a result of ubiquitination, and can be cyclic, with or without branching. Cyclic, branched, and branched cyclic antibodies can result from natural post-translational processes or can be prepared 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, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, RNA transfer-mediated addition of amino acids to proteins such as arginylation and ubiquitination; see, for example, Proteins - Structure And Molecular Properties, T.E. Creighton, W.H. Freeman and Company, New York 2nd Ed., (1993); Posttranslational Covalent Modification Of Proteins, B.C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Academy Sci. 663 (1992), 48-62).

[00234] The present invention also provides fusion proteins comprising an antibody, or antigen-binding fragment, variant or derivatives thereof, and a heterologous polypeptide. In one embodiment, a fusion protein of the invention comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of one or more of the VH regions of an antibody of the invention or the amino acid sequence of one or more of the regions VL 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 treatment methods disclosed herein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one, two, three VH-CDRs of an antibody, or fragments , variants or derivatives thereof, or the amino acid sequence of any one, two, three VL-CDRs of an antibody, or fragments, variants, or 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 an antibody of the invention, or fragment, derivative or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds the tau. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of at least one VH region of an antibody of the invention and the amino acid sequence of at least one VL region of an antibody of the invention or fragments, derivatives or variants of the same, and a sequence heterologous to the polypeptide. In one embodiment, the VH and VL regions of the fusion protein correspond to a single-source antibody (or scFv or Fab fragment) that specifically binds tau. In yet another embodiment, a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or more VH CDRs of an antibody and the amino acid sequence of any one, two, three or more VL CDRs of an antibody, or fragments or variants thereof, and a heterologous polypeptide sequence. In one embodiment, two, three, four, five, six or more of the VH-CDRs or VL-CDRs correspond to a single-source antibody (or scFv or Fab fragment) of the invention. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.

[00235] Exemplary fusion proteins reported in the literature include T cell receptor fusions (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84 (1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531; Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA Cell Biol.USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990), 667-670); L-selectin (feedback 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 (1991), 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), 1483-1489 (1991) and IgE receptor (Ridgway and Gorman, J. Cell. Biol. 115 (1991), Abstract No. 1448).

[00236] 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 known methods in technique. For example, in one embodiment, PEG can be conjugated to antibodies of the invention to increase their in vivo half-lives; see, for example, Leong et al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

[00237] Furthermore, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be fused to marker sequences such as a peptide to facilitate purification or detection thereof. In particular embodiments, the amino acid sequence of the tag is a hexahistidine peptide (HIS), such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. academic Sci. USA 86 (1989), 821-824, for example, hexahistidine provides convenient purification 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.

[00238] 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 exact site at which the fusion is prepared can be selected empirically to optimize the secretion or binding characteristics of the fusion protein. The DNA encoding the fusion protein is then transfected into a host cell for expression.

[00239] The antibodies of the present invention can be used in unconjugated form or can be conjugated to at least one of a variety of molecules, for example, to enhance the therapeutic properties of the molecule, to facilitate the detection of targets, or for the patient imaging or therapy. Antibodies, or antigen-binding fragments, variants or derivatives thereof of the invention may be labeled or conjugated before or after purification, when purification is performed. In particular, antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be conjugated with therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

[00240] Conjugates which are immunotoxins including conventional antibodies have been widely described in the art. Toxins can be coupled to antibodies by conventional coupling techniques or immunotoxins containing protein toxin parts can be produced as fusion proteins. Antibodies of the present invention can be correspondingly used to obtain these immunotoxins. Illustrations of these immunotoxins are described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.

[00241] Those skilled in the art will appreciate that conjugates can also be assembled using a variety of techniques depending on the agent selected to be conjugated. For example, biotin conjugates are prepared, for example, by reacting a tau-binding polypeptide with an activated biotin ester, such as the N-hydroxysuccinimide ester of biotin. Likewise, conjugates with a fluorescent label can be prepared in the presence of a coupling agent, for example those listed here, or by reaction with an isothiocyanate, or fluorescein isothiocyanate. Conjugates of the antibodies, or antigen-binding fragments, variants or derivatives thereof of the invention are prepared analogously.

[00242] The present invention further includes antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention conjugated to a diagnostic or therapeutic agent. Antibodies can be used diagnostically to, for example, demonstrate the presence of a neurological disease, to indicate the risk of contracting a neurological disease, to monitor the development or progression of a neurological disease, i.e. taupathic disease as part of of a clinical test procedure to, for example, determine the effectiveness of a given treatment and/or prevention scheme. Detection can be facilitated by coupling the antibody, or antigen-binding fragment, variant or derivative thereof to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, metals emitting positrons using various positron emission tomography scans, and non-radioactive paramagnetic metal ions; see, for example, US Patent 4,741,900 for metal ions that can be conjugated to antibodies for diagnostic use in accordance with the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ß-galactosidase or acetylcholinesterase; examples of suitable complex prosthetic groups include streptavidin/biotin and 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 aqueorin, and examples of suitable radioactive materials include 125I, 131I, 111In or 99Tc.

[00243] An antibody, or antigen-binding fragment, variant or derivative thereof may also be marked for detection by coupling it to a chemiluminescent compound. The presence of the chemiluminescently labeled antibody is 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, isoluminol, theromatic acridinium ester, imidazole acridinium salt and oxalate ester.

[00244] One of the ways in which an antibody, or antigen-binding fragment, variant or derivative thereof can be detectably labeled is by linking it to an enzyme and using the bound product in an enzyme immunoassay (EIA) (Voller , A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol. 31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The enzyme, which is bound to the antibody, will react with an appropriate substrate, preferably a chromogenic substrate, in order to produce a chemical fraction that can be detected, for example, spectrophotometrically, fluorimetrically or visually. Enzymes that can be used to detectably label an antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase , alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, detection can be performed by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection can also be performed by visually comparing the extent of enzymatic reaction of a substrate compared to similarly prepared standards.

[00245] Detection can also be performed using any of a variety of other immunoassays. For example, by radiolabelling the antibody, or antigen-binding fragment, variant, or derivatives thereof, it is possible to detect the antibody through the use of a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is incorporated herein by reference in its entirety). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.

[00246] An antibody, or antigen-binding fragment, variant or derivative thereof may also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[00247] Techniques for conjugating various moieties to an antibody, or antigen-binding fragment, variant, or derivative thereof are known; see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting 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 Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc. ., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475 -506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use 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 Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol Rev. 62 (1982), 119-158.

[00248] As mentioned, in certain embodiments, a moiety that enhances the stability or effectiveness of a binding molecule, e.g., a binding polypeptide, e.g., an antibody or immunospecific fragment thereof, may be conjugated. For example, in one embodiment, PEG can be conjugated with the binding molecules of the invention to increase their in vivo half-life. Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512. VI. Compositions and Methods of Use

[00249] The present invention relates to compositions comprising the aforementioned tau binding molecule, for example, antibody or antigen-binding fragment thereof of the present invention or derivative or variant thereof, or the polynucleotide, vector or cell of the invention. The composition of the present invention can further comprise a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical composition of the present invention may comprise other agents such as interleukins or interferons, depending on the intended use of the pharmaceutical composition. For use in treating taupathic disease, e.g., Alzheimer's disease, the additional agent may be selected from the group consisting of small organic molecules, anti-tau antibodies, and combinations thereof. Therefore, a particular embodiment of the present invention relates to the use of the tau binding molecule, e.g., antibody or antigen-binding fragment thereof of the present invention, or a binding molecule having substantially the same binding specificities as any of the same, the polynucleotide, the vector or the cell of the present invention for the preparation of a pharmaceutical or diagnostic composition for prophylactic and therapeutic treatment of a taupathic disease, monitoring the progression of a taupathic disease or a response to a disease treatment taupathic disease in a subject or to determine a subject's risk of developing a taupathic disease.

[00250] Therefore, in one embodiment, the invention relates to a method for treating a neurological disorder characterized by abnormal accumulation and/or deposition of tau in the brain and central nervous system, respectively, which method comprises administering to a subject in need thereof a therapeutically effective amount of any of the above-described tau-binding molecules, antibodies, polynucleotides, vectors or cells of the present invention. The term "neurological disorder" includes, but is not limited to, taupathic diseases such as Alzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex, argyrophilic grains dementia, British-type amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease , dementia pugilistica, diffuse neurofibrillary tangle with calcification, Down syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, multiple systems atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease, post-encephalitis parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, multi-infarct dementia, and ischemic stroke. Unless otherwise indicated, the terms neurodegenerative, neurological, or neuropsychiatric are used interchangeably herein.

[00251] A particular advantage of the therapeutic approach of the present invention lies in the fact that the antibodies of the present invention are derived from B cells or memory B cells from healthy human subjects without signs of a taupatic disease and therefore are, with a with a certain probability capable of preventing clinically manifest disease, or decreasing the risk of occurrence of clinically manifest disease, or delaying the onset or progression of clinically manifest disease. Typically, antibodies of the invention have also successfully undergone somatic maturation, i.e. optimization with regard to selectivity and efficiency in high affinity binding to the target tau molecule through somatic variation of the variable regions of the antibody.

[00252] The knowledge that these cells in vivo, for example in humans, have not been activated by means of related physiological or other proteins or cellular structures towards an autoimmune or allergic reaction is also of great medical importance, since this means a considerably greater chance of successfully living through the clinical trial phases. So to speak, efficiency, acceptability and tolerability have already been demonstrated prior to the preclinical and clinical development of the prophylactic or therapeutic antibody in at least one human subject. Thus, the human anti-tau antibodies of the present invention, both their target-specific structure efficiency as a therapeutic agent and their decreased likelihood of side effects can be expected to significantly increase their clinical likelihood of success.

[00253] The present invention also provides a pharmaceutical and diagnostic package or kit respectively comprising one or more containers filled with one or more of the above-described ingredients, for example anti-tau antibody, binding fragment, derivative or variant of same, polynucleotide, vector or cell of the present invention. Associated with these containers may be a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biologics, where the notice reflects approval by the agency of manufacture, use, or sale for human administration. In addition or alternatively the kit comprises reagents and/or instructions for use in appropriate diagnostic assays. The composition, e.g. kit of the present invention is particularly suitable for risk assessment, diagnosis, prevention and treatment of a disorder that is accompanied with the presence of tau, and in particular applicable for the treatment of Alzheimer's disease (AD) , amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC), argyrophilic dementia of the grains (AGD), British-type amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration (CBD), Creutzfeldt-Jakob disease (CJD), dementia pugilistica , diffuse neurofibrillary tangle with calcification, Down syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, body of myositis inclusion, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C (NP-C), non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease (PiD), post-encephalitis parkinsonism, protein cerebral amyloid angiopathy prion, progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-only dementia, multi-infarct dementia, and ischemic stroke.

