WO2014016737A9

WO2014016737A9 - Novel chicken monoclonal antibodies against human phosphorylated tau and uses thereof

Info

Publication number: WO2014016737A9
Application number: PCT/IB2013/055877
Authority: WO
Inventors: Wei Cao; Brian Joseph FENNELL; William James Jonathan Finlay; Heather Hongrong SHIH; Chao TU
Original assignee: Pfizer Inc.
Priority date: 2012-07-24
Filing date: 2013-07-17
Publication date: 2014-09-04
Also published as: WO2014016737A1

Abstract

The present invention provides isolated monoclonal antibodies, particularly chicken monoclonal antibodies, which specifically bind to phospho-tau with high affinity. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for producing the antibodies of the invention are also provided. Pharmaceutical compositions comprising the antibodies of the invention are also provided. The invention also provides methods for using the antibodies, including methods for detecting phospho-tau, as well as methods for treating diseases or disorders related to or mediated by phospho-tau, including Alzheimer's disease.

Description

NOVEL CHICKEN MONOCLONAL ANTIBODIES AGAINST HUMAN

PHOSPHORYLATED TAU AND USES THEREOF

FIELD OF THE INVENTION

This application relates to the field of monoclonal antibodies that specifically bind human phosphorylated tau polypeptides and methods of making and using the antibodies to, among other things, detect phosphorylated tau and treat diseases related thereto.

BACKGROUND OF THE INVENTION

The microtubule-associated protein tau, in a hyperphosphorylated state, makes up the protein constituent of the paired helical filaments (PHFs) of non-febrile tangles (NFTs) in the brains of Alzheimer's Disease (AD) patients. This observation has made phospho-Tau the focus of intensive research over the past two decades (Grundke-lqbal et al., 1996, J. Biol. Chem. 261 :6084-6089; Grundke-lqbal et al., 1986, Proc. Natl. Acad. Sci. USA 83:4913-4917). In humans, tau has six isoforms resulting from alternative splicing of mRNA encoded by a single gene. All six isoforms are present in tangles when in the hyperphosphorylated state (Grundke-lqbal et al., 1986, Proc. Natl. Acad. Sci. USA 83:4913-4917; Alonso et al., 2001 , J. Biol. Chem. 276:37967-37973; Brion et al., 1991 , Biochem. J. 273:127-133; Goedert et al., 1992, Neuron 8:159-168; Kosik et al., 1986, Proc. Natl. Acad. Sci. USA 83:4044-4048).

The phosphorylation of tau occurs under both normal and pathological conditions and is believed to negatively regulate the ability of tau to promote microtubule assembly (Lindwall & Cole, 1984, J. Biol. Chem. 259:5301-5305). In the brains of AD patients, tau is found to be phosphorylated at 3- to 4- fold higher levels than in normal brain (Kenessey & Yen, 1993, Brain. Res. 629:40-46; Kopke et al., 1993, J. Biol. Chem. 268:24374-24384; Ksiezak-Reding et al., 1992, Brain. Res. 597:209-219). As a result, significant efforts have been undertaken to determine the phosphorylation sites on the tau protein found in paired helical filaments of Alzheimer's Disease patient's brains. To date, approximately 40 tau phosphorylation sites have been identified in association with AD (Hanger et al., 2009, Expert. Rev. Neurother. 9:1647-1666). Pathological tau phosphorylation has been implicated in sequestering normal microtubule-associated proteins from microtubules, preventing axonal transport, generating protein aggregates in the cell body and eventually killing the neuron (Alonso et al., 2001 , J. Biol. Chem. 276:37967-37973; Abraha et al., 2000, J. Cell. Sci. 1 13:3737-3745; Haase et al., 2004, J. Neurochem. 88:1509-1520; Liu et al., 2007, Eur. J. Neurosci. 26:3429-3436).

As a result of the association between tau hyperphosphorylation and pathological phenotypes, tau has been proposed as both a diagnostic biomarker and a target for therapeutic intervention in AD (Hampel et al., 2010, Exp. Gerontol. 45:30-40). Therefore, there is a need for tools and assays to assess the phosphorylation events that occur at each of the potentially pathological tau phosphorylation sites.

The rapid, unambiguous recognition of phosphorylation events can be performed using immunoassays which rely on antibodies which specifically recognize the protein of interest in its phosphorylated form, but not the non-phosphorylated form. However, even though protein phosphorylation is one of the most extensively recognized and described events during normal and disease-related cell signaling, few significant advances have been made in the generation or molecular characterization of phospho-specific antibody reagents since initial reports 30 years ago (Nairn et al., 1982, Nature 299, 734-736; Ross et al., 1981 , Nature 294, 654-656).

Chickens are a historically reliable immune host due to their robust immune response against highly conserved mammalian proteins (Yamanaka et al., 1996, J. Immunol. 157:1 156-1162), the feasibility of co-immunizing single animals with multiple immunogens (Finlay et al., 2005, Clin. Exp. Allergy 35:1040-1048), and their proven ability to generate highly specific antibodies against both peptides (Nishibori et al., 2006, Mol. Immunol. 43:634-642), and haptens (Finlay et al., 2006, Appl. Environ. Microbiol. 72:3343-3349), via display technologies (Andris-Widhopf et al., 2000, J. Immunol. Methods 242:159-181 ).

There is a long felt need for high affinity monoclonal antibodies specific for different tau phosphorylation states, which have the potential to provide valuable research reagents to unravel normal versus pathological tau function, as well as providing potential diagnostic tools for AD. The present invention meets this need.

SUMMARY OF THE INVENTION

The invention includes an isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1 x10^~7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau.

In another aspect, the antibody is a chicken monoclonal antibody.

In yet another aspect, the antibody is chimeric or humanized.

In one aspect, the antibody, or antigen-biding fragment thereof, is selected from the group consisting of a Fab, a F(ab')2, a Fd, a Fv, a dAb, a CDR, a disulfide-linked Fv (dsFv), a scFv, and a diabody.

In another aspect, the antibody is a scFv.

In one aspect, the V_H and V_L regions are derived from chicken antibodies.

In yet another aspect, the V_H and V_L regions are derived from a chicken antibody and the constant regions are derived from human IgG C_H and a human kappa C_L or human lambda C_L.

In one aspect, the antibody binds to phospho-tau with a K_D of 5 x10^"8 M or less.

In another aspect, the antibody binds to phospho-tau with a K_D of 5 x10^"9 M or less.

In one aspect, the human phospho-tau comprises the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the protein is phosphorylated at least one amino acid selected from the group consisting of threonine 212 (T212), serine 214 (S214), threonine 231 (T231 ), serine 235 (S235), serine 396 (S396), serine 404 (S404), and serine 422 (S422) where the numbering of the amino acid residues is relative to the amino acid sequence provided in SEQ ID NO: 1 . In yet another aspect, the human phospho-tau comprises at least one phosphorylated amino acid selected from T231 and S235 relative to the numbering of the amino acid sequence of SEQ ID NO: 1 .

In one aspect, the human phospho-tau comprises a phosphorylated threonine 231 relative to the numbering of the amino acid sequence of SEQ ID NO:1.

In another aspect, the isolated antibody, or antigen binding portion thereof, binds to a human phospho-tau peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 5, 8, and 1 1 , wherein the peptide does not comprise the C-terminal cysteine, but does not bind to a human non-phosphorylated-tau peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:3, 6, 9, or 12, wherein the peptide does not comprise the C-terminal cysteine.

The invention also includes an isolated monoclonal antibody, or antigen binding portion thereof, comprising:

(a) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:14, 16, 18, 20, 22, and 24; and

a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 17, 19, 21 , 23 and 25;

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:14 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:15;

(c) a light chain variable region comprising the amino acid sequence of SEQ ID NO:16 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:17;

(d) a light chain variable region comprising the amino acid sequence of SEQ ID NO:18 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19;

(e) a light chain variable region comprising the amino acid sequence of SEQ ID NO:20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 ;

(f) a light chain variable region comprising the amino acid sequence of SEQ ID NO:22 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23; and

(g) a light chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25.

The invention further includes an isolated monoclonal antibody, or antigen binding portion thereof, comprising:

(a) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:26, 28, 30, 32, 34, and 36;

a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:38, 40, 42, 44, 46, and 48;

a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:50, 52, 54, 56, 58 and 60;

a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:27, 29, 31 , 33, 35 and 37; a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:39, 41 , 43, 45, 47, and 49; and

a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:51 , 53, 55, 57, 59 and 61.

(b) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 26, the CDR2 sequence of SEQ ID NO: 38, and the CDR3 sequence of SEQ ID NO: 50, and a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 27, the CDR2 sequence of SEQ ID NO: 39, and the CDR3 sequence of SEQ ID NO: 51 ;

(c) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 28, the CDR2 sequence of SEQ ID NO: 40, and the CDR3 sequence of SEQ ID NO: 52, and a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 29, the CDR2 sequence of SEQ ID NO: 41 , and the CDR3 sequence of SEQ ID NO: 53;

(d) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 32, the CDR2 sequence of SEQ ID NO: 44, and the CDR3 sequence of SEQ ID NO: 56, and a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 33, the CDR2 sequence of SEQ ID NO: 45, and the CDR3 sequence of SEQ ID NO: 57;

(e) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 34, the CDR2 sequence of SEQ ID NO: 46, and the CDR3 sequence of SEQ ID NO: 58, and a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 35, the CDR2 sequence of SEQ ID NO: 47, and the CDR3 sequence of SEQ ID NO: 59; and

(f) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 36, the CDR2 sequence of SEQ ID NO: 48, and the CDR3 sequence of SEQ ID NO: 60, and a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 37, the CDR2 sequence of SEQ ID NO: 49, and the CDR3 sequence of SEQ ID NO: 61 .

In one aspect, the antibody binds to human phospho-tau with a K_D of 1x10^~7 M or less, and does not substantially bind to human non-phosphorylated-tau, where the phospho-tau comprises a phosphorylated threonine at position 231 relative to the amino acid sequence of SEQ ID NO:1.

The invention also includes a composition comprising an isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1x10^~7 M or less, and further wherein the antibody does not substantially bind to human non- phosphorylated-tau, and a pharmaceutically acceptable carrier.

The invention further includes, an isolated nucleic acid molecule encoding an isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1 x10^~7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau.

The invention also includes an expression vector comprising the isolated nucleic acid molecule. The invention further includes, a host cell comprising the expression vector. The invention includes a method for producing a monoclonal antibody or an antigen-binding portion thereof that specifically bindings to human phospho-tau but does not bind to human non- phosphorylated-tau. The method comprises culturing the host cell under suitable conditions and recovering said antibody or antigen-binding portion.

The invention also includes a method for detecting the presence of human phospho-tau in a sample. The method comprises contacting a sample suspected of comprising phospho-tau with the isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1 x10^~7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau; and detecting the presence of a phospho-tau bound with the antibody thereby detecting phospho-tau in the sample.

The invention also includes, a kit for detecting the presence of human phospho-tau in a sample, the kit comprising the isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1x10^"7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau, an applicator, and an instructional material for the use thereof.

The invention further includes a method for determining the concentration of human phospho-tau in a sample, said method comprising:

(a) providing a labeled competitor comprising phospho-tau coupled to a detectable label;

(b) providing an antibody, or antigen binding fragment thereof, that specifically binds phospho-tau and does not substantially bind non-phospho-tau;

(c) combining the sample, the antibody and the labeled competitor, wherein the phospho-tau in the sample competes with the labeled competitor for binding to the antibody; and

(d) determining the concentration of phospho-tau in said sample by measuring the amount of labeled competitor not bound to antibody by detection of the label.