[00254] The pharmaceutical compositions of the present invention can be formulated according to methods known in the art; see, for example, Remington: The Science and Practice of Pharmacy (2000) by University of Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitable pharmaceutical carriers are known in the art and include phosphate buffer saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising these carriers can be formulated by known conventional methods. These pharmaceutical compositions can be administered to the subject in the proper dose. Administration of suitable compositions can be carried out in different ways, for example by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration or delivery to the spinal cord or brain. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. These formulations are adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Formulations for rectal or vaginal administration may be presented as a suppository with an appropriate carrier.

[00255] Furthermore, while 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 present invention, in one aspect, the binding molecule, especially the antibody or Antibody-based drug of the invention can cross the blood-brain barrier, which allows intravenous or oral administration.

[00256] The dosage schedule will be determined by the attending physician and clinical factors. As is known in the medical arts, dosages for any given patient will depend on many factors, including the patient's size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health and the like. drugs being administered simultaneously. A typical dose may be, for example, in the range 0.001 to 1000 µg (or of nucleic acid for expression or for inhibiting expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the dosage can range, for example, from about 0.0001 to 100 mg/kg, and more generally 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's body weight. Exemplary dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, or at least 1 mg/kg. Intermediate doses of the above ranges are also intended to be within the scope of the invention. Subjects may be administered these doses daily, on alternate days, weekly, or according to any other schedule determined by empirical analysis. Exemplary treatment involves administering multiple doses over a prolonged period, for example at least six months. Additional exemplary treatment regimens entail administering once every two weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg every other day, 30 mg/kg every other day, 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 indicated ranges. Progress can be monitored by periodic evaluation. Preparations for parental administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, aqueous/alcoholic solutions, emulsions or suspensions, including saline and buffered media. Parental vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include nutrient and fluid replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may further comprise other agents such as dopamine or psychopharmacological drugs, depending on the intended use of the pharmaceutical composition.

[00257] Furthermore, in a particular embodiment of the present invention, the pharmaceutical composition can be formulated as a vaccine, for example, if the pharmaceutical composition of the invention comprises an anti-tau antibody or binding fragment, derivative or variant thereof to passive immunization. As mentioned in the background section, phosphorus-tau species have been reported extracellularly in plasma and CSF (Aluise et al., Biochim. Biophys. Acta. 1782 (2008), 549-558) and studies in transgenic mouse lines using active vaccination with phosphorylated tau peptides revealed reduced brain levels of brain tau aggregates and delayed progression of behavioral impairment ( Sigurdsson, J. Alzheimers. Dis. 15 (2008), 157-168; Boimel et al., Exp Neurol. 224 (2010 ), 472-485). In this regard, it is prudent to expect that passive immunization with human anti-tau antibodies and equivalent tau binding molecules of the present invention would help to circumvent several adverse effects of active immunization therapy concepts as already discussed in the background section. Therefore, the present anti-tau antibodies and their equivalents of the present invention will be particularly useful as a vaccine for preventing or ameliorating taupathic diseases such as AD, ALS-PDC, AGD, CBD, CJD, FTD, FTDP-17, NP-C , PiD, PSP or other tauopathies as mentioned before.

[00258] In one embodiment, it may be beneficial to use recombinant bispecific or multispecific antibody constructs of the present invention. For a reference, see Fischer and Léger, Pathobiology 74 (2007), 3-14. Such a bispecific molecule can be designed to target tau with one binding arm and another pathological entity such as Aß or alpha-synuclein or a different pathological conformation of tau with a second binding arm. Alternatively, the second binding arm can be designed to target a protein present in the blood brain barrier to facilitate penetration of the antibody into the brain.

[00259] In one embodiment, it may be beneficial to use recombinant Fab (rFab) and single-chain fragments (scFvs) of the antibody of the present invention, which can more easily penetrate a cell membrane. For example, Robert et al., Protein Eng. Des. Sel. 2008) Oct 16; S1741-0134, published online, describes the use of recombinant chimeric Fab (rFab) and single chain fragments (scFvs) of WO-2 monoclonal antibody that recognizes an epitope in the N-terminal region of Aß. The engineered fragments were able to (i) prevent amyloid fibrillation, (ii) disrupt preformed Aß1-42 fibrils, and (iii) inhibit Aß1-42 oligomer-mediated neurotoxicity in vitro as efficiently as any whole IgG molecule. Perceived advantages using small Fab formats and scFv engineered antibodies devoid of effector function include more efficient passage across the blood-brain barrier and minimization of the risk of provoking inflammatory side reactions. Furthermore, in addition to scFv and single domain antibodies maintaining the binding specificity of whole antibodies, they can be expressed as single genes and intracellularly in mammalian cells such as intrabodies, with the potential to alter folding, interactions, modifications or subcellular location of their targets; see for review, eg, Miller and Messer, Molecular Therapy 12 (2005), 394-401.

[00260] In a different approach, Muller et al., Expert Opin. Biol. The R. (2005), 237-241, describes a technology platform, called 'SuperAntibody Technology', which allows antibodies to be carried in living cells without harming them. These cell-penetrating antibodies open new therapeutic and diagnostic windows. The term 'TransMabs' was coined for these antibodies.

[00261] In another embodiment, co-administration or sequential administration of other antibodies useful for treating a taupathic disease may be desirable. In one embodiment, the additional antibody is comprised of the pharmaceutical composition of the present invention. Examples of antibodies that can be used to treat a subject include, but are not limited to, antibodies directed at beta-amyloid, alpha-synuclein, TDP-43 and SOD-1.

[00262] In another embodiment, co-administration or sequential administration of other neuroprotective agents useful for treating a taupatic disease may be desirable. In one embodiment, the additional agent comprises the pharmaceutical composition of the present invention. Examples of neuroprotective agents that can be used to treat a subject include, but are not limited to, an acetylcholinesterase inhibitor, a glutamatergic receptor antagonist, kinase inhibitors, HDAC inhibitors, anti-inflammatory agents, divalproex sodium, or any combination thereof. Examples of other neuroprotective agents that can be used concomitantly with the pharmaceutical composition of the present invention are described in the art; see, for example, international publication WO2007/011907. In one embodiment, the additional agent is dopamine or a dopamine receptor agonist.

[00263] A therapeutically effective dose or amount refers to the amount of the active ingredient sufficient to improve the symptoms or condition. The therapeutic efficacy and toxicity of these compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, ED50 (the therapeutic dose effective in 50% of the population) and the LD50 (the lethal dose in 50% of the population) . The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. In one embodiment, the therapeutic agent in the composition is present in an amount sufficient to restore or preserve normal behavior and/or cognitive properties in the case of AD, ALS-PDC, AGD, CBD, CJD, FTD, FTDP-17, NP-C , PiD, PSP or other taupatic diseases as mentioned before.

[00264] From the foregoing, it is evident that the present invention includes any use of a tau binding molecule comprising at least one CDR of the antibody described above, in particular for diagnosing and/or treating a taupathic disease, as mentioned above, particularly disease of Alzheimer's. In one embodiment, said binding molecule is an antibody of the present invention or an immunoglobulin chain thereof. Furthermore, the present invention relates to anti-idiotypic antibodies of any of the mentioned antibodies described herein. These are antibodies or other binding molecules that bind to the unique antigenic peptide sequence located in the variable region of the antibody near the antigen-binding site and are useful, for example, for detecting anti-tau antibodies in a sample from a subject. .

[00265] In another embodiment, the present invention relates to a diagnostic composition comprising any of the tau binding molecules described above, antibodies, antigen binding fragments, polynucleotides, vectors or cells of the invention and optionally suitable means for detection , such as reagents conventionally used in diagnostic methods based on immunity or nucleic acid. Antibodies of the invention are, for example, suitable for use in immunoassays where they can be used in the liquid phase or linked to a solid phase carrier. Examples of immunoassays that can utilize the antibody of the invention are competitive and non-competitive immunoassays in a direct or indirect format. Examples of such immunoassays are radioimmunoassay (RIA), sandwich (immunometric assay), flow cytometry, and Western blot assay. Antigens and antibodies of the invention can be bound to many different carriers and used to isolate cells specifically bound thereto. Examples of known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified cellulose, polyacrylamides, agaroses and magnetite. The nature of the carrier can be soluble or insoluble for purposes of the invention. There are many different labels and methods for labeling known to those skilled in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds; see also the modalities discussed above.

[00266] Through another embodiment, the tau binding molecules, in particular antibodies of the present invention may also be used in a method of diagnosing a disorder in an individual by obtaining a sample of body fluid from the tested individual which may be a a blood sample, a lymph sample, or any other body fluid sample and contacting the body fluid sample with an antibody of the present invention under conditions that allow the formation of antigen-antibody complexes. The level of these complexes is determined by methods known in the art, a level significantly higher than that formed in a control sample indicating disease in the tested subject. Likewise, 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.

[00267] In this context, the present invention also relates to means designed specifically for this purpose. For example, an antibody-based matrix can be used which is loaded, for example, with antibodies or equivalent antigen-binding molecules of the present invention that specifically recognize tau. The design of microarray immunoassays is summarized in Kusnezow et al., Mol. Cell Proteomics 5 (2006), 1681-1696. In this regard, the present invention also relates to microarrays loaded with tau binding molecules identified in accordance with the present invention.

[00268] In one embodiment, the present invention relates to a method of diagnosing a taupathic disease in a subject, the method comprising determining the presence of tau and/or pathologically modified and/or aggregated tau in a sample of the subject to be diagnosed with at least one antibody of the present invention, a tau-binding fragment thereof, or a tau-binding molecule having substantially the same binding specificities as any of the same, wherein the presence of pathologically modified tau and/or or aggregated is indicative of a neurodegenerative tauopathy and an increase in the level of pathologically modified and/or aggregated tau compared to the level of physiological forms of tau is indicative of the progression of a neurodegenerative tauopathy in said subject.

[00269] The subject to be diagnosed may be asymptomatic or preclinical for the disease. In one embodiment, the control subject has a taupathic disease, e.g., AD, ALS-PDC, AGD, CBD, CJD, FTD, FTDP-17, NP-C, PiD, PSP or other tauopathies as mentioned before, wherein a similarity between the level of pathologically modified and/or aggregated tau and the reference standard indicates that the subject being diagnosed has a taupathic disease. Alternatively, or additionally as a second control, the control subject does not have a taupathic disease, where the difference between the level of tau and/or pathologically modified and/or aggregated tau and the reference standard indicates that the subject to be diagnosed have a taupatic disease. In one embodiment, the subject to be diagnosed and the control subjects are age matched. The sample to be analyzed can be any body fluid suspected of containing pathologically modified and/or aggregated tau, for example, a blood, CSF or urine sample.

[00270] The level of tau and/or pathologically modified and/or aggregated tau can be assessed by any appropriate method known in the art comprising, for example, analyzing tau by one or more chosen techniques of Western blot, immunoprecipitation, linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectroscopy (MS), matrix-assisted laser ionization/desorption time-of-flight (MALDI-TOF) , time-of-flight surface-enhanced laser ionization/desorption (SELDI-TOF), high performance liquid chromatography (HPLC), fast protein 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).

[00271] Methods for diagnosing a taupathic disease such as Alzheimer's disease, monitoring a taupathic disease progression, and monitoring a taupathic disease treatment using antibodies and related means that can be adapted in accordance with the present invention are also described in international applications WO93/08302, WO94/13795, WO95/17429, WO96/04309, WO2002/062851 and WO2004/016655. Likewise, antibody-based detection methods for tau are described in international application WO2005/080986, the contents of the disclosure all of which are incorporated herein by reference. These methods can be applied as described, but with a tau-specific antibody, binding fragment, derivative or variant of the present invention.

[00272] Another aspect of the present invention also relates to peptides having a tau epitope specifically recognized by any antibody of the present invention. In one embodiment, such a 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, 421427, 427-439, 1-158, 197-207, 57-67, 355-441, 313-319, 309-319, 221231 of SEQ ID NO:6, and any combination thereof, and a modified sequence wherein one, two, three, four, five, six, seven or more amino acids are substituted, deleted and/or added, whereby the peptide is recognized by any antibody of the present invention.

[00273] In one embodiment of this invention, this peptide can be used to diagnose a neurodegenerative tauopathy in a subject, comprising a step of determining the presence of an antibody that binds to a peptide in a biological sample of said subject, and being used for diagnosing a taupathy in said subject by measuring the levels of antibodies that recognize the above-described peptide of the present invention and comparing the measurements to the levels that are found in healthy subjects of comparable age and gender. A high level of measured antibodies specific for said peptide of the present invention would be indicative for the diagnosis of a tauopathy in said subject. The peptide of the present invention can be formulated into a matrix, kit and composition, respectively, as described above.

[00274] These and other embodiments are disclosed and included by the description and examples of the present invention. Additional literature on any of the materials, methods, uses and compounds to be employed in accordance with the invention may be obtained from public libraries and databases, using, for example, electronic 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. Other databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to those skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective research and current knowledge are given in Berks, TIBTECH 12 (1994), 352-364.

[00275] The above disclosure generally describes the present invention. Unless otherwise indicated, a term as used herein is given the definition given in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2. Various documents are cited throughout the text of this descriptive report. 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, granted patents, published patent applications, as cited throughout this application and manufacturer's specifications, instructions, etc.) are incorporated herein by reference in their entirety; however, there is no admission that any document cited is actually prior art concerning the invention.

[00276] A more complete understanding can be obtained with reference to the following specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLES

[00277] The examples that follow further illustrate the invention, but should not be construed to limit the scope of the invention in any way. The experiments in the examples below are illustrated and described with respect to NI-105.4E4, NI-105.24B2, and NI-105.4A3 antibodies as cloned, that is, the framework 1 Ig variable regions (FR1) without being adjusted for the germline (GL) sequences of human variable heavy and light chains; see Figure 1.

Materials and methods

[00278] Detailed descriptions of conventional methods such as those employed here can be found in the cited literature; see also "The Merck Manual of Diagnosis and Therapy" Seventeenth Ed. edited by Beers and Berkow (Merck & Co., Inc. 2003) and Patent Application Publication 2012/0087861, the contents of which are incorporated herein by reference in their entirety.

[00279] The practice of the present invention will employ, unless otherwise indicated, conventional techniques 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 techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews of cell biology and tissue culture; see also the references cited in the examples. General methods in molecular and cellular biochemistry can be found in such standard texts as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook 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); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription 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 Callos, eds.); Current Protocols in Molecular 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 Callos, 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 Molecular 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 Blackwell, 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 (Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and genetic manipulation kits referred to in this disclosure are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech. General techniques in cell culture and medium collection are described in Large Scale Mammalian Cell Culture ( Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148 ); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture of Mammalian Cells ( Birch et al., Bioprocess Technol. 19 (1990), 251 ); Extracting information from cDNA arrays, Herzel et al., CHAOS 11 (2001), 98-107.

Methods for identifying tau-specific B cells and cloning the respective antibodies

[00280] Unless otherwise indicated below, identification of tau-specific B cells and molecular cloning of anti-tau antibodies showing specificity of interest, as well as their recombinant expression and functional characterization was or may be performed as described in the Examples section and Complementary Methods of international application PCT/EP2008/000053 published as WO2008/081008, each of which are incorporated herein by reference in their entireties. See also US Patent Application Publication 2012/0087861, the contents of which are incorporated herein by reference in their entirety. A novel method for identifying tau-specific B cells and molecular cloning of tau antibodies having specificity of interest, as well as their recombinant expression and functional characterization is provided within this application. As described above, in one embodiment of the cultures of the present invention, single or oligoclonal B cells are cultured and the culture supernatant, which contains antibodies produced by said B cells, is screened for the presence and affinity of new anti-tau antibodies therein. The screening process comprises the steps of a sensitive tissue amyloid plaque immunoreactivity assay (TAPIR) as described in international application WO 2004/095031, the contents of the disclosure of which are incorporated herein by reference, and shown in Figure 3; screening brain extracts for binding to PHFTau as described in Example 2; screening for binding a tau-derived peptide of the amino acid sequence represented by SEQ ID NO:6 with phosphate groups on amino acids Ser-202 and Ser-205; on the amino acid Thr-231; and/or on amino acids Ser-396 and Ser-404 of said sequence as analogously described in Example 3 with non-phosphorylated peptides due to epitope confirmation experiments for antibody NI-105.4E4; a screening for full-length tau binding of the amino acid sequence represented by SEQ ID NO:6 and isolating the antibody for which binding is detected or the cell producing said antibody as described in international application WO2008/081008 and as described in Example 1.

Antigen purification

[00281] Recombinant human Tau40 was purchased from rPeptide (Bogart, GA, USA). PHFTau was extracted from AD brain.

[00282] Isolation of paired helical filaments containing pathologically phosphorylated tau (PHFTau) filaments was performed following the method by Goedert et al. (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 solution (100mM Tris pH 7.4, 6 mM EGTA, 1 mM Na3VO4 and 1 mM NaF) three times followed by homogenization with 20 ml lysis buffer (10mM Tris pH 7.4, 0.8M NaCl, 1mM EGTA, 1x Protease Inhibitor Cocktail, 1mM Na3VO4, 1mM NaF, 1mM AEBSF, 10% sucrose). The homogenate was centrifuged at 4°C at 20'000xg for 20 min. The supernatant was collected with the addition of N-lauroyl sarcosinate (Sigma, Switzerland) at 1% (w/v). After incubation for two hours at 37°C with shaking, the supernatant was then centrifuged at 4°C at 100'000xg for one hour. The pellet was collected and resuspended in PBS. After removing possible contaminating immunoglobulins with protein A magnetic granules, the PHFTau suspension was stored at -80°C before use. A control extract of healthy control human brain tissue was prepared accordingly.

Human tau antibody screening ELISA:

[00283] Half-area 96-well microplates (Corning) were coated with recombinant Tau protein (rPeptide, Bogart, USA) at a standard concentration of 1 µg/ml in carbonate ELISA coating buffer (pH 9.6) for overnight at 4°C. For auPHFT screening, Immobilizer 96-well microplates (Nunc, Denmark) were coated with auPHFT extracted from AD human 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 2hr in RT with PBS-T containing 2% BSA (Sigma, Buchs, Switzerland). B cell conditioned medium was transferred from B cell memory culture plates to ELISA plates and incubated for 1hr at RT. ELISA plates were washed in PBS-T and then incubated with horseradish peroxidase (HRP) conjugated donkey anti-human IgG (FCY fragment specific) polyclonal antibodies (Jackson ImmunoResearch, Newmarket, UK). After washing with PBS-T, binding of human antibodies was determined by measuring HRP activity in a standard colorimetric assay.

Screening on MULTI-ARRAY® microplates

[00284] Standard 96-well 10-spot MULTI-SPOT plates (Meso Scale Discovery, USA) were coated with 30 µg/ml rTau (rPeptide), PHFTau brain extract and healthy control brain extract in PBS. Non-specific binding sites were blocked for 1hr at RT with PBS-T containing 3% BSA followed by incubation with B cell conditioned medium for 1hr at RT. Plates were washed in PBS-T and then incubated with polyclonal anti-human antibody conjugated with SULFO-Tag (Meso Scale Discovery, USA). After washing with PBS-T, bound antibodies were detected by measuring electrochemiluminescence using a SECTOR Imager 6000 (Meso Scale Discovery, USA).

Molecular cloning of tau antibodies

[00285] Samples containing memory B cells were obtained from healthy humans. Live B cells from selected memory B cell cultures are harvested and mRNA is prepared. Immunoglobulin heavy and light chain sequences were then obtained using a nested PCR approach.

[00286] A combination of primers representing all families of the human immunoglobulin germline repertoire is used for leader peptide, V-segment, and J-segment amplifications. The first round of amplification is performed using leader peptide-specific primers at the 5-end ' and constant region specific primers at the 3' end ( Smith et al., Nat Protoc. 4 (2009), 372-384 ). For kappa heavy chains and light chains, the second round of amplification is performed using V segment specific primers at the 5' end and J segment specific primers at the 3' end. For lambda light chains, the second round of amplification is performed using V segment specific primers at the 5' end and C region specific primers at the 3' end ( Marks et al., Mol. Biol. 222 (1991), 581-597 de Haard et al., J. Biol. Chem. 26 (1999), 18218-18230).

[00287] Identification of the antibody clone with the desired specificity is performed by another screening in ELISA on recombinant expression of complete antibodies. Recombinant expression of full-length human IgG1 antibodies or chimeric IgG2a antibodies is achieved after insertion of the variable heavy and light chain sequences "in correct reading frame" into expression vectors that complement 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 domains. At that end, the primers contained restriction sites designed to facilitate cloning of the variable light and heavy chain sequences into antibody expression vectors. Heavy chain immunoglobulins are expressed by inserting the immunoglobulin heavy chain RT-PCR product in frame into a heavy chain expression vector having a signal peptide and the constant 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 inserting the lambda light chain RT-PCR product in frame into a lambda light chain expression vector providing a signal peptide and the constant domain of human or mouse lambda light chain immunoglobulin.