In one aspect, the selective antibody, or fragment thereof, binds to human phospho-tau with a K_D of 1x10^"7 M or less, and the antibody is derived from a chicken.

In another aspect, the labeled competitor comprises human phospho-tau comprising a phosphorylated threonine 231 relative to the numbering of the amino acid sequence of SEQ ID NO:1 .

The invention includes, a kit for determining the concentration of phospho-tau in a sample, the kit comprising

(a) a labeled competitor comprising phospho-tau coupled to a detectable label;

(b) an antibody, or antigen binding fragment thereof, that specifically binds phospho-tau but does not substantially bind non-phospho-tau;

(c) an applicator; and

(d) an instructional material for the use thereof.

In one aspect, the labeled competitor comprises a phospho-tau comprising a phosphorylated threonine 231 relative to the numbering of the amino acid sequence of SEQ ID NO:1. The invention also includes a competitive immunoassay kit for determining the concentration of phospho-tau in a test sample, the competitive immunoassay comprising:

(a) an antibody, or an antigen binding fragment thereof, that specifically binds phospho-tau and does not substantially bind non-phospho-tau;

(b) a labeled competitor comprising a phospho-tau conjugated to a detectable label;

wherein the labeled competitor competes with the phospho-tau in the test sample for binding with the antibody, and further wherein the label provides a signal indicative of the concentration of phospho-tau in the test sample.

In one aspect, the decrease in labeled competitor bound by the antibody in the test sample compared with the labeled competitor bound by the antibody in an otherwise identical sample that does not contain phospho-tau is an indication of the amount of phospho-tau in the test sample.

The invention also includes a method for identifying a human patient at risk for Alzheimer's disease, comprising:

a) contacting a sample from the patient with the isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1x10^~7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau; b) detecting the presence of any phospho-tau bound by the antibody;

wherein the presence of phospho-tau in a sample indicates that the patient is at risk for Alzheimer's disease.

The invention further includes a method for identifying a human subject at risk for Alzheimer's disease, the method comprising:

a) contacting a sample from the subject with the isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1x10^~7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau; b) detecting the presence of phospho-tau bound by the antibody;

c) comparing the level of phospho-tau in the sample with the level of phospho-tau in an otherwise identical sample from a subject not afflicted with Alzheimer's disease;

d) wherein a higher level of phospho-tau in the sample from the subject at risk compared with the level of phospho-tau in the sample from the subject not afflicted with Alzheimer's disease is an indication that the subject is at risk for Alzheimer's disease.

The invention also includes a kit for identifying a human patient at risk for Alzheimer's disease comprising the isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1x10^"7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau., an applicator, and an instructional material for the use thereof.

The invention includes a method for identifying a compound that inhibits production of phospho- tau, the method comprising contacting a cell producing phospho-tau with a compound and comparing the level of phospho-tau produced by the cell contacted with the compound with the level of phospho-tau produced by an otherwise identical cell not contacted with the compound, wherein the level of phospho- tau is assessed using the isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1x10^"7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau, and wherein a lower level of phospho-tau produced by the cell contacted with the compound compared with the level of phospho-tau produced by the cell not contacted with the compound, is an indication that the compound inhibits production of phospho-tau, thereby identifying a compound that inhibits production of phospho-tau.

The invention also includes a method for treating a disease or disorder associated with increased level of phospho-tau in a patient in need thereof, the method comprising administering a potential therapeutic compound to the patient and comparing the level of phospho-tau in a sample from the patient with the level of phospho tau in an otherwise identical sample obtained from the patient prior to administration of the compound, wherein the level of phospho-tau in a sample is assessed using the isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1 x10^~7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau, and wherein a lower level of phospho-tau in the sample after administration of the compound compared with the level of phospho-tau in the sample before treatment with the compound, is an indication that the compound is a potential therapeutic for treating a disease or disorder associated with an increased level of phospho-tau, thereby treating the disease or disorder.

The invention further includes a method for assessing the effectiveness of a treatment for a disease or disorder associated with an increased level of phospho-tau in a subject, the method comprising administering a treatment to the subject and comparing the level of phospho-tau in a sample obtained from the subject prior to the treatment with the level of phospho-tau in an otherwise identical sample obtained from the subject after the treatment, wherein the level of phospho-tau in a sample is assessed using the isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1x10^~7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau, and further wherein a lower, higher or equal level of phospho-tau in the sample collected from the subject after the treatment compared with the level of phospho-tau in a sample collected from the subject prior to treatment is an indication of the effectiveness of the course of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing the sequence alignment of anti-ptau peptide antibody v-regions with the VH and VL germline clones. CDR and framework definitions follow the Kabat numbering scheme. Figure 2, comprising panels A through D, shows the expression and binding activity of chimeric IgG molecules converted from chicken scFv. Panel A is an image of the Western Blot analysis showing the chimeric IgG molecules converted from scFv clones that bind to pT231/pS235_1 , pS396/pS404_1 , pT212/pS214_1 , respectively. Panels B-D show the result of ELISA analysis of each purified IgG molecule for binding to the phosphopeptide compared with the binding to non-phosphopeptide, and scrambled peptide.

Figure 3, comprising panels A through E, shows the sensorgrams demonstrating specificity of the anti- pTau IgG pT231/pS235_1 for the phosphorylated peptide pT231/pS235. Surface plasmon resonance (SPR)-based kinetic analysis was carried out using an anti-lgG Fc coated CM5-HEL chip (flow cell 1 and 2) upon which pT231/pS235_1 IgG was captured on flow-cell 2 only. Figure 3A shows peptide pT231/pS235, 7.8-0.49nM in a 2-fold dilution series. Figure 3B shows 500nM of the following peptides: non-phosphorylated pT231/pS235; both phosphorylated and non-phosphorylated versions of pT212/pS214 and pS396/pS404 were flowed at a rate of 50μΙ/ιηίη over both flow cells. No binding was observed for any of these peptides. Figure 3C shows peptide pT231 , 62.5nM - 3.91 nM. Figure 3D shows peptide pS235, 250 nM - 3.9nM. Figure 3E shows peptide ρΤ231Δ, 31.25nM - 1 .95 nM. Sensorgram data shown in panels A, C and E fit well to the 1 :1 interaction model (χ² < 0.1 ).

Figure 4, comprising panels A through D, shows photographs depicting the binding of pathological phospho-tau by pT231/pS235_1 antibody in AD and Tg4510 transgenic mouse brains. Figures 4A-4C show immunohistochemical staining of AD brain (Figures 4A and 4B) and healthy brain (Figure 4C). Figure 4B shows photographs depicting a magnification of the boxed area in Figure 4A. Figure 4D depicts a Western blot analysis of Tg4510 transgenic and wild type brain lysates with anti-pT231/pS235 chimeric IgG derived from scFv clone pT231/pS235_1 (plus control anti-GAPDH antibody). Each lane shows a different animal and the lysate samples are: Lanes 1 -2: 3-month old wild type mice; Lanes 3-4: 3-month old transgenic mice; Lanes 5-6: 6-month old wild type mice; Lanes 7-8: 6-month old transgenic mice. Arrowheads indicate phospho-Tau and loading control GAPDH, respectively.

Figure 5, comprising panels 5A through 5C, are drawings of models depicting the structure of the anti- ptau Fab (pT231/pS235_1 ) in complex with phosphoepitope pT231/pS235. Figure 5A shows a cartoon view of the complex. Fab heavy chain (VH+CH1 ) and light chain (VL+CL) are shown in black and gray cartoon, respectively. The phosphoepitope is shown as gray sticks on top with phosphorylated Thr231 (pT231 ) highlighted in black spheres. The solvent P043- ion is labeled and shown in gray spheres. Figure 5B shows a transparent electrostatic surface view of the CDR regions, showing the strong electrostatic interactions between CDR residues and phosphoepitope. The backbone of the bound phosphoepitope is shown as cartoon in gray; the side chains are shown as a stick model in atomic colors (carbon, gray; nitrogen, black; oxygen, darker gray). Side chains of P232, P233 and K234 are omitted for clarity with C shown in spheres. The residues within CDRs that form strong electrostatic interactions with the phosphoepitope are shown as black sticks. A water molecule is labeled as W1 . Hydrogen bonds are shown as dashed lines. Figure 5C shows the interaction details between CDR-H2 and pT231 . Two conformations of R53 side chain are shown as R53 and R53', respectively. Hydrogen bonds are shown as dashed lines. A water molecule is labeled as W2.

Figure 6, comprising panels 6A through 6C, depicts drawings illustrating Fab-phosphoepitope interaction details. Figure 6A shows a schematic representation of all contacts between the Fab (pT231/pS235_1 ) and the phosphoepitope (pT231/pS235) in the Fab phosphoepitope co-crystal structure. Figures 6B and 6C show the interaction details between 225KVAVVR230 and the Fab. Figures 6B and 6C have the same orientation. In Figure 6B, the CDR-H3 is shown as an electrostatic surface model, with positively charged areas in gray and negatively charged areas in black. In Figure 6C, the CDR-H3 is shown as thicker cartoon in darker gray and sticks in atomic colors (carbon, gray; nitrogen, black; oxygen, darker gray). The CDRs from the light chain are shown as cartoon in gray with side chains shown as sticks (carbon, gray; oxygen, darker gray). The peptide 225KVAVVR230 is shown as gray sticks (carbon, gray; nitrogen, black; oxygen, darker gray). Hydrogen bonds are shown as dashed lines. The disulfide bond within the CDR-H3 is shown and labeled as SS bond in Figure 6C.

Figure 7, comprising panels 7A through 7C, depicts diagrams demonstrating Chicken-specific conformations of CDR-L1 and CDR-H3 from anti-ptau Fab (pT231/pS235_1 ). Figure 7A is a ribbon view showing the superposition of chicken CDR-L1 (gray) with canonical mammalian CDR-L1 s, L1 -10-1 (black, Protein Data Bank ID 1YQV) and L1-10-2 (white, Protein Data Bank ID 1AY1 ), respectively. L1 -10-1 and L1-10-2 represent two clusters of mammalian CDR-L1 conformations and show the best structural similarity to chicken CDR-L1. Figure 7B shows a cartoon view showing the conformation of chicken CDR- H3 stabilized by the intramolecular disulfide bond shown as ball and stick model in gray. Two cysteines are labeled as C100B and C100I, respectively. Figure 7C is a cartoon view depicting models of chicken CDRs. CDR-L2, L3, H1 , and H2 are shown.

Figure 8, comprising panels 8A through 8D, shows diagrams depicting sensorgrams demonstrating the specificity of the anti-ptau IgG pT231/pS235_1 for the phosphorylated peptide pT231/pS235. Surface plasmon resonance (SPR)-based kinetic analysis was carried out using an anti-lgG Fc coated CM5-HEL chip (flow cell 1 and 2) upon which pT231/pS235_1 IgG was captured on flow-cell 2 only (flow cell 1 used for referencing). Figure 8A shows peptide pT231/pS235, 7.8-0.49nM in a 2-fold dilution series. Figure 8B shows peptide ρΤ231Δ, 31 .25nM - 1 .95 nM. Figure 8C shows peptide pT231 , 62.5nM - 3.91 nM. Figure 8D shows peptide pS235, 250 nM - 3.9nM.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to isolated monoclonal antibodies, particularly chicken monoclonal antibodies, which bind specifically to phosphorylated tau protein ("phospho-tau", "phosphotau", or "ptau") with high affinity, and, more preferably, that do not substantially bind non-phosphorylated tau protein (non-phospho-tau). In certain embodiments, the antibodies of the invention are derived from particular heavy and light chain germline sequences. The invention provides isolated antibodies, methods of making such antibodies, and pharmaceutical compositions comprising the antibodies. The invention also relates to methods of using the antibodies, such as to detect phospho-tau, as well as to treat diseases associated with increased level of phospho-tau, such as Alzheimer's disease.