[00288] Functional recombinant monoclonal antibodies are obtained by co-transfecting into HEK293 or CHO cells (or any other appropriate target cell line of human or mouse origin) an Ig heavy chain expression vector and a light chain expression vector of Ig kappa or lambda. Recombinant human monoclonal antibody is further purified from the conditioned medium using a standard protein A column purification. Recombinant human monoclonal antibody can be produced in unlimited quantities using transiently or stably transfected cells. Cell lines that produce recombinant human monoclonal antibody can be established using the Ig expression vectors directly or by recloning 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

[00289] Mouse monoclonal anti-human tau antibody Tau12 (Covance, California, USA.) and mouse monoclonal tau antibody AT180 (Thermo Scientific, USA.) were used according to the manufacturer's protocol. Recombinant human tau antibodies NI-105.4E4, NI-105.24B2 and NI-105.4A3 are described in US Patent Application Publication 2012/0087861, the contents of which are incorporated herein by reference in their entirety. 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, NI-105.22 E1, NI-105.26B12, NI-105.12E12, NI-105.60E7, NI-105.14E2, NI-105.39E2, NI-105.19C6, and NI-105.9C4 are antibodies of this invention. They were expressed in HEK293 or CHO cells, purified from conditioned media and directly used in subsequent applications, unless otherwise indicated.

direct ELISA

[00290] 96-well microplates (Costar, Corning, USA) were coated with recombinant Tau protein (hTau40, rPeptide, Bogart, USA) diluted at a concentration of 1 µg/ml in carbonate ELISA coating buffer (50mM, pH9 .6) at 4°C overnight. Non-specific binding sites were blocked for 2hr. in RT with PBS containing 2% BSA (Sigma, Buchs, Switzerland) and 0.5% Tween20. Binding of human antibodies of the present invention was determined using HRP-conjugated goat anti-human IgG FCY (Jackson immunoResearch, Newmarket, UK), followed by measurement of HRP activity in a standard colorimetric assay. EC50 values were estimated by non-linear regression using GraphPad Prism software (San Diego, USA).

Western blotting protein staining

[00291] PHFTau and recombinant hTau40 were resolved by gradient SDS-PAGE (NuPAGE 4-12%; Invitrogen, Basel, Switzerland) followed by electroblotting on nitrocellulose membranes. After blocking non-specific binding with 2% BSA at room temperature for one hour, the blots were incubated overnight with primary human anti-tau antibodies or Tau12 (mouse monoclonal antibody, Covance, California, USA.), followed by by an HRP-conjugated goat anti-human IgGFcY (for human primary antibodies) or an HRP-conjugated goat anti-mouse IgG secondary antibody.

[00292] Blots were developed using ECL and detection by ImageQuant 350 (GE Healthcare, Otelfingen, Switzerland).

AD brain PHFTau extraction

[00293] The isolation of paired helical filaments containing pathologically phosphorylated tau (PHFTau) filaments was performed following the method by Goedert et al. (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 solution (100mM Tris pH 7.4, 6 mM EGTA, 1 mM Na3VO4 and 1 mM NaF) three times followed by homogenization with 20 ml lysis buffer (10mM Tris pH 7.4, 0.8M NaCl, 1mM EGTA, 1x Protease Inhibitor Cocktail, 1mM Na3VO4, 1mM NaF, 1mM AEBSF, 10% sucrose). The homogenate was centrifuged at 4°C at 20'000xg for 20 min. The supernatant was collected with the addition of N-lauroyl sarcosinate (Sigma, Switzerland) at 1% (w/v). After incubation for two hours at 37°C with shaking, the supernatant was then centrifuged at 4°C at 100'000xg for one hour. The pellet was collected and resuspended in PBS. After removing possible contaminating immunoglobulins with protein A magnetic granules, the PHFTau suspension was stored at -80°C before use. A control extract of healthy control human brain tissue was prepared accordingly. Synthesis of tau peptides

[00294] A peptide corresponding to amino acids 333-346 of hTau40 (333GGGQVEVKSEKLDF346) that includes the NI-105.4E4 epitope identified by Pepspot mapping (amino acids 337-343) was synthesized by Schafer-N (Copenhagen, Denmark). An additional cysteine was added to the C-terminus to allow covalent attachment to Immobilizer Microplates (Nunc, Denmark). A second peptide corresponding to amino acids 226-239 of human tau (226VAVVRpTPPKSPSSA239), the cognate epitope of the commercially available mouse monoclonal tau antibody AT180 (Thermo Scientific, USA) was synthesized accordingly and used as a control.

transgenic mice

[00295] Three different animal models for tauopathies are used to validate the tau antibodies (and molecules with the binding specificities thereof) of the present invention.

[00296] 1. TauP301L transgenic mice (strain 183): expressing human Tau40 with P301L mutation under the murine Thy1.2 promoter (The generation of these transgenic animals is described in Gotz et al., J. Biol. Chem. 276 (2001), 529-534 and in international application WO 2003/017918, the contents of which are incorporated herein by reference)

[00297] 2. JNPL3 mice expressing the shorter 4R human tau isoform with P301L mutation under the murine PrP promoter (available from Taconic, Hudson, NY, USA).

[00298] 3. P301STau mice (PS19 strain) expressing human tau with P301S mutation under murine PrP promoter (available from Jackson Laboratory, Bar Harbor, Maine, USA).

[00299] Mouse models of tauopathies and corresponding wild-type mice are maintained under standard housing conditions on a 12h:12h inverted light/dark cycle with free access to food and water. Treatment groups are balanced for age and sex.

Example 1 Validation of target specificity and binding of human tau antibodies

[00300] To validate tau as a recognized target of isolated antibodies, direct ELISA assays were performed as described above. 96-well microplates of exemplary recombinant human NI-105.4A3 antibody (Costar, Corning, USA) were coated with recombinant human tau (hTau40, rPeptide, Bogart, USA) diluted to a concentration of 3 µg/ml or with BSA in carbonate ELISA coating buffer (pH 9.6) and antibody binding efficiency was tested. Exemplary NI-105.4A3 antibody specifically bound human tau by ELISA. No binding was observed for BSA.

[00301] For the determination of the half maximum effective concentration (EC50) of the exemplary antibodies NI-105.4E4 and NI-105.24B2, additional direct ELISA experiments with different concentrations of antibodies were performed. 96-well microplates (Costar, Corning, USA) were coated with recombinant human tau (hTau40, rPeptide, Bogart, USA) diluted to a concentration of 1µg/ml (for the NI-105.4E4 antibody assay), or 3µg/ ml (for NI-105.24B2 antibody assay) in carbonate ELISA coating buffer and antibody binding efficiency was tested. EC50 values were estimated by non-linear regression using GraphPad Prism software. Recombinant human-derived antibody NI-105.4E4 bound to hTau40 with high affinity in the low nanomolar range at 2.4 nM EC50. NI-105.24B2 bound to hTau40 with high affinity in the low nanomolar range at 6.6 nM EC50.

[00302] The half maximum effective concentration (EC50) of the exemplary antibody NI-105.4A3 was also determined using direct ELISA experiments. ELISA plates were coated with recombinant human tau (hTau40, 1ug/ml), PHFTau (1:100) and control preparation (1:100), and incubated with different concentrations of antibodies. NI-105.4A3 bound to rTau with high affinity in the low nanomolar range at 1.4 nM EC50. NI-105.4A3 bound to PHFTau with high affinity in the low nanomolar range at 1.2 nM EC50.

Example 2 Binding analysis of recombinant human antibodies to recombinant tau and pathological tau extracted from AD brain

[00303] To determine the binding capacity of NI-105.4E4 and NI-105.24B2 to pathological tau species extracted from brain AD. SDS-PAGE and Western 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 antibody, Covance, California, USA.), followed by an anti-human IgGFcY of HRP-conjugated goat (for human antibodies) or an HRP-conjugated goat anti-mouse IgG secondary antibody.

[00304] Recombinant antibodies NI-105.4E4 and NI-105.24B2 recognized recombinant hTau40 as well as pathologically modified PHFTau extracted from AD brain in Western blot. The Tau12 control antibody recognized both species of tau as well.

[00305] Additionally, as discussed in Example 1 above, the half maximum effective concentration (EC50) of the exemplary antibody NI-105.4A3 was determined using direct ELISA experiments. NI-105. 4A3 bound to PHFTau with high affinity in the low nanomolar range at 1.2 nM EC50.

Example 3 Mapping of the NI-105.4E4 and NI-105.4A3 binding epitope on hTau40

[00306] A peptide array of 118 peptide sequences covering the entire hTau40 (amino acids 1-441) with an overlap of 11 amino acids between two adjacent peptides was dripped onto a nitrocellulose membrane (JPT Peptide Technologies GmbH, Berlin, Germany). Antibody immunostaining as well as membrane regeneration was performed according to the manufacturer's instructions. To rule out nonspecific detection antibody binding, 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).

[00307] Two groups of adjacent peptide spots (peptide 83, 84 and 85; peptide 96 and 97) were specifically identified by NI105.4E4 when compared with 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 NI-105.4E4 recognized a discontinuous epitope comprising two linear sequences: one within the R4 microtubule binding domain and the other within the C-terminal domain.

[00308] The sequence shared by peptides 83-85 comprises amino acid residues 337-343 of hTau40. The Pepspot (JPT) data suggested that NI-105.4E4 recognized an epitope on hTau comprising amino acids 337-343 of human tau. This region lies within the microtubule binding domain of tau and is conserved among all neuronal human tau isoforms, as well as in other species including mouse and rat.

[00309] As this domain is linked to microtubules in physiological microtubule-associated tau, NI-105.4E4 is expected to preferentially target the pathologically relevant pool of tau that is taken from microtubules.

[00310] To determine the major residues within the NI-105.4E4 binding peptides, alanine scanning was performed to replace each residue with alanine one at a time. Alanine residues in the original sequence (A384 and A390) were substituted for proline and glycine. Points 35-50 and 51-68 are the original peptides (point 35 and point 51) and their substituted alanine variants. Alanine scanning suggested that V339, E342, D387, E391 and K395 were required for NI-105.4E4 binding.

[00311] An additional experiment was performed testing the binding of NI-105.4E4 to tau peptides. Direct ELISA showed that NI-105.4E4 specifically recognized a peptide corresponding to amino acid 333-346 of hTau40, which contains amino acid residues 337-343 identified by Pepspot mapping. No NI-105.4E4 cross-reactivity was observed for the control peptide covering the AT180 epitope. Vice versa, AT180 recognized its cognate epitope containing peptides, but failed to bind the specific peptide NI-105.4E4. 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 amino acid residues 337-343 of human Tau identified by Pepspot mapping.

[00312] To roughly map the binding epitope of NI-105.4A3 to hTau40, four tau domain polypeptides (Tau domain I, domain II, domain III and domain IV) were generated. DNA fragments, synthesized using GeneArt® (Invitrogen), encoding each Tau domain with a 6xHis tag at the N-terminus, were cloned into the pRSET-A expression vector (Invitrogen), were transfected into E. Coli BL21 (DE3) (New England Biolabs. Expressions of His-tagged Tau domains were induced by 0.5 mM IPTG for six hours before the bacteria were lysed with lysozyme with sonication. The lysate was boiled for five minutes before being further purified with Ni-NTA Columns Superflow (Qiagen). Eluted His-tagged Tau domains were coated onto ELISA plates or loaded onto a polyacrylamide gel for Western blotting. These sequentially overlapping tau domain polypeptides covered the entire length of hTau40. Purified Tau domains were coated on the ELISA plate and the binding of NI-105.4A3 was tested. NI-105.4A3 binds only to the tau I domain, eu and the entire hTau40, indicating that the epitope was within the N-terminal part of hTau40 (aa1- 136). Western blotting confirmed the specific binding of NI-105.4A3 to the tau I domain. NI-105.4A3 epitope mapping with PepSpot technology (JPT) identified amino acids Q35-Q49 of human Tau40. To determine the major residues within the epitopes for NI-105.4A3 binding, alanine scanning was performed to replace each residue with alanine one at a time. The alanine residue in the original sequence (A41) was replaced with glycine or proline. Alanine scanning showed that D40, A41 and K44 are key residues for NI-105.4A3 binding.

Example 4 Evaluation of NI-105.4E4 binding to physiological forms as well as pathological aggregates of tau AD and brain tissues in human tau transgenic mice

[00313] Neurofibrillary tangles (NFT) composed of hyperphosphorylated tau filaments are a neuropathological hallmark of AD. Hyperphosphorylated tau filaments are also major components of dystrophic neurites and neuropil hairs, both of which are common neuropathologic features in AD. Overexpression of human tau containing the familial P301L tau mutation in mice induces NFT formation at six months of age (Gotz et al., 2001a).

[00314] To assess binding of recombinant human tau antibody to physiological forms as well as pathological aggregates of tau, immunohistological stains were performed on AD brain tissues and on TauP301L transgenic mice with the exemplary NI-105.4E4 antibody of this invention.

[00315] Mice were perfused with 20ml 100 mM TrisCl/6 mM EGTA (pH7.4) at room temperature under deep anesthesia. Brains were removed and immersed in 4% PFA in PBS (pH 7.4) at 4°C overnight for fixation followed by paraffin embedding. For human tissue, paraffin blocks of brain tissue from AD and healthy control subjects were used. DAB staining was performed following standard protocols. As a positive control, mouse monoclonal antibody Tau-12 (Covance, California, USA.) was used. HRP-conjugated detection antibodies without primary antibodies were also included.

[00316] Recombinant human antibody NI-105.4E4 identified numerous NFTs and neuropil hairs in the AD brain (Figure 2A), which were absent in the healthy control brain (Figure 2B). Secondary antibody alone gave no signals in AD (Figure 2C) and control (Figure 2D) brain. In P301L tau transgenic mouse brain, NI-105.4E4 bound strongly to pathological tau resembling NFT (Figure 2E, F and H), neuropil hairs (Figure 2E and G) and dystrophic neurites (Figure 2E and H) . Furthermore, NI-105.4E4 also identified tau aggregates in the pre-entanglement phase (Figure 2I). In the brain of transgenic mice overexpressing human P301L tau and human APP with Swedish and Arctic mutations, NI-105.4E4 specifically bound to dystrophic neurites surrounding beta-amyloid plaques (Figure 2J).

Example 5 In vivo testing of antibodies of the present invention

[00317] As already described above, studies in transgenic mouse strains using active vaccination with phosphorylated tau peptides revealed reduced brain levels of brain tau aggregates and delayed progression of behavioral impairment (Sigurdsson, J. Alzheimers Dis. 15 (2008) , 157-168; Boimel et al., Exp. Neurol., 224 (2010), 472-485 ). However, active vaccination may not be particularly useful in humans because a significant fraction of the elderly population is expected to be non-respondent to vaccination. Furthermore, the potential side effects associated with a tau-directed immune response can be difficult to control. The tau binding molecules of the present invention can reasonably be expected to achieve similar reductions in brain levels of tau aggregates as described above for the mouse antibodies, because of their similar binding specificities against pathologically similar tau species. However, due to evolutionary optimization and affinity maturation within the human immune system antibodies of the present invention provide a valuable therapeutic tool due to being isolated from healthy humans, 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 in various possible routes to the animals such as intraperitoneal antibody injection, intracranial injection, intraventricular cerebral infusion and tested for treatment purposes. Any of the aforementioned application possibilities can also be used after anterior brain injection of amyloid-beta preparations into the brain of tau-transgenic mice to evaluate treatment effects on amyloid-beta-induced tau pathology.

[00318] Assessment of treatment effects can be performed by histochemical methods comprising quantification of Gallyas positive cell counts, total human tau staining, brain burden of phosphorylated tau and/or a biochemical determination of soluble and insoluble tau in the brain and levels of phosphorus after sequential brain extraction. Even more, behavioral testing of the treated mice can be performed, for example, 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 (2006), 369- 79, Pennanen Neurobiol Dis. 15 (2004), 500-9.)

Example 6 Chimerization of NI-105.4E4 and NI-105.4A3 antibodies with mouse IgG2a constant domains

[00319] In order to generate antibodies with reduced immunogenicity for use in chronic treatment studies, chimeric mouse versions of NI-105.4E4 and NI-105.4A3 antibodies were generated using recombinant DNA technology. A mouse IgG2a/lambda isotype was selected for these chimeric antibodies in order to generate a molecule that bound with high affinity to the mouse Fc-gamma receptors and therefore capable of inducing an effector immune response. The amino acid sequences of the chimeric NI-105.4E4 ("ch4E4") and NI-105.4A3 ("ch4A3") heavy and light chain constructs are shown below. Table 5: Amino acid sequences of chimeric NI-105.4E4 (ch4E4) and chimeric NI-105.4A3 (ch4A3)

Example 7 Removal of the consensus N-linked glycosylation site from the heavy chain of ch4E4 (mouse IgG2a)

[00320] A consensus N-linked glycosylation site has been identified in the CDR1 region of the heavy chain of NI-105.4E4. After mammalian cell expression (CHO), the predicted N-glycosylation site (Asn-30) was fully occupied by glycan, as demonstrated by mass spectrometry. To eliminate N-glycosylation in this region and reduce product heterogeneity, heavy chain Asn-30 from ch4E4 was changed to Gln (Table 4). When produced and purified from CHO cells, the modified antibody bound recombinant tau with ~4-fold greater apparent binding affinity than the original glycosylated antibody. Table 6: Amino acid sequences of mature ch4E4(N30Q) heavy chain (mouse IgG2a). The substituted residue Gln is in bold, underlined.

Example 8 Production of chimeric aglycosylated NI-105.4E4(N30Q) (ch4E4(N30Q) mIgG1 Agly)

[00321] A germline mouse chimeric aglycosylated variant NI-105.4E4 was produced ("ch4E4(N30Q) IgG1-Agly") in order to evaluate the relationship between antibody 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 IgG1 heavy chain constant region containing an Asn, Gln mutation to eliminate the of consensus Fc glycosylation. The heavy chain variable region contained the N30Q change to delete the consensus N-glycosylation site in CDR1 (Example 7). The light chain was the ch4E4 lambda light chain described above (SEQ ID 21). Example 9

Acute Cerebral Penetration Study of Human 4E4 and 4A3

[00322] Human NI-105.4E4 and NI-105.4A3 germline antibodies were produced by transient transfection of CHO cells and purified by affinity purification. Endotoxin levels were controlled and were all below 1 EU/mg. TauP301L mice were injected intraperitoneally with 30 mg/kg NI-105.4E4 antibody (n=7), 4A3 (n=7) or equal volume of PBS (n=7) on day 1 and day 4. On day 5, mice mice are perfused under anesthesia with PBS containing 1 Unit/ml heparin. Blood, brain and spinal cord are collected for analysis. The right hemisphere of the brain is frozen at -80°C, the left hemisphere of the brain and the spinal cord are post-fixed in 10% neutralized formalin at 4°C for two days before being embedded in a paraffin block and sectioned. Plasma was stored at -80°C in aliquots.

[00323] Brain protein extraction: the 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% diethylamine, protease inhibitors (Roche Diagnostics GmbH ) and phosphatase inhibitor (Roche Diagnostics GmbH). The samples are then transferred to polycarbonate tubes and another 5 volumes of homogenization solution are added and kept on ice for 30 min. The soluble fraction is then collected after centrifugation at 100,000 g, 4°C for 30 min. This soluble fraction is used in the human IgG assay. The pellet is resuspended in 3 volumes of PBS with a phosphatase and protease inhibitor. After centrifugation at 16,000g, 4°C for 30min, supernatants and pellets are stored separately at -80°C for further extraction of insoluble tau. The pellets are further extracted with modified sarkosyl extraction (Goedert M, Spillantini MG, Cairns NJ, Crowther RA. Neuron 8, 159 (1992)).

[00324] Human IgG Specific Sandwich ELISA: 2 µg/ml goat anti-human IgG Fab (Jackson), in 50 mM carbonate ELISA coating buffer (pH9.6) is used as capture antibody. 96-well half-area microtiter plates are coated with 30 µl/well of capture antibodies at 4°C overnight. The plate is then washed 4 times with PBS containing 0.1% Tween 20 before incubation with 50 µl/well PBS containing 2% BSA at room temperature for one hour. Soluble fractions of brain extracts, plasma samples and standard human antibody (4A3) are diluted in PBS containing 2% BSA and 0.1% Tween 20. 30 µl of the diluted samples are added to each well and incubated at room temperature for one hour. The plate is then washed with 200 µl/well PBS containing 0.1% Tween 20 four times before being incubated with HRP-conjugated donkey anti-human FCY (Jackson, diluted 1:10,000 in PBS containing 2% BSA and 0.1% Tween 20) at room temperature for one hour. The plate is then washed with 200 µl/well PBS containing 0.1% Tween 20 four times before adding 20 µl/well TMB (1:20 in 10 mM citrate solution pH=4.1). The reaction is then stopped by adding 10 µl 1M H2SO4 to each well. The antibody standard curve was obtained from serial dilutions of NI-105.4A3. Antibody concentrations in plasma and brain samples are calculated according to guidelines. The level of human IgG in the brain was then converted to µg of antibody/gram of fresh brain tissue (assuming 1g/10 ml) as indicated in Figure 6.