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The terms "tau" and "MAPT, DDPAC, FTDP-17, MAPTL, MSTD, MTBT1 , MTBT2 and PPND" all refer to the tau protein, the human version of which has Genbank accession number NP_058519 (SEQ ID NO: 1 ). The terms are used interchangeably, and include variants, isoforms and species homologs of human tau. Accordingly, chicken antibodies of the invention may, in certain cases, cross-react with tau from species other than chicken. In other cases, the antibodies may be completely specific for human tau and may not exhibit species or other types of cross-reactivity.

The terms "phospho-tau" and "ptau" refer to a tau protein that has a phosphate group attached to at least one amino acid.

The terms "hyperphosphorylated tau", "aberrantly phosphorylated tau", and "pathologically phosphorylated tau" refer to a tau protein that has a phosphate group attached to at least one amino acid selected from threonine 212 (T212), serine 214 (S214), threonine 231 (T231 ), serine 235 (S235), serine 396 (S396), serine 404 (S404), and serine 422 (S422), where the numbering of the amino acid residues is relative to the amino acid sequence provided in SEQ ID NO: 1 .

The term "unphosphorylated tau" or "non-phosphorylated tau" refers to a tau protein that does not have phosphate groups attached to an amino acid.

The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of, invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. An "antibody" also refers to an IgA, IgD, IgE, IgG, IgM antibody subtype. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region (C_H). The heavy chain constant region is typically comprised of three domains, C_Hi , C_H2 and C_H3. Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors.

The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply

"antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., tau). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and C_Hi domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and C_Hi domains; (iv) a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., 1989, Nature 341 :544-546), which consists of a V_H domain; and (vi) an isolated complementarity determining region. Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988, Science 242:423-426; and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds phospho-tau is substantially free of antibodies that specifically bind antigens other than phospho-tau). An isolated antibody that specifically binds phospho-tau may, however, have cross-reactivity to other antigens, such as phospho-tau molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The term "chicken antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from chicken germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from chicken germline immunoglobulin sequences. The chicken antibodies of the invention may include amino acid residues not encoded by chicken germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "chicken antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another species, such as a mouse, have been grafted onto chicken framework sequences.

By an "antibody derived from a chicken", is meant an antibody where the antigen-binding fragment thereof is derived from a chicken antibody. That is, an antibody derived from a chicken typically comprises at least one CDR derived from a chicken antibody, and, more preferably, comprises at least two CDRs derived from a chicken antibody, and even more preferably, comprises at least three CDRs derived from a chicken antibody, yet more preferably, comprises at least four CDRs derived from a chicken antibody, more preferably, comprises at least five CDRs derived from a chicken antibody, and even more preferably, comprises six CDRs derived from a chicken antibody.

The term "chicken monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from chicken germline immunoglobulin sequences.

The term "recombinant chicken antibody", as used herein, includes all chicken antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from a host cell transformed to express the chicken antibody, (c) antibodies isolated from a recombinant, combinatorial chicken antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of chicken immunoglobulin gene sequences to other DNA sequences. Such recombinant chicken antibodies comprise variable regions in which the framework and CDR regions are derived from chicken germline immunoglobulin sequences. In certain embodiments, however, such recombinant chicken antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V_H and V_L regions of the recombinant antibodies are sequences that, while derived from and related to chicken germline V_H and V_L sequences, may not naturally exist within the chicken antibody germline repertoire in vivo.

As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG) that is encoded by the heavy chain constant region genes.

The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."

The term "chicken antibody derivatives" refers to any modified form of the chicken antibody, e.g., a conjugate of the antibody and another agent or antibody.

The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of a non-human species, such as a chicken, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a chicken antibody and the constant region sequences are derived from a human antibody. Chimeric antibody can also include an antibody where the V domain and C domain are each derived two different sources even if both are from the same species.

As used herein, an antibody that "specifically binds to phospho-tau" is intended to refer to an antibody that binds to phospho-tau with a K_D of 1 x 10^~7 M or less, alternatively 5 x 10^~8 M or less, alternatively 3 x 10^"8 M or less, alternatively 1 x 10^"8 M or less, or alternatively 5 x 10^"9 M or less.

The term "K_assoc" or "K_a", as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "K_di_s" or "K_d," as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term "K_D", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of K_d to K_a (i.e.,. K_d/K_a) and is expressed as a molar concentration (M). K_D values for antibodies can be determined using methods well established in the art. Another method for determining the K_D of an antibody is by using surface plasmon resonance, optionally using a biosensor system such as a Biacore system.

As used herein, the term "high affinity" for an antibody refers to an antibody having a K_D of 1 x 10^" ⁷ M or less, alternatively 5 x 10^~8 M or less and alternatively 5 x 10^~9 M or less for a target antigen.

As used herein, the term "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows chickens, amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in the following subsections.

Anti-Phospho-Tau Antibodies

The antibodies of the present invention are characterized by the fact that they bind specifically to human phospho-tau. Optionally, an antibody of the invention binds to phospho-tau with high affinity, for example with a K_D of 1 x 10^~7 M or less. The anti-phospho antibody of the invention may bind to phospho- tau with a K_D of 1x10^"7 M or less, and/or does not substantially bind to human non-phosphorylated tau.

Optionally, the antibody binds to phospho-tau with a K_D of 5 x 10^~8 M or less, binds to phospho- tau with a K_D of 2 x 10^"8 M or less, binds to phospho-tau with a K_D of 5x10^"9 M or less, binds to phospho- tau with a K_D of 4x10^"9 M or less, binds to phospho-tau with a K_D of 3x10^"9 M or less, or binds to phospho-tau with a K_D of 2.1 x 10^~9 M or less, a K_D of 1 x 10^~9 M or less, a K_D of 1 x 10^~10 M or less, a K_D of 1 x 10^" M or less, or a K_D of 1 x 10^~12 M or less.

Standard assays to evaluate the binding ability of the antibodies toward phospho-tau are known in the art, including for example, ELISAs, Western blots, radioimmunoassays, and flow cytometry analysis. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by Biacore SPR analysis and Octet analysis.

The antibodies of the invention include chicken monoclonal antibodies pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2. The V_H amino acid sequences of pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2 are shown in SEQ ID NOs: 15, 17, 19, 21 , 23, and 25, respectively. The V_L amino acid sequences of pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2 are shown in SEQ ID NOs: 14, 16, 18, 20, 22 and 24, respectively.

The V_H and V_L sequences may be "mixed and matched" to create other anti-phospho-tau binding molecules of the invention. Phospho-tau binding of such mixed and matched antibodies can be tested using the binding assays described above and in the Examples. Optionally, when V_H and V_L chains are mixed and matched, a V_H sequence from a particular V_H V_L pairing is replaced with a structurally similar V_H sequence. Likewise, optionally a V_L sequence from a particular V_H V_L pairing is replaced with a structurally similar V_L sequence.

Accordingly, in one aspect, the invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising:

In another aspect, the invention provides antibodies that comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2, or combinations thereof. The amino acid sequences of the V_H CDR1s of pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2 are shown in SEQ ID NOs: 27, 29, 31 , 33, 35 and 37, respectively. The amino acid sequences of the V_H CDR2s of pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2 are shown in SEQ ID NOs: 39, 41 , 43, 45, 47 and 49, respectively. The amino acid sequences of the V_H CDR3s of pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2 are shown in SEQ ID NOs: 51 , 53, 55, 57, 59 and 61 , respectively. The amino acid sequences of the V_L CDR1 s of pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2 are shown in SEQ ID NOs: 26, 28, 30, 32, 34 and 36, respectively. The amino acid sequences of the V_L CDR2s of pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2 are shown in SEQ ID NOs: 38, 40, 42, 44, 46 and 48, respectively. The amino acid sequences of the V_L CDR3s of pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , and pS396/pS404_2 are shown in SEQ ID NOs: 50, 52, 54, 56, 58 and 60, respectively. The CDR regions are delineated using the Kabat system (Kabat, E. A., ef al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242 (1991 )).

The heavy and light chain CDRs for the antibody pT231/S235_1 comprise SEQ ID NOs: 26, 27,

38, 39, 50 and 51. The heavy and light chain CDRs for the antibody pT231/S235_2 comprise SEQ ID NOs: 28, 29, 40, 41 , 52 and 53. The heavy and light chain CDRs for the antibody pT212/pS214_1 comprise SEQ ID NOs: 30, 31 , 42, 43, 54, and 55. The heavy and light chain CDRs for the antibody pT212/pS214_2 comprise SEQ ID NOs: 32, 33, 44, 45, 56 and 57. The heavy and light chain CDRs for the antibody pS396/pS404_1 comprise SEQ ID NOs: 34, 35, 46, 47, 58 and 59. The heavy and light chain CDRs for the antibody pS396/pS404_2 comprise SEQ ID NOs: 36, 37, 48, 49, 60 and 61 .

In yet another embodiment, an antibody of the invention comprises heavy and light chain variable regions and/or heavy chain and light chain CDR1s, CDR2s and CDR3s comprising amino acid sequences that are homologous to the amino acid sequences of the antibodies previously described herein, and wherein the antibodies retain the desired functional properties of the anti-phospho-tau antibodies of the invention.

In an alternative embodiment the phospho-tau specific antibody of the invention specifically binds to at least one amino acid selected from threonine 212 (T212), serine 214 (S214), threonine 231 (T231 ), serine 235 (S235), serine 396 (S396), serine 404 (S404), and serine 422 (S422) where the numbering of the amino acid residues is relative to the amino acid sequence provided in SEQ ID NO: 1 .

In another embodiment, the phospho-tau specific antibody of the invention may bind a phospho- tau phosphorylated at least two, preferably at least three, more preferably four, even more preferably five, yet more preferably six, preferably seven, or even more preferably, eight amino acid residues selected from selected from threonine 212 (T212), serine 214 (S214), threonine 231 (T231 ), serine 235 (S235), serine 396 (S396), serine 404 (S404), and serine 422 (S422) where the numbering of the amino acid residues is relative to the amino acid sequence provided in SEQ ID NO: 1. In one embodiment, the antibody pT231/pS235_1 or pT231/pS235_2 may bind a phospho-tau phosphorylated at both amino acid residues threonine 231 (T231 ) and serine 235 (S235), where the numbering of the amino acid residues is relative to the amino acid sequence provided in SEQ ID NO: 1. In another embodiment, the antibody pT231/pS235_1 or pT231/pS235_2 may bind a phospho-tau phosphorylated at either amino acid residue threonine 231 (T231 ) or serine 235 (S235), where the numbering of the amino acid residues is relative to the amino acid sequence provided in SEQ ID NO: 1.

In yet another embodiment, the phospho-tau specific antibody of the invention specifically binds a phosphorylated tau peptide comprising a phosphorylated threonine 231 (T231 ), but does not bind a tau peptide where threonine 231 is not phosphorylated, where the numbering of the amino acid residues is relative to the amino acid sequence provided in SEQ ID NO: 1 . That is, the antibody will bind where T231 is phosphorylated (pT231 ), but will not substantially bind a tau peptide where T231 is not phosphorylated, even if other amino acid residues of the tau peptide are phosphorylated.

In various embodiments, the antibody can be, for example, a chicken antibody, a humanized antibody or a chimeric antibody derived from a chicken antibody. In other embodiments, the V_H and/or V_L amino acid sequences may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences set forth above. As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

In certain embodiments, an antibody of the invention comprises heavy and light chain variable regions and/or heavy chain and light chain CDR1 s, CDR2s and CDR3s, wherein one or more of these sequences comprise specified amino acid sequences based on the antibodies described herein, or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-phospho-tau antibodies of the invention.