[00325] High levels of human IgG were detected in the plasma of all mice treated with NI-105.4E4 and NI-105.4A3. In contrast, no human IgG was detected in the plasma of mice treated with PBS (Figure 5). A significant amount of human IgG was detected in brain homogenates from mice treated with 4E4 and 4A3 (Figure 6).

Example 10 Chronic study with NI-105.4E4 and chimeric NI-105.4A3

[00326] Chimeric NI-105.4E4 and NI-105.4A3 containing the variable domains of the original human antibody and the constant regions of mouse IgG2a can be produced by transient transfection of CHO cells and purified by affinity purification. Endotoxin levels in each batch of antibodies will be controlled and kept below 1 Eu/mg. Sex-balanced TauP301L mice aged 7.5-8 months will be injected intraperitoneally with 10 mg/kg, 3 mg/kg antibody solution, or equal volume of PBS control. Each treatment group will have 20-25 mice. The treatment will be carried out once a week for 26 weeks. Alternatively, the treatment is carried out twice a week for 13 weeks. Body weight will be monitored every two weeks. Mice are perfused under anesthesia at the end of the treatment period. Brain, spinal cord and blood are collected. Half of the brain and spinal cord are post-fixed in 10% formalin for three days before being embedded in a paraffin block. 4-6 µm thick sections cut from these tissue blocks can be used for immunohistochemical studies. The other half of the brain is weighed and deep frozen at -80°C for biochemical analyses.

[00327] The effects of drugs will be evaluated by comparing the level of neurofibrillary tangles (NFT) and the level of tau with different solubility characteristics in control and treated samples. NFT can be visualized by Gallyas silver staining (F Gallyas Acta Morphol. Acad. Sci. Hung 19.1 (1971)), or by immunostaining with mouse monoclonal antibodies AT100 and AT180, which recognize pathologically phosphorylated tau in NFT. The number or frequency of Gallyas positive neurons and/or AT100, AT180 labeled neurons in the brain and spinal cord in antibody-treated mice and control animals can be determined to assess the effect of antibody treatment.

[00328] Soluble and insoluble tau can be extracted following the brain protein extraction protocol described here. Alternatively, soluble and insoluble tau can be extracted with modified sarkosyl extraction (Goedert M, Spillantini MG, Cairns NJ, Crowther RA. Neuron 8, 159 (1992)). Briefly, the frozen brain is homogenized in 10 volumes (w/vol) of 10% sucrose homogenate buffer consisting of 10 mM Tris^HCl (pH 7.4), 0.8 M NaCl, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, 1 mM AEBSF, protease inhibitors (Roche Diagnostics GmbH) and phosphatase inhibitor (Roche Diagnostics GmbH). The homogenate is spun for 20 min in 20,000g, and the supernatant retained. The pellet is homogenized in 10 volumes of homogenization buffer and centrifuged a second time. The supernatants can be combined together, and N-lauryl sarcosinate (Sigma) is added to a final concentration of 1% (w/vol), and incubated at 37°C, rotating at 300 rpm for 1.5 hours, followed by centrifugation at 100,000 g for 1 h. The supernatant is collected as a soluble sarkosyl fraction and the 1g brain tissue pellet is resuspended in 0.2 ml 50 mM Tris^HCl (pH 7.4) as a PHF fraction.

[00329] Soluble and insoluble tau levels will be measured with commercially available Tau ELISA kits (Invitrogen). Furthermore, brain protein extracts will be separated with 4-12% Bis-Tris SDS-PAGE followed by immunostaining with antibodies Tau12 (human tau), AT8 (pS202/pT205), AT100 (pT212/pS214), AT180 (pT231) and E178 (pS396). Semi-quantitative analysis will be performed by measuring the integrated density of each sample against standards of known amounts of tau.

[00330] In addition, behavioral tests can be performed as indicated in Example 5, above. For example, improvement of working memory in antibody-treated TauP301L mice can be tested using a two-study Y-maze task (e.g., Pennanen, Genes Brain Behav. 5 (2006), 36979, which is incorporated herein by reference in its entirety). The three arms of the maze are 22 cm long, 5 cm wide and 15 cm deep. Black and white abstract clues are placed in a black curtain around the maze. The experiments are performed with an ambient light level of 6 lux during the dark phase. Each experiment comprises a training 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 for 4 min without access to the third arm (the new arm). The mouse is then removed from the maze and held in a holding cage for 1.5-5 min while the maze is thoroughly cleaned with 70% ethanol to remove any olfactory cues. The mouse is placed back in the maze for observation with all three arms accessible for 4 min. The entry sequence, the entry number for each arm, and the time spent on each arm is recorded. From these, the ratio of time spent in the third new arm over the average time spent in the other two arms (starting arm and the second arm) is calculated and compared between different treatment groups in a taupathy mouse model and a mouse model of corresponding control wild type. Rodents usually prefer to investigate a new branch of the maze rather than returning to one that was previously visited. The effects of the antibodies can be monitored with respect to recovering this preference for the treated tauopathy model mice compared to the non-discriminative behavior of the untreated mice due to their disorder-related working memory impairment. Therefore, a ratio close to 1 indicates compromised working memory. A higher ratio indicates better working memory. Impaired working memory in TauP301L mice is thought to be due to tau pathology resulting from overexpression of human tau. Therefore, a significantly higher ratio observed in TauP301L mice treated with anti-tau antibody than in control TauP301L mice will indicate that the anti-tau antibody has therapeutic effect on tau pathology.

Example 11 Identification of human anti-tau antibodies.

[00331] Recombinant human tau antibodies NI-105.17C1, NI-105.6C5, NI-105.29G10, NI-105.6L9, NI-105.40E8, NI-105.48E5, NI-105.6E3, NI-105.22E1, NI-105.26 B12, NI-105.12E12, NI-105.60E7, NI-105.14E2, NI-105.39E2, NI-105.19C6, and NI-105.9C4 were isolated according to the methods described herein. The target specificity and binding 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-105.17C, were germline. Table 7. In vitro characterization of human anti-tau antibodies. *: Binding region in hTau40 was identified by combined approaches of PepSPOTs western blot tau fragments and ELISA, tau peptide ELISA and Alanine scan. **: it was verified whether antibody binding requires phosphorylation on certain amino acids in tau protein by comparing antibody binding to rTau, PHFTau, PHFTau dephosphorylated by calf intestine phosphatase, rTau in vitro phosphorylated by GSK3ß or GSK3ß/CDK5/p35, and phosphorylated tau peptides in PepSPOTs and direct ELISA. Example 12

Chimerization of human antibodies with mouse IgG2a constant domains

[00332] In order to generate antibodies with reduced immunogenicity for use in chronic treatment studies, mouse chimeric versions of antibodies NI-105.17C1 ("ch17C1"), NI-105.6C5 ("ch6C5"), NI-105.40E8 ( "ch40E8"), and NI-105.6E3 ("ch6E3") were generated using recombinant DNA technology. A mouse IgG2a/lambda isotype was selected for these chimeric antibodies in order to generate a molecule that bound with high affinity to the mouse Fc-gamma receptors and therefore capable of inducing an effector immune response. The amino acid sequences of heavy and light chain constructs ch17C1, ch6C5, ch40E8, ch40E8(R104W), and ch6E3 are shown below.

Example 13 Deletion of the CDR glycosylation site in the NI-105.17C1 light chain.

[00333] A consensus N-linked glycosylation site was identified in the CDR1 region of the light chain of NI-105.17C1. After mammalian cell expression (CHO), the predicted N-glycosylation site (Asn-31) was fully occupied by glycan, as demonstrated by mass spectrometry. To eliminate N-glycosylation in this region and increase product heterogeneity, light chain Asn-31 from ch17C1 was changed to Gln (see sequence below). When produced and purified from CHO cells, the modified antibody (ch17C1(N31Q) mIgG2a) bound recombinant tau with similar apparent binding affinity 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.

Example 14 Production of antibodies with reduced effector function.

[00334] Antibody variants containing 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 functions. The amino acid sequences of Agly variants of tau antibodies are provided below.

Example 15 Comparison of Binding Activity of ch17C1-mIgG2a and ch17C1-mIgG1-Agly.

[00335] The relationship between effector function and antibody activity was assessed by ch17C1 Agly (Figure 8A). The ch17C1 antibody comprised the heavy chain of ch17C1 (SEQ ID NO:203), and the light chain of ch17C1 (SEQ ID NO:204). The ch17C1(N31Q) mIgG2a antibody comprised the heavy chain of ch17C1 (SEQ ID NO:203), and the light chain of ch17C1(N31Q) (SEQ ID NO:212), which incorporates the N31Q mutation in CDR1 to eliminate the binding site. CDR glycosylation. The ch17C1(N31Q) mIgG1 Agly antibody comprised ch17C1-mIgG1-Agly heavy chain (SEQ ID NO:216), in which the 17C1 variable domains are fused to a mouse IgG1 heavy chain containing an Asn to Gln mutation at position 294 (Kabat residue 297) to eliminate the consensus Fc glycosylation site, and the light chain of ch17C1(N31Q) (SEQ ID NO:212). When produced and purified from CHO cells, ch17C1(N31Q) mIgG1 Agly bound recombinant tau with similar apparent binding affinity to the original glycosylated antibody (see Figure 8A).

Example 16 Comparison of Valina vs. Isoleucine at position 48 of the 17C1 light chain.

[00336] In the process of generating the mouse chimeric IgG2a version of the germline antibody NI-105.17C1, residue 48 of the light chain was also changed from valine to isoleucine. To confirm that this substitution did not affect the binding affinity of NI-105.17C1, a chimeric mouse IgG2a version of NI-105.17C1 with valine at position 48 was prepared. When produced and purified from CHO cells, the ch17C1(N31Q, I48V) mIgG2a antibody bound recombinant tau with similar apparent binding affinity to ch17C1(N31Q) mIgG2a (see Figure 8B). 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).

Example 17 Comparison of Arg vs. Trp at position 104 of NI-105.40E8.

[00337] Antibody NI-105.40E8, 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 NI-105.40E8 VH. Normally this position within the human immunoglobulin repertoire is occupied by a tryptophan residue. A heavy chain form of NI-105.40E8, NI-105.40E8(R104W)-hIgG1, was generated in which the Arg104 residue was replaced with tryptophan. When produced and purified from CHO cells, the NI-105.40E8(R104W)-hIgG1 antibody bound human PHF tau with similar apparent binding affinity to NI-105.40E8-hIgG1 (see Figure 9). The light chain of the two antibodies was identical .

Example 18 Human anti-tau antibodies bind to pathologically aggregated tau in the AD brain and in the brain of transgenic mouse model of taupathy.

[00338] Brain tissue samples obtained from control and Alzheimer's disease patients, as well as the brain of taupathy transgenic mice and wild-type control were stained with the anti-human germline tau antibodies provided herein. Representative images of human germline anti-tau antibody NI-105.40E8, NI-105.48E5, NI-105.6C5, and NI-105.17C1 binding to pathological tau aggregates in Alzheimer's disease (AD) brain and mouse brain taupathy (Tg) transgenic antibodies are shown in Figure 10. None of these antibodies bind to normal tau in mentally healthy subject (Ctr) or wild-type (Wt) mouse brain. The different patterns among these antibodies reflected their differences in epitope specificity and binding affinity.

Example 19 Brain penetration of antibodies in TauP301L mice.

[00339] Animals and Antibody Treatments: Human anti-tau antibodies NI-105.6C5, NI-105.40E8 and NI-105.6E3 were produced by transient transfection of CHO cells and purified using standard methods. A humanized antibody without cross-reactivity with mouse antigens was used as an isotype control (hIgG1). In the first experiment, half of the injected NI-105.6C5, NI-105.6E3, and hIgG1 antibodies were Cy3-labelled (GE Healthcare, PA13105) with an approximate antibody: Cy3 ratio of 1:3. Labeling with Cy3 did not change antibody binding, as confirmed by ELISA and immunostaining with TauP301L brain (data not shown). In the second experiment, unlabeled NI-105.6C5 and NI-105.40E8 anti-tau antibodies were used.

[00340] TauP301L mice between 18-22 months of age received two doses of 30 mg/kg NI-105.6C5, NI-105.6E3 or hIgG1 human isotype control IgG1 via i.p. injection within seven days. Tissue samples were collected from three mice from each treated group at time points one day, eight days and 22 days after the second dosing. In the second experiment, TauP301L mice were injected with 30 mg/kg h40E8 and h6C5 twice within seven days and tissue samples were collected from these mice one day after the second injection.

[00341] For tissue sample collection, mice were deeply anesthetized with ketamine/xylazine before blood was collected through the right atrium. CSF was then collected by puncture of the cisterna magna. The brain and spine were subsequently harvested after perfusion for 2 min with ice-cold PBS containing 10 IU/ml heparin and five minutes with 10% neutralized formalin via the left ventricle. The brain was fixed in 10% neutralized formalin for another 3 h at 4°C, after immersion in 30% sucrose for 48 h. The brain was then frozen on dry ice and subsequently sectioned in a series of 30 µm thick coronal sections. The section series was stored at -20°C in antifreeze solution containing 1M glucose, 37.5% ethylene glycol in 50 mM pH7.4 sodium phosphate buffer with 0.025% sodium azide before use. The spine was post-fixed in 10% neutralized formalin for two days and embedded in paraffin blocks.

[00342] Immunohistochemistry: Coronal sections of 30 µm thickness were analyzed with biotinylated donkey anti-human IgG (H+L) by free floating stain. Free-floating sections were washed in Tris-Triton pH7.4 (50mM Tris, 150 mM NaCl, 0.05% Triton X-100), incubated in 1% H2O2 PBS for 30 min, and incubated with a blocking solution containing 2 % normal horse and goat serum in Tris-Triton and with additional 0.2% Triton X-100 for 1 h at room temperature. Sections were then incubated with biotinylated (H+L) donkey anti-human IgG (Jackson Immunoresearch Labs, 709-065-149) in 1:200 blocking solution for 16 h at 4°C with shaking at 100 rpm to detect IgG neural human. Biotinylated tissue-bound antibody was visualized by peroxidase chromogenic reaction using the Vectastain Elite ABC kit (Vector Laboratories, PK6100, 1:100). The enzymatic reaction was stopped with ice cold PBS and the sections were washed in PBS 3 times. Sections were then mounted on glass slides and air dried overnight before being counterstained with hemalum solution to visualize nuclei (Carl Roth GmbH + Co., T865.1). After the dehydration steps, the slides were covered with coverslips before being explored with the Olympus dotSlide 2.1 virtual microscopy system.

[00343] Human antibodies were detected in the brains of TauP301L mice, which received human anti-tau antibodies or hIgG1 control antibody through i.p. injection, but not in TauP301L mice and wild-type mice without antibody treatment ( Fig. 11 ). However, neuronal staining was observed only in the hippocampus of mice treated with anti-tau antibody NI-105.6C5, NI-105.6E3, and NI-105.40E8, but not in mice treated with hIgG1. Neuronal staining with anti-tau antibodies was readily detectable one day after injection, less pronounced eight days after injection, and was undetectable 22 days after injection (data not shown). TauP301L mice produce high levels of transgenic human tau in the formation of the hippocampus, and the hippocampus is one of the first regions to develop neurofibrillary tangles. Thus, peripherally injected anti-tau antibodies not only entered the brain, but also likely entered neurons that contained high levels of tau.

Example 20 Effects of chronic treatment of TauP301L mice with ch4E4(N30Q) and ch17C1(N31Q).

[00344] Animals and antibody treatments: chimeric NI-105.4E4(N30Q) ("ch4E4(N30Q)") and chimeric NI-105.17C1(N31Q) ("ch17C1(N31Q)") containing the variable domains of human antibody and mouse IgG2a constant regions were produced by transient transfection of CHO cells and purified using standard methods.

[00345] Sex-balanced TauP301L mice at ages 7.5-8 months were administered weekly with 10 mg/kg ch17C1(N31Q) (n=20), 10 mg/kg ch4E4(N30Q) (n=20) or a equal volume of PBS (n=20) via 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 ch17C1(N31Q)-treated group died prematurely. The mice were anesthetized one day after the 25th treatment for tissue collection. Blood was collected through the orbital cavity. The brain and spine were subsequently harvested after perfusion for 2 min with ice-cold PBS containing 10 IU/ml heparin through the left ventricle. The left brain half was then weighed, frozen by dry ice immersion and stored at -80°C before use. The right half of the brain and spine were post-fixed in 10% neutralized formalin at 4°C for two days, followed by further storage in PBS, before being processed into paraffin-embedded brain and spinal cord blocks.

[00346] Brain Protein Extraction: Brain protein was extracted sequentially on the basis of solubility. The left half of the brain was first homogenized in 10 times w/v of 50 mM NaCl containing 0.2% diethylamine, 1X protease inhibitor (Roche Diagnostics GmbH) and 1X phosphatase inhibitor (Roche Diagnostics GmbH). After incubating for 30 min on ice, the homogenate was centrifuged at 100,000 g at 4°C for 30 min. Subsequently, the supernatant was collected and defined as the soluble fraction. The remaining pellet was homogenized in 12 times w/v of 10% sucrose lysis buffer containing 10 mM Tris 7.4, 0.8 M NaCl, 1 mM EGTA, 1X phosphatase inhibitor, 1x protease inhibitor, 1 mM Na3VO4, 1 mM NaF and 1 mM AEBSF. After 30 min of incubation on ice, the homogenate was centrifuged at 20,500 g at 4°C for 20 min. 95% of the supernatant was carefully collected for sarkosyl extraction. The pellet was stored at -80°C. N-lauryl-sarcosinate was added to the supernatant (1% (w/v) final concentration). After incubation for 1 h at 37°C with 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 soluble fraction of sarkosyl. 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 insoluble PHF fraction, which was stored at -80 °C until use.

[00347] ELISA measurements of phosphorylated and total human Tau in three brain protein fractions were quantified with commercial ELISA kits (Life Technologies) following the manufacturer's protocol. Total human tau, Threonine 231 phosphorylated human tau (pT231 tau), Serine 199 phosphorylated human tau (pS199 tau) and Threonine 181 phosphorylated human tau (pT181) were detected. Soluble fraction samples were standardized to 1 mg/ml based on total protein content measured with BCA protein assay (Pierce) with 50 mM Tris pH7.4. Aliquots of standardized samples were prepared for ELISA and Western blotting measurements. To prepare solubilized PHF insoluble fraction for ELISA, 10 µl PHFTau was incubated with 10 µl 8M guanidine hydrochloride at room temperature for one hour followed by the addition of 180 µl 50 mM Tris 7.4. ELISA measurements were performed following standard protocols. Tau levels in each sample measured by ELISA were normalized to baseline brain weight for final analysis.

[00348] Plasma drug levels at endpoint were measured using sandwich ELISA. Briefly, 3 µg/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. 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 at 1:200, 1:400 and 1:800. Serial dilutions of ch17C1(N31Q) and ch4E4(N30Q) were used to generate standard curves. After the 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, bound antibody was further determined by a standard colorimetric assay. Standard curves were generated by sigmoidal curve fitting with GraphPad Prism 5.

[00349] Western blot Protein samples from 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® gel 4-12% (w/v). After semi-dry transfer of proteins to PVDF membrane, the membrane was blocked in 3% BSA containing 0.1% Tween-20 in TBS and further analyzed with different anti-tau antibodies at 4°C overnight. Peroxidase-conjugated secondary antibodies were then incubated at room temperature for 1 h after 4 washes with TBST. Subsequently, bound antibodies were detected by enhanced chemiluminescence (ECL) (Pierce). Densitometric analysis of immunoblots was performed using the National Institutes of Health ImageJ program.

[00350] Two-study Maze: Mice were tested for short-term spatial memory using a two-study Y-maze test. The arms of the maze are 35 cm long, 5 cm wide and 10 cm deep. Abstract clues were placed on the curtain around the maze. The experiments were carried out with an ambient light level of 6 lux. During the exposure phase, mice were assigned to two arms (the starting arm and another arm), which could be freely explored for 4 min, with no access to the third arm (new arm), blocked by a door made of the same maze material. The mice were then removed from the maze and kept in the cage for 2 min, when the maze was cleaned with 50% ethanol. During the test phase, mice were placed at the end of the start arm and allowed to freely explore all three arms for 4 min. The test phase was recorded using the TSE videoMot2 software for video monitoring and analysis of animal behavior (TSE Systems, Bad Homburg, Germany). The number of arm entries and the time spent in the new arm were recorded. The average number of arm entries and the time spent in the other two arms that were open during the training session were calculated. A ratio between the number of arm entries in (or time spent in) the new arm and the average of the other two arms was calculated. Wild-type control animals that do not have a deficit in spatial working memory will typically have a ratio between 1.5 and 2 on this test. Pennanen et al., Genes Brain Behavior 5(5):369-79 (2006 ).