In alternative embodiments, the antibody can be, for example, chicken antibodies, humanized antibodies or chimeric antibodies.

As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar 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, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function using the functional assays described herein.

In another embodiment, the invention provides an antibody that binds to the same epitope on human phospho-tau as the phospho-tau antibody of the invention (i.e., an antibody that has the ability to compete for binding to phospho-tau with an antibody of the invention). In alternative embodiments, the reference antibody for competition studies can be the monoclonal antibody pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , or pS396/pS404_2. Such competing antibodies can be identified based on their ability to compete with pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , or pS396/pS404_2 in standard phospho-tau binding assays. For example, Biacore analysis, Octect analysis, ELISA assays or flow cytometry may be used to demonstrate cross-competition with the antibodies of the current invention. The ability of a test antibody to inhibit the binding of, for example, pT231/S235_1 or pT231/S235_2, to human phospho-tau demonstrates that the test antibody can compete with pT231/S235_1 or pT231/S235_2 for binding to human phospho-tau and thus may bind the same epitope on human phospho-tau as pT231/S235_1 or pS231/T235_2. In a further embodiment, the antibody that binds to the same epitope on human phospho-tau as pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , or pS396/pS404_2 is a chicken monoclonal antibody. Such chicken monoclonal antibodies can be prepared and isolated as described in the Examples or by a wide variety of methods well-known in the art. In other embodiments, the antibody that competes with the antibody is a human, humanized or mouse antibody.

An antibody of the invention can be prepared using an antibody comprising at least one of the V_H and/or V_L sequence disclosed herein as starting material to engineer a modified antibody, which may have properties that differ from the starting antibody but which may bind the same, or substantially the same, epitope as the starting material antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., V_H and/or V_L), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

The present invention provides nucleic acids encoding the phospho-tau specific antibody of the invention. Nucleic acids encoding the antibodies of the invention can be generated by methods known in the art. Also, as would be understood by one skilled in the art, due to the degeneracy of the nucleic acid code, a wide variety of nucleic acid sequences can encode the amino acid sequence of the antibody of the invention.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., Nature 332:323-327 (1998); Jones et al. Nature 321 :522-525 (1986); Queen et al,. Proc. Natl. Acad. See. U.S.A. 86:10029-10033 (1989); U.S. Patent Nos. 5,225,539; 5,530,101 ; 5,585,089; 5,693,762 and 6,180,370)).

Accordingly, another embodiment of the invention pertains to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1 , CDR2, and CDR3 sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 29, 31 , 33, 35 and 37; SEQ ID NOs: 39, 41 , 43, 45, 47 and 49; and SEQ ID NOs: 51 , 53, 55, 57, 59 and 61 , respectively, and a light chain variable region comprising CDR1 , CDR2, and CDR3 sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, 30, 32, 34 and 36; SEQ ID NOs: 38, 40, 42, 44, 46, and 48; and SEQ ID NOs: 50, 52, 54, 56, 58 and 60, respectively. Such antibodies contain the V_H and V_L CDR sequences of monoclonal antibodies pT231/S235_1 , pT231/S235_2, pT212/pS214_1 , pT212/pS214_2, pS396/pS404_1 , or pS396/pS404_2, which may contain a different framework sequence from the antibodies. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.

Another type of variable region modification is to mutate amino acid residues within the V_H and/or V_L CDR1 , CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. Optionally conservative modifications (as discussed above) are introduced. The mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

In yet another embodiment, the invention provides isolated anti-phospho-tau monoclonal antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising: (a) a V_H CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 29, 31 , 33, 35 and 37, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs: 27, 29, 31 , 33, 35 and 37; (b) a V_H CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 41 , 43, 45, 47 and 49, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs: 39, 41 , 43, 45, 47 and 49; (c) a V_H CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 51 , 53, 55, 57, 59 and 61 , or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs: 51 , 53, 55, 57, 59 and 61 ; (d) a V_L CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, 30, 32, 34 and 36, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs: 26, 28, 30, 32, 34 and 36; (e) a V_L CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 40, 42, 44, 46, and 48, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs: 38, 40, 42, 44, 46, and 48; and (f) a V_L CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 52, 54, 56, 58 and 60, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs: 50, 52, 54, 56, 58 and 60.

Engineered antibodies of the invention include those in which modifications have been made to framework residues within V_H and/or V_L, e.g., to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence (also referred to as "germlining"). More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 2003/0153043. In addition to, or alternative to, modifications made within the framework or CDR regions, antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the invention may be chemically modified (e.g. , one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation. Each of these embodiments is described in further detail below.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made. Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen.

Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation.

Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g. , serum) half life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polymers including polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Alternatively, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. Methods of Engineering Antibodies

As discussed above, the anti-phospho-tau antibodies having V_H and V_L sequences disclosed herein can be used to create new anti-phospho-tau antibodies by modifying the V_H and/or V_L sequences, or the constant region(s) attached thereto. Thus, in another aspect of the invention, the structural features of an anti-phospho-tau antibody of the invention are used to create structurally related anti- phospho-tau antibodies that retain at least one functional property of the antibodies of the invention, such as binding to human phospho-tau. For example, one or more CDR regions of pS231/T235_1 or pS231/T235_2, or mutations thereof, can be combined recombinantly with known framework regions and/or other CDRs to create additional, recombinantly-engineered, anti-phospho-tau antibodies, as discussed above. Other types of modifications include those described in the previous section. The starting material for the engineering method is one or more of the V_H and/or V_L sequences provided herein, or one or more CDR regions thereof. To create the engineered antibody, it is not necessary to actually prepare (i.e. , express as a protein) an antibody having one or more of the V_H and/or V_L sequences provided herein, or one or more CDR regions thereof. Rather, the information contained in the sequence(s) is used as the starting material to create a "second generation" sequence(s) derived from the original sequence(s) and then the "second generation" sequence(s) is prepared and expressed as a protein.

Standard molecular biology techniques can be used to prepare and express the altered antibody sequence.

Optionally, the antibody encoded by the altered antibody sequence(s) is one that retains one, some, or all of the functional properties of the anti-phospho-tau antibodies described herein, which functional properties include, but are not limited to:

(i) binds to human phospho-tau with a K_D of 1x10^~7 M or less; and

(ii) does not substantially bind to non-phosphorylated tau.

The functional properties of the altered antibodies can be assessed using standard assays available in the art and/or described herein, as well as those known in the art or those discovered in the future.

In certain embodiments of the methods of engineering antibodies of the invention, mutations can be introduced randomly or selectively along all or part of an anti-phospho-tau antibody coding sequence and the resulting modified anti-phospho-tau antibodies can be screened for binding activity and/or other functional properties as described herein. Mutational methods are well-known in the art.

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology. Chicken monoclonal antibodies of the invention can also be prepared using phage display methods for screening libraries of chicken immunoglobulin genes. Such phage display methods for isolating chicken antibodies are established in the art and encompass phage display libraries prepared from immunized chickens or naive phage display libraries where the original starting material is not derived from immunized chickens.

The chicken monoclonal antibodies of the invention can also be prepared by culturing a host cell comprising a nucleic acid encoding the chicken monoclonal antibody under suitable conditions and recovering said antibody, or antigen binding portion thereof. Such host cell culturing methods are well- known in the art.

Immunization of Chickens

When chickens are used to raise antibodies of the invention, such chickens can be immunized with a purified or enriched preparation of phospho-tau antigen and/or recombinant phospho-tau, or an phospho-tau fusion protein (Lonberg et al., 1994, Nature 368(6474):856-859; Fishwild et al., 1996, Nature Biotechnology 14:845-851 ; PCT Publication WO 98/24884). Pharmaceutical Compositions

In another aspect, the present invention provides a composition (e.g., a pharmaceutical composition) containing one or a combination of monoclonal antibodies, or antigen-binding portion(s) thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) antibodies. For example, a pharmaceutical composition of the invention can comprise a combination of antibodies that bind to different epitopes on the target antigen or that have complementary activities.

Pharmaceutical compositions of the invention also can be administered in combination therapy. For example, the combination therapy can include an anti-phospho-tau antibody of the present invention combined with at least one other anti-inflammatory or immunosuppressant agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the antibodies of the invention.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Optionally, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration. Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as Ν,Ν'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, alternative methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, from about 0.1 per cent to about 70 per cent, or from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.

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

For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Other dosage regimens for an anti-phospho-tau antibody of the invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg /ml and in some methods about 25-300 μg /ml.

Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dosage" of an anti-phospho-tau antibody of the invention results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom- free periods, or a prevention of impairment or disability due to the disease affliction One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

A composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Alternative routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, an antibody of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Therapeutic compositions can be administered with medical devices known in the art. For example, in another embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device.

In certain embodiments, an antibody of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery. Exemplary targeting moieties include folate or biotin; mannosides; antibodies; surfactant protein A receptor; and p120, among many others.

Uses and Methods of the Invention

The antibodies, antibody compositions, and methods of the present invention have numerous in vitro and in vivo utilities including immunoassays, and use for the assessment and treatment of phospho- tau mediated disorders. As used herein, the term "subject" is intended to include human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non- human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Other subjects include human patients having disorders mediated by or associated with phospho-tau level. The methods are particularly suitable for assessing and treating human patients having a disorder associated with the existence of phospho-tau and, more preferably, with an increased level of phospho-tau, where increased level of phospho-tau encompasses an increased level of the amount of tau comprising at least one phosphorylated amino acid residue and/or an increase in the number of phosphorylated amino acid residues in a tau peptide.

The invention provides a method for detecting the presence of human phospho-tau in a sample, the method comprising contacting a sample suspected of comprising phospho-tau an antibody specific for phospho-tau, and detecting the presence of a phospho-tau bound with the antibody thereby detecting phospho-tau in the sample. Methods for detecting a phospho-tau bound with the antibody are well-known in the art including, but not limited to, an assay where an anti-phospho-tau is bound to a solid support and a sample is added thereto allowing the antibody to bind phospho-tau in the sample. A second anti- phospho-tau antibody that is either the same or different from the antibody bound to the solid support is added and can be detected by either direct labeling (i.e., the second antibody is conjugated to a detectable label) or by adding a third antibody, e.g., from another species which reacts with the constant domain of the second antibody and which comprises a detectable label. Thus, the assay can be used to detect the presence or absence of phospho-tau in a sample.

In another embodiment, the invention includes a kit for detecting the presence of human phospho-tau in a sample, the kit comprising an antibody specific for phospho-tau, an applicator, and an instructional material for the use thereof.

The invention also provides a method for determining the concentration of human phospho-tau in a sample, said method comprising providing a labeled competitor comprising phospho-tau coupled to a detectable label; providing an antibody, or antigen binding fragment thereof, that specifically binds phospho-tau and does not substantially bind non-phospho-tau; combining the sample, the antibody and the labeled competitor, wherein the phospho-tau in the sample competes with the labeled competitor for binding to the antibody; and determining the concentration of phospho-tau in said sample by measuring the amount of labeled competitor not bound to antibody by detection of the label. That is, the labeled competitor, which is configured such that it directly competes with phospho-tau which is not labeled, is not able to bind the antibody because non-labeled phospho-tau present in the sample is bound thereto. The amount of labeled competitor bound to the antibody in the absence of the sample is compared with the amount of labeled competitor bound to the antibody when the sample is added. Thus, the amount of decrease of bound labeled-competitor in the presence of the sample is an indicator of the amount of non- labeled phospho-tau present in the sample such that the assay can be used to assess the presence and level of phospho-tau in a sample.

In an alternative embodiment, the selective antibody, or fragment thereof, wherein the antibody binds to human phospho-tau with a K_D of 1x10^~7 M or less, and the antibody is derived from a chicken.