[00351] Data analysis: The ELISA data were log-transformed to meet the hypothesis of normality for the two-way analysis of variance. The difference was considered significant when p<0.05.

[00352] ch4E4(N30Q) significantly reduced soluble human tau in TauP301L mice: Levels of human tau in the DEA-soluble, sarkosyl-soluble, and insoluble fractions of brain protein extracts were quantified by ELISA. The majority of human tau was found in the soluble fraction of DEA (data not shown). Total human tau (hTau) was reduced in the soluble fraction in DEA-treated mice of ch17C1(N31Q) and ch4E4(N30Q) compared to PBS-treated mice (mean 29% reduction in ch17C1(N31Q) and 37 % in ch4E4(N30Q), Fig. 12A). Reductions were also seen in phosphorylated tau (pT231, pS199 and pT181) in the DEA-soluble fraction (Fig. 12B, C and D respectively). We previously observed lower expression of human tau in female TauP301L 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 measurements of soluble human tau (total human tau, human tau, pS199, pT181 and pT231). There was no interaction between gender and treatment (0.49 <p< 0.91 in all human soluble tau measurements). Importantly, there was a significant effect of soluble human tau on DEA (p<0.05 for hTau, pS199 and pT231, and p=0.06 for pT181). The treatment effect was predominantly driven by ch4E4(N30Q). When compared to the PBS control, ch4E4(N30Q) significantly reduced hTau (p<0.05), pS199 (p<0.01), pT231 (p<0.01) and pT181 (p<0.05). ELISA measurements using the sarkosyl-insoluble fraction showed a high variability between animals, and no significant gender effect was observed. No significant treatment effects were observed on the sarkosyl-insoluble fraction. Likewise, no significant treatment effect was observed by ELISA on the sarkosyl-soluble fraction.

[00353] Western blots using human tau-specific monoclonal antibody Tau12 showed whole human tau, as a single band at 62 kDa, as the major tau immunoreactive component in the soluble fraction of DEA. A clear reduction of full-length human tau was seen in most mice treated with ch4E4(N30Q) and ch17C1(N31Q). In the insoluble sarkosyl fraction, a 64 kDa band and several other higher molecular weight bands were observed, as well as lower molecular weight bands presumably corresponding to human tau fragments. Densitometric analysis showed a high degree of variability between individual animals, which precluded any quantitative comparison. However, there was an overall qualitative reduction in all human tau proteins in the insoluble fraction of sarkosyl detected by Tau12 in mice treated with ch17C1(N31Q) and ch4E4(N30Q) (representative Western blot shown in Fig. 13).

[00354] Plasma Drug Levels: Mice were treated with ch17C1(N31Q) and ch4E4(N30Q) at 10 mg/kg weekly by i.p. To assess drug exposure, plasma samples were collected 24 h after the last treatment. Plasma drug levels were measured with ELISA using rTau as the capture agent. Mean plasma levels of ch17C1(N31Q) and ch4E4(N30Q) were 200 µg/ml and 145 µg/ml, respectively, suggesting that both antibodies had good blood exposure ( Fig. 14 ).

[00355] Antibody treatment and spatial memory: A previous study suggested deficits in spatial reference memory, which is hippocampus-dependent, in TauP301L mice (Pennanen et al., Genes Brain Behav 5(5):369-79 (2006) ). The Y-maze from two studies was reported as a sensitive test for detecting short-term spatial memory deficits 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, spending a similar amount of time on each available arm (data not shown). No differences were detected comparing the displaced distance. During the test phase, PBS-treated TauP301L mice produced almost equal numbers of entries into the new arm as the average of the other two arms explored during the exposure phase, (ratio=1.18), suggesting a low spatial working memory in TauP301L mice treated with PBS. Mice treated with ch17C1(N31Q) and ch4E4(N30Q) showed a preference for the new arm over the other two arms, and made more visits to the new arm than the average of the other two arms (ratio=1.50 for ch17C1(N31Q) ) and 1.30 for ch4E4(N30Q)). The ratio of entry into the new arm in TauP301L mice treated with ch17C1(N31Q) and ch4E4(N30Q) compared to the PBS-treated group is shown in Fig. 15.

[00356] The present invention is not to be limited in scope by the specific embodiments described which are intended as simple illustrations of the individual aspects of the invention, and any compositions or methods which are functionally equivalent are within the scope of the present invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and accompanying figures. These modifications are intended to fall within the scope of the appended claims.

Claims (13)

1. Pharmaceutical composition, characterized in that it comprises an anti-tau antibody or tau-binding fragment thereof, wherein the anti-tau antibody or tau-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises the amino acid sequence shown in SEQ ID NO: 48 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (b) the VH comprises the amino acid sequence shown in SEQ ID NO: 47 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (c) the VH comprises the amino acid sequence shown in SEQ ID NO: 54 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55; or (d) the VH comprises the amino acid sequence shown in SEQ ID NO: 220 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier which is phosphate-buffered saline. 2. Use of an anti-tau antibody, or tau-binding fragment thereof, wherein the anti-tau antibody or tau-binding fragment thereof comprises a heavy chain (VH) variable region and a tau chain variable region. lightweight (VL), wherein: (a) the VH comprises the amino acid sequence shown in SEQ ID NO: 48 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (b) the VH comprises the amino acid sequence shown in SEQ ID NO: 47 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (c) the VH comprises the amino acid sequence shown in SEQ ID NO: 54 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55; or (d) the VH comprises the amino acid sequence shown in SEQ ID NO: 220 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55, or of the pharmaceutical composition, as defined in claim 1, characterized in that is for the manufacture of a drug to treat a neurodegenerative tauopathy. 3. Use of an anti-tau antibody, or tau-binding fragment thereof, wherein the anti-tau antibody or tau-binding fragment thereof comprises a heavy chain (VH) variable region and a tau chain variable region. lightweight (VL), wherein: (a) the VH comprises the amino acid sequence shown in SEQ ID NO: 48 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (b) the VH comprises the amino acid sequence shown in SEQ ID NO: 47 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (c) the VH comprises the amino acid sequence shown in SEQ ID NO: 54 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55; or (d) the VH comprises the amino acid sequence shown in SEQ ID NO: 220 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55, or of the pharmaceutical composition, as defined in claim 1, characterized in that is for the manufacture of a medicament for treating abnormal accumulation and/or deposition of tau in the central nervous system in a human subject in need thereof. 4. Use according to claim 2, characterized in that the neurodegenerative tautopathy is selected from the group consisting of Alzheimer's disease, dementia-parkinsonism complex/amyotrophic lateral sclerosis, argyrophilic grains dementia, British-type amyloid angiopathy, angiopathy cerebral amyloid, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle with calcification, Down syndrome, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick type C disease, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease, post-encephalitis parkinsonism, cerebral amyloid angiopathy of the prion protein, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, multi-infarct dementia, and ischemic stroke. 5. Use according to claim 2, characterized by the fact that the neurodegenerative tautopathy is Alzheimer's disease. 6. Use according to claim 2, characterized by the fact that the neurodegenerative tautopathy is frontotemporal dementia parkinsonism linked to chromosome 17. 7. Use according to claim 2, characterized by the fact that the neurodegenerative tautopathy is lobar frontotemporal degeneration. 8. Composition, characterized in that it comprises an anti-tau antibody or tau-binding fragment thereof and a pharmaceutically acceptable carrier which is phosphate saline buffer solution, wherein the anti-tau antibody or tau-binding fragment of it comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises the amino acid sequence shown in SEQ ID NO: 48 and the VL comprises the sequence of amino acids shown in SEQ ID NO: 49; (b) the VH comprises the amino acid sequence shown in SEQ ID NO: 47 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (c) the VH comprises the amino acid sequence shown in SEQ ID NO: 54 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55; or (d) the VH comprises the amino acid sequence shown in SEQ ID NO: 220 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55, for use in in vivo diagnosis of a tautopathy in a human subject in need of the same. 9. Composition according to claim 8, characterized by the fact that the tauopathy is Alzheimer's disease. 10. Composition, according to claim 8, characterized by the fact that the tauopathy is frontotemporal dementia with parkinsonism linked to chromosome 17. 11. Composition, according to claim 8, characterized by the fact that the tauopathy is frontotemporal lobar degeneration. 12. In vitro method of monitoring the progression of a neurodegenerative tautopathy, comprising: (a) measuring the level of pathologically modified or aggregated tau in a sample obtained from a human subject with an antibody or tau-binding fragment the same; and (b) comparing the level of modified or aggregated tau to a reference standard that indicates the level of pathologically modified or aggregated tau in one or more control subjects, where the difference or similarity between the level of pathologically modified or aggregated tau and the reference standard indicates that the subject has a neurodegenerative tautopathy, and wherein the antibody or tau-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (c) the VH comprises the amino acid sequence shown in SEQ ID NO: 48 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (d) the VH comprises the amino acid sequence shown in SEQ ID NO: 47 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (e) the VH comprises the amino acid sequence shown in SEQ ID NO: 54 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55; or (f) the VH comprises the amino acid sequence shown in SEQ ID NO: 220 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55. 13. In vitro method of using the level of pathologically modified or aggregated tau as an indicator of neurodegenerative tautopathy, the method characterized in that it comprises: (a) measuring the level of pathologically modified or aggregated tau in a sample obtained from an individual human with an antibody or tau-binding fragment thereof; and (b) comparing the level of modified or aggregated tau to a reference standard that indicates the level of pathologically modified or aggregated tau in one or more control subjects, where the difference or similarity between the level of pathologically modified or aggregated tau and the reference standard indicates that the subject has a neurodegenerative tautopathy, and wherein the antibody or tau-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (c) the VH comprises the amino acid sequence shown in SEQ ID NO: 48 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (d) the VH comprises the amino acid sequence shown in SEQ ID NO: 47 and the VL comprises the amino acid sequence shown in SEQ ID NO: 49; (e) the VH comprises the amino acid sequence shown in SEQ ID NO: 54 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55; or (f) the VH comprises the amino acid sequence shown in SEQ ID NO: 220 and the VL comprises the amino acid sequence shown in SEQ ID NO: 55.