In yet another embodiment, the method of claim 25, wherein the labeled competitor comprises human phospho-tau comprising a phosphorylated threonine 231 relative to the numbering of the amino acid sequence of SEQ ID NO:1.

In one embodiment, the invention provides a kit for determining the concentration of phospho-tau in a sample, the kit comprising a labeled competitor comprising phospho-tau coupled to a detectable label; an antibody, or antigen binding fragment thereof, that specifically binds phospho-tau but does not substantially bind non-phospho-tau; an applicator; and an instructional material for the use thereof. In a further embodiment, the labeled competitor comprises a phospho-tau comprising a phosphorylated threonine 231 relative to the numbering of the amino acid sequence of SEQ ID NO:1.

The invention further provides, a competitive immunoassay kit for determining the amount of phospho-tau in a test sample, the competitive immunoassay comprising an antibody, or an antigen binding fragment thereof, that specifically binds phospho-tau and does not substantially bind non- phospho-tau; a labeled competitor comprising a phospho-tau conjugated to a detectable label; wherein the labeled competitor competes with the phospho-tau in the test sample for binding with the antibody, and further wherein the label provides a signal indicative of the amount of phospho-tau in the test sample. In an exemplary embodiment, the decrease in label bound by the antibody in the test sample compared with the label bound by the antibody in an otherwise identical sample that does not contain phospho-tau is an indication of the amount of phospho-tau in the test sample.

The invention includes a method for identifying a human patient at risk for Alzheimer's disease, comprising contacting a sample from the patient with a phospho-tau specific antibody; detecting the presence of any phospho-tau bound by the antibody; wherein the presence of phospho-tau in a sample indicates that the patient is at risk for Alzheimer's disease.

The invention also provides a method for identifying a human subject at risk for Alzheimer's disease, the method comprising contacting a sample from the subject with a phospho-tau specific antibody; detecting the presence of phospho-tau bound by the antibody; comparing the level of phospho- tau in the sample with the level of phospho-tau in an otherwise identical sample from a subject not afflicted with Alzheimer's disease; wherein a higher level of phospho-tau in the sample from the subject at risk compared with the level of phospho-tau in the sample from the subject not afflicted with Alzheimer's disease is an indication that the subject is at risk for Alzheimer's disease.

In an alternative embodiment, the invention provides a kit for identifying a human patient at risk for Alzheimer's disease comprising a phospho-tau specific antibody, an applicator, and an instructional material for the use thereof.

The invention further provides a method for identifying a compound that inhibits production of phospho-tau, the method comprising contacting a cell producing phospho-tau with a compound and comparing the level of phospho-tau produced by the cell contacted with the compound with the level of phospho-tau produced by an otherwise identical cell not contacted with the compound, wherein the level of phospho-tau is assessed using a phospho-tau specific antibody, and wherein a lower level of phospho- tau produced by the cell contacted with the compound compared with the level of phospho-tau produced by the cell not contacted with the compound, is an indication that the compound inhibits production of phospho-tau, thereby identifying a compound that inhibits production of phospho-tau.

The invention also alternative provides a method for treating a disease or disorder associated with increased level of phospho-tau in a patient in need thereof, the method comprising administering a potential therapeutic compound to the patient and comparing the level of phospho-tau in a sample from the patient with the level of phospho tau in an otherwise identical sample obtained from the patient prior to administration of the compound, wherein the level of phospho-tau in a sample is assessed using phospho-tau specific antibody, and wherein a lower level of phospho-tau in the sample after

administration of the compound compared with the level of phospho-tau in the sample before treatment with the compound, is an indication that the compound is a potential therapeutic for treating a disease or disorder associated with an increased level of phospho-tau, thereby treating the disease or disorder.

In one embodiment, the invention provides a method for assessing the effectiveness of a treatment for a disease or disorder associated with an increased level of phospho-tau in a subject, the method comprising administering a treatment to the subject and comparing the level of phospho-tau in a sample obtained from the subject prior to the treatment with the level of phospho-tau in an otherwise identical sample obtained from the subject after the treatment, wherein the level of phospho-tau in a sample is assessed using phospho-tau specific antibody, and further wherein a lower, higher or equal level of phospho-tau in the sample collected from the subject after the treatment compared with the level of phospho-tau in a sample collected from the subject prior to treatment is an indication of the effectiveness of the course of treatment.

The term "labeled," with regard to the phospho-tau specific antibody or labeled competitor, includes direct labeling by coupling (i.e., physically linking) a detectable substance to the antibody or labeled competitor, as well as indirect labeling of the antibody or labeled competitor by coupling it with another reagent that is directly labeled. An example of indirect labeling includes detection of a primary antibody using a fluorescent-labeled secondary antibody. In vitro techniques for detection of a polypeptides of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitation, and immunofluorescence.

The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject.

The antibodies, labeled competitors, and potential therapeutic compounds described herein are also suitable for use with any of a number of other homogeneous and heterogeneous immunoassays with a range of detection systems.

The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLES Example 1. Phaqemid Library Constructions, Selection and Screening

Phospho, non-phospho, and scrambled peptides were synthesized by Open Biosystems. A cysteine, which is not present in the sequence of natural human tau, was added to the C-terminus of each peptide to facilitate linking the peptide to other moieties, including biotinylation and KLH conjugation of the peptide. The sequences of these peptides are shown in the Table 1 below.

Table 1.

Sequences of peptides used in the study

pS396/pS404

Phospho EIVYK(pS)PVVSGDT(pS)PRHLC SEQ ID NO:2

Non-phospho EIVYKSPVVSGDTSPRHLC SEQ ID NO:3

Scrambled RIGVH(pS)PELKVSY(pS)PDVTC SEQ ID NO:4

pS422 Phospho GSIDMVD(pS)PQLATLC SEQ ID NO:5

Non-phospho GSIDMVDSPQLATLC SEQ ID NO:6

Scrambled TLDLSAG(pS)PDMIQVC SEQ ID NO:7

pT212/pS214

Phospho GSRSR(pT)P(pS)LPTPPTRC SEQ ID NO:8

Non-phospho GSRSRTPSLPTPPTRC SEQ ID NO:9

Scrambled TRGPS(pT)P(pS)PRSRTPLC SEQ ID NO:10

pT231/pS235

Phospho KKVAVVR(pT)PPK(pS)PSSAKC SEQ ID NO:11

Non-phospho KKVAVVRTPPKSPSSAKC SEQ ID NO:12

Scrambled KAVKSKP(pT)PSR(pS)PAVKVC SEQ ID NO:13

The immunogen preparation consisted of all four phospho-peptides, which had been separately conjugated to the carrier protein KLH, then pooled at equal concentration. Three chickens were then immunized with a mixture of 200 μg pooled antigen in CFA (Charles River Laboratories). The chickens received three additional injections with the same antigen mixed with IFA at 21 -day intervals. Serum antibody response to the immunogens was monitored using a standard direct ELISA (Finlay et al., Methods Mol Biol 681 , 383-401 (2011 )). Spleen and bone marrow from the two responding chickens were isolated and RNA extraction, V_H and V_L repertoire PCR amplification and scFv phagemid library construction were performed as previously described (Finlay et al., Methods Mol Biol 681 , 383-401 (2011 )).

Selections were carried out using a solution-phase protocol using biotinylated peptides and streptavidin bead capture. In brief: after blocking the phage library with 3% (w/v) skim milk/PBS, the library was subsequently incubated with biotinylated phosphopeptide, in the presence of 10-fold molar excess cognate non-phospho and scrambled phosphopeptides as competitors (both non-biotinylated). The phage-scFv/phosphopeptide complex was captured onto streptavidin beads and washing/elution/reinfection steps were performed as described previously (Finlay et. al., Methods Mol Biol 681 :383-401 (201 1 )). A total of three to four rounds of selections were carried out per peptide. Single bacterial clones were then picked from each selection round and cultured to produce phage-scFv or periplasmic extracts and to perform direct binding ELISA as described previously (Finlay et. al., Methods Mol Biol 681 :383-401 (201 1 ); Cummins et al., J Immunol Methods 339:38-46 (2008)).

From each of the chickens that developed an antibody response to ptau peptides, spleen and bone marrow RNA was isolated and a pooled scFv phagemid library of 3.8 x 10⁸ cfu was constructed. This library was rescued and a series of solution-phase selections were then carried out using biotinylated versions of each of the ptau peptides in the presence of a 10-fold excess of their respective non-biotinylated scrambled phosphopeptide and non-phosphopeptide. Screening of 100-800 clones after the third and fourth rounds of selections was carried out by ELISA for binding of phage scFv to plate-immobilized phospho and non-phospho tau peptides. For each of the three peptides, binders specific to phosphopeptide were isolated.

The DNA sequences of the phospho-specific clones were then obtained. A total of 12 unique binders for pT231/pS235, 5 unique binders for pS396/pS404, and 3 unique binders for pT212/pS214 were identified, respectively. The alignment of the V_H and V_L sequences for the two most dominant binders for each phosphoepitope in comparison with chicken V_H and V_L germline sequences is shown in Figure 1 . Each of these clones was found to have the high framework uniformity that is typical to chicken antibodies (Wu et al., 2011 , J. Immunol. 188:322-333). They are highly divergent in their V_H CDR3 sequences, however, with the exception of pT231/pS235_1 and pT231/pS235_2, which have identical heavy chain sequences, but exhibit promiscuity in light chain usage. Clones pT212/pS214_1 , pT212/pS214_2 and pS396/pS404_1 are "Type 2" chicken V_H, while clones pT231/pS235_1 , pT231/pS235_2 and pS396/pS404_2 all contain a pair of non-canonical cysteine residues in the CDR-H3 and are representatives of the major structural "Type 1 " chicken V_H (Wu et al., 2011 , J. Immunol. 188:322-333).

During the phage selection and screening processes, a small number of clones that bound to the non-phospho peptides were also obtained. Modification of the phage selection conditions and competition with non-phosphopeptide and scrambled peptide was critical for the isolation of phospho-specific binders. Phospho-specific binders were not isolated in the absence of competitive peptides. By simple modification of the selection pressures applied, antibodies with the desired properties were enriched and those that were not desired were actively deselected. Affinity analyses using Biacore clearly demonstrated that the failure of non-deselected phage pannings was not due to the lack of high affinity antibodies in the library. These observations highlight the utility of the combined immunization and selection approach, proving that highly specific clones of low nM binding affinity can be generated against phosphoepitopes, despite the tendency of the immune system to predominantly generate a non-phospho- specific response. As described below, the analyses showed clearly that the lead clones identified are truly specific and incapable of binding other phospho or non-phospho peptides in either avid solid-state or highly sensitive biosensor 1 :1 binding methods. Example 2. Conversion of scFv to Chimeric IqG

V_H and V_L sequences were amplified by PCR from each scFv clone and inserted into expression vectors containing the coding sequences for the constant regions of human lgG1 (Finlay, et al., J Mol Biol 388:541-558 (2009)). Expression vectors encoding the converted full-length heavy and light chains of IgG were co-transfected into COS cells using Lipofectamin (InVitrogen). Twenty four hours post transfection, the media was changed to 293 serum-free media (InVitrogen) which was harvested after a further 48 h. The expression of IgG in the conditioned media (CM) was analyzed by Western blot using HRP conjugated anti-Fc to detect the heavy chain. The phospho-peptide binding activity of the CM was also determined by ELISA, as previously described in Example 1 .

As shown in Fig. 2A, the converted IgG molecules are well expressed as examined by Western analysis of the conditioned media from transiently transfected COS cells. Importantly, these chimeric antibodies preserved the binding specificity to each phospho-peptide when tested in ligand binding ELISA. Each of the IgGs showed avid, dose dependent binding to phospho-peptide, but no detectable binding to either non-phospho or control scrambled peptides (Fig. 2B-D).

Example 3. Immunostaininq of AD Brain

To test whether these phospho-tau specific chicken antibodies can recognize the phospho-tau epitopes in full-length tau, the antibodies were tested in immunohistochemistry studies using human AD brain samples. Formalin-fixed paraffin-embedded tissue sections (5 μιη thick) from Braak stage VI human AD brain (Sun Health Research Institute) were analyzed by immunohistochemistry. Slides were dehydrated and heat-induced antigen (epitope) retrieval was performed using a wet-heat treatment at 122 °C in Reveal buffer solution, pH 6 (Biocare Medical). After blocking for endogenous peroxidase activity with 3% H₂0₂ and for unspecific protein interactions with 10% (v/v) goat serum (Jackson ImmunoResearch), sections were incubated with chimeric anti-phospho tau IgG at a concentration of 1 μg ml. Primary antibody was detected with 0.1 μg ml Rabbit anti-human IgG (Jackson ImmunoResearch) and detected using an Envision System+ Kit (Dako). Labeled slides were counterstained with Mayer's Hematoxylin nuclear stain (Dako).

As shown in Fig. 4, the chimeric antibody pT231/pS235_1 showed highly specific staining patterns only in the AD brain samples but not in normal patient brain samples (compare Fig. 4A with 4C). The staining of neurofibrillary tangles was also observed (Fig. 4B). Example 4. Preparation and Western blot analysis of mouse brain lysates.

Mouse brains were harvested, cut in half, immediately frozen on dry ice and kept at -80 °C until use. The hemibrains without cerebellum were rinsed in PBS, homogenized using a Dounce homogenizer in a lysis buffer from the Roche Immunoprecipitation kit (Roche) and processed according to the manufacturer's protocol. Protein measurements were performed using a BCA kit (Pierce). Brain homogenates (28 μg total protein/well) were loaded onto 4-12% NuPage Novex Bis-Tris mini gels (Invitrogen) and electophoresed according to the manufacturer's instructions. The gels were then blotted onto nitrocellulose membrane using the iBIot™ transfer system (Invitrogen). After blocking with Blocking buffer (Li-Cor), the blot was incubated with anti-phospho tau antibodies and anti-GAPDH (LifeSpan Biosciences), the latter of which was used as a loading control. Blots were washed three times with 0.1 % (v/v) TPBS, and incubated with the rabbit anti-human IgG secondary antibody (Jackson ImmunoResearch Labs). The blot was washed and incubated with fluorescently labeled detection antibodies (AlexaFluor680, Invitrogen and IRDye800, Rockland). The blot was washed and visualized using Li-Cor's Odyssey infrared imaging system.

In western blot analysis, the antibody pT231/pS235_1 recognized phospho-tau in the brain of 6- month old tauP301 L Tg4510 transgenic mice (Fig. 4D, Lanes 7-8), but not in young (3-month old) transgenic mice (Fig. 4D, Lanes 3-4) or wildtype control mice at either 3 months or 6 months of age (Fig. 4D, Lanes 1-2 and 5-6). The results are consistent with the report that these transgenic mice carrying the P301 L mutation develop tau tangles constituting hyperphosphorylated tau at 4-6 months of age (Santacruz Science 309, 476-481 (2005)). Example 5. Affinity determination by BIAcore analysis

To establish the affinities of the IgGs for their respective phosphopeptide epitopes, binding kinetics analyses for the 6 clones shown in Figure 1 were performed using Biacore technology. To ensure 1 :1 interaction kinetics, the IgGs were captured on anti-human IgG CM5 chips and binding responses were measured with peptide in the mobile phase.

BIAcore analysis was performed using the T100 biosensor, series S CM5 chips, an amine- coupling kit, 10 mM Sodium acetate immobilization buffer at pH 5.0, 1 X HBS-EP running buffer, and 3M MgCI₂ (GE Healthcare). A targeted immobilization program was set to immobilize approximately 8000 response units of anti-human IgG (Fc) (GE Healthcare) at pH 5 on flow cells 1 and 2 only, followed by the capture of 1000-2500 RUs of each respective anti-phospho tau IgG on flow cell 2 only. For calculation of kinetic constants using global fit analysis, the phospho tau peptides were diluted in 1x HBS-EP running buffer (1500 -15.62 nM (2-fold dilution series)). Each concentration was injected over both flow cells 1 and 2 for 3 min at a flow rate of 50 μΙ/min (in order to minimize the potential for mass transfer or rebinding events) and allowed to dissociate for 3 minutes, followed by a 5 second regeneration pulse with 5 M MgCI₂. Reference subtracted sensorgrams (flow cell 2 minus 1 ) for each concentration were analyzed using the kinetics 1 :1 binding program within the BIAcore T100 evaluation software 1 .1.1.

These analyses showed that the 1 :1 affinities ranged from ~2 μΜ up to a high of 3 nM (Table 2).

Table 2.

Comparative kinetic analysis of anti-pTAU IgGs for their respective targets

Clone Interacting Peptide k_a (1/Ms) 1/s) K_D (nM)

Hit 1 p231 2.390 x 10³ 7.344 x 10^~4 3.073

Hit 2 p231 5.260 x 10³ 0.005 8.765

Hit 4 p396 1.518 x 10° 0.31 1 2045

Hit 5 p396 6.343 x 10³ 1.216 1917

Hit 6 p212 8.297 x 10⁴ 0.008 97.91

Hit 7 p212 2.738 x 10³ 0.226 825.1 As final confirmation of the specificity data set forth in Figure 2, a secondary Biacore analysis was performed in which the non-phospho and scrambled versions of all peptides used in this study were tested for binding compared to the highest affinity clone pT231/pS235_1 (Fig. 3A). At 31.5nM, a classical binding profile was observed for the pT231/pS235 peptide, but no binding signal at all could be seen for any other peptide, despite the use of 500nM peptide in the mobile phase (Fig. 3B).

Based on the co-crystal structure of the pT231/pS235_1 Fab and pT231/pS235 peptides described in Examples 7 and 8, the necessity for pT231 and pS235 phosphorylation in the binding of pT231/pS235_1 was examined. A series of modified peptides were synthesized and subjected to binding affinity analyses, as outlined above. Peptide pT231 lacked the phosphorylation at position S235, while ρΤ231Δ had the full c-terminal sequence after position K234 was removed. Both peptides retained binding at a slightly reduced affinity in comparison to the pT231/pS235 peptide (Table 3, Figures 3C, 3E, 8B, and 8E). Peptide pS235 lacked the phosphorylation at position T231 and showed no measurable binding signal, proving the necessity of pT231 for binding affinity of the antibody (Figures 3D and 8D).

Table 3

SPR kinetic analyses of anti-ptau IgGs for their respective targets

This finding confirmed that the identified clones of exemplary phosphospecificity do not detectably bind to non-cognate phospho or non-phospho epitopes under either avid solid-state (Fig. 2), or 1 :1 binding conditions (Figures 3 and 8). Further the biosensor studies corroborated the binding mechanism suggested by the co-crystal structure described in Examples 7 and 8, which showed the phosphorylation event at position T231 to be the critical element in the interaction between the paratope of pT231/pS235_1 and the phosphoepitope. When this residue was de-phosphorylated, all affinity for the peptide was lost. These data demonstrate for the first time, an antibody demonstrating exquisite sensitivity wherein the antibody specifically binds the tau peptide when T231 is phosphorylated but which does not bind the otherwise identical tau peptide where T231 is not phosphorylated.

Example 6. Antibody (IqG) expression and Fab Preparation. DNA expression vectors encoding the converted full-length heavy and light chains of anti-ptau antibody IgG were used to transiently co-transfect CHO-S cells (InVitrogen) using CHO-MAX™ transfection reagent according to the manufacturer's protocol. After transfection, the cells were grown serum-free in suspension cultures using FreeStyle™ CHO Expression Medium (Cat#12651022) containing 8mM L-glutamine at 37 C in a humidified 8% C0₂ environment. After 120 hours post- transfection, the cells were removed from the culture by centrifugation. Anti-ptau IgG conditioned media (CM) was filtered through 0.22 μιη filter before loading onto 5 ml HiTrap rProtein A FF column (GE Healthcare). The column was pre-equilibrated with binding buffer (100 mM NaCI, 20 mM Tris, pH 7.0). After loading the CM, the column was washed with 5 column volumes (CV) of binding buffer, then washed with 5 CV of elution buffer (100 mM NaCI, 100 mM Acetic Acid, pH 3.0). The elution fraction was neutralized by immediate addition of an equal volume of neutralization buffer (1 M Tris, pH 7.9). The pooled fraction (IgG) was then concentrated and dialyzed into Fab preparation buffer. Fab fragment was prepared and purified according to the standard protocol provided by Pierce Fab Preparation Kit (Thermo Scientific).

Example 7. Crystallization and Data Collection

pT231/pS235_1 Fab was formulated at 14.9 mg/ml in 20 mM Tris, 100 mM NaCI (pH 7.0), 10 mM phosphor-peptide (₂₂₄KKVAVVR-(pT₂₃i)-PPK-(pS₂₃₅)-PSSAKC₂4i ) was added and mixed before crystallization trials. The molar ratio of Fab-to-peptide was 1 :1.2. Crystals of Fab-peptide were grown at 18 ± 1 °C using the sitting-drop vapor diffusion method. Each drop contained 0.15 μΙ Fab-peptide mixture and 0.15 μΙ reservoir solution containing 200 mM NH₄H₂P0₄ and 40 % MPD. The crystals appeared after approximately two months, at a final size of 0.1 ^χ 0.4 ^χ 0.05 mm³. X-ray diffraction data were collected remotely at beam line 17-ID of the Industrial Macromolecular Crystallography Association Collaborative Access Team (IMCA-CAT) at the Advanced Photon Source (APS), Argonne National Laboratory. Data processing was carried out with the HKL2000 program (Otwinowski & Minor, Methods Enzymol 276:307- 326 (1997)) and AutoPROC™ (Vonrhein et al., 201 1 , Acta. Crystallogr. D. Biol. Crystallogr. 67:293-302). Final data statistics were generated using AutoPROC. Crystal data and processing statistics are summarized in Table 4.

Table 4

X-ray data collection and refinement statistics

Crystal anti-pTau Fab phosphoepitope

Data collection

Space group Ρ2^2

Unit cell parameters

a (A) 68.5

b (A) 216.5

^*Values in parentheses are for the highest resolution shell.

†R_merge ⁼∑ ( I -< I > )/ ( I ) , where I is the observed intensity.

tRfactor and R_free =∑ ||F_obs-F_ca|_C||/∑ F_obs where R_free was calculated over 5% of the amplitude chosen at random and not used in the refinement.

§RMSD, root-mean-square deviation.

The analysis of co-crystal structures between antibodies and their antigens, combined with natural repertoire characterization, has greatly informed the understanding of antibody structure-function relationships (Almagro, 2004, J. Mol. Recognit. 17:132-143; Zemlin et al., 2003, J. Mol. Biol. 334:733- 749). These analyses have led to the recognition that the structural topography and chemical content of the paratope can be distinctly different between antibodies, depending on the size and nature of the antigen (MacCallum et al., 1996, J. Mol. Biol. 262:732-745). These structural insights have been critical to the adoption of the 'designer compound libraries' approach in antibody drug discovery (Valler & Green, 2000, Drug. Discov. Today 5:286-293), leading to the creation of synthetic antibody libraries that are tailored to recognize therapeutically or diagnostically relevant proteins (Almagro et al, 2006, J. Mol. Recognit. 19:413-422), haptens (Persson et al, 2012, J. Mol. Biol. 415:1 18-127) or peptides (Cobaugh et al., 2008, J. Mol. Biol. 378:622-633). The identification of the high affinity antibody pT231/pS235_1 facilitated the generation of the first Fab-phosphopeptide crystal structure. As truly phospho-specific monoclonal antibody generation is challenging (Brumbaugh et al., Methods Mol Biol 717:3-43 (201 1 )), it may be rare to find such clones with single-digit nanomolar 1 :1 binding affinity that subsequently aid the generation of high quality crystals. The high resolution pT231/pS235_1 co-crystal structure showed that the mechanism of interaction used by the antibody does not fit strictly into any one of the classical protein, peptide or hapten binding modes.

Example 8. Structure Solution and Refinement

The anti-ptau Fab-peptide complex was solved by molecular replacement using the PHASER (McCoy et al., 2007, J. Appl. Crystallogr. 40:658-674) program, with an ensemble of 3GJE, 3BN9, and 3KYM (pdb ID) for the heavy chain and 3MA9, 3G6D and 3H42 for the light chain, as the search models. The structure was refined using PHENIX (Adams et al., 2002, Acta. Crystallogr. D. Biol. Crystallogr. 58:1948-1954) in the beginning stage and finalized with BUSTER (Bricogne et al., 201 1 , Cambridge, UK: Global Phasing Ltd.). Bulk solvent correction was used. The 2F₀-F_C and F₀-F_c electron density maps were calculated for the inspection and improvement of the structure during refinement. The peptide was built at later stage of the refinement. Solvent molecules, as peaks greater than or equal to 3σ on the F₀-F_c electron density map with reasonable hydrogen bond networks, were included as water molecules. Graphic work was carried out using COOT (Emsley & Cowtan, 2004, Acta. Crystallogr. D. Biol. Crystallogr. 60:2126-2132). The structure was verified with annealed omit maps and assessed using PROCHECK (Laskowski et al., 1993, J. Appl. Crystallogr. 26:283-291 ). Illustrations were prepared with PyMOL (DeLano Scientific LLC).

In the Fab-peptide co-crystal structure, 10 amino acids (₂25KVAVVR-(pT)-PPK₂34) are visible, of which 6 (225KAVVR-(pT₂3i )) interact directly with the Fab. The remaining 8 residues of the peptide are disordered. The phosphoepitope adopts a specific conformation on top of the CDRs, with two sharp turns at V228 and pT231 (Fig. 5A & 5B). This conformation appears to be maintained by an intramolecular hydrogen bond between the side chain nitrogen of K225 and the carbonyl oxygen of V226 and by a water-mediated hydrogen bonding network between a phosphate oxygen and a side chain nitrogen of R230 (Fig. 5B). It is not clear whether this conformation reflects the phosphoepitope's natural state within full-length Tau, but the immunohistochemistry data shows that the antibody does recognize the phosphorylated epitope in the intact molecule (Fig. 4).

The mechanism of epitope recognition involves 8 hydrogen bonds (hb), 3 salt bridges, and 6 hydrophobic interactions (Fig. 6A). The phosphoepitope is dominated by the CDR-H2 and CDR-H3 from the heavy chain, with secondary support from CDR-H1 and the light chain. CDR-H3 provides a platform for the binding of the non-phosphorylated 225KVAVVK230 sequence, while the light chain CDRs form a hydrophobic wall to support binding of V228 and K225 (Fig. 5B & 6B). A total of 16 residues make contact with antigen (Fig. 6A), which is in agreement with the average number of contacts found in anti-peptide antibodies (-17) (Almagro, J Mol Recognit 17:132-143 (2004)). In contrast, the CDR-H3 makes 6 contacts, more than the average (4) and the CDR-L2 also makes contact with antigen which is unusual amongst antibodies to haptens or peptides, where the paratope is usually too small (Almagro, 2004, J. Mol. Recognit. 17:132-143).

The critical phosphorylation site (pT231 ) is exclusively recognized by CDR-H2 (Fig. 5C), which forms a positively charged pocket to accommodate the phosphate. The phosphate group forms hydrogen bonds with the backbone nitrogens of R53 and G55 and the side chain of T52, and forms a water- mediated hydrogen bonding network with S52A (Kabat numbering scheme; Kabat, Public Health Service, National Institutes of Health, Bethesda, MD). The side chain of R53 is well positioned to form a charge- charge interaction with the phosphate but the guanidinium moiety is not visible in the electron density, suggesting that this interaction is not stable. The fact that 4 of the 8 hydrogen bonds as well as the water mediated interaction between the Fab and the phosphoepitope are involved in phosphate recognition (Fig. 6A), might explain why the antibody does not bind to non-phosphorylated peptides with the same sequence (Fig. 4). Interestingly, a phosphate-binding motif in which the backbone nitrogens of consecutive glycines coordinate the bound phosphate has been reported in a number of enzymes (Andreeva et al., 2007, Nucleic. Acids. Res. 35:D253-259). The phosphate recognition motif from the CDR-H2 of pT231/pS235_1 also includes two consecutive Glycines (₅₂TSRGGV₅₆, Fig. 6A), demonstrating a surprising convergence between an evolutionarily conserved enzyme sequence motif and a sequence with similar function in an immunoglobulin selected by phage display.

In addition to the hydrogen bonding network between CDR-H2 and pT231 , three salt bridges appear to provide further anchor points for the phosphoepitope binding, forming two charge complementary areas around positively charged side chains of K225 and R230 (Fig. 6B). Two salt bridges (E100F-K225 and D100-R230) involve CDR-H3, and one salt bridge (D32-K225) engages CDR- L1. Only one interaction is found between CDR-H1 and the phosphoepitope (Fig. 6A & 6B).

Fab pT231/pS235_1 in complex with its cognate phosphoepitope represents the first chicken antibody structure to be deposited in the Protein Data Bank. The CDR sequences and conformations of this clone were therefore systematically compared with the canonical CDRs from humans and rodents, as classified by North et al. (North et al., 2011 , J. Mol. Biol. 406:228-256). CDR-H3 was not included in the analysis since it is known to have diverse lengths and structures (North et al., 201 1 , J. Mol. Biol. 406:228- 256). We found that the chicken CDR-L2, L3, H1 , and H2 all adhere to canonical conformations seen in mammalian structures, and could be classified into clusters L2-8, L3-11 , H1 -13, and H2-10, respectively (North et al., 201 1 , J. Mol. Biol. 406:228-256). In contrast, the chicken pT231/pS235_1 CDR-L1 has a conformation that is distinct from all canonical CDR-L1 clusters described in humans and rodents, to date (Fig. 7A). To establish whether or not this conformation was a repeated phenomenon in chicken antibodies, pT231/pS235_1 CDR-L1 was compared with three other chicken CDR-L1 structures from protein-binding antibodies. The data demonstrated 3 of the 4 antibodies examined share very similar conformations, thus defining a novel, chicken-specific canonical conformation. Disulfide bonds are often seen in chicken CDR-H3 loops and are proposed to play important roles in creating chicken paratopes of great stability and structural complexity in the context of a single framework repertoire (Wu et al., 201 1 , J. Immunol. 188:322-333). A large insertion within CDR-H3 (18 amino acids in length) forms a surface that makes numerous interactions with the non-phosphorylated part of the epitope (Fig. 6B). The disulfide bond between C100B and C100I may help to maintain and stabilize the compact conformation of this large loop (Fig. 7B). The combined CDR-H3 and CDR-L1 structures therefore create a unique binding site topology, not previously described in human or murine antibodies (Fig. 7C).

In general terms, anti-protein antibodies tend to use broad, flat interfaces with antigen (Almagro et al., 2006, J. Mol. Recognit. 19:413-422), while anti-hapten antibodies tend to have more limited interaction through a smaller paratope that is buried deep in the V_H-V_L interface (Persson et al., 2006, J. Mol. Biol. 357:607-620; Tars et al., 2012, J. Mol. Biol. 415:1 18-127). Peptide-binding antibodies meanwhile, are thought to use a 'grooved' paratope that is in-between the size of the surfaces observed in anti-protein and anti-hapten clones (Almagro, 2004, J. Mol. Recognit. 17:132-143). Antibody pT231/pS235_1 contains an unusual 'bowl like' recess in its CDR-H2 surface, into which the phosphate group of residue pT231 inserts. This stabilizes the specific interaction with the phosphopeptide (but not the unmodified peptide), through a network of hydrogen bond interactions. Antibody pT231/pS235_1 also utilizes a long, but highly structured, CDR-H3 loop to make the majority of its definitive anti-peptide contacts. Without wishing to be bound by any particular theory, given the issue of entropic penalty during peptide binding (Zahnd et al., 2004, J. Biol. Chem. 279:18870-18877), it seems likely that the rigid, disulphide-constrained structure of the CDR-H3 may be an additional important factor in the high affinity ligand interaction observed for pT231/pS235_1 .

The sequences disclosed herein are set forth in Table 5 below.

Table 5

Name SEQ SEQUENCE

ID NO

Tau (Gen bank accession 1 MAE PRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQ number NP_058519) TPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTE I P

EGTTAEEAGIGDTPSLEDEAAGHVTQE PESGKVVQEGFLREPGPPGLSH QLMSGMPGAPLLPEGPREATRQPSGTGPEDTEGGRHAPELLKHQLLGDL HQEGPPLKGAGGKERPGSKEEVDEDRDVDESSPQDSPPSKASPAQDGRP PQTAAREATS I PGFPAEGAI PLPVDFLSKVSTE I PASEPDGPSVGRAKG QDAPLEFTFHVE I TPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLG EDTKEADLPE PSEKQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDDKK AKTSTRSSAKTLKNRPCLSPKHPTPGSSDPLIQPSSPAVCPEPPSSPKH VSSVTSRTGSSGAKEMKLKGADGKTKIATPRGAAPPGQKGQANATRI PA KTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTRE PKKVAWRTPPKS PSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPG GGKVQI INKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTS KCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNI THVPGGGNK KIETHKLTFRENAKAKTDHGAEIVYKSPWSGDTSPRHLSNVSSTGS ID

MVDSPQLATLADEVSASLAKQGL

pS396/pS404 Phospho 2 EIVYK (pS) PWSGDT (pS) PRHLC

pS396/pS404 Non-phospho 3 EIVYKSPWSGDTSPRHLC

pS396/pS404 Scrambled 4 RIGVH (pS) PELKVSY (pS) PDVTC

pS422 Phospho 5 GSIDMVD (pS) PQLATLC

pS422 Non-phospho 6 GSIDMVDSPQLATLC

pS422 Scrambled 7 TLDLSAG (pS) PDMIQVC

pT212/pS214 Phospho 8 GSRSR (pT) P (pS) LPTPPTRC

pT212/pS214 Non-phospho 9 GSRSRTPSLPTPPTRC

pT212/pS214 Scrambled 10 TRGPS (pT) P (pS) PRSRTPLC

pT231/pS235 Phospho 11 KKVAWR (pT) PPK (pS) PSSAKC

pT231/pS235 Non-phospho 12 KKVAWRTPPKSPSSAKC

pT231/pS235 Scrambled 13 KAVKSKP (pT) PSR(pS) PAVKVC

pT231/pS235_1 VL 14 ALTQPTSVSANLGGSVE ITCSGSDYDYGWYQQKAPGSAPVTVIYWNDKR

PSDIPSRFSGSTSGSTSTLTITGVQAEDEAVYYCGAYDGSAGGGIFGAG TTLTVL

pT231/pS235_1 VH 15 AVTLDESGGGLQTPGGGLSLVCKASGFTLSSYQMMWVRQAPGKGLEWVA

GITSRGGVTGYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAK PALDSDQCGFPEAGCIDAWGHGTEVIVSS

pT231/pS235_2 VL 16 ALTQPTSVSANPGETVKITCSGDSSWYGWYQQKSPGSAPVTLIYNNNNR

PSNIPSRFSGSKSGSTATLTITGVQAEDEAVYFCGGHDSSYAGIFGAGT TLTVL

pT231/pS235_2 VH 17 AVTLDESGGGLQTPGGGLSLVCKASGFTLSSYQMMWVRQAPGKGLEWVA

GITSRGGVTGYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAK PALDSDQCGFPEAGCIDAWGHGTEVIVSS

pT212/pS214_1 VL 18 ALTQPTSVSANLGGTVKITCSGGGSYAGSYYYGWYQQKSPGSAPVTVIY

DNTNRPSNIPSRFSGSLSGSTSTLTITGVQAEDEAVYFCGGYDSSTYAG IFGAGTTLTVL

pT212/pS214_1 VH 19 AVTLDESGGGLQTPGGALSLVCKASGFTFSSFNMFWVRQAPGKGLEFVA

GIGKTGRSTYYGAAVKG

RATISRDNGQTTVRLQLNNLRAEDTGIYFCAKGATGPYI IDTWGHGTEV IVSS

pT212/pS214_2 VL 20 ALTQPTSVSANPGETVKITCSGGGSGYGYGWFQQKSPGSAPVTVIYYND

KRPSDIPSRFSGSTSGSTSTLTITGVQVEDEAVYYCGSGDSSGAGIFGA GTTLTVL

pT212/pS214_2 VH 21 AVTLDESGGGLQTPGGGLSLVCKASGFTFSSYPMVWVRQVPGKGLEWVA

DIDAAGSGTWYATAVKGRATISRDNGQSTLRLQLNNLRAEDTGTYFCAK TTPSTSGGWSVGSIDTWGHGTEVIVSS

pS396/pS404_1 VL 22 ALTQPTSVSANLGETVKITCSGESGNYGWYQQKAPGSAPVTVIYQNTQR

PSDIPSRFSGSKSGSTGTLTITGVQADDEAVYFCGSGDGNFVGIFGAGT TLTVL

pS396/pS404_1 VH 23 AVTLDESGGGLQTPGGGLSLVCKASGFTFSSYGMAWVRQAPGKGLEFVA

GISSSGTYTNYAPAVKGRATISRDNGQSTSRLQLNNLRAEDTGIYYCAK TTGRGAGEIDAWGHGTEVIVSS

pS396/pS404_2 VL 24 ALTQPTSVSANPGETVKITCSGGDSYYGWYQQKSPGSAPVTVIYDNDKR

PSDIPSRFSGSKSGSTGTLTITGVQADDEAVYFCGTEDRSGTPIFGAGT TLTVL

pS396/pS404_2 VH 25 DRALDESGGGLQTPGGTISLVCKASGFTMSSYGMNWVRQAPGKGLEWVG

VISSRGSSTGYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCGK DSGGRCGGRSYSGGCIDAWGHGTEVIVSS

pT231/pS235 1 VL CDR1 26 SGSDYDYG

pT231/pS235 1 VH CDR1 27 SYQMM

pT231/pS235 2 VL CDR1 28 SGDSSWYG

pT231/pS235 2 VH CDR1 29 SYQMM pT212/pS214 1 VL CDR1 30 SGGGSYAGSYYYG

pT212/pS214 1 VH CDR1 31 SFNMF

pT212/pS214 2 VL CDR1 32 SGGGSGYGYG

pT212/pS214 2 VH CDR1 33 SYPMV

pS396/pS404 1 VL CDR1 34 SGESGNYG

pS396/pS404 1 VH CDR1 35 SYGMA

pS396/pS404 2 VL CDR1 36 SGGDSYYG

pS396/pS404 2 VH CDR1 37 SYGMN

pT231/pS235 1 VL CDR2 38 WNDKRPS

pT231/pS235 1 VH CDR2 39 GITSRGGVTGYGSAVKG

pT231/pS235 2 VL CDR2 40 NNNNRPS

pT231/pS235 2 VH CDR2 41 GITSRGGVTGYGSAVKG

pT212/pS214 1 VL CDR2 42 DNTNRPS

pT212/pS214 1 VH CDR2 43 GIGKTGRSTYYGAAVKG

pT212/pS214 2 VL CDR2 44 YNDKRPS

pT212/pS214 2 VH CDR2 45 DIDAAGSGTWYATAVKG

pS396/pS404 1 VL CDR2 46 QNTQRPS

pS396/pS404 1 VH CDR2 47 GISSSGTYTNYAPAVKG

pS396/pS404 2 VL CDR2 48 DNDKRPS

pS396/pS404 2 VH CDR2 49 VISSRGSSTGYGAAVKG

pT231/pS235 1 VL CDR3 50 GAYDGSAGGGI

pT231/pS235 1 VH CDR3 51 PALDSDQCGFPEAGCIDA

pT231/pS235 2 VL CDR3 52 GGHDSSYAGI

pT231/pS235 2 VH CDR3 53 PALDSDQCGFPEAGCIDA

pT212/pS214 1 VL CDR3 54 GGYDSSTYAGI

pT212/pS214 1 VH CDR3 55 GATGPYI IDT

pT212/pS214 2 VL CDR3 56 GSGDSSGAGI

pT212/pS214 2 VH CDR3 57 TTPSTSGGWSVGSIDT

pS396/pS404 1 VL CDR3 58 GSGDGNFVGI

pS396/pS404 1 VH CDR3 59 TTGRGAGEIDA

pS396/pS404 2 VL CDR3 60 GTEDRSGTPI

pS396/pS404 2 VH CDR3 61 DSGGRCGGRSYSGGCIDA

Germline VL 62 ALTQPSSVSANPGGTVKITCSGDSSYYGWYQQKAPGSAPVTVIYDNTNR

PSNIPSRFSGSKSGSTATLTITGVRADDNAVYYCASTDSSTAGI

FGAGTTLTVL

Germline VH 63 AVTLDESGGGLQTPGRALSLVCKASGFTFS SYNMG

WVRQAPGKGLEFVA GIDNTGRYTGYGSAVKG RATISRDNGQSTVRLQLNNLRAEDTGTYYCAK AAGIDA WGHGTEVIVSS

Germline VL CDR1 64 SGDSSYYG

Germline VH CDR1 65 SYNMG

Germline VL CDR2 66 DNTNRPS

Germline VH CDR2 67 GIDNTGRYTGYGSAVKG

Germline VL CDR3 68 ASTDSSTAGI

Germline VH CDR3 69 AAGIDA

pT231/pS235 Heavy Chain 70 AVTLDESGGGLQTPGGGLSLVCKASGFTLSSYQMMWVRQAPGKGLEWVA

(VH-CH1 -CH2-CH3) GITSRGGVTGYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAK

PALDSDQCGFPEAGCIDAWGHGTEVIVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAA GAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

pT231/pS235 Light Chain (VL- 71 ALTQPTSVSANLGGSVE ITCSGSDYDYGWYQQKAPGSAPVTVIYWNDKR CL, lambda) PSDIPSRFSGSTSGSTSTLTITGVQAEDEAVYYCGAYDGSAGGGIFGAG

TTLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK ADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTEC

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS We claim:

1. An isolated antibody, or an antigen-binding fragment thereof, wherein the antibody binds to human phosphorylated-tau (phospho-tau) with a K_D of 1 x10^"7 M or less, and further wherein the antibody does not substantially bind to human non-phosphorylated-tau.

2. The antibody of claim 1 , wherein the antibody is a chicken monoclonal antibody.

3. The antibody of claim 2, wherein the antibody is chimeric or humanized.

4. The antibody of claim 1 , wherein the antibody, or antigen-biding fragment thereof, is selected from the group consisting of a Fab, a F(ab')2, a Fd, a Fv, a dAb, a CDR, a disulfide-linked Fv (dsFv), a scFv, and a diabody.

5. The antibody of claim 1 , wherein the V_H and V_L regions are derived from chicken antibodies.

6. The antibody of claim 5, wherein the V_H and V_L regions are derived from a chicken antibody and the constant regions are derived from human IgG C_H and a human kappa C_L or human lambda C_L.

7. The antibody of claim 2, wherein human phospho-tau comprises the amino acid sequence as set forth in SEQ ID NO: 1 , wherein the protein is phosphorylated at least one amino acid selected from the group consisting of threonine 212 (T212), serine 214 (S214), threonine 231 (T231 ), serine 235 (S235), serine 396 (S396), serine 404 (S404), and serine 422 (S422) where the numbering of the amino acid residues is relative to the amino acid sequence provided in SEQ ID NO: 1.

8. The antibody of claim 7, wherein the human phospho-tau comprises at least one phosphorylated amino acid selected from T231 and S235 relative to the numbering of the amino acid sequence of SEQ ID NO: 1 .

9. The antibody of claim 8, wherein the human phospho-tau comprises a phosphorylated threonine 231 relative to the numbering of the amino acid sequence of SEQ ID NO:1.

10. The isolated antibody, or antigen binding portion thereof, of claim 2, which binds to a human phospho-tau peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 5, 8, and 1 1 , wherein the peptide does not comprise the C-terminal cysteine, but does not bind to a human non-phosphorylated-tau peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:3, 6, 9, or 12, wherein the peptide does not comprise the C-terminal cysteine.

11. An isolated monoclonal antibody, or antigen binding portion thereof, comprising:

(f) a light chain variable region comprising the amino acid sequence of SEQ ID NO:22 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23; and (g) a light chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25.

12. An isolated monoclonal antibody, or antigen binding portion thereof, comprising:

a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:27, 29, 31 , 33, 35 and 37;

a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:39, 41 , 43, 45, 47, and 49; and

(b) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 26, the CDR2 sequence of SEQ ID NO: 38, and the CDR3 sequence of SEQ ID NO: 50, and a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 27, the CDR2 sequence of SEQ ID NO: 39, and the CDR3 sequence of SEQ ID NO: 51 ; (c) a light chain variable region comprising the CDR1 sequence of SEQ ID NO: 28, the CDR2 sequence of SEQ ID NO: 40, and the CDR3 sequence of SEQ ID NO: 52, and a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO: 29, the CDR2 sequence of SEQ ID NO: 41 , and the CDR3 sequence of SEQ ID NO: 53;

13. The isolated antibody of claim 10, wherein the antibody binds to human phospho-tau with a K_D of 1x10^"7 M or less, and does not substantially bind to human non-phosphorylated-tau, where the phospho- tau comprises a phosphorylated threonine at position 231 relative to the amino acid sequence of SEQ ID NO:1.

14. A composition comprising the antibody, or antigen-binding portion thereof, of claim 1 , and a pharmaceutically acceptable carrier.

15. An isolated nucleic acid molecule encoding the antibody of claim 1 , or an antigen-binding portion thereof.

16. An expression vector comprising the nucleic acid molecule of claim 15.

17. A host cell comprising the expression vector of claim 16.

18. A method for producing a monoclonal antibody or an antigen-binding portion thereof that specifically bindings to human phospho-tau but does not bind to human non-phosphorylated-tau, comprising culturing the host cell according to claim 20 under suitable conditions and recovering said antibody or antigen-binding portion.

19. A method for detecting the presence of human phospho-tau in a sample, comprising:

a) contacting a sample suspected of comprising phospho-tau with the antibody of claim 1 ; and b) detecting the presence of a phospho-tau bound with the antibody thereby detecting phospho-tau in the sample.

20. A kit for detecting the presence of human phospho-tau in a sample, the kit comprising the antibody of claim 1 , an applicator, and an instructional material for the use thereof.